AU2005201389A1

AU2005201389A1 - RNA interference mediated treatment of polyglutamine (polyQ) repeat expansion diseases using short interfering nucleic acid (siNA)

Info

Publication number: AU2005201389A1
Application number: AU2005201389A
Authority: AU
Inventors: James Mcswiggen
Original assignee: Sirna Therapeutics Inc
Current assignee: Sirna Therapeutics Inc
Priority date: 2004-02-20
Filing date: 2005-04-01
Publication date: 2005-09-08

Description

S&F Ref: 715679

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT Name and Address of Applicant: Actual Inventor(s): Address for Service: Invention Title: Sima Therapeutics, Inc., of 2950 Wilderness Place, Boulder, Colorado, 80301, United States of America James McSwiggen Spruson Ferguson St Martins Tower Level 31 Market Street Sydney NSW 2000 (CCN 3710000177) RNA interference mediated treatment of polyglutamine (polyQ) repeat expansion diseases using short interfering nucleic acid (siNA) The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5845c t RNA INTERFERENCE MEDIATED TREATMENT OF POLYGLUTAMINE O (POLYQ) REPEAT EXPANSION DISEASES USING SHORT INTERFERING NUCLEIC ACID (siNA) a This application is a continuation-in-part of U.S. Patent Application No.

10/824,036, filed April 14, 2004, which is continuation-in-part of U.S. Patent Application No. 10/783,128, filed February 20, 2004. This patent application is also a continuation-in-part of U.S. Patent Application No. 10/923,536, filed August 20, 2004, 00 M which is continuation-in-part of International Patent Application No. PCT/US04/16390, O filed May 24, 2004, which is a continuation-in-part of U.S. Patent Application No.

10/826,966, filed April 16, 2004, which is continuation-in-part of U.S. Patent Application No. 10/757,803, filed January 14, 2004, which is a continuation-in-part of U.S. Patent Application No. 10/720,448, filed November 24, 2003, which is a continuation-in-part of U.S. Patent Application No. 10/693,059, filed October 23, 2003, which is a continuation-in-part of U.S. Patent Application No. 10/444,853, filed May 23, 2003, which is a continuation-in-part of International Patent Application No.

PCT/US03/05346, filed February 20, 2003, and a continuation-in-part of International Patent Application No. PCT/US03/05028, filed February 20, 2003, both of which claim the benefit of U.S. Provisional Application No. 60/358,580 filed February 20, 2002, U.S.

Provisional Application No. 60/363,124 filed March 11, 2002, U.S. Provisional Application No. 60/386,782 filed June 6, 2002, U.S. Provisional Application No.

60/406,784 filed August 29, 2002, U.S. Provisional Application No. 60/408,378 filed September 5, 2002, U.S. Provisional Application No. 60/409,293 filed September 9, 2002, and U.S. Provisional Application No. 60/440,129 filed January 15, 2003. This application is also a continuation-in-part of International Patent Application No.

PCT/US04/13456, filed April 30, 2004, which is a continuation-in-part of Patent Application No. 10/780,447, filed February 13, 2004, which is a continuation-in-part of US Patent Application No. 10/427,160, filed April 30, 2003, which is a continuation-inpart of International Patent Application No. PCT/US02/15876 filed May 17, 2002, which claims the benefit of U.S. Provisional Application No. 60/362,016, filed March 6, 2002, U.S. Provisional Application No. 60/292,217, filed May 18, 2001, U.S. Provisional Application No. 60/306,883 filed July 20, 2001, and U.S. Provisional Application No.

60/311,865 filed August 13, 2001. This application is also a continuation-in-part of U.S.

Patent Application No. 10/727,780 filed December 3, 2003. This application also claims t the benefit of U.S. Provisional Application No. 60/543,480 filed February 10, 2004.

,i The instant application claims the benefit of all the listed applications, which are hereby Sincorporated by reference herein in their entireties, including the drawings.

Field Of The Invention The present invention concerns compounds, compositions, and methods for the 00oO study, diagnosis, and treatment of diseases and conditions associated with polyglutamine repeat (polyQ) allelic variants that respond to the modulation of gene expression and/or ,In activity. The present invention also concerns compounds, compositions, and methods relating to diseases and conditions associated with polyglutamine repeat (polyQ) allelic S 10 variants that respond to the modulation of expression and/or activity of genes involved in polyQ repeat gene expression pathways or other cellular processes that mediate the maintenance or development of polyQ repeat diseases and conditions. 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) against the expression disease related genes or alleles having polyQ repeat sequences.

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.

RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs) (Zamore et al., 2000, Cell, 101, 25-33; Fire et al., 1998, Nature, 391, 806; Hamilton et al., 1999, Science, 286, 950-951; Lin et al., 1999, Nature, 402, 128-129; Sharp, 1999, Genes Dev., 13:139-141; and Strauss, 1999, Science, 286, 886). The corresponding process in plants (Heifetz et al., International PCT Publication No. WO 99/61631) 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 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 through a mechanism that has yet to be fully characterized. This mechanism appears to be different from other known mechanisms involving double stranded RNA-specific ribonucleases, such as the interferon response that results from dsRNA-mediated activation of protein kinase PKR and 2',5'-oligoadenylate synthetase resulting in nonspecific cleavage of mRNA by ribonuclease L (see for example US Patent Nos.

6,107,094; 5,898,031; Clemens et al., 1997, J. Interferon Cytokine Res., 17, 503-524; Adah et al., 2001, Curr. Med. Chem., 8, 1189).

The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as dicer (Bass, 2000, Cell, 101, 235; Zamore et al., 2000, Cell, 101, 25-33; Hammond et al., 2000, Nature, 404, 293). Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs) (Zamore et al., 2000, Cell, 101, 25-33; Bass, 2000, Cell, 101, 235; 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 (Zamore et al., 2000, Cell, 101, 25-33; 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 siRNA duplex. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex (Elbashir et al., 2001, Genes Dev., 15, 188).

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. Bahramian and Zarbl, 1999, Molecular and Cellular Biology, 19, 274-283 and Wianny and Goetz, 1999, Nature Cell Biol., 2, 3 describe RNAi mediated by dsRNA in mammalian systems. Hammond et al., 2000, Nature, 404, 293, describe RNAi in Drosophila cells transfected with dsRNA. Elbashir 2 et al., 2001, Nature, 411, 494 and Tuschl et al., International PCT Publication No. WO S01/75164, 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 20, 6877 00 and Tuschl et al., International PCT Publication No. WO 01/75164) has revealed certain Srequirements for siRNA length, structure, chemical composition, and sequence that are essential to mediate efficient RNAi activity. These studies have shown that 21- 0 10 nucleotide siRNA duplexes are most active when containing 3'-terminal dinucleotide C1 overhangs. Furthermore, complete substitution of one or both siRNA strands with 2'deoxy or 2'-O-methyl nucleotides abolishes RNAi activity, whereas substitution of the 3'-terminal siRNA overhang nucleotides with 2'-deoxy nucleotides 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 target RNA is defined by the 5'-end of the siRNA guide sequence rather than the 3'-end of the guide sequence (Elbashir et al., 2001, EMBO J., 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).

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 20, 6877 and Tuschl et al., International PCT Publication No. WO 01/75164). In addition, Elbashir et al., 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 al., International PCT Publication No. WO 01/68836 preliminarily suggest that siRNA may include modifications to either the phosphate-sugar backbone or the nucleoside to include at least one of a nitrogen or sulfur heteroatom, however, neither application Spostulates to what extent such modifications would be tolerated in siRNA molecules, nor Sprovides any further guidance or examples of such modified siRNA. Kreutzer et al., SCanadian Patent Application No. 2,359,180, also describe certain chemical modifications for use in dsRNA constructs in order to counteract activation of double-stranded RNAdependent protein kinase PKR, specifically 2'-amino or 2'-O-methyl nucleotides, and nucleotides containing a 2'-O or 4'-C methylene bridge. However, Kreutzer et al.

similarly fails to provide examples or guidance as to what extent these modifications c would be tolerated in dsRNA molecules.

n Parrish et al., 2000, Molecular Cell, 6, 1077-1087, 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-thiouracil, 5-bromouracil, 5-iodouracil, and 3-(aminoallyl)uracil for uracil, and inosine for guanosine. Whereas 4-thiouracil and 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-iodouracil and 3-(aminoallyl)uracil in the antisense strand resulted in a substantial decrease in RNAi activity as well.

The use of longer dsRNA has been described. For example, Beach et al., International PCT Publication No. WO 01/68836, describes specific methods for attenuating gene expression using endogenously-derived dsRNA. Tuschl et al., 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 t certain therapeutic applications; although Tuschl, 2001, Chem. Biochem., 2, 239-245, N doubts that RNAi can be used to cure genetic diseases or viral infection due to the danger Sof activating interferon response. Li et al., International PCT Publication No. WO 00/44914, describe the use of specific long (141 bp-488 bp) enzymatically synthesized or vector expressed dsRNAs for attenuating the expression of certain target genes.

Zernicka-Goetz et al., International PCT Publication No. WO 01/36646, describe certain 00 methods for inhibiting the expression of particular genes in mammalian cells using Scertain long (550 bp-714 bp), enzymatically synthesized or vector expressed dsRNA I molecules. Fire et al., International PCT Publication No. WO 99/32619, describe 0 10 particular methods for introducing certain long dsRNA molecules into cells for use in (N inhibiting gene expression in nematodes. 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 long dsRNA molecules.

Mello et al., International PCT Publication No. WO 01/29058, describe the identification of specific genes involved in dsRNA-mediated RNAi. Pachuck et al., International PCT Publication No. WO 00/63364, describe certain long (at least 200 nucleotide) dsRNA constructs. Deschamps Depaillette et al., International PCT Publication No. WO 99/07409, describe specific compositions consisting of particular dsRNA molecules combined with certain anti-viral agents. Waterhouse et al., International PCT Publication No. 99/53050 and 1998, PNAS, 95, 13959-13964, 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 expression 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, 1077-1087, describe specific chemically-modified dsRNA 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. WO 01/68836, describe certain methods for gene silencing in plants. Honer et al., SInternational PCT Publication No. WO 01/70944, describe certain methods of drug screening using transgenic nematodes as Parkinson's Disease models using certain dsRNAs. Deak et al., International PCT Publication No. WO 01/72774, describe certain Drosophila-derived gene products that may be related to RNAi in Drosophila. Arndt et al., International PCT Publication No. WO 01/92513 describe certain methods for mediating gene suppression by using factors that enhance RNAi. Tuschl et al., International PCT Publication No. WO 02/44321, describe certain synthetic siRNA constructs. Pachuk et al., International PCT Publication No. WO 00/63364, and C( Satishchandran et al., International PCT Publication No. WO 01/04313, describe certain methods and compositions for inhibiting the function of certain polynucleotide sequences using certain long (over 250 bp), vector expressed dsRNAs. Echeverri et al., International PCT Publication No. WO 02/38805, describe certain C. elegans genes identified via RNAi. Kreutzer et al., International PCT Publications Nos. WO 02/055692, WO 02/055693, and EP 1144623 Bl describes certain methods for inhibiting gene expression using dsRNA. Graham et al., International PCT Publications Nos. WO 99/49029 and WO 01/70949, and AU 4037501 describe certain vector expressed siRNA molecules. Fire et al., US 6,506,559, describe certain methods for inhibiting gene expression in vitro using certain long dsRNA (299 bp-1033 bp) constructs that mediate RNAi. Martinez et al., 2002, Cell, 110, 563-574, describe certain single stranded siRNA constructs, including certain 5'-phosphorylated single stranded siRNAs that mediate RNA interference in Hela cells. Harborth et al., 2003, Antisense Nucleic Acid Drug Development, 13, 83-105, describe certain chemically and structurally modified siRNA molecules. Chiu and Rana, 2003, RNA, 9, 1034-1048, describe certain chemically and structurally modified siRNA molecules. Woolf et al., International PCT Publication Nos. WO 03/064626 and WO 03/064625 describe certain chemically modified dsRNA constructs. Miller et al., 2003, PNAS, 100, 7195-7200, describe certain transcribed siRNA molecules targeting certain allele specific RNA transcripts associated with trinucleotide reapeat/polyQ nuerodegenerative disorders such as Machado Joseph Disease, spinocerebellar ataxia, and frontotemporaral dementia. Davidson et al., WO 04/013280, describe certain siRNA molecules targeting certain allele specific RNA transcripts including certain polyQ repeat gene transcripts associated with certain 7 neurodegenerative diseases. Xia et al., 2004, Nature Medicine, 10, 816 820, describe RNAi suppressesion of polyglutamine-induced neurodegeneration in a model of spinocerebellar ataxia.

SUMMARY OF THE INVENTION This invention relates to compounds, compositions, and methods useful for modulating the expression of repeat expansion genes associated with the maintenance or development of neurodegenerative disease, for example polyglutamine repeat expansion genes and variants thereof, including single nucleotide polymorphism (SNP) variants associated with disease related trinucleotide repeat expansion genes, using short interfering nucleic acid (siNA) molecules. This invention also relates to compounds, compositions, and methods useful for modulating the expression and activity of repeat expansion genes, or other genes involved in pathways of repeat expansion genes expression and/or activity by RNA interference (RNAi) using small nucleic acid molecules. In particular, the instant invention features small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), doublestranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules and methods used to modulate the expression repeat expansion genes.

A siNA of the invention can be unmodified or chemically-modified. A siNA of the instant invention can be chemically synthesized, expressed from a vector or enzymatically synthesized. The instant invention also features various chemicallymodified synthetic short interfering nucleic acid (siNA) molecules capable of modulating repeat expansion (RE) gene expression or activity in cells by RNA interference (RNAi).

The use of chemically-modified siNA improves various properties of native siNA molecules through increased resistance to nuclease degradation in vivo and/or through improved cellular uptake. Further, contrary to earlier published studies, siNA having multiple chemical modifications retains its RNAi activity. The siNA molecules of the instant invention provide useful reagents and methods for a variety of therapeutic, cosmetic, veterinary, diagnostic, target validation, genomic discovery, genetic engineering, and pharmacogenomic applications.

In one embodiment, the invention features one or more siNA molecules and methods that independently or in combination modulate the expression of repeat expansion genes encoding proteins, such as proteins comprising polyglutamine repeat I expansions, associated with the maintenance and/or development of neurodegenerative Sdiseases, such as genes encoding sequences comprising those sequences referred to by GenBank Accession Nos. shown in Table I, referred to herein generally as repeat expansion (RE) genes. The description below of the various aspects and embodiments of the invention is provided with reference to exemplary Huntingtin gene referred to herein 00 as HD. However, the various aspects and embodiments are also directed to other repeat Sexpansion genes, such spinocerebellar ataxia genes including SCA1, SCA2, SCA3, SCA5, SCA7, SCA12, and SCA17, spinal and bulbar muscular atrophy genes such as androgen receptor (AR) locus Xqll-ql2 genes, and dentatorubropallidoluysian atrophy CNI genes such as DRPLA, as well as other mutant gene variants having trinucleotide repeat expansions and SNPs associated with such trinucleotide repeat expansions. The various aspects and embodiments are also directed to other genes that are involved in RE mediated pathways of signal transduction or gene expression that are involved in the progression, development, and/or maintenance of disease Huntington disease, spinocerebellar ataxia, spinal and bulbar muscular dystrophy, and dentatorubropallidoluysian atrophy), including enzymes involved in processing RE proteins. These additional genes can be analyzed for target sites using the methods described for HD genes herein. Thus, the modulation of other genes and the effects of such modulation of the other genes can be performed, determined, and measured as described herein.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a repeat expansion (RE) gene, wherein said siNA molecule comprises about 15 to about 28 base pairs.

In one embodiment, the invention features a double stranded short interfering nucleic acid (siNA) molecule that directs cleavage of a repeat expansion (RE) RNA via RNA interference (RNAi), wherein the double stranded siNA molecule comprises a first and a second strand, each strand of the siNA molecule is about 18 to about 28 nucleotides in length, the first strand of the siNA molecule comprises nucleotide sequence having sufficient complementarity to the repeat expansion (RE) RNA for the siNA molecule to direct cleavage of the repeat expansion (RE) RNA via RNA interference, and the second strand of said siNA molecule comprises nucleotide sequence 9 that is complementary to the first strand. The repeat expansion (RE) RNA can be Sderived from a gene, for example, huntingtin, SCA1, SCA2, SCA3, SCA6, SCA7, SSCA12, SCA17, SBMA, or DRPLA (see for example Table including both mutant and wild-type alleles thereof.

In one embodiment, the invention features a double stranded short interfering 0 nucleic acid (siNA) molecule that directs cleavage of a repeat expansion (RE) RNA via 00 RNA interference (RNAi), wherein the double stranded siNA molecule comprises a first 0and a second strand, each strand of the siNA molecule is about 18 to about 23 t nucleotides in length, the first strand of the siNA molecule comprises nucleotide 0 10 sequence having sufficient complementarity to the repeat expansion (RE) RNA for the siNA molecule to direct cleavage of the repeat expansion (RE) RNA via RNA interference, and the second strand of said siNA molecule comprises nucleotide sequence that is complementary to the first strand. The repeat expansion (RE) RNA can be derived from a gene, for example, huntingtin, SCAl, SCA2, SCA3, SCA6, SCA7, SCA12, SCA17, SBMA, or DRPLA (see for example Table including both mutant and wild-type alleles thereof.

In one embodiment, the invention features a chemically synthesized double stranded short interfering nucleic acid (siNA) molecule that directs cleavage of a repeat expansion (RE) RNA via RNA interference (RNAi), wherein each strand of the siNA molecule is about 18 to about 28 nucleotides in length; and one strand of the siNA molecule comprises nucleotide sequence having sufficient complementarity to the repeat expansion (RE) RNA for the siNA molecule to direct cleavage of the repeat expansion (RE) RNA via RNA interference. The repeat expansion (RE) RNA can be derived from a gene, for example, huntingtin, SCA1, SCA2, SCA3, SCA6, SCA7, SCA12, SCA17, SBMA, or DRPLA (see for example Table including both mutant and wild-type alleles thereof.

In one embodiment, the invention features a chemically synthesized double stranded short interfering nucleic acid (siNA) molecule that directs cleavage of a repeat expansion (RE) RNA via RNA interference (RNAi), wherein each strand of the siNA molecule is about 18 to about 23 nucleotides in length; and one strand of the siNA molecule comprises nucleotide sequence having sufficient complementarity to the repeat Sexpansion (RE) RNA for the siNA molecule to direct cleavage of the repeat expansion RNA via RNA interference. The repeat expansion (RE) RNA can be derived from a gene, for example, huntingtin, SCA1, SCA2, SCA3, SCA6, SCA7, SCA12, SCA17, SBMA, or DRPLA (see for example Table including both mutant and wild-type alleles thereof.

In one embodiment, the invention features a siNA molecule that down-regulates 00 expression of a repeat expansion (RE) gene or that directs cleavage of a repeat expansion (RE) RNA, for example, wherein the repeat expansion (RE) gene or RNA comprises Srepeat expansion (RE) encoding sequence. In one embodiment, the invention features a siNA molecule that down-regulates expression of a repeat expansion (RE) gene or that directs cleavage of a repeat expansion (RE) RNA, for example, wherein the repeat expansion (RE) gene or RNA comprises repeat expansion (RE) non-coding sequence or regulatory elements involved in repeat expansion (RE) gene expression.

In one embodiment, a siNA of the invention is used to inhibit the expression of repeat expansion (RE) genes or a repeat expansion (RE) gene family, wherein the genes or gene family sequences share sequence homology. Such homologous sequences can be identified as is known in the art, for example using sequence alignments. siNA molecules can be designed to target such homologous sequences, for example using perfectly complementary sequences or by incorporating non-canonical base pairs, for example mismatches and/or wobble base pairs, that can provide additional target sequences. In instances where mismatches are identified, non-canonical base pairs (for example, mismatches and/or wobble bases) can be used to generate siNA molecules that target more than one gene sequence. In a non-limiting example, non-canonical base pairs such as UU and CC base pairs are used to generate siNA molecules that are capable of targeting sequences for differing repeat expansion (RE) targets that share sequence homology. As such, one advantage of using siNAs of the invention is that a single siNA can be designed to include nucleic acid sequence that is complementary to the nucleotide sequence that is conserved between the homologous genes. In this approach, a single siNA can be used to inhibit expression of more than one gene instead of using more than one siNA molecule to target the different genes.

In one embodiment, the invention features a siNA molecule having RNAi activity (against repeat expansion (RE) RNA, wherein the siNA molecule comprises a sequence Scomplementary to any RNA having repeat expansion (RE) encoding sequence, such as those sequences having GenBank Accession Nos. shown in Table I. In another embodiment, the invention features a siNA molecule having RNAi activity against repeat expansion (RE) RNA, wherein the siNA molecule comprises a sequence complementary 00 to an RNA having variant repeat expansion (RE) encoding sequence, for example other Smutant repeat expansion (RE) genes not shown in Table I but known in the art to be (N associated with the maintenance and/or development of Huntington disease, spinocerebellar ataxia, spinal and bulbar muscular dystrophy, and (N dentatorubropallidoluysian atrophy. Chemical modifications as shown in Tables III and IV or otherwise described herein can be applied to any siNA construct of the invention.

In another embodiment, a siNA molecule of the invention includes a nucleotide sequence that can interact with nucleotide sequence of a repeat expansion (RE) gene and thereby mediate silencing of repeat expansion (RE) gene expression, for example, wherein the siNA mediates regulation of repeat expansion (RE) gene expression by cellular processes that modulate the chromatin structure or methylation patterns of the repeat expansion (RE) gene and prevent transcription of the repeat expansion (RE) gene.

In one embodiment, siNA molecules of the invention are used to down regulate or inhibit the expression of proteins arising from repeat expansion (RE) haplotype polymorphisms that are associated with a trait, disease or condition such as Huntington disease, spinocerebellar ataxia, spinal and bulbar muscular dystrophy, and dentatorubropallidoluysian atrophy in a subject or organism. Analysis of genes, or protein or RNA levels can be used to identify subjects with such repeat expansion genes and/or polymorphisms or those subjects who are at risk of developing traits, conditions, or diseases described herein, such as Huntington disease. These subjects are amenable to treatment, for example, treatment with siNA molecules of the invention and any other composition useful in treating diseases related to repeat expansion (RE) gene expression.

As such, analysis of repeat expansion (RE) protein or RNA levels can be used to determine treatment type and the course of therapy in treating a subject. Monitoring of repeat expansion (RE) protein or RNA levels can be used to predict treatment outcome and to determine the efficacy of compounds and compositions that modulate the level t and/or activity of certain repeat expansion (RE) proteins associated with a trait, condition, or disease.

In one embodiment, siNA molecules of the invention are used to down regulate or inhibit the expression of mutant repeat expansion (RE) proteins that are neurotoxic, such as mutant repeat expansion (RE) proteins resulting from polyglutamine repeat C, expansions and fragments or portions of such mutant repeat expansion (RE) proteins that 00 M€ are processed by cellular enzymes resulting in neurotoxic proteins or peptides.

In one embodiment of the invention a siNA molecule comprises an antisense strand comprising a nucleotide sequence that is complementary to a nucleotide sequence S 10 or a portion thereof encoding a repeat expansion (RE) protein. The siNA further comprises a sense strand, wherein said sense strand comprises a nucleotide sequence of a repeat expansion (RE) gene or a portion thereof.

In another embodiment, a siNA molecule comprises an antisense region comprising a nucleotide sequence that is complementary to a nucleotide sequence encoding a repeat expansion (RE) protein or a portion thereof. The siNA molecule further comprises a sense region, wherein said sense region comprises a nucleotide sequence of a repeat expansion (RE) gene or a portion thereof.

In another embodiment, the invention features a siNA molecule comprising nucleotide sequence, for example, nucleotide sequence in the antisense region of the siNA molecule that is complementary to a nucleotide sequence or portion of sequence of a repeat expansion (RE) gene. In another embodiment, the invention features a siNA molecule comprising a region, for example, the antisense region of the siNA construct, complementary to a sequence comprising a repeat expansion (RE) gene sequence or a portion thereof.

In one embodiment, the antisense region of siNA constructs comprises a sequence complementary to sequence having any of target SEQ ID NOs. shown in Tables II and III. In one embodiment, the antisense region of siNA constructs of the invention comprises sequence having any of antisense (lower) SEQ ID NOs. in Tables II and III and Figures 4 and 5. In another embodiment, the sense region of siNA constructs of the invention comprises sequence having any of sense (upper) SEQ ID NOs. in Tables II and III and Figures 4 and In one embodiment, a siNA molecule of the invention comprises any of SEQ ID NOs. 1-3575. The sequences shown in SEQ ID NOs: 1-3575 are not limiting. A siNA molecule of the invention can comprise any contiguous repeat expansion (RE) sequence about 15 to about 25 or more, or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 00 Mc, or more contiguous repeat expansion (RE) nucleotides).

,IC In yet another embodiment, the invention features a siNA molecule comprising a sequence, for example, the antisense sequence of the siNA construct, complementary to a ,I 10 sequence or portion of sequence comprising sequence represented by GenBank Accession Nos. shown in Table I. Chemical modifications in Tables III and IV and described herein can be applied to any siNA construct of the invention.

In one embodiment of the invention a siNA molecule comprises an antisense strand having about 15 to about 30 about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 27, 28, 29, or 30) nucleotides, wherein the antisense strand is complementary to a RNA sequence or a portion thereof encoding repeat expansion and wherein said siNA further comprises a sense strand having about 15 to about 30 about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, and wherein said sense strand and said antisense strand are distinct nucleotide sequences where at least about 15 nucleotides in each strand are complementary to the other strand.

In another embodiment of the invention a siNA molecule of the invention comprises an antisense region having about 15 to about 30 about 15, 16, 17, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, wherein the antisense region is complementary to a RNA sequence encoding repeat expansion and wherein said siNA further comprises a sense region having about 15 to about 30 about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, wherein said sense region and said antisense region are comprised in a linear molecule where the sense region comprises at least about 15 nucleotides that are complementary to the antisense region.

In one embodiment, a siNA molecule of the invention has RNAi activity that 0 modulates expression of RNA encoded by a repeat expansion (RE) gene. Because repeat Sexpansion (RE) genes can share some degree of sequence homology with each other, siNA molecules can be designed to target a class of repeat expansion (RE) genes or alternately specific repeat expansion (RE) genes polymorphic variants) by selecting sequences that are either shared amongst different repeat expansion (RE) targets or 00 alternatively that are unique for a specific repeat expansion (RE) target. Therefore, in Sone embodiment, the siNA molecule can be designed to target conserved regions of Ni, repeat expansion (RE) RNA sequences having homology among several repeat expansion (RE) gene variants so as to target a class of repeat expansion (RE) genes with one siNA molecule RE variants having differing trinucleotide repeat expansions).

Accordingly, in one embodiment, the siNA molecule of the invention modulates the expression of one or both alleles of a repeat expansion (RE) associated gene both mutant and wildtype HD alleles) in a subject. In another embodiment, the siNA molecule can be designed to target a sequence that is unique to a specific RE RNA sequence a single repeat expansion allele or repeat expansion SNP) due to the high degree of specificity that the siNA molecule requires to mediate RNAi activity. As such, in one embodiment, a siNA molecule of the invention is used to target only the mutant repeat expansion (RE) allele mutant HD allele) in a subject or organism.

In one embodiment, nucleic acid molecules of the invention that act as mediators of the RNA interference gene silencing response are double-stranded nucleic acid molecules. In another embodiment, the siNA molecules of the invention consist of duplex nucleic acid molecules containing about 15 to about 30 base pairs between oligonucleotides comprising about 15 to about 30 about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides. In yet another embodiment, siNA molecules of the invention comprise duplex nucleic acid molecules with overhanging ends of about 1 to about 3 about 1, 2, or 3) nucleotides, for example, about 21nucleotide duplexes with about 19 base pairs and 3'-terminal mononucleotide, dinucleotide, or trinucleotide overhangs. In yet another embodiment, siNA molecules of the invention comprise duplex nucleic acid molecules with blunt ends, where both ends are blunt, or alternatively, where one of the ends is blunt.

I

SIn one embodiment, the invention features one or more chemically-modified siNA constructs having specificity for repeat expansion (RE) expressing nucleic acid f molecules, such as RNA encoding a repeat expansion (RE) protein or non-coding RNA associated with the expression of repeat expansion (RE) genes. In one embodiment, the 0 5 invention features a RNA based siNA molecule a siNA comprising 2'-OH nucleotides) having specificity for repeat expansion (RE) expressing nucleic acid 0 molecules that includes one or more chemical modifications described herein. Non- Slimiting examples of such chemical modifications include without limitation phosphorothioate intemucleotide linkages, 2'-deoxyribonucleotides, 2'-O-methyl 0 10 ribonucleotides, 2'-deoxy-2'-fluoro ribonucleotides, 4'-thio ribonucleotides, 2'-O- C trifluoromethyl nucleotides, 2'-0-ethyl-trifluoromethoxy nucleotides, difluoromethoxy-ethoxy nucleotides (see for example USSN 10/981,966 filed November 2004, incorporated by reference herein), "universal base" nucleotides, "acyclic" nucleotides, 5-C-methyl nucleotides, and terminal glyceryl and/or inverted deoxy abasic residue incorporation. These chemical modifications, when used in various siNA constructs, RNA based siNA constructs), are shown to preserve RNAi activity in cells while at the same time, dramatically increasing the serum stability of these compounds. Furthermore, contrary to the data published by Parrish et al., supra, applicant demonstrates that multiple (greater than one) phosphorothioate substitutions are well-tolerated and confer substantial increases in serum stability for modified siNA constructs.

In one embodiment, a siNA molecule of the invention comprises modified nucleotides while maintaining the ability to mediate RNAi. The modified nucleotides can be used to improve in vitro or in vivo characteristics such as stability, activity, toxicity, immune response, and/or bioavailability. For example, a siNA molecule of the invention can comprise modified nucleotides as a percentage of the total number of nucleotides present in the siNA molecule. As such, a siNA molecule of the invention can generally comprise about 5% to about 100% modified nucleotides about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 95% or 100% modified nucleotides). The actual percentage of modified nucleotides present in a given siNA molecule will depend on the total number of nucleotides present in the siNA. If the siNA molecule is single stranded, the percent t modification can be based upon the total number of nucleotides present in the single ,I stranded siNA molecules. Likewise, if the siNA molecule is double stranded, the percent Smodification can be based upon the total number of nucleotides present in the sense strand, antisense strand, or both the sense and antisense strands.

A siNA molecule of the invention can comprise modified nucleotides at various locations within the siNA molecule. In one embodiment, a double stranded siNA 00 Mn molecule of the invention comprises modified nucleotides at internal base paired Spositions within the siNA duplex. For example, internal positions can comprise n positions from about 3 to about 19 nucleotides from the 5'-end of either sense or O 10 antisense strand or region of a 21 nucleotide siNA duplex having 19 base pairs and two nucleotide 3'-overhangs. In another embodiment, a double stranded siNA molecule of the invention comprises modified nucleotides at non-base paired or overhang regions of the siNA molecule. For example, overhang positions can comprise positions from about to about 21 nucleotides from the 5'-end of either sense or antisense strand or region of a 21 nucleotide siNA duplex having 19 base pairs and two nucleotide 3'-overhangs. In another embodiment, a double stranded siNA molecule of the invention comprises modified nucleotides at terminal positions of the siNA molecule. For example, such terminal regions include the 3'-position, 5'-position, for both 3' and 5'-positions of the sense and/or antisense strand or region of the siNA molecule. In another embodiment, a double stranded siNA molecule of the invention comprises modified nucleotides at basepaired or internal positions, non-base paired or overhang regions, and/or terminal regions, or any combination thereof.

One aspect of the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a repeat expansion (RE) gene or that directs cleavage of a repeat expansion (RE) RNA. In one embodiment, the double stranded siNA molecule comprises one or more chemical modifications and each strand of the double-stranded siNA is about 21 nucleotides long. In one embodiment, the double-stranded siNA molecule does not contain any ribonucleotides. In another embodiment, the double-stranded siNA molecule comprises one or more ribonucleotides.

In one embodiment, each strand of the double-stranded siNA molecule independently comprises about 15 to about 30 about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, wherein each strand comprises about 15 to about 30 17 t about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides that are complementary to the nucleotides of the other strand. In one embodiment, one of the 2 strands of the double-stranded siNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof of the repeat expansion (RE) gene, and the second strand of the double-stranded siNA molecule comprises a nucleotide sequence substantially similar to the nucleotide sequence of the repeat 0expansion (RE) gene or a portion thereof.

e¢3 In another embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a repeat expansion (RE) gene or that directs cleavage of a repeat expansion (RE) RNA, comprising an antisense region, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of the repeat expansion (RE) gene or a portion thereof, and a sense region, wherein the sense region comprises a nucleotide sequence substantially similar to the nucleotide sequence of the repeat expansion (RE) gene or a portion thereof. In one embodiment, the antisense region and the sense region independently comprise about 15 to about 30 about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, wherein the antisense region comprises about 15 to about 30 about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides that are complementary to nucleotides of the sense region.

In another embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a repeat expansion (RE) gene or that directs cleavage of a repeat expansion (RE) RNA, comprising a sense region and an antisense region, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of RNA encoded by the repeat expansion (RE) gene or a portion thereof and the sense region comprises a nucleotide sequence that is complementary to the antisense region.

In one embodiment, a siNA molecule of the invention comprises blunt ends, i.e., ends that do not include any overhanging nucleotides. For example, a siNA molecule comprising modifications described herein comprising nucleotides having Formulae I-VII or siNA constructs comprising "Stab 00"-"Stab 34" or "Stab 3F"-"Stab t 34F" (Table IV) or any combination thereof (see Table IV)) and/or any length described herein can comprise blunt ends or ends with no overhanging nucleotides.

In one embodiment, any siNA molecule of the invention can comprise one or more blunt ends, i.e. where a blunt end does not have any overhanging nucleotides. In one embodiment, the blunt ended siNA molecule has a number of base pairs equal to the number of nucleotides present in each strand of the siNA molecule. In another 00 M embodiment, the siNA molecule comprises one blunt end, for example wherein the Send of the antisense strand and the 3'-end of the sense strand do not have any i overhanging nucleotides. In another example, the siNA molecule comprises one blunt S 10 end, for example wherein the 3'-end of the antisense strand and the 5'-end of the sense strand do not have any overhanging nucleotides. In another example, a siNA molecule comprises two blunt ends, for example wherein the 3'-end of the antisense strand and the of the sense strand as well as the 5'-end of the antisense strand and 3'-end of the sense strand do not have any overhanging nucleotides. A blunt ended siNA molecule can comprise, for example, from about 15 to about 30 nucleotides about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides). Other nucleotides present in a blunt ended siNA molecule can comprise, for example, mismatches, bulges, loops, or wobble base pairs to modulate the activity of the siNA molecule to mediate RNA interference.

By "blunt ends" is meant symmetric termini or termini of a double stranded siNA molecule having no overhanging nucleotides. The two strands of a double stranded siNA molecule align with each other without over-hanging nucleotides at the termini.

For example, a blunt ended siNA construct comprises terminal nucleotides that are complementary between the sense and antisense regions of the siNA molecule.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a repeat expansion (RE) gene or that directs cleavage of a repeat expansion (RE) RNA, wherein the siNA molecule is assembled from two separate oligonucleotide fragments wherein one fragment comprises the sense region and the second fragment comprises the antisense region of the siNA molecule. The sense region can be connected to the antisense region via a linker molecule, such as a polynucleotide linker or a non-nucleotide linker.

In one embodiment, the invention features double-stranded short interfering N, nucleic acid (siNA) molecule that down-regulates expression of a repeat expansion (RE) Sgene or that directs cleavage of a repeat expansion (RE) RNA, wherein the siNA molecule comprises about 15 to about 30 about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) base pairs, and wherein each strand of the siNA molecule comprises one or more chemical modifications. In another embodiment, one of the 00 strands of the double-stranded siNA molecule comprises a nucleotide sequence that is Scomplementary to a nucleotide sequence of a repeat expansion (RE) gene or a portion ,I thereof, and the second strand of the double-stranded siNA molecule comprises a 0 10 nucleotide sequence substantially similar to the nucleotide sequence or a portion thereof ,IC of the repeat expansion (RE) gene. In another embodiment, one of the strands of the double-stranded siNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence of a repeat expansion (RE) gene or portion thereof, and the second strand of the double-stranded siNA molecule comprises a nucleotide sequence substantially similar to the nucleotide sequence or portion thereof of the repeat expansion (RE) gene. In another embodiment, each strand of the siNA molecule comprises about to about 30 about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or nucleotides, and each strand comprises at least about 15 to about 30 about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides that are complementary to the nucleotides of the other strand. The repeat expansion (RE) gene can comprise, for example, sequences referred to in Table I.

In one embodiment, the repeat expansion (RE) gene can comprise, for example, huntingtin, SCA1, SCA2, SCA3, SCA6, SCA7, SCA12, SCA17, SBMA, or DRPLA (see for example Table including both mutant and wild type versions of such genes.

In one embodiment, a siNA molecule of the invention comprises no ribonucleotides. In another embodiment, a siNA molecule of the invention comprises ribonucleotides.

In one embodiment, a siNA molecule of the invention comprises an antisense region comprising a nucleotide sequence that is complementary to a nucleotide sequence of a repeat expansion (RE) gene or a portion thereof, and the siNA further comprises a sense region comprising a nucleotide sequence substantially similar to the nucleotide t sequence of the repeat expansion (RE) gene or a portion thereof. In another embodiment, the antisense region and the sense region each comprise about 15 to about about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides and the antisense region comprises at least about 15 to about 30 about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides that are complementary to nucleotides of the sense region. The repeat expansion (RE) gene can comprise, for 00 example, sequences referred to in Table I. In another embodiment, the siNA is a double Sstranded nucleic acid molecule, where each of the two strands of the siNA molecule independently comprise about 15 to about 40 about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 23, 33, 34, 35, 36, 37, 38, 39, or 40) nucleotides, and where one of the strands of the siNA molecule comprises at least about 15 about 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 or more) nucleotides that are complementary to the nucleic acid sequence of the repeat expansion (RE) gene or a portion thereof.

In one embodiment, a siNA molecule of the invention comprises a sense region and an antisense region, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of RNA encoded by a repeat expansion (RE) gene, or a portion thereof, and the sense region comprises a nucleotide sequence that is complementary to the antisense region. In one embodiment, the siNA molecule is assembled from two separate oligonucleotide fragments, wherein one fragment comprises the sense region and the second fragment comprises the antisense region of the siNA molecule. In another embodiment, the sense region is connected to the antisense region via a linker molecule. In another embodiment, the sense region is connected to the antisense region via a linker molecule, such as a nucleotide or nonnucleotide linker. The repeat expansion (RE) gene can comprise, for example, sequences referred in to Table I.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a repeat expansion (RE) gene or that directs cleavage of a repeat expansion (RE) RNA, comprising a sense region and an antisense region, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of RNA encoded by the repeat expansion (RE) gene or a portion thereof and the sense region comprises a nucleotide sequence that is complementary to the antisense region, and wherein the siNA molecule has one or 21 0 more modified pyrimidine and/or purine nucleotides. In one embodiment, the 1 pyrimidine nucleotides in the sense region are 2'-O-methyl pyrimidine nucleotides or 2'deoxy-2'-fluoro pyrimidine nucleotides and the purine nucleotides present in the sense region are 2'-deoxy purine nucleotides. In another embodiment, the pyrimidine nucleotides in the sense region are 2'-deoxy-2'-fluoro pyrimidine nucleotides and the purine nucleotides present in the sense region are 2'-O-methyl purine nucleotides. In 0 another embodiment, the pyrimidine nucleotides in the sense region are 2'-deoxy-2'- Sfluoro pyrimidine nucleotides and the purine nucleotides present in the sense region are I 2'-deoxy purine nucleotides. In one embodiment, the pyrimidine nucleotides in the 8 10 antisense region are 2'-deoxy-2'-fluoro pyrimidine nucleotides and the purine nucleotides N present in the antisense region are 2'-O-methyl or 2'-deoxy purine nucleotides. In another embodiment of any of the above-described siNA molecules, any nucleotides present in a non-complementary region of the sense strand overhang region) are 2'deoxy nucleotides.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a repeat expansion (RE) gene or that directs cleavage of a repeat expansion (RE) RNA, wherein the siNA molecule is assembled 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 wherein the fragment comprising the sense region includes a terminal cap moiety at the 5'-end, the 3'-end, or both of the 5' and 3' ends of the fragment. In one embodiment, the terminal cap moiety is an inverted deoxy abasic moiety or glyceryl moiety. In one embodiment, each of the two fragments of the siNA molecule independently comprise about 15 to about 30 about 15, 16, 17, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides. In another embodiment, each of the two fragments of the siNA molecule independently comprise about 15 to about 40 (e.g.

about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 23, 33, 34, 35, 36, 37, 38, 39, or 40) nucleotides. In a non-limiting example, each of the two fragments of the siNA molecule comprise about 21 nucleotides.

In one embodiment, the invention features a siNA molecule comprising at least one modified nucleotide, wherein the modified nucleotide is a 2'-deoxy-2'-fluoro nucleotide, 2'-O-trifluoromethyl nucleotide, 2'-O-ethyl-trifluoromethoxy nucleotide, or 22 0 difluoromethoxy-ethoxy nucleotide or any other modified nucleoside/nucleotide N described in USSN 10/981,966 filed November 5, 2004, incorporated by reference 4 herein. The siNA can be, for example, about 15 to about 40 nucleotides in length. In one embodiment, all pyrimidine nucleotides present in the siNA are 2'-deoxy-2'-fluoro, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy, 4'thio pyrimidine nucleotides. In one embodiment, the modified nucleotides in the siNA 0 include at least one 2'-deoxy-2'-fluoro cytidine or 2'-deoxy-2'-fluoro uridine nucleotide.

SIn another embodiment, the modified nucleotides in the siNA include at least one 2'fluoro cytidine and at least one 2'-deoxy-2'-fluoro uridine nucleotides. In one embodiment, all uridine nucleotides present in the siNA are 2'-deoxy-2'-fluoro uridine nucleotides. In one embodiment, all cytidine nucleotides present in the siNA are 2'deoxy-2'-fluoro cytidine nucleotides. In one embodiment, all adenosine nucleotides present in the siNA are 2'-deoxy-2'-fluoro adenosine nucleotides. In one embodiment, all guanosine nucleotides present in the siNA are 2'-deoxy-2'-fluoro guanosine nucleotides. The siNA can further comprise at least one modified intemucleotidic linkage, such as phosphorothioate linkage. In one embodiment, the 2'-deoxy-2'fluoronucleotides are present at specifically selected locations in the siNA that are sensitive to cleavage by ribonucleases, such as locations having pyrimidine nucleotides.

In one embodiment, the invention features a method of increasing the stability of a siNA molecule against cleavage by ribonucleases comprising introducing at least one modified nucleotide into the siNA molecule, wherein the modified nucleotide is a 2'deoxy-2'-fluoro nucleotide. In one embodiment, all pyrimidine nucleotides present in the siNA are 2'-deoxy-2'-fluoro pyrimidine nucleotides. In one embodiment, the modified nucleotides in the siNA include at least one 2'-deoxy-2'-fluoro cytidine or 2'deoxy-2'-fluoro uridine nucleotide. In another embodiment, the modified nucleotides in the siNA include at least one 2'-fluoro cytidine and at least one 2'-deoxy-2'-fluoro uridine nucleotides. In one embodiment, all uridine nucleotides present in the siNA are 2'-deoxy-2'-fluoro uridine nucleotides. In one embodiment, all cytidine nucleotides present in the siNA are 2'-deoxy-2'-fluoro cytidine nucleotides. In one embodiment, all adenosine nucleotides present in the siNA are 2'-deoxy-2'-fluoro adenosine nucleotides.

In one embodiment, all guanosine nucleotides present in the siNA are 2'-deoxy-2'-fluoro guanosine nucleotides. The siNA can further comprise at least one modified 3 internucleotidic linkage, such as a phosphorothioate linkage. In one embodiment, the 2'deoxy-2'-fluoronucleotides are present at specifically selected locations in the siNA that Sare sensitive to cleavage by ribonucleases, such as locations having pyrimidine nucleotides.

In one embodiment, the invention features a double-stranded short interfering C nucleic acid (siNA) molecule that down-regulates expression of a repeat expansion (RE) M gene or that directs cleavage of a repeat expansion (RE) RNA, comprising a sense region Sand an antisense region, wherein the antisense region comprises a nucleotide sequence t that is complementary to a nucleotide sequence of RNA encoded by the repeat expansion S 10 (RE) gene or a portion thereof and the sense region comprises a nucleotide sequence that is complementary to the antisense region, and wherein the purine nucleotides present in the antisense region comprise 2'-deoxy- purine nucleotides. In an alternative embodiment, the purine nucleotides present in the antisense region comprise 2'-O-methyl purine nucleotides. In either of the above embodiments, the antisense region can comprise a phosphorothioate internucleotide linkage at the 3' end of the antisense region.

Alternatively, in either of the above embodiments, the antisense region can comprise a glyceryl modification at the 3' end of the antisense region. In another embodiment of any of the above-described siNA molecules, any nucleotides present in a noncomplementary region of the antisense strand overhang region) are 2'-deoxy nucleotides.

In one embodiment, the antisense region of a siNA molecule of the invention comprises sequence complementary to a portion of an endogenous transcript having sequence unique to a particular repeat expansion (RE) disease or trait related allele in a subject or organism, such as sequence comprising a single nucleotide polymorphism (SNP) associated with the disease or trait specific allele. As such, the antisense region of a siNA molecule of the invention can comprise sequence complementary to sequences that are unique to a particular allele to provide specificity in mediating selective RNAi against the disease, condition, or trait related allele.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a repeat expansion (RE) gene or that directs cleavage of a repeat expansion (RE) RNA, wherein the siNA molecule is assembled from two separate oligonucleotide fragments wherein one N fragment comprises the sense region and the second fragment comprises the antisense Sregion of the siNA molecule. In another embodiment, the siNA molecule is a double stranded nucleic acid molecule, where each strand is about 21 nucleotides long and 0 5 where about 19 nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule, wherein at least 0 two 3' terminal nucleotides of each fragment of the siNA molecule are not base-paired to Sthe nucleotides of the other fragment of the siNA molecule. In another embodiment, the N siNA molecule is a double stranded nucleic acid molecule, where each strand is about 19 O 10 nucleotide long and where the nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule to form at least about 15 15, 16, 17, 18, or 19) base pairs, wherein one or both ends of the siNA molecule are blunt ends. In one embodiment, each of the two 3' terminal nucleotides of each fragment of the siNA molecule is a 2'-deoxy-pyrimidine nucleotide, such as a 2'-deoxy-thymidine. In another embodiment, all nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule. In another embodiment, the siNA molecule is a double stranded nucleic acid molecule of about 19 to about 25 base pairs having a sense region and an antisense region, where about 19 nucleotides of the antisense region are base-paired to the nucleotide sequence or a portion thereof of the RNA encoded by the repeat expansion (RE) gene. In another embodiment, about 21 nucleotides of the antisense region are base-paired to the nucleotide sequence or a portion thereof of the RNA encoded by the repeat expansion (RE) gene. In any of the above embodiments, the of the fragment comprising said antisense region can optionally include a phosphate group.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits the expression of a repeat expansion (RE) RNA sequence wherein said target RNA sequence is encoded by a repeat expansion (RE) gene involved in the repeat expansion (RE) pathway), wherein the siNA molecule does not contain any ribonucleotides and wherein each strand of the doublestranded siNA molecule is about 15 to about 30 nucleotides. In one embodiment, the siNA molecule is 21 nucleotides in length. Examples of non-ribonucleotide containing t siNA constructs are combinations of stabilization chemistries shown in Table IV in any combination of Sense/Antisense chemistries, such as Stab 7/8, Stab 7/11, Stab 8/8, Stab S18/8, Stab 18/11, Stab 12/13, Stab 7/13, Stab 18/13, Stab 7/19, Stab 8/19, Stab 18/19, Stab 7/20, Stab 8/20, Stab 18/20, Stab 7/32, Stab 8/32, or Stab 18/32 any siNA 0 5 having Stab 7, 8, 11, 12, 13, 14, 15, 17, 18, 19, 20, or 32 sense or antisense strands or any combination thereof). Herein, numeric Stab chemistries can include both 2'-fluoro 00 and 2'-OCF3 versions of the chemistries shown in Table IV. For example, "Stab 7/8" refers to both Stab 7/8 and Stab 7F/8F etc. In one embodiment, the invention features a chemically synthesized double stranded RNA molecule that directs cleavage of a repeat 0 10 expansion (RE) RNA via RNA interference, wherein each strand of said RNA molecule NI is about 15 to about 30 nucleotides in length; one strand of the RNA molecule comprises nucleotide sequence having sufficient complementarity to the repeat expansion (RE) RNA for the RNA molecule to direct cleavage of the repeat expansion (RE) RNA via RNA interference; and wherein at least one strand of the RNA molecule optionally comprises one or more chemically modified nucleotides described herein, such as without limitation deoxynucleotides, 2'-O-methyl nucleotides, 2'-deoxy-2'-fluoro nucleotides, 2'-O-methoxyethyl nucleotides, 4'-thio nucleotides, 2'-O-trifluoromethyl nucleotides, 2'-O-ethyl-trifluoromethoxy nucleotides, nucleotides, etc.

In one embodiment, the invention features a medicament comprising a siNA molecule of the invention.

In one embodiment, the invention features an active ingredient comprising a siNA molecule of the invention.

In one embodiment, the invention features the use of a double-stranded short interfering nucleic acid (siNA) molecule to inhibit, down-regulate, or reduce expression of a repeat expansion (RE) gene, wherein the siNA molecule comprises one or more chemical modifications and each strand of the double-stranded siNA is independently about 15 to about 30 or more about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 or more) nucleotides long. In one embodiment, the siNA molecule of the invention is a double stranded nucleic acid molecule comprising one or more chemical modifications, where each of the two fragments of the siNA molecule

I

independently comprise about 15 to about 40 about 15, 16, 17, 18, 19, 20, 21, 22, (23, 24, 25, 26, 27, 28, 29, 30, 31, 23, 33, 34, 35, 36, 37, 38, 39, or 40) nucleotides and 2 where one of the strands comprises at least 15 nucleotides that are complementary to nucleotide sequence of repeat expansion (RE) encoding RNA or a portion thereof. In a non-limiting example, each of the two fragments of the siNA molecule comprise about 21 nucleotides. In another embodiment, the siNA molecule is a double stranded nucleic 00 acid molecule comprising one or more chemical modifications, where each strand is Sabout 21 nucleotide long and where about 19 nucleotides of each fragment of the siNA (molecule are base-paired to the complementary nucleotides of the other fragment of the 0 10 siNA molecule, wherein at least two 3' terminal nucleotides of each fragment of the siNA molecule are not base-paired to the nucleotides of the other fragment of the siNA molecule. In another embodiment, the siNA molecule is a double stranded nucleic acid molecule comprising one or more chemical modifications, where each strand is about 19 nucleotide long and where the nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule to form at least about 15 15, 16, 17, 18, or 19) base pairs, wherein one or both ends of the siNA molecule are blunt ends. In one embodiment, each of the two 3' terminal nucleotides of each fragment of the siNA molecule is a 2'-deoxy-pyrimidine nucleotide, such as a 2'-deoxy-thymidine. In another embodiment, all nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule. In another embodiment, the siNA molecule is a double stranded nucleic acid molecule of about 19 to about 25 base pairs having a sense region and an antisense region and comprising one or more chemical modifications, where about 19 nucleotides of the antisense region are base-paired to the nucleotide sequence or a portion thereof of the RNA encoded by the repeat expansion (RE) gene. In another embodiment, about 21 nucleotides of the antisense region are base-paired to the nucleotide sequence or a portion thereof of the RNA encoded by the repeat expansion (RE) gene. In any of the above embodiments, the 5'-end of the fragment comprising said antisense region can optionally include a phosphate group.

In one embodiment, the invention features the use of a double-stranded short interfering nucleic acid (siNA) molecule that inhibits, down-regulates, or reduces expression of a repeat expansion (RE) gene, wherein one of the strands of the doublestranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of repeat expansion (RE) RNA or a portion thereof, the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand and wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits, down-regulates, or reduces expression of a repeat expansion (RE) gene, wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of repeat expansion (RE) RNA or a portion thereof, wherein the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand and wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits, down-regulates, or reduces expression of a repeat expansion (RE) gene, wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of repeat expansion (RE) RNA that encodes a protein or portion thereof, the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand and wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification. In one embodiment, each strand of the siNA molecule comprises about 15 to about 30 or more about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more) nucleotides, wherein each strand comprises at least about 15 nucleotides that are complementary to the nucleotides of the other strand. In one embodiment, the siNA molecule is assembled from two oligonucleotide fragments, wherein one fragment comprises the nucleotide sequence of the antisense strand of the siNA molecule and a second fragment comprises nucleotide sequence of the sense region of the siNA molecule. In one embodiment, the sense strand is connected to the antisense strand via a linker molecule, such as a polynucleotide linker or a non- 28 3 nucleotide linker. In a further embodiment, the pyrimidine nucleotides present in the Ssense strand are 2'-deoxy-2'fluoro pyrimidine nucleotides and the purine nucleotides 2 present in the sense region are 2'-deoxy purine nucleotides. In another embodiment, the pyrimidine nucleotides present in the sense strand are 2'-deoxy-2'fluoro pyrimidine 0 5 nucleotides and the purine nucleotides present in the sense region are 2'-O-methyl purine nucleotides. In still another embodiment, the pyrimidine nucleotides present in the 00 antisense strand are 2'-deoxy-2'-fluoro pyrimidine nucleotides and any purine nucleotides Spresent in the antisense strand are 2'-deoxy purine nucleotides. In another embodiment, Sthe antisense strand comprises one or more 2'-deoxy-2'-fluoro pyrimidine nucleotides 0 10 and one or more 2'-O-methyl purine nucleotides. In another embodiment, the N1 pyrimidine nucleotides present in the antisense strand are 2'-deoxy-2'-fluoro pyrimidine nucleotides and any purine nucleotides present in the antisense strand are 2'-O-methyl purine nucleotides. In a further embodiment the sense strand comprises a 3'-end and a end, wherein a terminal cap moiety an inverted deoxy abasic moiety or inverted deoxy nucleotide moiety such as inverted thymidine) is present at the 5'-end, the 3'-end, or both of the 5' and 3' ends of the sense strand. In another embodiment, the antisense strand comprises a phosphorothioate intemucleotide linkage at the 3' end of the antisense strand. In another embodiment, the antisense strand comprises a glyceryl modification at the 3' end. In another embodiment, the 5'-end of the antisense strand optionally includes a phosphate group.

In any of the above-described embodiments of a double-stranded short interfering nucleic acid (siNA) molecule that inhibits expression of a repeat expansion (RE) gene, wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification, each of the two strands of the siNA molecule can comprise about 15 to about 30 or more about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more) nucleotides. In one embodiment, about 15 to about or more about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more) nucleotides of each strand of the siNA molecule are base-paired to the complementary nucleotides of the other strand of the siNA molecule. In another embodiment, about 15 to about 30 or more about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more) nucleotides of each strand of the siNA molecule are base-paired to the complementary nucleotides of the other strand of the siNA molecule, t wherein at least two 3' terminal nucleotides of each strand of the siNA molecule are not NI base-paired to the nucleotides of the other strand of the siNA molecule. In another Sembodiment, each of the two 3' terminal nucleotides of each fragment of the siNA molecule is a 2'-deoxy-pyrimidine, such as 2'-deoxy-thymidine. In one embodiment, each strand of the siNA molecule is base-paired to the complementary nucleotides of the other strand of the siNA molecule. In one embodiment, about 15 to about 30 about 00 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides of the Santisense strand are base-paired to the nucleotide sequence of the repeat expansion (RE) I RNA or a portion thereof. In one embodiment, about 18 to about 25 about 18, 19, 0 10 20, 21, 22, 23, 24, or 25) nucleotides of the antisense strand are base-paired to the NI nucleotide sequence of the repeat expansion (RE) RNA or a portion thereof.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits expression of a repeat expansion (RE) gene, wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of repeat expansion (RE) RNA or a portion thereof, the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand and wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification, and wherein the of the antisense strand optionally includes a phosphate group.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits expression of a repeat expansion (RE) gene, wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of repeat expansion (RE) RNA or a portion thereof, the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand and wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification, and wherein the nucleotide sequence or a portion thereof of the antisense strand is complementary to a nucleotide sequence of the untranslated region or a portion thereof of the repeat expansion (RE) RNA.

I

t In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits expression of a repeat expansion (RE) gene, 2 wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of repeat expansion (RE) RNA or a portion thereof, wherein the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide 00 sequence of the antisense strand, wherein a majority of the pyrimidine nucleotides Spresent in the double-stranded siNA molecule comprises a sugar modification, and wherein the nucleotide sequence of the antisense strand is complementary to a nucleotide sequence of the repeat expansion (RE) RNA or a portion thereof that is present in the 1 repeat expansion (RE) RNA.

In one embodiment, the invention features a composition comprising a siNA molecule of the invention in a pharmaceutically acceptable carrier or diluent.

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 vivo stability and bioavailability inherent to native RNA molecules that are delivered exogenously. For example, the use of chemically-modified nucleic acid molecules can enable a lower dose of a particular nucleic acid molecule for a given therapeutic effect since chemically-modified nucleic acid molecules tend to have a longer half-life in serum. Furthermore, certain chemical modifications can improve the bioavailability of nucleic acid molecules by targeting particular cells or tissues and/or improving cellular uptake of the nucleic acid molecule. Therefore, even if the activity of a chemically-modified nucleic acid molecule is reduced as compared to a native nucleic acid molecule, for example, when compared to an all-RNA nucleic acid molecule, the overall activity of the modified nucleic acid molecule can be greater than that of the native molecule due to improved stability and/or delivery of the molecule. Unlike native unmodified siNA, chemically-modified siNA can also minimize the possibility of activating interferon activity or immunostimulation in humans.

In any of the embodiments of siNA molecules described herein, the antisense region of a siNA molecule of the invention can comprise a phosphorothioate internucleotide linkage at the 3'-end of said antisense region. In any of the embodiments t of siNA molecules described herein, the antisense region can comprise about one to Sabout five phosphorothioate intemucleotide linkages at the 5'-end of said antisense Sregion. In any of the embodiments of siNA molecules described herein, the 3'-terminal nucleotide overhangs of a siNA molecule of the invention can comprise ribonucleotides C 5 or deoxyribonucleotides that are chemically-modified at a nucleic acid sugar, base, or backbone. In any of the embodiments of siNA molecules described herein, the 3'- 0 terminal nucleotide overhangs can comprise one or more universal base ribonucleotides.

SIn any of the embodiments of siNA molecules described herein, the 3'-terminal cnucleotide overhangs can comprise one or more acyclic nucleotides.

One embodiment of the invention provides an expression vector comprising a nucleic acid sequence encoding at least one siNA molecule of the invention in a manner that allows expression of the nucleic acid molecule. Another embodiment of the invention provides a mammalian cell comprising such an expression vector. The mammalian cell can be a human cell. The siNA molecule of the expression vector can comprise a sense region and an antisense region. The antisense region can comprise sequence complementary to a RNA or DNA sequence encoding repeat expansion (RE) and the sense region can comprise sequence complementary to the antisense region. The siNA molecule can comprise two distinct strands having complementary sense and antisense regions. The siNA molecule can comprise a single strand having complementary sense and antisense regions.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against repeat expansion (RE) inside a cell or reconstituted in vitro system, wherein the chemical modification comprises one or more about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides comprising a backbone modified internucleotide linkage having Formula I:

Z

II

RI-X-P-Y-R

2

W

wherein each R1 and R2 is independently any nucleotide, non-nucleotide, or polynucleotide which can be naturally-occurring or chemically-modified, each X and Y S is independently O, S, N, alkyl, or substituted alkyl, each Z and W is independently O, S, I N, alkyl, substituted alkyl, O-alkyl, S-alkyl, alkaryl, aralkyl, or acetyl and wherein W, X, SY, and Z are optionally not all O. In another embodiment, a backbone modification of the invention comprises a phosphonoacetate and/or thiophosphonoacetate internucleotide linkage (see for example Sheehan et al., 2003, Nucleic Acids Research, 31, 4109-4118).

The chemically-modified internucleotide linkages having Formula I, for example, M wherein any Z, W, X, and/or Y independently comprises a sulphur atom, can be present Sin one or both oligonucleotide strands of the siNA duplex, for example, in the sense t strand, the antisense strand, or both strands. The siNA molecules of the invention can comprise one or more about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) chemicallymodified intemucleotide linkages having Formula I at the 3'-end, the 5'-end, or both of the 3' and 5'-ends of the sense strand, the antisense strand, or both strands. For example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more about 1, 2, 3, 4, 5, or more) chemically-modified intemucleotide linkages having Formula I at the 5'-end of the sense strand, the antisense strand, or both strands. In another non-limiting example, an exemplary siNA molecule of the invention can comprise one or more about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) pyrimidine nucleotides with chemically-modified intemucleotide linkages having Formula I in the sense strand, the antisense strand, or both strands. In yet another non-limiting example, an exemplary siNA molecule of the invention can comprise one or more about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purine nucleotides with chemically-modified interucleotide linkages having Formula I in the sense strand, the antisense strand, or both strands. In another embodiment, a siNA molecule of the invention having internucleotide linkage(s) of Formula I also comprises a chemically-modified nucleotide or non-nucleotide having any of Formulae I-VII.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against repeat expansion (RE) inside a cell or reconstituted in vitro system, wherein the chemical modification comprises one or more about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides or non-nucleotides having Formula II: 00 M_ wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ON02, N02, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid,

O-

aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or group having Formula I or II; R9 is O, S, CH2, S=0, CHF, or CF2, and B is a nucleosidic base such as adenine, guanine, uracil, cytosine, thymine, 2aminoadenosine, 5-methylcytosine, 2,6-diaminopurine, or any other non-naturally occurring base that can be complementary or non-complementary to target RNA or a non-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole, 5-nitroindole, nebularine, pyridone, pyridinone, or any other non-naturally occurring universal base that can be complementary or non-complementary to target RNA. In one embodiment, R3 and/or R7 comprises a conjugate moiety and a linker a nucleotide or non-nucleotide linker as described herein or otherwise known in the art). Non-limiting examples of conjugate moieties include ligands for cellular receptors, such as peptides derived from naturally occurring protein ligands; protein localization sequences, including cellular ZIP code sequences; antibodies; nucleic acid aptamers; vitamins and other co-factors, such as folate and N-acetylgalactosamine; polymers, such as polyethyleneglycol

(PEG);

phospholipids; cholesterol; steroids, and polyamines, such as PEI, spermine or spermidine.

The chemically-modified nucleotide or non-nucleotide of Formula II can be present in one or both oligonucleotide strands of the siNA duplex, for example in the sense strand, the antisense strand, or both strands. The siNA molecules of the invention can comprise one or more chemically-modified nucleotides or non-nucleotides of Formula II at the 3'-end, the 5'-end, or both of the 3' and 5'-ends of the sense strand, the 34 antisense strand, or both strands. For example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more about 1, 2, 3, 4, 5, or more) chemically-modified nucleotides or non-nucleotides of Formula II at the 5'-end of the sense strand, the antisense strand, or both strands. In anther non-limiting example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more about 1, 2, 3, 4, 5, or more) chemically-modified nucleotides or non-nucleotides of Formula II at the 3'-end of the sense strand, the antisense strand, or both strands.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against repeat expansion (RE) inside a cell or reconstituted in vitro system, wherein the chemical modification comprises one or more about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides or non-nucleotides having Formula III: R7 R11 R8 B 3 Rs R3 wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ON02, N02, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, Oaminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or group having Formula I or II; R9 is O, S, CH2, S=O, CHF, or CF2, and B is a nucleosidic base such as adenine, guanine, uracil, cytosine, thymine, 2aminoadenosine, 5-methylcytosine, 2,6-diaminopurine, or any other non-naturally occurring base that can be employed to be complementary or non-complementary to target RNA or a non-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole, nitroindole, nebularine, pyridone, pyridinone, or any other non-naturally occurring universal base that can be complementary or non-complementary to target RNA. In one 3 embodiment, R3 and/or R7 comprises a conjugate moiety and a linker a nucleotide N or non-nucleotide linker as described herein or otherwise known in the art). Nonlimiting examples of conjugate moieties include ligands for cellular receptors, such as peptides derived from naturally occurring protein ligands; protein localization sequences, including cellular ZIP code sequences; antibodies; nucleic acid aptamers; vitamins and other co-factors, such as folate and N-acetylgalactosamine; polymers, such as 0 polyethyleneglycol (PEG); phospholipids; cholesterol; steroids, and polyamines, such as PEI, spermine or spermidine.

The chemically-modified nucleotide or non-nucleotide of Formula III can be 0 10 present in one or both oligonucleotide strands of the siNA duplex, for example, in the sense strand, the antisense strand, or both strands. The siNA molecules of the invention can comprise one or more chemically-modified nucleotides or non-nucleotides of Formula III at the 3'-end, the 5'-end, or both of the 3' and 5'-ends of the sense strand, the antisense strand, or both strands. For example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more about 1, 2, 3, 4, 5, or more) chemically-modified nucleotide(s) or non-nucleotide(s) of Formula III at the 5'-end of the sense strand, the antisense strand, or both strands. In anther non-limiting example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more about 1, 2, 3, 4, 5, or more) chemically-modified nucleotide or non-nucleotide of Formula III at the 3'-end of the sense strand, the antisense strand, or both strands.

In another embodiment, a siNA molecule of the invention comprises a nucleotide having Formula II or III, wherein the nucleotide having Formula II or III is in an inverted configuration. For example, the nucleotide having Formula II or III is connected to the siNA construct in a or configuration, such as at the 3'-end, the end, or both of the 3' and 5'-ends of one or both siNA strands.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against repeat expansion (RE) inside a cell or reconstituted in vitro system, wherein the chemical modification comprises a 5'-terminal phosphate group having Formula IV:

Z

X-P-Y-

W

wherein each X and Y is independently O, S, N, alkyl, substituted alkyl, or alkylhalo; wherein each Z and W is independently O, S, N, alkyl, substituted alkyl, O-alkyl, Salkyl, alkaryl, aralkyl, alkylhalo, or acetyl; and wherein W, X, Y and Z are not all O.

In one embodiment, the invention features a siNA molecule having a phosphate group having Formula IV on the target-complementary strand, for example, a strand complementary to a target RNA, wherein the siNA molecule comprises an all RNA siNA molecule. In another embodiment, the invention features a siNA molecule having a 5'-terminal phosphate group having Formula IV on the target-complementary strand wherein the siNA molecule also comprises about 1 to about 3 about 1, 2, or 3) nucleotide 3'-terminal nucleotide overhangs having about 1 to about 4 about 1, 2, 3, or 4) deoxyribonucleotides on the 3'-end of one or both strands. In another embodiment, a 5'-terminal phosphate group having Formula IV is present on the targetcomplementary strand of a siNA molecule of the invention, for example a siNA molecule having chemical modifications having any of Formulae I-VII.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against repeat expansion (RE) inside a cell or reconstituted in vitro system, wherein the chemical modification comprises one or more phosphorothioate intemucleotide linkages. For example, in a non-limiting example, the invention features a chemically-modified short interfering nucleic acid (siNA) having about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages in one siNA strand. In yet another embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) individually having about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages in both siNA strands. The phosphorothioate internucleotide linkages can be present in one or both oligonucleotide strands of the siNA duplex, for example in the sense strand, the antisense strand, or both strands. The siNA molecules of the invention can comprise one or more phosphorothioate internucleotide linkages at the 3'-end, the 5'-end, or both of the and 5'-ends of the sense strand, the antisense N strand, or both strands. For example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more about 1, 2, 3, 4, 5, or more) consecutive phosphorothioate intemucleotide linkages at the 5'-end of the sense strand, the antisense 0 5 strand, or both strands. In another non-limiting example, an exemplary siNA molecule of the invention can comprise one or more about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 00 more) pyrimidine phosphorothioate intemucleotide linkages in the sense strand, the Santisense strand, or both strands. In yet another non-limiting example, an exemplary C1 siNA molecule of the invention can comprise one or more about 1, 2, 3, 4, 5, 6, 7, S 10 8, 9, 10, or more) purine phosphorothioate intemucleotide linkages in the sense strand, C the antisense strand, or both strands.

In one embodiment, the invention features a siNA molecule, wherein the sense strand comprises one or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/or one or more about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, 2'-O-trifluoromethyl, 2'- O-ethyl-trifluoromethoxy, 2'-O-difluoromethoxy-ethoxy and/or about one or more about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3'-end, the 5'-end, or both of the and of the sense strand; and wherein the antisense strand comprises about 1 to about 10 or more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/or one or more about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, 2'-O-trifluoromethyl, 2'-O-ethyltrifluoromethoxy, 2'-O-difluoromethoxy-ethoxy, 4'-thio and/or one or more about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3'-end, the 5'-end, or both of the and 5'-ends of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides of the sense and/or antisense siNA strand are chemically-modified with 2'-deoxy, 2'-O-methyl, 2'-O-trifluoromethyl, 2'-O-ethyltrifluoromethoxy, 2'-O-difluoromethoxy-ethoxy, 4'-thio and/or 2'-deoxy-2'-fluoro nucleotides, with or without one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3't end, the 5'-end, or both of the and 5'-ends, being present in the same or different N strand.

In another embodiment, the invention features a siNA molecule, wherein the sense strand comprises about 1 to about 5, specifically about 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages, and/or one or more about 1, 2, 3, 4, 5, or more) 2'-deoxy, C, 2'-O-methyl, 2'-deoxy-2'-fluoro, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, 2'- M O-difluoromethoxy-ethoxy, 4'-thio and/or one or more about 1, 2, 3, 4, 5, or more) Suniversal base modified nucleotides, and optionally a terminal cap molecule at the 3-end, Sthe 5'-end, or both of the and 5'-ends of the sense strand; and wherein the antisense S 10 strand comprises about 1 to about 5 or more, specifically about 1, 2, 3, 4, 5, or more phosphorothioate intemucleotide linkages, and/or one or more about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, 2'-O-trifluoromethyl, 2'- O-ethyl-trifluoromethoxy, 2'-O-difluoromethoxy-ethoxy, 4'-thio and/or one or more about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3'-end, the 5'-end, or both of the and of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides of the sense and/or antisense siNA strand are chemically-modified with 2'-deoxy, 2'-O-methyl, 2'-O-trifluoromethyl, ethyl-trifluoromethoxy, 2'-O-difluoromethoxy-ethoxy, 4'-thio and/or 2'-deoxy-2'-fluoro nucleotides, with or without about 1 to about 5 or more, for example about 1, 2, 3, 4, or more phosphorothioate intemucleotide linkages and/or a terminal cap molecule at the 3'-end, the 5'-end, or both of the and 5'-ends, being present in the same or different strand.

In one embodiment, the invention features a siNA molecule, wherein the antisense strand comprises one or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/or about one or more about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, 2'-O-difluoromethoxy-ethoxy, 4'-thio and/or one or more about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3'-end, the 5'-end, or both of the and 5'-ends of the sense strand; and wherein the antisense strand comprises about 1 to about 10 or more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 39 more phosphorothioate internucleotide linkages, and/or one or more about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, 2'-O-difluoromethoxy-ethoxy, 4'-thio and/or one or more about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3'-end, the 5'-end, or both of the and 5'-ends of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides of the sense and/or antisense siNA strand are chemically-modified with 2'-deoxy, 2'-O-methyl, trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, 2'-O-difluoromethoxy-ethoxy, 4'-thio and/or 2'-deoxy-2'-fluoro nucleotides, with or without one or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate intemucleotide linkages and/or a terminal cap molecule at the 3'-end, the 5'-end, or both of the 3' and 5'-ends, being present in the same or different strand.

In another embodiment, the invention features a siNA molecule, wherein the antisense strand comprises about 1 to about 5 or more, specifically about 1, 2, 3, 4, 5 or more phosphorothioate interucleotide linkages, and/or one or more about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, 2'-O-difluoromethoxy-ethoxy, 4'-thio and/or one or more about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3'-end, the 5'-end, or both of the and 5'-ends of the sense strand; and wherein the antisense strand comprises about 1 to about 5 or more, specifically about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages, and/or one or more about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, 2'-O-trifluoromethyl, 2'- O-ethyl-trifluoromethoxy, 2'-O-difluoromethoxy-ethoxy, 4'-thio and/or one or more about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3'-end, the 5'-end, or both of the and ends of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides of the sense and/or antisense siNA strand are chemically-modified with 2'-deoxy, 2'-O-methyl, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, 2'-O-difluoromethoxy-ethoxy, 4'-thio and/or 2'-deoxy-2'fluoro nucleotides, with or without about 1 to about 5, for example about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3'- Send, the 5'-end, or both of the and 5'-ends, being present in the same or different strand.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule having about 1 to about 5 or more (specifically about 1, 2, 03, 4, 5 or more) phosphorothioate intemucleotide linkages in each strand of the siNA 00 Mr^ molecule.

C1 In another embodiment, the invention features a siNA molecule comprising Sinternucleotide linkages. The internucleotide linkage(s) can be at the 3'-end, the c 10 end, or both of the and 5'-ends of one or both siNA sequence strands. In addition, the intemucleotide linkage(s) can be present at various other positions within one or both siNA sequence strands, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more including every internucleotide linkage of a pyrimidine nucleotide in one or both strands of the siNA molecule can comprise a intemucleotide linkage, or about 1, 2, 3, 4, 6, 7, 8, 9, 10, or more including every intemucleotide linkage of a purine nucleotide in one or both strands of the siNA molecule can comprise a internucleotide linkage.

In another embodiment, a chemically-modified siNA molecule of the invention comprises a duplex having two strands, one or both of which can be chemicallymodified, wherein each strand is independently about 15 to about 30 about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length, wherein the duplex has about 15 to about 30 about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) base pairs, and wherein the chemical modification comprises a structure having any of Formulae I-VII. For example, an exemplary chemicallymodified siNA molecule of the invention comprises a duplex having two strands, one or both of which can be chemically-modified with a chemical modification having any of Formulae I-VII or any combination thereof, wherein each strand consists of about 21 nucleotides, each having a 2-nucleotide 3'-terminal nucleotide overhang, and wherein the duplex has about 19 base pairs. In another embodiment, a siNA molecule of the invention comprises a single stranded hairpin structure, wherein the siNA is about 36 to about 70 about 36, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length having about to about 30 about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or base pairs, and wherein the siNA can include a chemical modification comprising a I structure having any of Formulae I-VII or any combination thereof. For example, an Sexemplary chemically-modified siNA molecule of the invention comprises a linear oligonucleotide having about 42 to about 50 about 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides that is chemically-modified with a chemical modification having any of Formulae I-VII or any combination thereof, wherein the linear oligonucleotide forms a 00 hairpin structure having about 19 to about 21 19, 20, or 21) base pairs and a 2- Snucleotide 3'-terminal nucleotide overhang. In another embodiment, a linear hairpin i siNA molecule of the invention contains a stem loop motif, wherein the loop portion of 8 10 the siNA molecule is biodegradable. For example, a linear hairpin siNA molecule of the I invention is designed such that degradation of the loop portion of the siNA molecule in vivo can generate a double-stranded siNA molecule with 3'-terminal overhangs, such as 3'-terminal nucleotide overhangs comprising about 2 nucleotides.

In another embodiment, a siNA molecule of the invention comprises a hairpin structure, wherein the siNA is about 25 to about 50 about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in length having about 3 to about 25 about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs, and wherein the siNA can include one or more chemical modifications comprising a structure having any of Formulae I-VII or any combination thereof. For example, an exemplary chemically-modified siNA molecule of the invention comprises a linear oligonucleotide having about 25 to about 35 about 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleotides that is chemically-modified with one or more chemical modifications having any of Formulae I-VII or any combination thereof, wherein the linear oligonucleotide forms a hairpin structure having about 3 to about 25 about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs and a 5'-terminal phosphate group that can be chemically modified as described herein (for example a 5'-terminal phosphate group having Formula IV). In another embodiment, a linear hairpin siNA molecule of the invention contains a stem loop motif, wherein the loop portion of the siNA molecule is biodegradable. In one embodiment, a linear hairpin siNA molecule of the invention comprises a loop portion comprising a non-nucleotide linker.

3 In another embodiment, a siNA molecule of the invention comprises an I asymmetric hairpin structure, wherein the siNA is about 25 to about 50 about S26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in length having about 3 to about 25 about 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs, and wherein the siNA can include one or more chemical modifications comprising a structure having any 00 of Formulae I-VII or any combination thereof. For example, an exemplary chemically- Smodified siNA molecule of the invention comprises a linear oligonucleotide having about 25 to about 35 about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleotides 0 10 that is chemically-modified with one or more chemical modifications having any of (NI Formulae I-VII or any combination thereof, wherein the linear oligonucleotide forms an asymmetric hairpin structure having about 3 to about 25 about 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs and a phosphate group that can be chemically modified as described herein (for example a terminal phosphate group having Formula IV). In one embodiment, an asymmetric hairpin siNA molecule of the invention contains a stem loop motif, wherein the loop portion of the siNA molecule is biodegradable. In another embodiment, an asymmetric hairpin siNA molecule of the invention comprises a loop portion comprising a nonnucleotide linker.

In another embodiment, a siNA molecule of the invention comprises an asymmetric double stranded structure having separate polynucleotide strands comprising sense and antisense regions, wherein the antisense region is about 15 to about 30 about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length, wherein the sense region is about 3 to about 25 about 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides in length, wherein the sense region and the antisense region have at least 3 complementary nucleotides, and wherein the siNA can include one or more chemical modifications comprising a structure having any of Formulae I-VII or any combination thereof. For example, an exemplary chemically-modified siNA molecule of the invention comprises an asymmetric double stranded structure having separate polynucleotide strands comprising sense and antisense regions, wherein the antisense region is about 18 to about 23 about 18, 19, 20, 21, 22, or 23) nucleotides in length and wherein the sense region is about 3 to about 15 t about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) nucleotides in length, wherein the sense N region the antisense region have at least 3 complementary nucleotides, and wherein the L siNA can include one or more chemical modifications comprising a structure having any of Formulae I-VII or any combination thereof. In another embodiment, the asymmetric double stranded siNA molecule can also have a 5'-terminal phosphate group that can be chemically modified as described herein (for example a 5'-terminal phosphate group 00 having Formula IV).

0In another embodiment, a siNA molecule of the invention comprises a circular i nucleic acid molecule, wherein the siNA is about 38 to about 70 about 38, 40, 0 10 50, 55, 60, 65, or 70) nucleotides in length having about 15 to about 30 about 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) base pairs, and wherein the siNA can include a chemical modification, which comprises a structure having any of Formulae I-VII or any combination thereof. For example, an exemplary chemicallymodified siNA molecule of the invention comprises a circular oligonucleotide having about 42 to about 50 about 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides that is chemically-modified with a chemical modification having any of Formulae I-VII or any combination thereof, wherein the circular oligonucleotide forms a dumbbell shaped structure having about 19 base pairs and 2 loops.

In another embodiment, a circular siNA molecule of the invention contains two loop motifs, wherein one or both loop portions of the siNA molecule is biodegradable.

For example, a circular siNA molecule of the invention is designed such that degradation of the loop portions of the siNA molecule in vivo can generate a double-stranded siNA molecule with 3'-terminal overhangs, such as 3'-terminal nucleotide overhangs comprising about 2 nucleotides.

In one embodiment, a siNA molecule of the invention comprises at least one about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) abasic moiety, for example a compound having Formula V:

I

oo Mc wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, Salkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, Oalkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ON02, N02, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or group having Formula I or II; R9 is O, S, CH2, S=O, CHF, or CF2.

In one embodiment, R3 and/or R7 comprises a conjugate moiety and a linker a nucleotide or non-nucleotide linker as described herein or otherwise known in the art).

Non-limiting examples of conjugate moieties include ligands for cellular receptors, such as peptides derived from naturally occurring protein ligands; protein localization sequences, including cellular ZIP code sequences; antibodies; nucleic acid aptamers; vitamins and other co-factors, such as folate and N-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol; steroids, and polyamines, such as PEI, spermine or spermidine.

In one embodiment, a siNA molecule of the invention comprises at least one about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) inverted abasic moiety, for example a compound having Formula VI: F3 Rs R13 R8

R

R9 R 12 RI, R7 RIo

I

r3 wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, Salkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, Oalkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, 0 substituted silyl, or group having Formula I or II; R9 is O, S, CH2, S=0, CHF, or CF2, Sand either R2, R3, R8 or R13 serve as points of attachment to the siNA molecule of the invention. In one embodiment, R3 and/or R7 comprises a conjugate moiety and a linker a nucleotide or non-nucleotide linker as described herein or otherwise known in the C1 art). Non-limiting examples of conjugate moieties include ligands for cellular receptors, such as peptides derived from naturally occurring protein ligands; protein localization sequences, including cellular ZIP code sequences; antibodies; nucleic acid aptamers; vitamins and other co-factors, such as folate and N-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol; steroids, and polyamines, such as PEI, spermine or spermidine.

In another embodiment, a siNA molecule of the invention comprises at least one about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) substituted polyalkyl moieties, for example a compound having Formula VII: RI n n R3 R2 wherein each n is independently an integer from 1 to 12, each R1, R2 and R3 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, Oaminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or a group having Formula I, and R1, R2 or R3 serves as points of attachment to the siNA molecule of the invention. In one embodiment, R3 and/or R1 comprises a conjugate moiety and a linker a nucleotide or non-nucleotide linker as described herein or otherwise known in the art).

N Non-limiting examples of conjugate moieties include ligands for cellular receptors, such Sas peptides derived from naturally occurring protein ligands; protein localization sequences, including cellular ZIP code sequences; antibodies; nucleic acid aptamers; 0 5 vitamins and other co-factors, such as folate and N-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol; steroids, and polyamines, such 0 as PEI, spermine or spermidine.

0 By "ZIP code" sequences is meant, any peptide or protein sequence that is i involved in cellular topogenic signaling mediated transport (see for example Ray et al., 2004, Science, 306(1501): 1505) In another embodiment, the invention features a compound having Formula VII, wherein R1 and R2 are hydroxyl (OH) groups, n 1, and R3 comprises O and is the point of attachment to the 3'-end, the 5'-end, or both of the 3' and 5'-ends of one or both strands of a double-stranded siNA molecule of the invention or to a single-stranded siNA molecule of the invention. This modification is referred to herein as "glyceryl" (for example modification 6 in Figure In another embodiment, a chemically modified nucleoside or non-nucleoside (e.g.

a moiety having any of Formula V, VI or VII) of the invention is at the 3'-end, the or both of the 3' and 5'-ends of a siNA molecule of the invention. For example, chemically modified nucleoside or non-nucleoside a moiety having Formula V, VI or VII) can be present at the 3'-end, the 5'-end, or both of the 3' and 5'-ends of the antisense strand, the sense strand, or both antisense and sense strands of the siNA molecule. In one embodiment, the chemically modified nucleoside or non-nucleoside a moiety having Formula V, VI or VII) is present at the 5'-end and 3'-end of the sense strand and the 3'-end of the antisense strand of a double stranded siNA molecule of the invention. In one embodiment, the chemically modified nucleoside or nonnucleoside a moiety having Formula V, VI or VII) is present at the terminal position of the 5'-end and 3'-end of the sense strand and the 3'-end of the antisense strand of a double stranded siNA molecule of the invention. In one embodiment, the chemically modified nucleoside or non-nucleoside a moiety having Formula V, VI or VII) is present at the two terminal positions of the 5'-end and 3'-end of the sense strand and the 3'-end of the antisense strand of a double stranded siNA molecule of the invention. In one embodiment, the chemically modified nucleoside or non-nucleoside a moiety having Formula V, VI or VII) is present at the penultimate position of the and 3'-end of the sense strand and the 3'-end of the antisense strand of a double 0 5 stranded siNA molecule of the invention. In addition, a moiety having Formula VII can be present at the 3'-end or the 5'-end of a hairpin siNA molecule as described herein.

00 M In another embodiment, a siNA molecule of the invention comprises an abasic Sresidue having Formula V or VI, wherein the abasic residue having Formula VI or VI is t connected to the siNA construct in a or configuration, such as at the O 10 3'-end, the 5'-end, or both of the 3' and 5'-ends of one or both siNA strands.

In one embodiment, a siNA molecule of the invention comprises one or more about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) locked nucleic acid (LNA) nucleotides, for example, at the 5'-end, the 3'-end, both of the 5' and 3'-ends, or any combination thereof, of the siNA molecule.

In one embodiment, a siNA molecule of the invention comprises one or more about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 4'-thio nucleotides, for example, at the the 3'-end, both of the 5' and 3'-ends, or any combination thereof, of the siNA molecule.

In another embodiment, a siNA molecule of the invention comprises one or more about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) acyclic nucleotides, for example, at the 5'-end, the 3'-end, both of the 5' and 3'-ends, or any combination thereof, of the siNA molecule.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising a sense region, wherein any one or more or all) pyrimidine nucleotides present in the sense region are 2'-deoxy- 2'-fluoro pyrimidine nucleotides wherein all pyrimidine nucleotides are 2'-deoxy- 2'-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides), and wherein any one or more or all) purine nucleotides present in the sense region are 2'-deoxy purine nucleotides wherein all purine nucleotides are 2'-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2'-deoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising a sense region, wherein any one or more or all) pyrimidine nucleotides present in the sense region are 2'-deoxy- 2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or difluoromethoxy-ethoxy pyrimidine nucleotides wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or 2'- 0 O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of 2 pyrimidine nucleotides are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyltrifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and wherein 0 10 any one or more or all) purine nucleotides present in the sense region are 2'-deoxy Cl purine nucleotides wherein all purine nucleotides are 2'-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2'-deoxy purine nucleotides), wherein any nucleotides comprising a 3'-terminal nucleotide overhang that are present in said sense region are 2'-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising a sense region, wherein any one or more or all) pyrimidine nucleotides present in the sense region are 2'-deoxy- 2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2'-0-ethyl-trifluoromethoxy, or difluoromethoxy-ethoxy pyrimidine nucleotides wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or 2'- O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyltrifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and wherein any one or more or all) purine nucleotides present in the sense region are methyl purine nucleotides wherein all purine nucleotides are 2'-O-methyl, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy purine nucleotides or alternately a plurality of purine nucleotides are 2'-O-methyl, 4'thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxyethoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising a sense region, wherein any one or more or all) pyrimidine nucleotides present in the sense region are 2'-deoxy- 49 2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2'-0-ethyl-trifluoromethoxy, or difluoromethoxy-ethoxy pyrimidine nucleotides wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or 2'- O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyltrifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy pyrimidine nucleotides), wherein any one or more or all) purine nucleotides present in the sense region are 2'-O-methyl, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxyethoxy purine nucleotides wherein all purine nucleotides are 2'-O-methyl, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy purine nucleotides or alternately a plurality of purine nucleotides are 2'--methyl, 4'thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxyethoxy purine nucleotides), and wherein any nucleotides comprising a 3'-terminal nucleotide overhang that are present in said sense region are 2'-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising an antisense region, wherein any one or more or all) pyrimidine nucleotides present in the antisense region are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or difluoromethoxy-ethoxy pyrimidine nucleotides wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or 2'- O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyltrifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and wherein any one or more or all) purine nucleotides present in the antisense region are methyl, 4'-thio, 2'-O-trifluoromethyl, 2'-0-ethyl-trifluoromethoxy, or difluoromethoxy-ethoxy purine nucleotides wherein all purine nucleotides are 2'- O-methyl, 4'-thio, 2'-O-trifluoromethyl, 2'-0-ethyl-trifluoromethoxy, or difluoromethoxy-ethoxy purine nucleotides or alternately a plurality of purine nucleotides are 2'-O-methyl, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising an antisense region, wherein any one or more or all) pyrimidine nucleotides present in the antisense region are N 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or difluoromethoxy-ethoxy pyrimidine nucleotides wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or 2'- O 5 O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl- 00 trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy pyrimidine nucleotides), wherein any one or more or all) purine nucleotides present in the antisense region are Ni methyl, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or S 10 difluoromethoxy-ethoxy purine nucleotides wherein all purine nucleotides are 2'- N 0-methyl, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or difluoromethoxy-ethoxy purine nucleotides or alternately a plurality of purine nucleotides are 2'-O-methyl, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy purine nucleotides), and wherein any nucleotides comprising a 3'-terminal nucleotide overhang that are present in said antisense region are 2'-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising an antisense region, wherein any one or more or all) pyrimidine nucleotides present in the antisense region are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or difluoromethoxy-ethoxy pyrimidine nucleotides wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or 2'- O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyltrifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and wherein any one or more or all) purine nucleotides present in the antisense region are 2'deoxy purine nucleotides wherein all purine nucleotides are 2'-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2'-deoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising an antisense region, wherein any one or more or all) pyrimidine nucleotides present in the antisense region are 51 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or difluoromethoxy-ethoxy pyrimidine nucleotides wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or 2'- O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyltrifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and wherein 00 any one or more or all) purine nucleotides present in the antisense region are methyl, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or N difluoromethoxy-ethoxy purine nucleotides wherein all purine nucleotides are 2'- S 10 O-methyl, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or N difluoromethoxy-ethoxy purine nucleotides or alternately a plurality of purine nucleotides are 2'-0-methyl, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention capable of mediating RNA interference (RNAi) against repeat expansion (RE) inside a cell or reconstituted in vitro system comprising a sense region, wherein one or more pyrimidine nucleotides present in the sense region are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyltrifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy pyrimidine nucleotides wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro, 4'-thio, trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and one or more purine nucleotides present in the sense region are 2'-deoxy purine nucleotides wherein all purine nucleotides are 2'-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2'-deoxy purine nucleotides), and an antisense region, wherein one or more pyrimidine nucleotides present in the antisense region are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2'- O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy pyrimidine nucleotides wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro, 4'-thio, trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and one or more purine nucleotides present in the antisense region are 2'-O-methyl, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy purine nucleotides wherein all purine nucleotides are 2'-O-methyl, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or difluoromethoxy-ethoxy purine nucleotides or alternately a plurality of purine nucleotides are 2'-O-methyl, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy purine nucleotides). The sense region and/or the antisense region can have a terminal cap modification, such as any modification described herein or shown in Figure 10, that is optionally present at the 3'-end, the end, or both of the 3' and 5'-ends of the sense and/or antisense sequence. The sense and/or antisense region can optionally further comprise a 3'-terminal nucleotide overhang having about 1 to about 4 about 1, 2, 3, or 4) 2'-deoxynucleotides. The overhang nucleotides can further comprise one or more about 1, 2, 3, 4 or more) phosphorothioate, phosphonoacetate, and/or thiophosphonoacetate internucleotide linkages. Non-limiting examples of these chemically-modified siNAs are shown in Figures 4 and 5 and Tables III and IV herein. In any of these described embodiments, the purine nucleotides present in the sense region are alternatively 2'-0-methyl, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy purine nucleotides wherein all purine nucleotides are 2'-O-methyl, 4'-thio, trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy purine nucleotides or alternately a plurality of purine nucleotides are 2'-O-methyl, 4'-thio, trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy purine nucleotides) and one or more purine nucleotides present in the antisense region are methyl, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or difluoromethoxy-ethoxy purine nucleotides wherein all purine nucleotides are 2'- O-methyl, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or difluoromethoxy-ethoxy purine nucleotides or alternately a plurality of purine nucleotides are 2'-O-methyl, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy purine nucleotides). Also, in any of these embodiments, one or more purine nucleotides present in the sense region are alternatively purine ribonucleotides wherein all purine nucleotides are purine ribonucleotides or alternately a plurality of purine nucleotides are purine ribonucleotides) 53 and any purine nucleotides present in the antisense region are 2'-O-methyl, 4'-thio, 2'-O- N trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy purine nucleotides wherein all purine nucleotides are 2'-O-methyl, 4'-thio, trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy purine nucleotides or alternately a plurality of purine nucleotides are 2'-O-methyl, 4'-thio, trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy purine 00 nucleotides). Additionally, in any of these embodiments, one or more purine nucleotides Spresent in the sense region and/or present in the antisense region are alternatively 1 selected from the group consisting of 2'-deoxy nucleotides, locked nucleic acid (LNA) S 10 nucleotides, 2'-methoxyethyl nucleotides, 4'-thionucleotides, 2'-O-trifluoromethyl C, nucleotides, 2'-O-ethyl-trifluoromethoxy nucleotides, 2'-O-difluoromethoxy-ethoxy nucleotides and 2'-O-methyl nucleotides wherein all purine nucleotides are selected from the group consisting of 2'-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2'-methoxyethyl nucleotides, 4'-thionucleotides, 2'-O-trifluoromethyl nucleotides, 2'-O-ethyl-trifluoromethoxy nucleotides, 2'-O-difluoromethoxy-ethoxy nucleotides and 2'-0-methyl nucleotides or alternately a plurality of purine nucleotides are selected from the group consisting of 2'-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2'-methoxyethyl nucleotides, 4'-thionucleotides, trifluoromethyl nucleotides, 2'-O-ethyl-trifluoromethoxy nucleotides, difluoromethoxy-ethoxy nucleotides and 2'-0-methyl nucleotides).

In another embodiment, any modified nucleotides present in the siNA molecules of the invention, preferably in the antisense strand of the siNA molecules of the invention, but also optionally in the sense and/or both antisense and sense strands, comprise modified nucleotides having properties or characteristics similar to naturally occurring ribonucleotides. For example, the invention features siNA molecules including modified nucleotides having a Northern conformation Northern pseudorotation cycle, see for example Saenger, Principles of Nucleic Acid Structure, Springer-Verlag ed., 1984). As such, chemically modified nucleotides present in the siNA molecules of the invention, preferably in the antisense strand of the siNA molecules of the invention, but also optionally in the sense and/or both antisense and sense strands, are resistant to nuclease degradation while at the same time maintaining the capacity to mediate RNAi. Nonlimiting examples of nucleotides having a northern configuration include locked nucleic acid (LNA) nucleotides 4'-C-methylene-(D-ribofuranosyl) nucleotides); 2'- N methoxyethoxy (MOE) nucleotides; 2'-methyl-thio-ethyl, 2'-deoxy-2'-fluoro nucleotides, 2'-deoxy-2'-chloro nucleotides, 2'-azido nucleotides, 2'-O-trifluoromethyl nucleotides, 2'-O-ethyl-trifluoromethoxy nucleotides, 2'-O-difluoromethoxy-ethoxy 0 5 nucleotides, 4'-thio nucleotides and 2'-O-methyl nucleotides.

o0 In one embodiment, the sense strand of a double stranded siNA molecule of the invention comprises a terminal cap moiety, (see for example Figure 10) such as an Sinverted deoxyabaisc moiety, at the 3'-end, 5'-end, or both 3' and 5'-ends of the sense t strand.

1 10 In one embodiment, the invention features a chemically-modified short interfering nucleic acid molecule (siNA) capable of mediating RNA interference (RNAi) against repeat expansion (RE) inside a cell or reconstituted in vitro system, wherein the chemical modification comprises a conjugate covalently attached to the chemically-modified siNA molecule. Non-limiting examples of conjugates contemplated by the invention include conjugates and ligands described in Vargeese et al., USSN 10/427,160, filed April 2003, incorporated by reference herein in its entirety, including the drawings. In another embodiment, the conjugate is covalently attached to the chemically-modified siNA molecule via a biodegradable linker. In one embodiment, the conjugate molecule is attached at the 3'-end of either the sense strand, the antisense strand, or both strands of the chemically-modified siNA molecule. In another embodiment, the conjugate molecule is attached at the 5'-end of either the sense strand, the antisense strand, or both strands of the chemically-modified siNA molecule. In yet another embodiment, the conjugate molecule is attached both the 3'-end and 5'-end of either the sense strand, the antisense strand, or both strands of the chemically-modified siNA molecule, or any combination thereof. In one embodiment, a conjugate molecule of the invention comprises a molecule that facilitates delivery of a chemically-modified siNA molecule into a biological system, such as a cell. In another embodiment, the conjugate molecule attached to the chemically-modified siNA molecule is a ligand for a cellular receptor, such as peptides derived from naturally occurring protein ligands; protein localization sequences, including cellular ZIP code sequences; antibodies; nucleic acid aptamers; vitamins and other co-factors, such as folate and N-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol; steroids, and polyamines, such S as PEI, spermine or spermidine. Examples of specific conjugate molecules contemplated i by the instant invention that can be attached to chemically-modified siNA molecules are Sdescribed in Vargeese et al., U.S. Serial No. 10/201,394, filed July 22, 2002 incorporated by reference herein. The type of conjugates used and the extent of conjugation of siNA molecules of the invention can be evaluated for improved pharmacokinetic profiles, bioavailability, and/or stability of siNA constructs while at the same time maintaining the 00 ability of the siNA to mediate RNAi activity. As such, one skilled in the art can screen SsiNA constructs that are modified with various conjugates to determine whether the "1 siNA conjugate complex possesses improved properties while maintaining the ability to mediate RNAi, for example in animal models as are generally known in the art.

In one embodiment, the invention features a short interfering nucleic acid (siNA) molecule of the invention, wherein the siNA further comprises a nucleotide, nonnucleotide, or mixed nucleotide/non-nucleotide linker that joins the sense region of the siNA to the antisense region of the siNA. In one embodiment, a nucleotide, nonnucleotide, or mixed nucleotide/non-nucleotide linker is used, for example, to attach a conjugate moiety to the siNA. In one embodiment, a nucleotide linker of the invention can be a linker of 2 2 nucleotides in length, for example about 3, 4, 5, 6, 7, 8, 9, or nucleotides in length. In another embodiment, the nucleotide linker can be a nucleic acid aptamer. By "aptamer" or "nucleic acid aptamer" as used herein is meant a nucleic acid molecule that binds specifically to a target molecule wherein the nucleic acid molecule has sequence that comprises a sequence recognized by the target molecule in its natural setting. Alternately, an aptamer can be a nucleic acid molecule that binds to a target molecule where the target molecule does not naturally bind to a nucleic acid. The target molecule can be any molecule of interest. For example, the aptamer can be used to bind to a ligand-binding domain of a protein, thereby preventing interaction of the naturally occurring ligand with the protein. This is a non-limiting example and those in the art will recognize that other embodiments can be readily generated using techniques generally known in the art. (See, for example, Gold et al., 1995, Annu. Rev. Biochem., 64, 763; Brody and Gold, 2000, J. Biotechnol., 74, 5; Sun, 2000, Curr. Opin. Mol. Ther., 2, 100; Kusser, 2000, J. Biotechnol., 74, 27; Hermann and Patel, 2000, Science, 287, 820; and Jayasena, 1999, Clinical Chemistry, 45, 1628.) In yet another embodiment, a non-nucleotide linker of the invention comprises abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, Spolyhydrocarbon, or other polymeric compounds polyethylene glycols such as those having between 2 and 100 ethylene glycol units). Specific examples include those described by Seela and Kaiser, Nucleic Acids Res. 1990, 18:6353 and Nucleic Acids Res.

1987, 15:3113; Cload and Schepartz, J. Am. Chem. Soc. 1991, 113:6324; Richardson and 0 Schepartz, J. Am. Chem. Soc. 1991, 113:5109; Ma et al., Nucleic Acids Res. 1993, 21:2585 and Biochemistry 1993, 32:1751; Durand et al., Nucleic Acids Res. 1990, 18:6353; McCurdy et al., Nucleosides Nucleotides 1991, 10:287; Jschke et al., 0 10 Tetrahedron Lett. 1993, 34:301; Ono et al., Biochemistry 1991, 30:9914; Arnold et al., N International Publication No. WO 89/02439; Usman et al., International Publication No.

WO 95/06731; Dudycz et al., International Publication No. WO 95/11910 and Ferentz and Verdine, J. Am. Chem. Soc. 1991, 113:4000, all hereby incorporated by reference herein. A "non-nucleotide" further means any group or compound that can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity. The group or compound can be abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine, for example at the Cl position of the sugar.

In one embodiment, the invention features a short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) inside a cell or reconstituted in vitro system, wherein one or both strands of the siNA molecule that are assembled from two separate oligonucleotides do not comprise any ribonucleotides. For example, a siNA molecule can be assembled from a single oligonculeotide where the sense and antisense regions of the siNA comprise separate oligonucleotides that do not have any ribonucleotides nucleotides having a 2'-OH group) present in the oligonucleotides.

In another example, a siNA molecule can be assembled from a single oligonculeotide where the sense and antisense regions of the siNA are linked or circularized by a nucleotide or non-nucleotide linker as described herein, wherein the oligonucleotide does not have any ribonucleotides nucleotides having a 2'-OH group) present in the oligonucleotide. Applicant has surprisingly found that the presense of ribonucleotides nucleotides having a 2'-hydroxyl group) within the siNA molecule is not required 3 or essential to support RNAi activity. As such, in one embodiment, all positions within Sthe siNA can include chemically modified nucleotides and/or non-nucleotides such as nucleotides and or non-nucleotides having Formula I, II, III, IV, V, VI, or VII or any combination thereof to the extent that the ability of the siNA molecule to support RNAi activity in a cell is maintained.

o\ In one embodiment, a siNA molecule of the invention is a single stranded siNA 00 M molecule that mediates RNAi activity in a cell or reconstituted in vitro system O comprising a single stranded polynucleotide having complementarity to a target nucleic Sacid sequence. In another embodiment, the single stranded siNA molecule of the O 10 invention comprises a 5'-terminal phosphate group. In another embodiment, the single stranded siNA molecule of the invention comprises a 5'-terminal phosphate group and a 3'-terminal phosphate group a 2',3'-cyclic phosphate). In another embodiment, the single stranded siNA molecule of the invention comprises about 15 to about 30 about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides. In yet another embodiment, the single stranded siNA molecule of the invention comprises one or more chemically modified nucleotides or non-nucleotides described herein. For example, all the positions within the siNA molecule can include chemically-modified nucleotides such as nucleotides having any of Formulae I-VII, or any combination thereof to the extent that the ability of the siNA molecule to support RNAi activity in a cell is maintained.

In one embodiment, a siNA molecule of the invention is a single stranded siNA molecule that mediates RNAi activity in a cell or reconstituted in vitro system comprising a single stranded polynucleotide having complementarity to a target nucleic acid sequence, wherein one or more pyrimidine nucleotides present in the siNA are 2'deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or difluoromethoxy-ethoxy pyrimidine nucleotides wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or 2'- O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyltrifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and wherein any purine nucleotides present in the antisense region are 2'-O-methyl, 4'-thio, trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy purine 58 nucleotides wherein all purine nucleotides are 2'-O-methyl, 4'-thio, trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy purine Snucleotides or alternately a plurality of purine nucleotides are 2'-O-methyl, 4'-thio, 2'-Otrifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy purine nucleotides), and a terminal cap modification, such as any modification described herein or shown in Figure 10, that is optionally present at the 3'-end, the 5'-end, or both of the 0 3' and 5'-ends of the antisense sequence. The siNA optionally further comprises about 1 Sto about 4 or more about 1, 2, 3, 4 or more) terminal 2'-deoxynucleotides at the 3'- N end of the siNA molecule, wherein the terminal nucleotides can further comprise one or more 1, 2, 3, 4 or more) phosphorothioate, phosphonoacetate, and/or thiophosphonoacetate internucleotide linkages, and wherein the siNA optionally further comprises a terminal phosphate group, such as a 5'-terminal phosphate group. In any of these embodiments, any purine nucleotides present in the antisense region are alternatively 2'-deoxy purine nucleotides wherein all purine nucleotides are 2'deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2'-deoxy purine nucleotides). Also, in any of these embodiments, any purine nucleotides present in the siNA purine nucleotides present in the sense and/or antisense region) can alternatively be locked nucleic acid (LNA) nucleotides wherein all purine nucleotides are LNA nucleotides or alternately a plurality of purine nucleotides are LNA nucleotides). Also, in any of these embodiments, any purine nucleotides present in the siNA are alternatively 2'-methoxyethyl purine nucleotides wherein all purine nucleotides are 2'-methoxyethyl purine nucleotides or alternately a plurality of purine nucleotides are 2'-methoxyethyl purine nucleotides). In another embodiment, any modified nucleotides present in the single stranded siNA molecules of the invention comprise modified nucleotides having properties or characteristics similar to naturally occurring ribonucleotides. For example, the invention features siNA molecules including modified nucleotides having a Northern conformation Northern pseudorotation cycle, see for example Saenger, Principles of Nucleic Acid Structure, Springer-Verlag ed., 1984). As such, chemically modified nucleotides present in the single stranded siNA molecules of the invention are preferably resistant to nuclease degradation while at the same time maintaining the capacity to mediate RNAi.

t In one embodiment, a siNA molecule of the invention comprises chemically Smodified nucleotides or non-nucleotides having any of Formulae I-VII, such as 2'- 2 deoxy, 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, 2'-O-difluoromethoxy-ethoxy or 2'-O-methyl nucleotides) at alternating positions within one or more strands or regions of the siNA molecule. For example, such chemical modifications can be introduced at every other position of a RNA based siNA molecule, 0 starting at either the first or second nucleotide from the 3'-end or 5'-end of the siNA. In Sa non-limiting example, a double stranded siNA molecule of the invention in which each N strand of the siNA is 21 nucleotides in length is featured wherein positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21 of each strand are chemically modified with compounds N having any of Formulae I-VII, such as such as 2'-deoxy, 2'-deoxy-2'-fluoro, 4'-thio, 2'- O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, 2'-O-difluoromethoxy-ethoxy or methyl nucleotides). In another non-limiting example, a double stranded siNA molecule of the invention in which each strand of the siNA is 21 nucleotides in length is featured wherein positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 of each strand are chemically modified with compounds having any of Formulae I-VII, such as such as 2'-deoxy, 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, difluoromethoxy-ethoxy or 2'-O-methyl nucleotides). Such siNA molecules can further comprise terminal cap moieties and/or backbone modifications as described herein.

In one embodiment, the invention features a method for modulating the expression of a repeat expansion (RE) gene within a cell comprising: synthesizing a siNA molecule of the invention, which can be chemically-modified or unmodified, wherein one of the siNA strands comprises a sequence complementary to RNA of the repeat expansion (RE) gene; and introducing the siNA molecule into a cell under conditions suitable to modulate inhibit) the expression of the repeat expansion (RE) gene in the cell.

In one embodiment, the invention features a method for modulating the expression of a repeat expansion (RE) gene within a cell comprising: synthesizing a siNA molecule of the invention, which can be chemically-modified or unmodified, wherein one of the siNA strands comprises a sequence complementary to RNA of the repeat expansion (RE) gene and wherein the sense strand sequence of the siNA comprises a sequence identical or substantially similar to the sequence of the target RNA; and (b)

I

introducing the siNA molecule into a cell under conditions suitable to modulate inhibit) the expression of the repeat expansion (RE) gene in the cell.

In another embodiment, the invention features a method for modulating the expression of more than one repeat expansion (RE) gene within a cell comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified or unmodified, wherein one of the siNA strands comprises a sequence complementary to 00 M RNA of the repeat expansion (RE) genes; and introducing the siNA molecules into a Scell under conditions suitable to modulate inhibit) the expression of the repeat i expansion (RE) genes in the cell.

S 10 In another embodiment, the invention features a method for modulating the expression of two or more repeat expansion (RE) genes within a cell comprising: (a) synthesizing one or more siNA molecules of the invention, which can be chemicallymodified or unmodified, wherein the siNA strands comprise sequences complementary to RNA of the repeat expansion (RE) genes and wherein the sense strand sequences of the siNAs comprise sequences identical or substantially similar to the sequences of the target RNAs; and introducing the siNA molecules into a cell under conditions suitable to modulate inhibit) the expression of the repeat expansion (RE) genes in the cell.

In another embodiment, the invention features a method for modulating the expression of more than one repeat expansion (RE) gene within a cell comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified or unmodified, wherein one of the siNA strands comprises a sequence complementary to RNA of the repeat expansion (RE) gene and wherein the sense strand sequence of the siNA comprises a sequence identical or substantially similar to the sequences of the target RNAs; and introducing the siNA molecule into a cell under conditions suitable to modulate inhibit) the expression of the repeat expansion (RE) genes in the cell.

In another embodiment, the invention features a method for modulating the expression of a repeat expansion (RE) gene within a cell comprising: synthesizing a siNA molecule of the invention, which can be chemically-modified or unmodified, wherein one of the siNA strands comprises a sequence complementary to RNA of the repeat expansion (RE) gene, wherein the sense strand sequence of the siNA comprises a 61 t sequence identical or substantially similar to the sequences of the target RNA; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate inhibit) the expression of the repeat expansion (RE) gene in the cell.

In one embodiment, siNA molecules of the invention are used as reagents in ex vivo applications. For example, siNA reagents are introduced into tissue or cells that are transplanted into a subject for therapeutic effect. The cells and/or tissue can be derived 00 M€ from an organism or subject that later receives the explant, or can be derived from another organism or subject prior to transplantation. The siNA molecules can be used to r modulate the expression of one or more genes in the cells or tissue, such that the cells or 0 10 tissue obtain a desired phenotype or are able to perform a function when transplanted in vivo. In one embodiment, certain target cells from a patient are extracted. These extracted cells are contacted with siNAs targeting a specific nucleotide sequence within the cells under conditions suitable for uptake of the siNAs by these cells using delivery reagents such as cationic lipids, liposomes and the like or using techniques such as electroporation to facilitate the delivery of siNAs into cells). The cells are then reintroduced back into the same patient or other patients.

In one embodiment, the invention features a method of modulating the expression of a repeat expansion (RE) gene in a tissue explant comprising: synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the repeat expansion (RE) gene; and introducing the siNA molecule into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate inhibit) the expression of the repeat expansion (RE) gene in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate inhibit) the expression of the repeat expansion (RE) gene in that organism.

In one embodiment, the invention features a method of modulating the expression of a repeat expansion (RE) gene in a tissue explant comprising: synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the repeat expansion (RE) gene and wherein the sense strand sequence of the siNA comprises a sequence identical or substantially similar to the sequence of the target RNA; and introducing the siNA molecule into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate inhibit) the expression of the repeat expansion (RE) gene in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate inhibit) the expression of the repeat expansion (RE) gene in that organism.

In another embodiment, the invention features a method of modulating the expression of more than one repeat expansion (RE) gene in a tissue explant comprising: synthesizing siNA molecules of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the repeat expansion (RE) genes; and introducing the siNA molecules into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate inhibit) the expression of the repeat expansion (RE) genes in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate inhibit) the expression of the repeat expansion (RE) genes in that organism.

In one embodiment, the invention features a method of modulating the expression of a repeat expansion (RE) gene in a subject or organism comprising: synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the repeat expansion (RE) gene; and introducing the siNA molecule into the subject or organism under conditions suitable to modulate inhibit) the expression of the repeat expansion (RE) gene in the subject or organism. The level of repeat expansion (RE) protein or RNA can be determined using various methods well-known in the art.

In another embodiment, the invention features a method of modulating the expression of more than one repeat expansion (RE) gene in a subject or organism comprising: synthesizing siNA molecules of the invention, which can be chemicallymodified, wherein one of the siNA strands comprises a sequence complementary to RNA of the repeat expansion (RE) genes; and introducing the siNA molecules into the 63 subject or organism under conditions suitable to modulate inhibit) the expression of the repeat expansion (RE) genes in the subject or organism. The level of repeat 2 expansion (RE) protein or RNA can be determined as is known in the art.

In one embodiment, the invention features a method for modulating the expression of a repeat expansion (RE) gene within a cell comprising: synthesizing a siNA C molecule of the invention, which can be chemically-modified, wherein the siNA 00 M€ comprises a single stranded sequence having complementarity to RNA of the repeat expansion (RE) gene; and introducing the siNA molecule into a cell under conditions suitable to modulate inhibit) the expression of the repeat expansion (RE) gene in the cell.

In another embodiment, the invention features a method for modulating the expression of more than one repeat expansion (RE) gene within a cell comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the repeat expansion (RE) gene; and contacting the cell in vitro or in vivo with the siNA molecule under conditions suitable to modulate inhibit) the expression of the repeat expansion (RE) genes in the cell.

In one embodiment, the invention features a method of modulating the expression of a repeat expansion (RE) gene in a tissue explant a brain, spinal cord, neuron or any other organ, tissue or cell as can be transplanted from one organism to another or back to the same organism from which the organ, tissue or cell is derived) comprising: synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the repeat expansion (RE) gene; and contacting a cell of the tissue explant derived from a particular subject or organism with the siNA molecule under conditions suitable to modulate inhibit) the expression of the repeat expansion (RE) gene in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the subject or organism the tissue was derived from or into another subject or organism under conditions suitable to modulate inhibit) the expression of the repeat expansion (RE) gene in that subject or organism.

In another embodiment, the invention features a method of modulating the .I expression of more than one repeat expansion (RE) gene in a tissue explant a brain, Sspinal cord, neuron, or any other organ, tissue or cell as can be transplanted from one organism to another or back to the same organism from which the organ, tissue or cell is derived) comprising: synthesizing siNA molecules of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having 00 complementarity to RNA of the repeat expansion (RE) gene; and introducing the SsiNA molecules into a cell of the tissue explant derived from a particular subject or organism under conditions suitable to modulate inhibit) the expression of the repeat 0 10 expansion (RE) genes in the tissue explant. In another embodiment, the method further .I comprises introducing the tissue explant back into the subject or organism the tissue was derived from or into another subject or organism under conditions suitable to modulate inhibit) the expression of the repeat expansion (RE) genes in that subject or organism.

In one embodiment, the invention features a method of modulating the expression of a repeat expansion (RE) gene in a subject or organism comprising: synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the repeat expansion (RE) gene; and introducing the siNA molecule into the subject or organism under conditions suitable to modulate inhibit) the expression of the repeat expansion (RE) gene in the subject or organism.

In another embodiment, the invention features a method of modulating the expression of more than one repeat expansion (RE) gene in a subject or organism comprising: synthesizing siNA molecules of the invention, which can be chemicallymodified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the repeat expansion (RE) gene; and introducing the siNA molecules into the subject or organism under conditions suitable to modulate inhibit) the expression of the repeat expansion (RE) genes in the subject or organism.

In one embodiment, the invention features a method of modulating the expression of a repeat expansion (RE) gene in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate inhibit) the expression of the repeat expansion (RE) gene in the subject or N organism.

In one embodiment, the invention features a method for treating or preventing Huntington's diease in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the repeat expansion (RE) gene both mutant and wild type HD 00 M€ alleles, or alternately the mutant HD allele) in the subject or organism whereby the treatment or prevention of Huntington's diease can be achieved. In one embodiment, the Vn invention features contacting the subject or organism with a siNA molecule of the invention via local administration to relevant tissues or cells, such as brain tissue or brain cells, for example cortex and striatum. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via systemic administration (such as via intravenous or subcutaneous administration of siNA) to relevant tissues or cells, such as tissues or cells involved in the maintenance or development of Huntington's diease. The siNA molecule of the invention can be formulated or conjugated as described herein or otherwise known in the art to target appropriate tisssues or cells in the subject or organism.

In one embodiment, the invention features a method for treating or preventing spinocerebellar ataxia in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the repeat expansion (RE) gene both mutant and wild type SCA alleles, such as wild type and mutant SCA1, SCA2, SCA3, SCAS, SCA7, SCA12, and SCA17, or alternately the mutant SCA allele such as mutant SCA1, SCA2, SCA3, SCA7, SCA12, and SCA17) in the subject or organism whereby the treatment or prevention of spinocerebellar ataxia can be achieved. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via local administration to relevant tissues or cells, such as CNS tissue or CNS cells, for example the spinal cord, dorsal ganglia, or cerebellum. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via systemic administration (such as via intravenous or subcutaneous administration of siNA) to relevant tissues or cells, such as tissues or cells involved in the maintenance or development of spinocerebellar ataxia. The siNA molecule of the 66

I

t invention can be formulated or conjugated as described herein or otherwise known in the N art to target appropriate tissues or cells in the subject or organism.

In one embodiment, the invention features a method for treating or preventing spinal muscular dystrophy in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate Cthe expression of the repeat expansion (RE) gene both mutant and wild type 00 M androgen receptor (AR) locus Xqll-ql2 alleles, or alternately the mutant androgen receptor (AR) locus Xq 11 -q 12 allele) in the subject or organism whereby the treatment or prevention of spinal muscular dystrophy can be achieved. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via local administration to relevant tissues or cells, such as CNS tissue or CNS cells, for example the spinal cord, dorsal ganglia, or cerebellum or PNS cells and tissue such as motor neurons. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via systemic administration (such as via intravenous or subcutaneous administration of siNA) to relevant tissues or cells, such as tissues or cells involved in the maintenance or development of spinal muscular dystrophy. The siNA molecule of the invention can be formulated or conjugated as described herein or otherwise known in the art to target appropriate tisssues or cells in the subject or organism.

In one embodiment, the invention features a method for treating or preventing bulbar muscular dystrophy in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the repeat expansion (RE) gene both mutant and wild type androgen receptor (AR) locus Xqll-ql2 alleles, or alternately the mutant androgen receptor (AR) locus Xq 1 -q12 allele) in the subject or organism whereby the treatment or prevention of bulbar muscular dystrophy can be achieved. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via local administration to relevant tissues or cells, such as CNS tissue or CNS cells, for example the spinal cord, dorsal ganglia, or cerebellum or PNS cells and tissue such as motor neurons. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via systemic administration (such as via intravenous or subcutaneous administration of siNA) to relevant tissues or cells, such 67 O as tissues or cells involved in the maintenance or development of bulbar muscular NI dystrophy. The siNA molecule of the invention can be formulated or conjugated as Sdescribed herein or otherwise known in the art to target appropriate tisssues or cells in the subject or organism.

In one embodiment, the invention features a method for treating or preventing dentatorubropallidoluysian atrophy in a subject or organism comprising contacting the 00 M subject or organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the repeat expansion (RE) gene both mutant and wild Stype DRPLA alleles, or alternately the mutant DRPLA allele) in the subject or organism whereby the treatment or prevention of dentatorubropallidoluysian atrophy can be achieved. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via local administration to relevant tissues or cells, such as CNS tissue or CNS cells, for example the spinal cord, dorsal ganglia, or cerebellum or PNS cells and tissue such as motor neurons. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via systemic administration (such as via intravenous or subcutaneous administration of siNA) to relevant tissues or cells, such as tissues or cells involved in the maintenance or development of dentatorubropallidoluysian atrophy. The siNA molecule of the invention can be formulated or conjugated as described herein or otherwise known in the art to target appropriate tisssues or cells in the subject or organism.

In any of the methods of treatment of the invention, the siNA can be administered to the subject as a course of treatment, for example administration at various time intervals, such as once per day over the course of treatment, once every two days over the course of treatment, once every three days over the course of treatment, once every four days over the course of treatment, once every five days over the course of treatment, once every six days over the course of treatment, once per week over the course of treatment, once every other week over the course of treatment, once per month over the course of treatment, etc. In one embodiment, the course of treatment is from about one to about 52 weeks or longer indefinitely). In one embodiment, the course of treatment is from about one to about 48 months or longer indefinitely). In the case of inner ear implants, the course of treatment may comprise one day to one month or 68 more. In the case of inner ear surgery, the course of treatment may comprise a single administration or multiple administrations as is required In any of the methods of treatment of the invention, the siNA can be administered to the subject systemically as described herein or otherwise known in the art. Systemic administration can include, for example, intravenous, subcutaneous, intramuscular, catheterization, nasopharangeai, transdermal, or gastrointestinal administration as is 00 M€ generally known in the art. In one embodiment, approaches to opening the blood brain 0 barrier or penetrating the blood brain barrier are utilized, see for example Pardridge, 2002, Nat Rev Drug Discov. 131-9 and Schlachetzki et al., 2004, Neurology, 62(8), 1275-81.

In one embodiment, in any of the methods of treatment or prevention of the invention, the siNA can be administered to the subject locally or to local tissues as described herein or otherwise known in the art. Local administration can include, for example, convection enhanced delivery, intrathecal administration, catheterization, implantation, direct injection, stenting, or other administration to relevant tissues, or any other local administration technique, method or procedure, as is generally known in the art.

In one embodiment, the invention features a method for administering siNA molecules and compositions of the invention to the CNS, including cortex, striatum, hippocampus, cerebellum, or spinal cord, comprising, contacting the siNA with such cells, tissues, or structures, under conditions suitable for the administration.

In one embodiment, the siNA, vector, or expression cassette is administered to the subject or organism by stereotactic or convection enhanced delivery to the brain. For example, US Patent No. 5,720,720 provides methods and devices useful for stereotactic and convection enhanced delivery of reagents to the brain. Such methods and devices can be readily used for the delivery of siNAs, vectors, or expression cassettes of the invention to a subject or organism, and is incorporated by reference herein in its entirety.

US Patent Application Nos. 2002/0141980; 2002/0114780; and 2002/0187127 all provide methods and devices useful for stereotactic and convection enhanced delivery of reagents that can be readily adapted for delivery of siNAs, vectors, or expression cassettes of the invention to a subject or organism, and are incorporated by reference 69 O herein in their entirety. Particular devices that may be useful in delivering siNAs, vectors, or expression cassettes of the invention to a subject or organism are for example Sdescribed in US Patent Application No. 2004/0162255, which is incorporated by reference herein in its entirety.

In another embodiment, the invention features a method of modulating the expression of more than one repeat expansion (RE) gene in a subject or organism 00 M comprising contacting the subject or organism with one or more siNA molecules of the invention under conditions suitable to modulate inhibit) the expression of the repeat t expansion (RE) genes in the subject or organism. In one embodiment, the repeat expansion (RE) genes, are for example, selected from the group consisting of huntingtin, SCA1, SCA2, SCA3, SCA6, SCA7, SCA12, SCA17, SBMA, or DRPLA (see for example Table including both mutant and wild-type alleles thereof.

The siNA molecules of the invention can be designed to down regulate or inhibit target repeat expansion gene expression through RNAi targeting of a variety of nucleic acid molecules. In one embodiment, the siNA molecules of the invention are used to target various DNA corresponding to a target gene, for example via heterochromatic silencing. In one embodiment, the siNA molecules of the invention are used to target various RNAs corresponding to a target gene, for example via RNA target cleavage or translational inhibition. Non-limiting examples of such RNAs include messenger RNA (mRNA), non-coding RNA or regulatory elements, alternate RNA splice variants of target gene(s), post-transcriptionally modified RNA of target gene(s), pre-mRNA of target gene(s), and/or RNA templates. If alternate splicing produces a family of transcripts 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 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 protein. Non-limiting examples of applications of the invention relating to targeting these RNA molecules include therapeutic pharmaceutical applications, cosmetic applications, veterinary applications, pharmaceutical discovery applications, molecular diagnostic and gene function applications, and gene mapping, for example ,I using single nucleotide polymorphism mapping with siNA molecules of the invention.

SSuch applications can be implemented using known gene sequences or from partial sequences available from an expressed sequence tag (EST).

In another embodiment, the siNA molecules of the invention are used to target C conserved sequences corresponding to a gene family or gene families such as repeat 00 M€ expansion (RE) family genes, including both wild type and mutant alleles of repeat Sexpansion genes. As such, siNA molecules targeting multiple repeat expansion (RE) V)targets can provide increased therapeutic effect. In one embodiment, the invention features the targeting (cleavage or inhibition of expression or function) of more than one repeat expansion (RE) gene sequence using a single siNA molecule, by targeting the conserved sequences of the targeted repeat expansion (RE) gene sequences that are unique to the mutant allele of a repeat expansion gene).

In addition, siNA 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 pathway to determine the function of uncharacterized gene(s) in gene function analysis, mRNA function analysis, or translational analysis.

The invention can be used to determine potential target gene pathways involved in various diseases and conditions toward pharmaceutical development. The invention can be used to understand pathways of gene expression involved in, for example, the progression and/or maintenance Huntington disease and related conditions such as progressive chorea, rigidity, dementia, and seizures, spinocerebellar ataxia, spinal and bulbar muscular dystrophy (SBMA), dentatorubropallidoluysian atrophy (DRPLA), and any other diseases or conditions that are related to or will respond to the levels of a repeat expansion (RE) protein in a cell, tissue, subject, or organism, alone or in combination with other therapies.

In one embodiment, siNA molecule(s) and/or methods of the invention are used to down regulate the expression of gene(s) that encode RNA referred to by Genbank Accession, for example, repeat expansion (RE) genes encoding RNA sequence(s) referred to herein by Genbank Accession number, for example, Genbank Accession Nos.

shown in Table I.

t In one embodiment, the invention features a method comprising: generating a (library of siNA constructs having a predetermined complexity; and assaying the siNA Sconstructs of above, under conditions suitable to determine RNAi target sites within the target RNA sequence. In one embodiment, the siNA molecules of have strands of a fixed length, for example, about 23 nucleotides in length. In another embodiment, the siNA molecules of are of differing length, for example having strands of about 15 to 00 about 30 about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or Snucleotides in length. In one embodiment, the assay can comprise a reconstituted in (vitro siNA assay as described herein. In another embodiment, the assay can comprise a cell culture system in which target RNA is expressed. In another embodiment, fragments Ni. of target RNA are analyzed for detectable levels of cleavage, for example by gel electrophoresis, northern blot analysis, or RNAse protection assays, to determine the most suitable target site(s) within the target RNA sequence. The target RNA sequence can be obtained as is known in the art, for example, by cloning and/or transcription for in vitro systems, and by cellular expression in in vivo systems.

In one embodiment, the invention features a method comprising: generating a randomized library of siNA constructs having a predetermined complexity, such as of 4N, where N represents the number of base paired nucleotides in each of the siNA construct strands (eg. for a siNA construct having 21 nucleotide sense and antisense strands with 19 base pairs, the complexity would be 419); and assaying the siNA constructs of (a) above, under conditions suitable to determine RNAi target sites within the target repeat expansion (RE) RNA sequence. In another embodiment, the siNA molecules of have strands of a fixed length, for example about 23 nucleotides in length. In yet another embodiment, the siNA molecules of are of differing length, for example having strands of about 15 to about 30 about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length. In one embodiment, the assay can comprise a reconstituted in vitro siNA assay as described in Example 6 herein. In another embodiment, the assay can comprise a cell culture system in which target RNA is expressed. In another embodiment, fragments of repeat expansion (RE) RNA are analyzed for detectable levels of cleavage, for example, by gel electrophoresis, northern blot analysis, or RNAse protection assays, to determine the most suitable target site(s) within the target repeat expansion (RE) RNA sequence. The target repeat expansion t (RE) RNA sequence can be obtained as is known in the art, for example, by cloning and/or transcription for in vitro systems, and by cellular expression in in vivo systems.

a In another embodiment, the invention features a method comprising: analyzing the sequence of a RNA target encoded by a target gene; synthesizing one or more sets of siNA molecules having sequence complementary to one or more regions of the RNA of and assaying the siNA molecules of under conditions suitable to determine 00 M€ RNAi targets within the target RNA sequence. In one embodiment, the siNA molecules Sof have strands of a fixed length, for example about 23 nucleotides in length. In t another embodiment, the siNA molecules of are of differing length, for example having strands of about 15 to about 30 about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 27, 28, 29, or 30) nucleotides in length. In one embodiment, the assay can comprise a reconstituted in vitro siNA assay as described herein. In another embodiment, the assay can comprise a cell culture system in which target RNA is expressed. Fragments of target RNA are analyzed for detectable levels of cleavage, for example by gel electrophoresis, northern blot analysis, or RNAse protection assays, to determine the most suitable target site(s) within the target RNA sequence. The target RNA sequence can be obtained as is known in the art, for example, by cloning and/or transcription for in vitro systems, and by expression in in vivo systems.

By "target site" is meant a sequence within a target RNA that is "targeted" for cleavage mediated by a siNA construct which contains sequences within its antisense region that are complementary to the target sequence.

By "detectable level of cleavage" is meant cleavage of target RNA (and formation of cleaved product RNAs) to an extent sufficient to discern cleavage products above the background of RNAs produced by random degradation of the target RNA. Production of cleavage products from 1-5% of the target RNA is sufficient to detect above the background for most methods of detection.

In one embodiment, the invention features a composition comprising a siNA molecule of the invention, which can be chemically-modified, in a pharmaceutically acceptable carrier or diluent. In another embodiment, the invention features a pharmaceutical composition comprising siNA molecules of the invention, which can be chemically-modified, targeting one or more genes in a pharmaceutically acceptable 73 t carrier or diluent. In another embodiment, the invention features a method for 0Ni diagnosing a disease, trait, or condition in a subject comprising administering to the Ssubject a composition of the invention under conditions suitable for the diagnosis of the disease, trait, or condition in the subject. In another embodiment, the invention features a method for treating or preventing a disease, trait, or condition, such as Huntington disease, spinocerebellar ataxia, spinal and bulbar muscular dystrophy, and 00 dentatorubropallidoluysian atrophy in a subject, comprising administering to the subject a composition of the invention under conditions suitable for the treatment or prevention (Ni of the disease, trait, or condition in the subject, alone or in conjunction with one or more Vt) other therapeutic compounds.

In another embodiment, the invention features a method for validating a repeat expansion (RE) gene target, comprising: synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands includes a sequence complementary to RNA of a repeat expansion (RE) target gene; (b) introducing the siNA molecule into a cell, tissue, subject, or organism under conditions suitable for modulating expression of the repeat expansion (RE) target gene in the cell, tissue, subject, or organism; and determining the function of the gene by assaying for any phenotypic change in the cell, tissue, subject, or organism.

In another embodiment, the invention features a method for validating a repeat expansion (RE) target comprising: synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands includes a sequence complementary to RNA of a repeat expansion (RE) target gene; introducing the siNA molecule into a biological system under conditions suitable for modulating expression of the repeat expansion (RE) target gene in the biological system; and determining the function of the gene by assaying for any phenotypic change in the biological system.

By "biological system" is meant, material, in a purified or unpurified form, from biological sources, including but not limited to human or animal, wherein the system comprises the components required for RNAi activity. The term "biological system" includes, for example, a cell, tissue, subject, or organism, or extract thereof. The term biological system also includes reconstituted RNAi systems that can be used in an in vitro setting.

I

By "phenotypic change" is meant any detectable change to a cell that occurs in N, response to contact or treatment with a nucleic acid molecule of the invention SsiNA). Such detectable changes include, but are not limited to, changes in shape, size, proliferation, motility, protein expression or RNA expression or other physical or chemical changes as can be assayed by methods known in the art. The detectable change can also include expression of reporter genes/molecules such as Green Florescent Protein 00 (GFP) or various tags that are used to identify an expressed protein or any other cellular component that can be assayed.

In one embodiment, the invention features a kit containing a siNA molecule of the invention, which can be chemically-modified, that can be used to modulate the expression of a repeat expansion (RE) target gene in a biological system, including, for example, in a cell, tissue, subject, or organism. In another embodiment, the invention features a kit containing more than one siNA molecule of the invention, which can be chemically-modified, that can be used to modulate the expression of more than one repeat expansion (RE) target gene in a biological system, including, for example, in a cell, tissue, subject, or organism.

In one embodiment, the invention features a cell containing one or more siNA molecules of the invention, which can be chemically-modified. In another embodiment, the cell containing a siNA molecule of the invention is a mammalian cell. In yet another embodiment, the cell containing a siNA molecule of the invention is a human cell.

In one embodiment, the synthesis of a siNA molecule of the invention, which can be chemically-modified, comprises: synthesis of two complementary strands of the siNA molecule; annealing the two complementary strands together under conditions suitable to obtain a double-stranded siNA molecule. In another embodiment, synthesis of the two complementary strands of the siNA molecule is by solid phase oligonucleotide synthesis. In yet another embodiment, synthesis of the two complementary strands of the siNA molecule is by solid phase tandem oligonucleotide synthesis.

In one embodiment, the invention features a method for synthesizing a siNA duplex molecule comprising: synthesizing a first oligonucleotide sequence strand of the siNA molecule, wherein the first oligonucleotide sequence strand comprises a cleavable linker molecule that can be used as a scaffold for the synthesis of the second oligonucleotide sequence strand of the siNA; synthesizing the second oligonucleotide sequence strand of siNA on the scaffold of the first oligonucleotide sequence strand, wherein the second oligonucleotide sequence strand further comprises a chemical moiety than can be used to purify the siNA duplex; cleaving the linker molecule of under conditions suitable for the two siNA oligonucleotide strands to hybridize and form a stable duplex; and purifying the siNA duplex utilizing the chemical moiety of the second oligonucleotide sequence strand. In one embodiment, cleavage of the linker molecule in above takes place during deprotection of the oligonucleotide, for example, under hydrolysis conditions using an alkylamine base such as methylamine. In one embodiment, the method of synthesis comprises solid phase synthesis on a solid support such as controlled pore glass (CPG) or polystyrene, wherein the first sequence of is synthesized on a cleavable linker, such as a succinyl linker, using the solid support as a scaffold. The cleavable linker in used as a scaffold for synthesizing the second strand can comprise similar reactivity as the solid support derivatized linker, such that cleavage of the solid support derivatized linker and the cleavable linker of takes place concomitantly. In another embodiment, the chemical moiety of that can be used to isolate the attached oligonucleotide sequence comprises a trityl group, for example a dimethoxytrityl group, which can be employed in a trityl-on synthesis strategy as described herein. In yet another embodiment, the chemical moiety, such as a dimethoxytrityl group, is removed during purification, for example, using acidic conditions.

In a further embodiment, the method for siNA synthesis is a solution phase synthesis or hybrid phase synthesis wherein both strands of the siNA duplex are synthesized in tandem using a cleavable linker attached to the first sequence which acts a scaffold for synthesis of the second sequence. Cleavage of the linker under conditions suitable for hybridization of the separate siNA sequence strands results in formation of the double-stranded siNA molecule.

In another embodiment, the invention features a method for synthesizing a siNA duplex molecule comprising: synthesizing one oligonucleotide sequence strand of the siNA molecule, wherein the sequence comprises a cleavable linker molecule that can be used as a scaffold for the synthesis of another oligonucleotide sequence; (b) synthesizing a second oligonucleotide sequence having complementarity to the first 76 t sequence strand on the scaffold of wherein the second sequence comprises the other strand of the double-stranded siNA molecule and wherein the second sequence further Scomprises a chemical moiety than can be used to isolate the attached oligonucleotide sequence; purifying the product of utilizing the chemical moiety of the second 0 5 oligonucleotide sequence strand under conditions suitable for isolating the full-length sequence comprising both siNA oligonucleotide strands connected by the cleavable 0 linker and under conditions suitable for the two siNA oligonucleotide strands to Shybridize and form a stable duplex. In one embodiment, cleavage of the linker molecule Sin above takes place during deprotection of the oligonucleotide, for example, under 0 10 hydrolysis conditions. In another embodiment, cleavage of the linker molecule in (c) N, above takes place after deprotection of the oligonucleotide. In another embodiment, the method of synthesis comprises solid phase synthesis on a solid support such as controlled pore glass (CPG) or polystyrene, wherein the first sequence of is synthesized on a cleavable linker, such as a succinyl linker, using the solid support as a scaffold. The cleavable linker in used as a scaffold for synthesizing the second strand can comprise similar reactivity or differing reactivity as the solid support derivatized linker, such that cleavage of the solid support derivatized linker and the cleavable linker of takes place either concomitantly or sequentially. In one embodiment, the chemical moiety of that can be used to isolate the attached oligonucleotide sequence comprises a trityl group, for example a dimethoxytrityl group.

In another embodiment, the invention features a method for making a doublestranded siNA molecule in a single synthetic process comprising: synthesizing an oligonucleotide having a first and a second sequence, wherein the first sequence is complementary to the second sequence, and the first oligonucleotide sequence is linked to the second sequence via a cleavable linker, and wherein a terminal 5'-protecting group, for example, a 5'-O-dimethoxytrityl group (5'-O-DMT) remains on the oligonucleotide having the second sequence; deprotecting the oligonucleotide whereby the deprotection results in the cleavage of the linker joining the two oligonucleotide sequences; and purifying the product of under conditions suitable for isolating the double-stranded siNA molecule, for example using a trityl-on synthesis strategy as described herein.

1 In another embodiment, the method of synthesis of siNA molecules of the invention comprises the teachings of Scaringe et al., US Patent Nos. 5,889,136; 2 6,008,400; and 6,111,086, incorporated by reference herein in their entirety.

In one embodiment, the invention features siNA constructs that mediate RNAi against repeat expansion wherein the siNA construct comprises one or more C* chemical modifications, for example, one or more chemical modifications having any of 00 M€ Formulae I-VII or any combination thereof that increases the nuclease resistance of the siNA construct.

SIn another embodiment, the invention features a method for generating siNA molecules with increased nuclease resistance comprising introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step under conditions suitable for isolating siNA molecules having increased nuclease resistance.

In another embodiment, the invention features a method for generating siNA molecules with improved toxicologic profiles having attenuated or no immunstimulatory properties) comprising introducing nucleotides having any of Formula I-VII siNA motifs referred to in Table IV) or any combination thereof into a siNA molecule, and assaying the siNA molecule of step under conditions suitable for isolating siNA molecules having improved toxicologic profiles.

In another embodiment, the invention features a method for generating siNA formulations with improved toxicologic profiles having attenuated or no immunstimulatory properties) comprising generating a siNA formulation comprising a siNA molecule of the invention and a delivery vehicle or delivery particle as described herein or as otherwise known in the art, and assaying the siNA formualtion of step (a) under conditions suitable for isolating siNA formulations having improved toxicologic profiles.

In another embodiment, the invention features a method for generating siNA molecules that do not stimulate an interferon response no interferon response or attenuated interferon response) in a cell, subject, or organism, comprising introducing nucleotides having any of Formula I-VII siNA motifs referred to in Table IV) or 78 any combination thereof into a siNA molecule, and assaying the siNA molecule of step under conditions suitable for isolating siNA molecules that do not stimulate an Sinterferon response.

In another embodiment, the invention features a method for generating siNA formulations that do not stimulate an interferon response no interferon response or attenuated interferon response) in a cell, subject, or organism, comprising generating 00 M€ a siNA formulation comprising a siNA molecule of the invention and a delivery vehicle or delivery particle as described herein or as otherwise known in the art, and assaying the siNA formualtion of step under conditions suitable for isolating siNA formulations that do not stimulate an interferon response.

By "improved toxicologic profile", is meant that the chemically modified or formulated siNA construct exhibits decreased toxicity in a cell, subject, or organism compared to an unmodified or unformulated siNA, or siNA molecule having fewer modifications or modifications that are less effective in imparting improved toxicology.

In a non-limiting example, siNA molecules and formulations with improved toxicologic profiles are associated with a decreased or attenuated immunostimulatory response in a cell, subject, or organism compared to an unmodified or unformulated siNA, or siNA molecule having fewer modifications or modifications that are less effective in imparting improved toxicology. In one embodiment, a siNA molecule or formulation with an improved toxicological profile comprises no ribonucleotides. In one embodiment, a siNA molecule or formulation with an improved toxicological profile comprises less than ribonucleotides 1, 2, 3, or 4 ribonucleotides). In one embodiment, a siNA molecule or formulation with an improved toxicological profile comprises Stab 7, Stab 8, Stab 11, Stab 12, Stab 13, Stab 16, Stab 17, Stab 18, Stab 19, Stab 20, Stab 23, Stab 24, Stab 25, Stab 26, Stab 27, Stab 28, Stab 29, Stab 30, Stab 31, Stab 32, Stab 33, Stab 34 or any combination thereof (see Table IV). Herein, numeric Stab chemistries include both 2'-fluoro and 2'-OCF3 versions of the chemistries shown in Table IV. For example, "Stab 7/8" refers to both Stab 7/8 and Stab 7F/8F etc. In one embodiment, a siNA molecule or formulation with an improved toxicological profile comprises a siNA molecule of the invention and a formulation as described in United States Patent Application Publication No. 20030077829, incorporated by reference herein in its entirety including the drawings. In one embodiment, the level of immunostimulatory 79

I

response associated with a given siNA molecule can be measured as is known in the art, for example by determining the level of PKR/interferon response, proliferation, B-cell activation, and/or cytokine production in assays to quantitate the immunostimulatory response of particular siNA molecules (see, for example, Leifer et al., 2003, J Immunother. 26, 313-9; and U.S. Patent No. 5,968,909, incorporated in its entirety by reference).

In one embodiment, the invention features siNA constructs that mediate RNAi against repeat expansion wherein the siNA construct comprises one or more chemical modifications described herein that modulates the binding affinity between the sense and antisense strands of the siNA construct.

In another embodiment, the invention features a method for generating siNA molecules with increased binding affinity between the sense and antisense strands of the siNA molecule comprising introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and assaying the siNA molecule of step under conditions suitable for isolating siNA molecules having increased binding affinity between the sense and antisense strands of the siNA molecule.

In one embodiment, the invention features siNA constructs that mediate RNAi against repeat expansion wherein the siNA construct comprises one or more chemical modifications described herein that modulates the binding affinity between the antisense strand of the siNA construct and a complementary target RNA sequence within a cell.

In one embodiment, the invention features siNA constructs that mediate RNAi against repeat expansion wherein the siNA construct comprises one or more chemical modifications described herein that modulates the binding affinity between the antisense strand of the siNA construct and a complementary target DNA sequence within a cell.

In another embodiment, the invention features a method for generating siNA molecules with increased binding affinity between the antisense strand of the siNA molecule and a complementary target RNA sequence comprising introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and assaying the siNA molecule of step under conditions suitable for Ni, isolating siNA molecules having increased binding affinity between the antisense strand of the siNA molecule and a complementary target RNA sequence.

In another embodiment, the invention features a method for generating siNA molecules with increased binding affinity between the antisense strand of the siNA molecule and a complementary target DNA sequence comprising introducing 00 M€ nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and assaying the siNA molecule of step under conditions suitable for isolating siNA molecules having increased binding affinity between the antisense strand of the siNA molecule and a complementary target DNA sequence.

In one embodiment, the invention features siNA constructs that mediate RNAi against repeat expansion wherein the siNA construct comprises one or more chemical modifications described herein that modulate the polymerase activity of a cellular polymerase capable of generating additional endogenous siNA molecules having sequence homology to the chemically-modified siNA construct.

In another embodiment, the invention features a method for generating siNA molecules capable of mediating increased polymerase activity of a cellular polymerase capable of generating additional endogenous siNA molecules having sequence homology to a chemically-modified siNA molecule comprising introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and assaying the siNA molecule of step under conditions suitable for isolating siNA molecules capable of mediating increased polymerase activity of a cellular polymerase capable of generating additional endogenous siNA molecules having sequence homology to the chemically-modified siNA molecule.

In one embodiment, the invention features chemically-modified siNA constructs that mediate RNAi against repeat expansion (RE) in a cell, wherein the chemical modifications do not significantly effect the interaction of siNA with a target RNA molecule, DNA molecule and/or proteins or other factors that are essential for RNAi in a manner that would decrease the efficacy of RNAi mediated by such siNA constructs.

t In another embodiment, the invention features a method for generating siNA molecules with improved RNAi specificity against repeat expansion (RE) targets a. comprising introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and assaying the siNA molecule of step under conditions suitable for isolating siNA molecules having improved RNAi specificity. In one embodiment, improved specificity comprises having reduced off target effects 00 compared to an unmodified siNA molecule. For example, introduction of terminal cap Smoieties at the 3'-end, 5'-end, or both 3' and 5'-ends of the sense strand or region of a 1 siNA molecule of the invention can direct the siNA to have improved specificity by preventing the sense strand or sense region from acting as a template for RNAi activity against a corresponding target having complementarity to the sense strand or sense region.

In another embodiment, the invention features a method for generating siNA molecules with improved RNAi activity against repeat expansion (RE) comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and assaying the siNA molecule of step under conditions suitable for isolating siNA molecules having improved RNAi activity.

In yet another embodiment, the invention features a method for generating siNA molecules with improved RNAi activity against repeat expansion (RE) target RNA comprising introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and assaying the siNA molecule of step under conditions suitable for isolating siNA molecules having improved RNAi activity against the target RNA.

In yet another embodiment, the invention features a method for generating siNA molecules with improved RNAi activity against repeat expansion (RE) target DNA comprising introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and assaying the siNA molecule of step under conditions suitable for isolating siNA molecules having improved RNAi activity against the target DNA.

In one embodiment, the invention features siNA constructs that mediate RNAi against repeat expansion wherein the siNA construct comprises one or more 82 S chemical modifications described herein that modulates the cellular uptake of the siNA construct, such as cholesterol conjugation of the siNA.

In another embodiment, the invention features a method for generating siNA molecules against repeat expansion (RE) with improved cellular uptake comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and assaying the siNA molecule of step under conditions suitable 00 M for isolating siNA molecules having improved cellular uptake.

I In one embodiment, the invention features siNA constructs that mediate RNAi Sagainst repeat expansion wherein the siNA construct comprises one or more

C

I 10 chemical modifications described herein that increases the bioavailability of the siNA construct, for example, by attaching polymeric conjugates such as polyethyleneglycol or equivalent conjugates that improve the pharmacokinetics of the siNA construct, or by attaching conjugates that target specific tissue types or cell types in vivo. Non-limiting examples of such conjugates are described in Vargeese et al., U.S. Serial No. 10/201,394 incorporated by reference herein.

In one embodiment, the invention features a method for generating siNA molecules of the invention with improved bioavailability comprising introducing a conjugate into the structure of a siNA molecule, and assaying the siNA molecule of step under conditions suitable for isolating siNA molecules having improved bioavailability. Such conjugates can include ligands for cellular receptors, such as peptides derived from naturally occurring protein ligands; protein localization sequences, including cellular ZIP code sequences; antibodies; nucleic acid aptamers; vitamins and other co-factors, such as folate and N-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol; cholesterol derivatives, polyamines, such as spermine or spermidine; and others.

In one embodiment, the invention features a double stranded short interfering nucleic acid (siNA) molecule that comprises a first nucleotide sequence complementary to a target RNA sequence or a portion thereof, and a second sequence having complementarity to said first sequence, wherein said second sequence is chemically modified in a manner that it can no longer act as a guide sequence for efficiently mediating RNA interference and/or be recognized by cellular proteins that facilitate 83 3 RNAi. In one embodiment, the first nucleotide sequence of the siNA is chemically modified as described herein. In one embodiment, the first nucleotide sequence of the 2 siNA is not modified is all RNA).

In one embodiment, the invention features a double stranded short interfering nucleic acid (siNA) molecule that comprises a first nucleotide sequence complementary C* to a target RNA sequence or a portion thereof, and a second sequence having 00 M€ complementarity to said first sequence, wherein the second sequence is designed or modified in a manner that prevents its entry into the RNAi pathway as a guide sequence or as a sequence that is complementary to a target nucleic acid RNA) sequence. In one embodiment, the first nucleotide sequence of the siNA is chemically modified as described herein. In one embodiment, the first nucleotide sequence of the siNA is not modified is all RNA). Such design or modifications are expected to enhance the activity of siNA and/or improve the specificity of siNA molecules of the invention.

These modifications are also expected to minimize any off-target effects and/or associated toxicity.

In one embodiment, the invention features a double stranded short interfering nucleic acid (siNA) molecule that comprises a first nucleotide sequence complementary to a target RNA sequence or a portion thereof, and a second sequence having complementarity to said first sequence, wherein said second sequence is incapable of acting as a guide sequence for mediating RNA interference. In one embodiment, the first nucleotide sequence of the siNA is chemically modified as described herein. In one embodiment, the first nucleotide sequence of the siNA is not modified is all RNA).

In one embodiment, the invention features a double stranded short interfering nucleic acid (siNA) molecule that comprises a first nucleotide sequence complementary to a target RNA sequence or a portion thereof, and a second sequence having complementarity to said first sequence, wherein said second sequence does not have a terminal 5'-hydroxyl or 5'-phosphate group.

In one embodiment, the invention features a double stranded short interfering nucleic acid (siNA) molecule that comprises a first nucleotide sequence complementary to a target RNA sequence or a portion thereof, and a second sequence having complementarity to said first sequence, wherein said second sequence comprises a 84 t terminal cap moiety at the 5'-end of said second sequence. In one embodiment, the terminal cap moiety comprises an inverted abasic, inverted deoxy abasic, inverted Snucleotide moiety, a group shown in Figure 10, an alkyl or cycloalkyl group, a heterocycle, or any other group that prevents RNAi activity in which the second sequence serves as a guide sequence or template for RNAi.

In one embodiment, the invention features a double stranded short interfering 00 0M nucleic acid (siNA) molecule that comprises a first nucleotide sequence complementary 0 to a target RNA sequence or a portion thereof, and a second sequence having complementarity to said first sequence, wherein said second sequence comprises a terminal cap moiety at the 5'-end and 3'-end of said second sequence. In one embodiment, each terminal cap moiety individually comprises an inverted abasic, inverted deoxy abasic, inverted nucleotide moiety, a group shown in Figure 10, an alkyl or cycloalkyl group, a heterocycle, or any other group that prevents RNAi activity in which the second sequence serves as a guide sequence or template for RNAi.

In one embodiment, the invention features a method for generating siNA molecules of the invention with improved specificity for down regulating or inhibiting the expression of a target nucleic acid a DNA or RNA such as a gene or its corresponding RNA), comprising introducing one or more chemical modifications into the structure of a siNA molecule, and assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved specificity. In another embodiment, the chemical modification used to improve specificity comprises terminal cap modifications at the 5'-end, 3'-end, or both 5' and 3'-ends of the siNA molecule. The terminal cap modifications can comprise, for example, structures shown in Figure 10 inverted deoxyabasic moieties) or any other chemical modification that renders a portion of the siNA molecule the sense strand) incapable of mediating RNA interference against an off target nucleic acid sequence. In a non-limiting example, a siNA molecule is designed such that only the antisense sequence of the siNA molecule can serve as a guide sequence for RISC mediated degradation of a corresponding target RNA sequence. This can be accomplished by rendering the sense sequence of the siNA inactive by introducing chemical modifications to the sense strand that preclude recognition of the sense strand as a guide sequence by RNAi machinery. In one embodiment, such chemical modifications comprise any chemical group at the 5'-end of the sense strand of the siNA, or any other group that serves to render the sense strand ,I inactive as a guide sequence for mediating RNA interference. These modifications, for Sexample, can result in a molecule where the 5'-end of the sense strand no longer has a free 5'-hydroxyl or a free 5'-phosphate group phosphate, diphosphate, triphosphate, cyclic phosphate etc.). Non-limiting examples of such siNA constructs are described herein, such as "Stab 9/10", "Stab "Stab 7/19", "Stab 17/22", "Stab 00 23/24", "Stab 24/25", and "Stab 24/26" any siNA having Stab 7, 9, 17, 23, or 24 sense strands) chemistries and variants thereof (see Table IV) wherein the 5'-end and 3'- I end of the sense strand of the siNA do not comprise a hydroxyl group or phosphate 0 10 group. Herein, numeric Stab chemistries include both 2'-fluoro and 2'-OCF3 versions CN of the chemistries shown in Table IV. For example, "Stab 7/8" refers to both Stab 7/8 and Stab 7F/8F etc.

In one embodiment, the invention features a method for generating siNA molecules of the invention with improved specificity for down regulating or inhibiting the expression of a target nucleic acid a DNA or RNA such as a gene or its corresponding RNA), comprising introducing one or more chemical modifications into the structure of a siNA molecule that prevent a strand or portion of the siNA molecule from acting as a template or guide sequence for RNAi activity. In one embodiment, the inactive strand or sense region of the siNA molecule is the sense strand or sense region of the siNA molecule, i.e. the strand or region of the siNA that does not have complementarity to the target nucleic acid sequence. In one embodiment, such chemical modifications comprise any chemical group at the 5'-end of the sense strand or region of the siNA that does not comprise a 5'-hydroxyl or 5'-phosphate group, or any other group that serves to render the sense strand or sense region inactive as a guide sequence for mediating RNA interference. Non-limiting examples of such siNA constructs are described herein, such as "Stab 9/10", "Stab "Stab 7/19", "Stab 17/22", "Stab 23/24", "Stab 24/25", and "Stab 24/26" any siNA having Stab 7, 9, 17, 23, or 24 sense strands) chemistries and variants thereof (see Table IV) wherein the and 3'-end of the sense strand of the siNA do not comprise a hydroxyl group or phosphate group. Herein, numeric Stab chemistries include both 2'-fluoro and 2'-OCF3 versions of the chemistries shown in Table IV. For example, "Stab 7/8" refers to both Stab 7/8 and Stab 7F/8F etc.

In one embodiment, the invention features a method for screening siNA molecules that are active in mediating RNA interference against a target nucleic acid sequence comprising generating a plurality of unmodified siNA molecules, screening the siNA molecules of step under conditions suitable for isolating siNA molecules that are active in mediating RNA interference against the target nucleic acid sequence, and introducing chemical modifications chemical modifications as described herein or as otherwise known in the art) into the active siNA molecules of In one embodiment, the method further comprises re-screening the chemically modified siNA molecules of step under conditions suitable for isolating chemically modified siNA molecules that are active in mediating RNA interference against the target nucleic acid sequence.

In one embodiment, the invention features a method for screening chemically modified siNA molecules that are active in mediating RNA interference against a target nucleic acid sequence comprising generating a plurality of chemically modified siNA molecules siNA molecules as described herein or as otherwise known in the art), and screening the siNA molecules of step under conditions suitable for isolating chemically modified siNA molecules that are active in mediating RNA interference against the target nucleic acid sequence.

The term "ligand" refers to any compound or molecule, such as a drug, peptide, hormone, or neurotransmitter, that is capable of interacting with another compound, such as a receptor, either directly or indirectly. The receptor that interacts with a ligand can be present on the surface of a cell or can alternately be an intercellular receptor. Interaction of the ligand with the receptor can result in a biochemical reaction, or can simply be a physical interaction or association.

In another embodiment, the invention features a method for generating siNA molecules of the invention with improved bioavailability comprising introducing an excipient formulation to a siNA molecule, and assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved bioavailability.

Such excipients include polymers such as cyclodextrins, lipids, cationic lipids, polyamines, phospholipids, nanoparticles, receptors, ligands, and others.

l n In another embodiment, the invention features a method for generating siNA 2i molecules of the invention with improved bioavailability comprising introducing Snucleotides having any of Formulae I-VII or any combination thereof into a siNA molecule, and assaying the siNA molecule of step under conditions suitable for isolating siNA molecules having improved bioavailability.

In another embodiment, polyethylene glycol (PEG) can be covalently attached to M siNA compounds of the present invention. The attached PEG can be any molecular 0 weight, preferably from about 100 to about 50,000 daltons (Da).

O The present invention can be used alone or as a component of a kit having at least C N 10 one of the reagents necessary to carry out the in vitro or in vivo introduction of RNA to test samples and/or subjects. For example, preferred components of the kit include a siNA molecule of the invention and a vehicle that promotes introduction of the siNA into cells of interest as described herein using lipids and other methods of transfection known in the art, see for example Beigelman et al, US 6,395,713). The kit can be used for target validation, such as in determining gene function and/or activity, or in drug optimization, and in drug discovery (see for example Usman et al., USSN 60/402,996).

Such a kit can also include instructions to allow a user of the kit to practice the invention.

The term "short interfering nucleic acid", "siNA", "short interfering RNA", "siRNA", "short interfering nucleic acid molecule", "short interfering oligonucleotide molecule", or "chemically-modified short interfering nucleic acid molecule" as used herein refers to any nucleic acid molecule capable of inhibiting or down regulating gene expression or viral replication, for example by mediating RNA interference "RNAi" or gene silencing in a sequence-specific manner; see for example Zamore et al., 2000, Cell, 101, 25-33; Bass, 2001, Nature, 411, 428-429; Elbashir et al., 2001, Nature, 411, 494- 498; and Kreutzer et al., International PCT Publication No. WO 00/44895; Zernicka- Goetz et al., International PCT Publication No. WO 01/36646; Fire, International

PCT

Publication No. WO 99/32619; Plaetinck et al., International PCT Publication No. WO 00/01846; Mello and Fire, International PCT Publication No. WO 01/29058; Deschamps-Depaillette, International PCT Publication No. WO 99/07409; and Li et al., International PCT Publication No. WO 00/44914; Allshire, 2002, Science, 297, 1818- 1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215- 3 2218; and Hall et al., 2002, Science, 297, 2232-2237; Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus et al., 2002, RNA, 8, 842-850; Reinhart et al., 2002, Gene Dev., 16, 1616-1626; and Reinhart Bartel, 2002, Science, 297, 1831). Non limiting examples of siNA molecules of the invention are shown in Figures 4-6, and Tables II and III herein. For example the siNA can be a double-stranded polynucleotide molecule comprising self-complementary sense and antisense regions, 0 wherein the antisense region comprises nucleotide sequence that is complementary to Snucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense 1 region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The siNA can be assembled from two separate oligonucleotides, where 1 one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary each strand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand; such as where the antisense strand and sense strand form a duplex or double stranded structure, for example wherein the double stranded region is about 15 to about 30, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 base pairs; the antisense strand comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense strand comprises nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof about 15 to about 25 or more nucleotides of the siNA molecule are complementary to the target nucleic acid or a portion thereof). Alternatively, the siNA is assembled from a single oligonucleotide, where the self-complementary sense and antisense regions of the siNA are linked by means of a nucleic acid based or non-nucleic acid-based linker(s).

The siNA can be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The siNA can be a circular singlestranded polynucleotide having two or more loop structures and a stem comprising selfcomplementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence 89 corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siNA molecule capable of mediating RNAi. The siNA can also comprise a single stranded polynucleotide having nucleotide sequence complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof (for example, where such siNA molecule does not require the presence within the siNA molecule of nucleotide 0 sequence corresponding to the target nucleic acid sequence or a portion thereof), wherein Sthe single stranded polynucleotide can further comprise a terminal phosphate group, such i as a 5'-phosphate (see for example Martinez et al., 2002, Cell., 110, 563-574 and Schwarz et al., 2002, Molecular Cell, 10, 537-568), or 5',3'-diphosphate. In certain embodiments, the siNA molecule of the invention comprises separate sense and antisense sequences or regions, wherein the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linkers molecules as is known in the art, or are alternately non-covalently linked by ionic interactions, hydrogen bonding, van der waals interactions, hydrophobic interactions, and/or stacking interactions. In certain embodiments, the siNA molecules of the invention comprise nucleotide sequence that is complementary to nucleotide sequence of a target gene. In another embodiment, the siNA molecule of the invention interacts with nucleotide sequence of a target gene in a manner that causes inhibition of expression of the target gene. As used herein, siNA molecules need not be limited to those molecules containing only RNA, but further encompasses chemically-modified nucleotides and non-nucleotides. In certain embodiments, the short interfering nucleic acid molecules of the invention lack 2'hydroxy containing nucleotides. Applicant describes in certain embodiments short interfering nucleic acids that do not require the presence of nucleotides having a 2'hydroxy group for mediating RNAi and as such, short interfering nucleic acid molecules of the invention optionally do not include any ribonucleotides nucleotides having a 2'-OH group). Such siNA molecules that do not require the presence of ribonucleotides within the siNA molecule to support RNAi can however have an attached linker or linkers or other attached or associated groups, moieties, or chains containing one or more nucleotides with 2'-OH groups. Optionally, siNA molecules can comprise ribonucleotides at about 5, 10, 20, 30, 40, or 50% of the nucleotide positions. The modified short interfering nucleic acid molecules of the invention can also be referred to as short interfering modified oligonucleotides "siMON." As used herein, the term siNA t is meant to be equivalent to other terms used to describe nucleic acid molecules that are N capable of mediating sequence specific RNAi, for example short interfering RNA S(siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid, short interfering modified oligonucleotide, chemically-modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA), and others. In addition, as used herein, the term RNAi 00 is meant to be equivalent to other terms used to describe sequence specific RNA Sinterference, such as post transcriptional gene silencing, translational inhibition, or N epigenetics. For example, siNA molecules of the invention can be used to epigenetically silence genes at both the post-transcriptional level or the pre-transcriptional level. In a (non-limiting example, epigenetic modulation of gene expression by siNA molecules of the invention can result from siNA mediated modification of chromatin structure or methylation pattern to alter gene expression (see, for example, Verdel et al., 2004, Science, 303, 672-676; Pal-Bhadra et al., 2004, Science, 303, 669-672; Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237). In another non-limiting example, modulation of gene expression by siNA molecules of the invention can result from siNA mediated cleavage of RNA (either coding or non-coding RNA) via RISC, or alternately, translational inhibition as is known in the art.

In one embodiment, a siNA molecule of the invention is a duplex forming oligonucleotide "DFO", (see for example Figures 14-15 and Vaish et al., USSN 10/727,780 filed December 3, 2003 and International PCT Application No. US04/16390, filed May 24, 2004).

In one embodiment, a siNA molecule of the invention is a multifunctional siNA, (see for example Figures 16-21 and Jadhav et al., USSN 60/543,480 filed February 2004 and International PCT Application No. US04/16390, filed May 24, 2004). In one embodiment, the multifunctional siNA of the invention can comprise sequence targeting, for example, two or more regions of repeat expansion (RE) RNA (see for example target sequences in Tables II and III).

By "asymmetric hairpin" as used herein is meant a linear siNA molecule comprising an antisense region, a loop portion that can comprise nucleotides or nont nucleotides, and a sense region that comprises fewer nucleotides than the antisense region to the extent that the sense region has enough complementary nucleotides to base 2 pair with the antisense region and form a duplex with loop. For example, an asymmetric hairpin siNA molecule of the invention can comprise an antisense region having length sufficient to mediate RNAi in a cell or in vitro system about 15 to about 30, or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and a 00 loop region comprising about 4 to about 12 about 4, 5, 6, 7, 8, 9, 10, 11, or 12) Snucleotides, and a sense region having about 3 to about 25 about 3, 4, 5, 6, 7, 8, 9, 1 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides that are complementary to the antisense region. The asymmetric hairpin siNA molecule can also C comprise a 5'-terminal phosphate group that can be chemically modified. The loop portion of the asymmetric hairpin siNA molecule can comprise nucleotides, nonnucleotides, linker molecules, or conjugate molecules as described herein.

By "asymmetric duplex" as used herein is meant a siNA molecule having two separate strands comprising a sense region and an antisense region, wherein the sense region comprises fewer nucleotides than the antisense region to the extent that the sense region has enough complementary nucleotides to base pair with the antisense region and form a duplex. For example, an asymmetric duplex siNA molecule of the invention can comprise an antisense region having length sufficient to mediate RNAi in a cell or in vitro system about 15 to about 30, or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 27, 28, 29, or 30 nucleotides) and a sense region having about 3 to about 25 about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or nucleotides that are complementary to the antisense region.

By "modulate" is meant that the expression of the gene, or level of a RNA molecule or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits is up regulated or down regulated, such that expression, level, or activity is greater than or less than that observed in the absence of the modulator. For example, the term "modulate" can mean "inhibit," but the use of the word "modulate" is not limited to this definition.

By "inhibit", "down-regulate", or "reduce", it is meant that the expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more t proteins or protein subunits, or activity of one or more proteins or protein subunits, is ,i reduced below that observed in the absence of the nucleic acid molecules siNA) of Sthe invention. In one embodiment, inhibition, down-regulation or reduction with an siNA molecule is below that level observed in the presence of an inactive or attenuated molecule. In another embodiment, inhibition, down-regulation, or reduction with siNA molecules is below that level observed in the presence of, for example, an siNA 00 molecule with scrambled sequence or with mismatches. In another embodiment, Sinhibition, down-regulation, or reduction of gene expression with a nucleic acid molecule of the instant invention is greater in the presence of the nucleic acid molecule than in its absence. In one embodiment, inhibition, down regulation, or reduction of gene expression is associated with post transcriptional silencing, such as RNAi mediated cleavage of a target nucleic acid molecule RNA) or inhibition of translation. In one embodiment, inhibition, down regulation, or reduction of gene expression is associated with pretranscriptional silencing, such as by alterations in DNA methylation patterns and DNA chromatin structure.

By "gene", or "target gene", is meant a nucleic acid that encodes an RNA, for example, nucleic acid sequences including, but not limited to, structural genes encoding a polypeptide. A gene or target gene can also encode a functional RNA (fRNA) or noncoding RNA (ncRNA), such as small temporal RNA (stRNA), micro RNA (miRNA), small nuclear RNA (snRNA), short interfering RNA (siRNA), small nucleolar RNA (snRNA), ribosomal RNA (rRNA), transfer RNA (tRNA) and precursor RNAs thereof.

Such non-coding RNAs can serve as target nucleic acid molecules for siNA mediated RNA interference in modulating the activity of fRNA or ncRNA involved in functional or regulatory cellular processes. Abberant fRNA or ncRNA activity leading to disease can therefore be modulated by siNA molecules of the invention. siNA molecules targeting fRNA and ncRNA can also be used to manipulate or alter the genotype or phenotype of a subject, organism or cell, by intervening in cellular processes such as genetic imprinting, transcription, translation, or nucleic acid processing transamination, methylation etc.). The target gene can be a gene derived from a cell, an endogenous gene, a transgene, or exogenous genes such as genes of a pathogen, for example a virus, which is present in the cell after infection thereof. The cell containing the target gene can be derived from or contained in any organism, for example a plant, animal, protozoan, virus, bacterium, or fungus. Non-limiting examples of plants include monocots, dicots, or gymnosperms. Non-limiting examples of animals include Svertebrates or invertebrates. Non-limiting examples of fungi include molds or yeasts.

For a review, see for example Snyder and Gerstein, 2003, Science, 300, 258-260.

By "non-canonical base pair" is meant any non-Watson Crick base pair, such as 0 mismatches and/or wobble base pairs, including flipped mismatches, single hydrogen 00 M bond mismatches, trans-type mismatches, triple base interactions, and quadruple base Sinteractions. Non-limiting examples of such non-canonical base pairs include, but are t not limited to, AC reverse Hoogsteen, AC wobble, AU reverse Hoogsteen, GU wobble, O 10 AA N7 amino, CC 2-carbonyl-amino(H1)-N3-amino(H 2 GA sheared, UC 4-carbonylamino, UU imino-carbonyl, AC reverse wobble, AU Hoogsteen, AU reverse Watson Crick, CG reverse Watson Crick, GC N3-amino-amino N3, AA N1-amino symmetric, AA N7-amino symmetric, GA N7-N1 amino-carbonyl, GA+ carbonyl-amino N7-N1, GG N1-carbonyl symmetric, GG N3-amino symmetric, CC carbonyl-amino symmetric, CC N3-amino symmetric, UU 2-carbonyl-imino symmetric, UU 4-carbonyl-imino symmetric, AA amino-N3, AA Nl-amino, AC amino 2-carbonyl, AC N3-amino, AC N7-amino, AU amino-4-carbonyl, AU Nl-imino, AU N3-imino, AU N7-imino, CC carbonyl-amino, GA amino-N1, GA amino-N7, GA carbonyl-amino, GA N3-amino, GC amino-N3, GC carbonyl-amino, GC N3-amino, GC N7-amino, GG amino-N7, GG carbonyl-imino, GG N7-amino, GU amino-2-carbonyl, GU carbonyl-imino, GU imino- 2-carbonyl, GU N7-imino, psiU imino-2-carbonyl, UC 4-carbonyl-amino, UC iminocarbonyl, UU imino-4-carbonyl, AC C2-H-N3, GA carbonyl-C2-H, UU imino-4carbonyl 2 carbonyl-C5-H, AC amino(A) N3(C)-carbonyl, GC imino amino-carbonyl, Gpsi imino-2-carbonyl amino-2- carbonyl, and GU imino amino-2-carbonyl base pairs.

By "repeat expansion" or "RE" as used herein is meant, any protein, peptide, or polypeptide comprising a trinucleotide repeat expansion that is associated with the maintenance or development of a polyQ disease, such as Huntington disease, spinocerebellar ataxia, spinal and bulbar muscular dystrophy, and dentatorubropallidoluysian atrophy, for example as encoded by Genbank Accession Nos.

shown in Table I huntingtin, SCA1, SCA2, SCA3, SCA6, SCA7, SCA12, SCA17, SBMA, or DRPLA genes). The terms "repeat expansion" or "RE" also refer to nucleic acid sequences encloding any protein, peptide, or polypeptide comprising a trinucleotide 94 repeat expansion, such as RNA or DNA comprising trinucleotide repeat expansion encoding sequence (see for example Wood et al., 2003, Neuropathol Appl Neurobiol., S29, 529-45). In certain embodiments, siNA molecules of the invention target both wild type and mutant forms of such repeat expansion disease genes. In certain embodiments, 0 5 siNA molecules of the invention target only mutant forms of such repeat expansion disease genes.

00 M By "Huntingtin" or "HD" as used herein is meant, any Huntingtin protein, peptide, 0 or polypeptide associated with the deveopment or maintenence of Huntington disease.

in The terms "Huntingtin" and "HD" also refer to nucleic acid sequences encloding any huntingtin protein, peptide, or polypeptide, such as Huntingtin RNA or Huntingtin DNA (see for example Van Dellen et al., January 24, 2004, Neurogenetics).

By "homologous sequence" is meant, a nucleotide sequence that is shared by one or more polynucleotide sequences, such as genes, gene transcripts and/or non-coding polynucleotides. For example, a homologous sequence can be a nucleotide sequence that is shared by two or more genes encoding related but different proteins, such as different members of a gene family, different protein epitopes, different protein isoforms or completely divergent genes, such as a cytokine and its corresponding receptors. A homologous sequence can be a nucleotide sequence that is shared by two or more noncoding polynucleotides, such as noncoding DNA or RNA, regulatory sequences, introns, and sites of transcriptional control or regulation. Homologous sequences can also include conserved sequence regions shared by more than one polynucleotide sequence.

Homology does not need to be perfect homology 100%), as partially homologous sequences are also contemplated by the instant invention 99%, 98%, 97%, 96%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% etc.).

By "conserved sequence region" is meant, a nucleotide sequence of one or more regions in a polynucleotide does not vary significantly between generations or from one biological system, subject, or organism to another biological system, subject, or organism. The polynucleotide can include both coding and non-coding DNA and RNA.

By "sense region" is meant a nucleotide sequence of a siNA molecule having complementarity to an antisense region of the siNA molecule. In addition, the sense

I

Sregion of a siNA molecule can comprise a nucleic acid sequence having homology with a target nucleic acid sequence.

By "antisense region" is meant a nucleotide sequence of a siNA molecule having complementarity to a target nucleic acid sequence. In addition, the antisense region of a siNA molecule can optionally comprise a nucleic acid sequence having complementarity Sto a sense region of the siNA molecule.

00 By "target nucleic acid" is meant any nucleic acid sequence whose expression or activity is to be modulated. The target nucleic acid can be DNA or RNA. In one embodiment, a target nucleic acid of the invention is repeat expansion (RE) RNA or S 10 DNA.

By "complementarity" is meant that a nucleic acid can form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types. In reference to the nucleic molecules of the present invention, the binding free energy for a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, RNAi activity.

Determination of binding free energies for nucleic acid molecules is well known in the art (see, Turner et al., 1987, CSH Symp. Quant. Biol. LII pp.123-13 3 Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc.

109:3783-3785). A percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds Watson-Crick base pairing) with a second nucleic acid sequence 5, 6, 7, 8, 9, or 10 nucleotides out of a total of 10 nucleotides in the first oligonucleotide being based paired to a second nucleic acid sequence having 10 nucleotides represents 50%, 60%, 70%, 80%, 90%, and 100% complementary respectively). "Perfectly complementary" means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. In one embodiment, a siNA molecule of the invention comprises about 15 to about 30 or more about 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more) nucleotides that are complementary to one or more target nucleic acid molecules or a portion thereof.

In one embodiment, the siNA molecules of the invention represent a novel therapeutic approach to treat Huntington disease and related conditions such as 96

I

t progressive chorea, rigidity, and dementia, and seizures, and any other diseases or conditions that are related to or will respond to the levels of huntingtin in a cell or tissue, alone or in combination with other therapies. The reduction of huntingtin expression (specifically alleles associated with Huntington disease, such as polyglutamine repeat expansion and related SNPs) and thus reduction in the level of the respective protein relieves, to some extent, the symptoms of the disease or condition.

00 M€ In one embodiment of the present invention, each sequence of a siNA molecule of the invention is independently about 15 to about 30 nucleotides in length, in specific Sembodiments about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or nucleotides in length. In another embodiment, the siNA duplexes of the invention independently comprise about 15 to about 30 base pairs about 15, 16, 17, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30). In another embodiment, one or more strands of the siNA molecule of the invention independently comprises about 15 to about nucleotides about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or that are complementary to a target nucleic acid molecule. In yet another embodiment, siNA molecules of the invention comprising hairpin or circular structures are about 35 to about 55 about 35, 40, 45, 50 or 55) nucleotides in length, or about 38 to about 44 about 38, 39, 40, 41, 42, 43, or 44) nucleotides in length and comprising about to about 25 about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs.

Exemplary siNA molecules of the invention are shown in Table II. Exemplary synthetic siNA molecules of the invention are shown in Table III and/or Figures As used herein "cell" is used in its usual biological sense, and does not refer to an entire multicellular organism, specifically does not refer to a human. The cell can be present in an organism, birds, plants and mammals such as humans, cows, sheep, apes, monkeys, swine, dogs, and cats. The cell can be prokaryotic bacterial cell) or eukaryotic mammalian or plant cell). The cell can be of somatic or germ line origin, totipotent or pluripotent, dividing or non-dividing. The cell can also be derived from or can comprise a gamete or embryo, a stem cell, or a fully differentiated cell.

The siNA molecules of the invention are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells or tissues. The nucleic acid or nucleic acid complexes can be locally administered to Srelevant tissues ex vivo, or in vivo through local delivery to the lung, with or without their incorporation in biopolymers. In particular embodiments, the nucleic acid molecules of the invention comprise sequences shown in Tables II-III and/or Figures 4- Examples of such nucleic acid molecules consist essentially of sequences defined in these tables and figures. Furthermore, the chemically modified constructs described in Table IV can be applied to any siNA sequence of the invention.

00 M In another aspect, the invention provides mammalian cells containing one or more S siNA molecules of this invention. The one or more siNA molecules can independently l t be targeted to the same or different sites.

C 10 By "RNA" is meant a molecule comprising at least one ribonucleotide residue. By "ribonucleotide" is meant a nucleotide with a hydroxyl group at the 2' position of a (3-Dribofuranose moiety. The terms include double-stranded RNA, single-stranded

RNA,

isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of the siNA or internally, for example at one or more nucleotides of the RNA.

Nucleotides in the RNA molecules of the instant invention can also comprise nonstandard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring

RNA.

By "subject" is meant an organism, which is a donor or recipient of explanted cells or the cells themselves. "Subject" also refers to an organism to which the nucleic acid molecules of the invention can be administered. A subject can be a mammal or mammalian cells, including a human or human cells.

The term "phosphorothioate" as used herein refers to an internucleotide linkage having Formula I, wherein Z and/or W comprise a sulfur atom. Hence, the term phosphorothioate refers to both phosphorothioate and phosphorodithioate internucleotide linkages.

The term "phosphonoacetate" as used herein refers to an intemucleotide linkage N having Formula I, wherein Z and/or W comprise an acetyl or protected acetyl group.

The term "thiophosphonoacetate" as used herein refers to an interucleotide linkage having Formula I, wherein Z comprises an acetyl or protected acetyl group and W comprises a sulfur atom or alternately W comprises an acetyl or protected acetyl group and Z comprises a sulfur atom.

00 The term "universal base" as used herein refers to nucleotide base analogs that N form base pairs with each of the natural DNA/RNA bases with little discrimination 0 between them. Non-limiting examples of universal bases include C-phenyl, C-naphthyl C 10 and other aromatic derivatives, inosine, azole carboxamides, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as known in the art (see for example Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).

The term "acyclic nucleotide" as used herein refers to any nucleotide having an acyclic ribose sugar, for example where any of the ribose carbons (Cl, C2, C3, C4, or C5), are independently or in combination absent from the nucleotide.

The nucleic acid molecules of the instant invention, individually, or in combination or in conjunction with other drugs, can be used to for preventing or treating Huntington disease, spinocerebellar ataxia, spinal and bulbar muscular dystrophy, and dentatorubropallidoluysian atrophy in a subject or organism.

In one embodiment, the siNA molecules of the invention can be administered to a subject or can be administered to other appropriate cells liver, intestine, pancreas) evident to those skilled in the art, individually or in combination with one or more drugs under conditions suitable for the treatment.

In a further embodiment, the siNA molecules can be used in combination with other known treatments to prevent or treat Huntington disease, spinocerebellar ataxia, spinal and bulbar muscular dystrophy, and dentatorubropallidoluysian atrophy in a subject or organism. For example, the described molecules could be used in combination with one or more known compounds, treatments, or procedures to prevent or treat 3 Huntington disease, spinocerebellar ataxia, spinal and bulbar muscular dystrophy, and N dentatorubropallidoluysian atrophy in a subject or organism as are known in the art.

In one embodiment, the invention features an expression vector comprising a nucleic acid sequence encoding at least one siNA molecule of the invention, in a manner which allows expression of the siNA molecule. For example, the vector can contain sequence(s) encoding both strands of a siNA molecule comprising a duplex. The vector 00 M can also contain sequence(s) encoding a single nucleic acid molecule that is self- Scomplementary and thus forms a siNA molecule. Non-limiting examples of such Sexpression vectors are described in Paul et al., 2002, Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002, Nature Biotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology, 19, 500; and Novina et al., 2002, Nature Medicine, advance online publication doi: 10.1038/nm725.

In another embodiment, the invention features a mammalian cell, for example, a human cell, including an expression vector of the invention.

In yet another embodiment, the expression vector of the invention comprises a sequence for a siNA molecule having complementarity to a RNA molecule referred to by a Genbank Accession numbers, for example Genbank Accession Nos. shown in Table I.

In one embodiment, an expression vector of the invention comprises a nucleic acid sequence encoding two or more siNA molecules, which can be the same or different.

In another aspect of the invention, siNA molecules that interact with target RNA molecules and down-regulate gene encoding target RNA molecules (for example target RNA molecules referred to by Genbank Accession numbers herein) are expressed from transcription units inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. siNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. The recombinant vectors capable of expressing the siNA molecules can be delivered as described herein, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of siNA molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the siNA molecules bind and down-regulate gene function or expression via RNA interference (RNAi). Delivery of siNA expressing 100 3 vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from a subject followed by reintroduction into the subject, or by any other means that would allow for introduction into the desired target cell.

By "vectors" is meant any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.

00 SIn one embodiment, a viral vector of the invention is an AAV vector. By an 1 "AAV vector" is meant a vector derived from an adeno-associated virus serotype, Sincluding without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAVX7, etc.

N 10 AAV vectors can have one or more of the AAV wild-type genes, preferably the rep and/or cap genes, deleted in whole or part, but retain functional flanking ITR sequences.

Functional ITR sequences can be necessary for the rescue, replication and packaging of the AAV virion. Thus, an AAV vector is defined herein to include at least those sequences required for example in cis for replication and packaging functional ITRs) of the virus. The ITRs need not be the wild-type nucleotide sequences, and may be altered, by the insertion, deletion or substitution of nucleotides, so long as the sequences provide for functional rescue, replication and packaging.

In one embodiment, the AAV expression vectors are constructed using known techniques to at least provide as operatively linked components in the direction of transcription, control elements including a transcriptional initiation region, the DNA of interest and a transcriptional termination region. The control elements are selected to be functional in a mammalian cell. The resulting construct which contains the operatively linked components is bounded and with functional AAV ITR sequences.

By "adeno-associated virus inverted terminal repeats" or "AAV ITRs" is meant the art-recognized regions found at each end of the AAV genome which function together in cis as origins of DNA replication and as packaging signals for the virus. AAV ITRs, together with the AAV rep coding region, provide for the efficient excision and rescue from, and integration of a nucleotide sequence interposed between two flanking ITRs into a mammalian cell genome.

The nucleotide sequences of AAV ITR regions are known. See for example Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Berns, K. I. "Parvoviridae and 101 t3 their Replication" in Fundamental Virology, 2nd Edition, N. Fields and D. M. Knipe, eds.). As used herein, an "AAV ITR" need not have the wild-type nucleotide sequence depicted, but may be altered, by the insertion, deletion or substitution of nucleotides. Additionally, the AAV ITR may be derived from any of several AAV serotypes, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAVX7, etc. Furthermore, 5' and 3' ITRs which flank a selected nucleotide sequence in 0 an AAV vector need not necessarily be identical or derived from the same AAV serotype Sor isolate, so long as they function as intended, to allow for excision and rescue of 1 the sequence of interest from a host cell genome or vector, and to allow integration of the heterologous sequence into the recipient cell genome when AAV Rep gene products are 1 present in the cell.

In one embodiment, AAV ITRs can be derived from any of several AAV serotypes, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAVX7, etc. Furthermore, 5' and 3' ITRs which flank a selected nucleotide sequence in an AAV expression vector need not necessarily be identical or derived from the same AAV serotype or isolate, so long as they function as intended, to allow for excision and rescue of the sequence of interest from a host cell genome or vector, and to allow integration of the DNA molecule into the recipient cell genome when AAV Rep gene products are present in the cell.

In one embodiment, suitable DNA molecules for use in AAV vectors will be less than about 5 kilobases (kb) in size and will include, for example, a stuffer sequence and a sequence encoding a siRNA molecule of the invention. For example, in order to prevent any packaging of AAV genomic sequences containing the rep and cap genes, a plasmid containing the rep and cap DNA fragment may be modified by the inclusion of a stuffer fragment as is known in the art into the AAV genome which causes the DNA to exceed the length for optimal packaging. Thus, the helper fragment is not packaged into AAV virions. This is a safety feature, ensuring that only a recombinant AAV vector genome that does not exceed optimal packaging size is packaged into virions. An AAV helper fragment that incorporates a stuffer sequence can exceed the wild-type genome length of 4.6 kb, and lengths above 105% of the wild-type will generally not be packaged. The stuffer fragment can be derived from, for example, such non-viral sources as the Lac-Z or beta-galactosidase gene.

O In one embodiment, the selected nucleotide sequence is operably linked to control elements that direct the transcription or expression thereof in the subject in vivo.

Such control elements can comprise control sequences normally associated with the selected gene. Alternatively, heterologous control sequences can be employed. Useful heterologous control sequences generally include those derived from sequences encoding mammalian or viral genes. Examples include, but are not limited to, the SV40 early 00 promoter, mouse mammary tumor virus LTR promoter; adenovirus major late promoter S(Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, pol II promoters, pol III promoters, synthetic promoters, hybrid Spromoters, and the like. In addition, sequences derived from nonviral genes, such as the murine metallothionein gene, will also find use herein. Such promoter sequences are commercially available from, Stratagene (San Diego, Calif.).

In one embodiment, both heterologous promoters and other control elements, such as CNS-specific and inducible promoters, enhancers and the like, will be of particular use. Examples of heterologous promoters include the CMB promoter.

Examples of CNS-specific promoters include those isolated from the genes from myelin basic protein (MBP), glial fibrillary acid protein (GFAP), and neuron specific enolase (NSE). Examples of inducible promoters include DNA responsive elements for ecdysone, tetracycline, hypoxia and aufin.

In one embodiment, the AAV expression vector which harbors the DNA molecule of interest bounded by AAV ITRs, can be constructed by directly inserting the selected sequence(s) into an AAV genome which has had the major AAV open reading frames ("ORFs") excised therefrom. Other portions of the AAV genome can also be deleted, so long as a sufficient portion of the ITRs remain to allow for replication and packaging functions. Such constructs can be designed using techniques well known in the art. See, U.S. Pat. Nos. 5,173,414 and 5,139,941; International Publication Nos.

WO 92/01070 (published Jan. 23, 1992) and WO 93/03769 (published Mar. 4 1993); Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter, B. J. (1992) Current Opinion in Biotechnology 3:533-539; Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol. 158:97-129; Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Shelling 103

I

t and Smith (1994) Gene Therapy 1:165-169; and Zhou et al. (1994) J. Exp. Med.

179:1867-1875.

S Alternatively, AAV ITRs can be excised from the viral genome or from an AAV vector containing the same and fused 5' and 3' of a selected nucleic acid construct that is present in another vector using standard ligation techniques, such as those described in Sambrook et al., supra. For example, ligations can be accomplished in 20 mM Tris-Cl 00 S pH 7.5, 10 mM MgCl.sub.2, 10 mM DTT, 33 ug/ml BSA, 10 mM-50 mM NaC1, and Seither 40 uM ATP, 0.01-0.02 (Weiss) units T4 DNA ligase at 0°C. (for "sticky end" Sligation) or 1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14 0 C. (for "blunt end" ligation). Intermolecular "sticky end" ligations are usually performed at 30-100 pg/ml total DNA concentrations (5-100 nM total end concentration). AAV vectors which contain ITRs have been described in, U.S. Pat. No. 5,139,941. In particular, several AAV vectors are described therein which are available from the American Type Culture Collection ("ATCC" under Accession Numbers 53222, 53223, 53224, 53225 and 53226.

Additionally, chimeric genes can be produced synthetically to include AAV ITR sequences arranged 5' and 3' of one or more selected nucleic acid sequences. Preferred codons for expression of the chimeric gene sequence in mammalian CNS cells can be used. The complete chimeric sequence is assembled from overlapping oligonucleotides prepared by standard methods. See, Edge, Nature (1981) 292:756; Nambair et al.

Science (1984) 223:1299; Jay et al. J. Biol. Chem. (1984) 259:6311.

In order to produce rAAV virions, an AAV expression vector is introduced into a suitable host cell using known techniques, such as by transfection. A number of transfection techniques are generally known in the art. See, Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197. Particularly suitable transfection methods include calcium phosphate co-precipitation (Graham et al. (1973) Virol. 52:456- 467), direct micro-injection into cultured cells (Capecchi, M. R. (1980) Cell 22:479- 488), electroporation (Shigekawa et al. (1988) BioTechniques 6:742-751), liposome mediated gene transfer (Mannino et al. (1988) BioTechniques 6:682-690), lipid-mediated t transduction (Feigner et al. (1987) Proc. Natl. Acad. Sci. USA 84:7413-7417), and Snucleic acid delivery using high-velocity microprojectiles (Klein et al. (1987) Nature 327:70-73).

In one embodiment, suitable host cells for producing rAAV virions include microorganisms, yeast cells, insect cells, and mammalian cells, that can be, or have been, used as recipients of a heterologous DNA molecule. The term includes the progeny of M the original cell which has been transfected. Thus, a "host cell" as used herein generally Srefers to a cell which has been transfected with an exogenous DNA sequence. Cells from the stable human cell line, 293 (readily available through, the American Type Culture Collection under Accession Number ATCC CRL1573) can be used in the practice of the present invention. Particularly, the human cell line 293 is a human embryonic kidney cell line that has been transformed with adenovirus type-5 DNA fragments (Graham et al. (1977) J. Gen. Virol. 36:59), and expresses the adenoviral Ela and Elb genes (Aiello et al. (1979) Virology 94:460). The 293 cell line is readily transfected, and provides a particularly convenient platform in which to produce rAAV virions.

In one embodiment, host cells containing the above-described AAV expression vectors are rendered capable of providing AAV helper functions in order to replicate and encapsidate the nucleotide sequences flanked by the AAV ITRs to produce rAAV virions. AAV helper functions are generally AAV-derived coding sequences which can be expressed to provide AAV gene products that, in turn, function in trans for productive AAV replication. AAV helper functions are used herein to complement necessary AAV functions that are missing from the AAV expression vectors. Thus, AAV helper functions include one, or both of the major AAV ORFs, namely the rep and cap coding regions, or functional homologues thereof.

The Rep expression products have been shown to possess many functions, including, among others: recognition, binding and nicking of the AAV origin of DNA replication; DNA helicase activity; and modulation of transcription from AAV (or other heterologous) promoters. The Cap expression products supply necessary packaging functions. AAV helper functions are used herein to complement AAV functions in trans that are missing from AAV vectors.

t The term "AAV helper construct" refers generally to a nucleic acid molecule that Sincludes nucleotide sequences providing AAV functions deleted from an AAV vector Swhich is to be used to produce a transducing vector for delivery of a nucleotide sequence of interest. AAV helper constructs are commonly used to provide transient expression of AAV rep and/or cap genes to complement missing AAV functions that are necessary for lytic AAV replication; however, helper constructs lack AAV ITRs and can neither 00 replicate nor package themselves. AAV helper constructs can be in the form of a Splasmid, phage, transposon, cosmid, virus, or virion. A number of AAV helper Sconstructs have been described, such as the commonly used plasmids pAAV/Ad and a 10 pIM29+45 which encode both Rep and Cap expression products. See, Samulski et Sal. (1989) J. Virol. 63:3822-3828; and McCarty et al. (1991) J. Virol. 65:2936-2945. A number of other vectors have been described which encode Rep and/or Cap expression products. See, U.S. Pat. No. 5,139,941.

By "AAV rep coding region" is meant the art-recognized region of the AAV genome which encodes the replication proteins Rep 78, Rep 68, Rep 52 and Rep These Rep expression products have been shown to possess many functions, including recognition, binding and nicking of the AAV origin of DNA replication, DNA helicase activity and modulation of transcription from AAV (or other heterologous) promoters.

The Rep expression products are collectively required for replicating the AAV genome.

For a description of the AAV rep coding region, see, Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol. 158:97-129; and Kotin, R. M. (1994) Human Gene Therapy 5:793-801. Suitable homologues of the AAV rep coding region include the human herpesvirus 6 (HHV-6) rep gene which is also known to mediate AAV-2 DNA replication (Thomson et al. (1994) Virology 204:304-311).

By "AAV cap coding region" is meant the art-recognized region of the AAV genome which encodes the capsid proteins VP1, VP2, and VP3, or functional homologues thereof. These Cap expression products supply the packaging functions which are collectively required for packaging the viral genome. For a description of the AAV cap coding region, see, Muzyczka, N. and Kotin, R. M. (supra).

In one embodiment, AAV helper functions are introduced into the host cell by transfecting the host cell with an AAV helper construct either prior to, or concurrently 3 with, the transfection of the AAV expression vector. AAV helper constructs are thus N used to provide at least transient expression of AAV rep and/or cap genes to complement missing AAV functions that are necessary for productive AAV infection. AAV helper constructs lack AAV ITRs and can neither replicate nor package themselves. These constructs can be in the form of a plasmid, phage, transposon, cosmid, virus, or virion. A number of AAV helper constructs have been described, such as the commonly used 0 plasmids pAAV/Ad and pIM29+45 which encode both Rep and Cap expression Sproducts. See, Samulski et al. (1989) J. Virol. 63:3822-3828; and McCarty et al.

N, (1991) J. Virol. 65:2936-2945. A number of other vectors have been described which encode Rep and/or Cap expression products. See, U.S. Pat. No. 5,139,941.

In one embodiment, both AAV expression vectors and AAV helper constructs can be constructed to contain one or more optional selectable markers. Suitable markers include genes which confer antibiotic resistance or sensitivity to, impart color to, or change the antigenic characteristics of those cells which have been transfected with a nucleic acid construct containing the selectable marker when the cells are grown in an appropriate selective medium. Several selectable marker genes that are useful in the practice of the invention include the hygromycin B resistance gene (encoding Aminoglycoside phosphotranferase (APH)) that allows selection in mammalian cells by conferring resistance to G418 (available from Sigma, St. Louis, Other suitable markers are known to those of skill in the art.

In one embodiment, the host cell (or packaging cell) is rendered capable of providing non AAV derived functions, or "accessory functions," in order to produce rAAV virions. Accessory functions are non AAV derived viral and/or cellular functions upon which AAV is dependent for its replication. Thus, accessory functions include at least those non AAV proteins and RNAs that are required in AAV replication, including those involved in activation of AAV gene transcription, stage specific AAV mRNA splicing, AAV DNA replication, synthesis of Cap expression products and AAV capsid assembly. Viral-based accessory functions can be derived from any of the known helper viruses.

In one embodiment, accessory functions can be introduced into and then expressed in host cells using methods known to those of skill in the art. Commonly, 3 accessory functions are provided by infection of the host cells with an unrelated helper I virus. A number of suitable helper viruses are known, including adenoviruses; Sherpesviruses such as herpes simplex virus types 1 and 2; and vaccinia viruses. Nonviral accessory functions will also find use herein, such as those provided by cell synchronization using any of various known agents. See, Buller et al. (1981) J.

Virol. 40:241-247; McPherson et al. (1985) Virology 147:217-222; Schlehofer et al.

S(1986) Virology 152:110-117.

SIn one embodiment, accessory functions are provided using an accessory function t vector. Accessory function vectors include nucleotide sequences that provide one or 0 10 more accessory functions. An accessory function vector is capable of being introduced into a suitable host cell in order to support efficient AAV virion production in the host cell. Accessory function vectors can be in the form of a plasmid, phage, transposon or cosmid. Accessory vectors can also be in the form of one or more linearized DNA or RNA fragments which, when associated with the appropriate control elements and enzymes, can be transcribed or expressed in a host cell to provide accessory functions.

See, for example, International Publication No. WO 97/17548, published May 15, 1997.

In one embodiment, nucleic acid sequences providing the accessory functions can be obtained from natural sources, such as from the genome of an adenovirus particle, or constructed using recombinant or synthetic methods known in the art. In this regard, adenovirus-derived accessory functions have been widely studied, and a number of adenovirus genes involved in accessory functions have been identified and partially characterized. See, Carter, B. J. (1990) "Adeno-Associated Virus Helper Functions," in CRC Handbook of Parvoviruses, vol. I Tijssen, and Muzyczka, N. (1992) Curr. Topics. Microbiol and Immun. 158:97-129. Specifically, early adenoviral gene regions El a, E2a, E4, VAI RNA and, possibly, Elb are thought to participate in the accessory process. Janik et al. (1981) Proc. Natl. Acad. Sci. USA 78:1925-1929.

Herpesvirus-derived accessory functions have been described. See, Young et al.

(1979) Prog. Med. Virol. 25:113. Vaccinia virus-derived accessory functions have also been described. See, Carter, B. J. (1990), supra., Schlehofer et al. (1986) Virology 152:110-117.

108 3 In one embodiment, as a consequence of the infection of the host cell with a helper virus, or transfection of the host cell with an accessory function vector, accessory Sfunctions are expressed which transactivate the AAV helper construct to produce AAV Rep and/or Cap proteins. The Rep expression products excise the recombinant

DNA

(including the DNA of interest) from the AAV expression vector. The Rep proteins also serve to duplicate the AAV genome. The expressed Cap proteins assemble into capsids, 00 and the recombinant AAV genome is packaged into the capsids. Thus, productive

AAV

Sreplication ensues, and the DNA is packaged into rAAV virions.

In one embodiment, following recombinant AAV replication, rAAV virions can be purified from the host cell using a variety of conventional purification methods, such as CsCl gradients. Further, if infection is employed to express the accessory functions, residual helper virus can be inactivated, using known methods. For example, adenovirus can be inactivated by heating to temperatures of approximately 60 0 C for, 20 minutes or more. This treatment effectively inactivates only the helper virus since AAV is extremely heat stable while the helper adenovirus is heat labile. The resulting rAAV virions are then ready for use for DNA delivery to the CNS cranial cavity) of the subject.

Methods of delivery of viral vectors include, but are not limited to, intra-arterial, intra-muscular, intravenous, intranasal and oral routes. Generally, rAAV virions may be introduced into cells of the CNS using either in vivo or in vitro transduction techniques.

If transduced in vitro, the desired recipient cell will be removed from the subject, transduced with rAAV virions and reintroduced into the subject. Alternatively, syngeneic or xenogeneic cells can be used where those cells will not generate an inappropriate immune response in the subject.

Suitable methods for the delivery and introduction of transduced cells into a subject have been described. For example, cells can be transduced in vitro by combining recombinant AAV virions with CNS cells in appropriate media, and screening for those cells harboring the DNA of interest can be screened using conventional techniques such as Southern blots and/or PCR, or by using selectable markers. Transduced cells can then be formulated into pharmaceutical compositions, described more fully below, and 109 O the composition introduced into the subject by various techniques, such as by grafting, N intramuscular, intravenous, subcutaneous and intraperitoneal injection.

a In one embodiment, for in vivo delivery, the rAAV virions are formulated into pharmaceutical compositions and will generally be administered parenterally, by intramuscular injection directly into skeletal or cardiac muscle or by injection into the

CNS.

00 SIn one embodiment, viral vectors of the invention are delivered to the CNS via .I convection-enhanced delivery (CED) systems that can efficiently deliver viral vectors, Se.g., AAV, over large regions of a subject's brain striatum and/or cortex). As 10 described in detail and exemplified below, these methods are suitable for a variety of viral vectors, for instance AAV vectors carrying therapeutic genes siRNAs).

Any convection-enhanced delivery device may be appropriate for delivery of viral vectors. In one embodiment, the device is an osmotic pump or an infusion pump.

Both osmotic and infusion pumps are commerically available from a variety of suppliers, for example Alzet Corporation, Hamilton Corporation, Aiza, Inc., Palo Alto, Calif.).

Typically, a viral vector is delivered via CED devices as follows. A catheter, cannula or other injection device is inserted into CNS tissue in the chosen subject. In view of the teachings herein, one of skill in the art could readily determine which general area of the CNS is an appropriate target. For example, when delivering AAV vector encoding a therapeutic gene to treat PD, the striatum is a suitable area of the brain to target.

Stereotactic maps and positioning devices are available, for example from ASI Instruments, Warren, Mich. Positioning may also be conducted by using anatomical maps obtained by CT and/or MRI imaging of the subject's brain to help guide the injection device to the chosen target. Moreover, because the methods described herein can be practiced such that relatively large areas of the brain take up the viral vectors, fewer infusion cannula are needed. Since surgical complications are related to the number of penetrations, the methods described herein also serve to reduce the side effects seen with conventional delivery techniques.

In one embodiment, pharmaceutical compositions will comprise sufficient genetic material to produce a therapeutically effective amount of the siRNA of interest, an amount sufficient to reduce or ameliorate symptoms of the disease state in 110 Vt) question or an amount sufficient to confer the desired benefit. The pharmaceutical 1 compositions will also contain a pharmaceutically acceptable excipient. Such excipients include any pharmaceutical agent that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, Tween80, and liquids such as water, saline, glycerol and ethanol.

0Pharmaceutically acceptable salts can be included therein, for example, mineral acid salts Ssuch as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.

Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering N substances, and the like, may be present in such vehicles. A thorough discussion of pharmaceutically acceptable excipients is available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991).

As is apparent to those skilled in the art in view of the teachings of this specification, an effective amount of viral vector which must be added can be empirically determined. Administration can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosages of administration are well known to those of skill in the art and will vary with the viral vector, the composition of the therapy, the target cells, and the subject being treated. Single and multiple administrations can be carried out with the dose level and pattern being selected by the treating physician.

It should be understood that more than one transgene could be expressed by the delivered viral vector. Alternatively, separate vectors, each expressing one or more different transgenes, can also be delivered to the CNS as described herein. Furthermore, it is also intended that the viral vectors delivered by the methods of the present invention be combined with other suitable compositions and therapies.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a non-limiting example of a scheme for the synthesis of siNA molecules. The complementary siNA sequence strands, strand 1 and strand 2, are synthesized in tandem and are connected by a cleavable linkage, such as a nucleotide succinate or abasic succinate, which can be the same or different from the cleavable linker used for solid phase synthesis on a solid support. The synthesis can be either solid 00 Mn phase or solution phase, in the example shown, the synthesis is a solid phase synthesis.

SThe synthesis is performed such that a protecting group, such as a dimethoxytrityl group, in remains intact on the terminal nucleotide of the tandem oligonucleotide. Upon cleavage O 10 and deprotection of the oligonucleotide, the two siNA strands spontaneously hybridize to form a siNA duplex, which allows the purification of the duplex by utilizing the properties of the terminal protecting group, for example by applying a trityl on purification method wherein only duplexes/oligonucleotides with the terminal protecting group are isolated.

Figure 2 shows a MALDI-TOF mass spectrum of a purified siNA duplex synthesized by a method of the invention. The two peaks shown correspond to the predicted mass of the separate siNA sequence strands. This result demonstrates that the siNA duplex generated from tandem synthesis can be purified as a single entity using a simple trityl-on purification methodology.

Figure 3 shows a non-limiting proposed mechanistic representation of target RNA degradation involved in RNAi. Double-stranded RNA (dsRNA), which is generated by RNA-dependent RNA polymerase (RdRP) from foreign single-stranded RNA, for example viral, transposon, or other exogenous RNA, activates the DICER enzyme that in turn generates siNA duplexes. Alternately, synthetic or expressed siNA can be introduced directly into a cell by appropriate means. An active siNA complex forms which recognizes a target RNA, resulting in degradation of the target RNA by the RISC endonuclease complex or in the synthesis of additional RNA by RNA-dependent RNA polymerase (RdRP), which can activate DICER and result in additional siNA molecules, thereby amplifying the RNAi response.

Figure 4A-F shows non-limiting examples of chemically-modified siNA constructs of the present invention. In the figure, N stands for any nucleotide (adenosine, 112 Sguanosine, cytosine, uridine, or optionally thymidine, for example thymidine can be N substituted in the overhanging regions designated by parenthesis (N Various Smodifications are shown for the sense and antisense strands of the siNA constructs.

Figure 4A: The sense strand comprises 21 nucleotides wherein the two terminal 3'-nucleotides are optionally base paired and wherein all nucleotides present are ribonucleotides except for (N N) nucleotides, which can comprise ribonucleotides, M deoxynucleotides, universal bases, or other chemical modifications described herein.

SThe antisense strand comprises 21 nucleotides, optionally having a 3'-terminal glyceryl i moiety wherein the two terminal 3'-nucleotides are optionally complementary to the target RNA sequence, and wherein all nucleotides present are ribonucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as optionally connects the (N N) nucleotides in the antisense strand.

Figure 4B: The sense strand comprises 21 nucleotides wherein the two terminal 3'-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2'deoxy-2'-fluoro modified nucleotides and all purine nucleotides that may be present are 2'-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, optionally having a 3'-terminal glyceryl moiety and wherein the two terminal 3'nucleotides are optionally complementary to the target RNA sequence, and wherein all pyrimidine nucleotides that may be present are 2'-deoxy-2'-fluoro modified nucleotides and all purine nucleotides that may be present are 2'-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as optionally connects the (N N) nucleotides in the sense and antisense strand.

l n Figure 4C: The sense strand comprises 21 nucleotides having and terminal Scap moieties wherein the two terminal 3'-nucleotides are optionally base paired and Swherein all pyrimidine nucleotides that may be present are 2'-O-methyl or 2'-deoxy-2'fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, optionally having a 3'- 00 terminal glyceryl moiety and wherein the two terminal 3'-nucleotides are optionally Scomplementary to the target RNA sequence, and wherein all pyrimidine nucleotides that may be present are 2'-deoxy-2'-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other ,I chemical modifications described herein. A modified interucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified intemucleotide linkage as described herein, shown as optionally connects the (N N) nucleotides in the antisense strand.

Figure 4D: The sense strand comprises 21 nucleotides having and terminal cap moieties wherein the two terminal 3'-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2'-deoxy-2'-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein and wherein and all purine nucleotides that may be present are 2'-deoxy nucleotides. The antisense strand comprises 21 nucleotides, optionally having a 3'-terminal glyceryl moiety and wherein the two terminal 3'-nucleotides are optionally complementary to the target RNA sequence, wherein all pyrimidine nucleotides that may be present are 2'deoxy-2'-fluoro modified nucleotides and all purine nucleotides that may be present are 2'-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified intemucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as optionally connects the (N N) nucleotides in the antisense strand.

Figure 4E: The sense strand comprises 21 nucleotides having and terminal cap moieties wherein the two terminal 3'-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2'-deoxy-2'-fluoro modified 114

I

nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, N deoxynucleotides, universal bases, or other chemical modifications described herein.

The antisense strand comprises 21 nucleotides, optionally having a 3'-terminal glyceryl moiety and wherein the two terminal 3'-nucleotides are optionally complementary to the target RNA sequence, and wherein all pyrimidine nucleotides that may be present are 2'deoxy-2'-fluoro modified nucleotides and all purine nucleotides that may be present are 0 2'-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise Sribonucleotides, deoxynucleotides, universal bases, or other chemical modifications Sdescribed herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown CN as optionally connects the (N N) nucleotides in the antisense strand.

Figure 4F: The sense strand comprises 21 nucleotides having and terminal cap moieties wherein the two terminal 3'-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2'-deoxy-2'-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein and wherein and all purine nucleotides that may be present are 2'-deoxy nucleotides. The antisense strand comprises 21 nucleotides, optionally having a 3'-terminal glyceryl moiety and wherein the two terminal 3'-nucleotides are optionally complementary to the target RNA sequence, and having one 3'-terminal phosphorothioate internucleotide linkage and wherein all pyrimidine nucleotides that may be present are 2'-deoxy-2'-fluoro modified nucleotides and all purine nucleotides that may be present are 2'-deoxy nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as optionally connects the (N N) nucleotides in the antisense strand. The antisense strand of constructs A-F comprise sequence complementary to any target nucleic acid sequence of the invention. Furthermore, when a glyceryl moiety is present at the 3'-end of the antisense strand for any construct shown in Figure 4 A-F, the modified internucleotide linkage is optional.

Figure 5A-F shows non-limiting examples of specific chemically-modified siNA sequences of the invention. A-F applies the chemical modifications described in Figure S4A-F to a Huntingtin siNA sequence. Such chemical modifications can be applied to any repeat expansion (RE) sequence.

Figure 6 shows non-limiting examples of different siNA constructs of the invention. The examples shown (constructs 1, 2, and 3) have 19 representative base 00 M€ pairs; however, different embodiments of the invention include any number of base pairs described herein. Bracketed regions represent nucleotide overhangs, for example, V comprising about 1, 2, 3, or 4 nucleotides in length, preferably about 2 nucleotides.

Constructs 1 and 2 can be used independently for RNAi activity. Construct 2 can comprise a polynucleotide or non-nucleotide linker, which can optionally be designed as a biodegradable linker. In one embodiment, the loop structure shown in construct 2 can comprise a biodegradable linker that results in the formation of construct 1 in vivo and/or in vitro. In another example, construct 3 can be used to generate construct 2 under the same principle wherein a linker is used to generate the active siNA construct 2 in vivo and/or in vitro, which can optionally utilize another biodegradable linker to generate the active siNA construct 1 in vivo and/or in vitro. As such, the stability and/or activity of the siNA constructs can be modulated based on the design of the siNA construct for use in vivo or in vitro and/or in vitro.

Figure 7A-C is a diagrammatic representation of a scheme utilized in generating an expression cassette to generate siNA hairpin constructs.

Figure 7A: A DNA oligomer is synthesized with a 5'-restriction site (R1) sequence followed by a region having sequence identical (sense region of siNA) to a predetermined repeat expansion (RE) target sequence, wherein the sense region comprises, for example, about 19, 20, 21, or 22 nucleotides in length, which is followed by a loop sequence of defined sequence comprising, for example, about 3 to about 10 nucleotides.

Figure 7B: The synthetic construct is then extended by DNA polymerase to generate a hairpin structure having self-complementary sequence that will result in a siNA transcript having specificity for a repeat expansion (RE) target sequence and having self-complementary sense and antisense regions.

116 ~t Figure 7C: The construct is heated (for example to about 95 0 C) to linearize the I sequence, thus allowing extension of a complementary second DNA strand using a Sprimer to the 3'-restriction sequence of the first strand. The double-stranded DNA is then inserted into an appropriate vector for expression in cells. The construct can be designed such that a 3'-terminal nucleotide overhang results from the transcription, for example, by engineering restriction sites and/or utilizing a poly-U termination region as described 0 in Paul et al., 2002, Nature Biotechnology, 29, 505-508.

SFigure 8A-C is a diagrammatic representation of a scheme utilized in generating tn an expression cassette to generate double-stranded siNA constructs.

10 Figure 8A: A DNA oligomer is synthesized with a 5'-restriction (R1) site sequence followed by a region having sequence identical (sense region of siNA) to a predetermined repeat expansion (RE) target sequence, wherein the sense region comprises, for example, about 19, 20, 21, or 22 nucleotides in length, and which is followed by a 3'-restriction site (R2) which is adjacent to a loop sequence of defined sequence Figure 8B: The synthetic construct is then extended by DNA polymerase to generate a hairpin structure having self-complementary sequence.

Figure 8C: The construct is processed by restriction enzymes specific to R1 and R2 to generate a double-stranded DNA which is then inserted into an appropriate vector for expression in cells. The transcription cassette is designed such that a U6 promoter region flanks each side of the dsDNA which generates the separate sense and antisense strands of the siNA. Poly T termination sequences can be added to the constructs to generate U overhangs in the resulting transcript.

Figure 9A-E is a diagrammatic representation of a method used to determine target sites for siNA mediated RNAi within a particular target nucleic acid sequence, such as messenger RNA.

Figure 9A: A pool of siNA oligonucleotides are synthesized wherein the antisense region of the siNA constructs has complementarity to target sites across the target S3 nucleic acid sequence, and wherein the sense region comprises sequence complementary i to the antisense region of the siNA.

a Figure 9B&C: (Figure 9B) The sequences are pooled and are inserted into vectors such that (Figure 9C) transfection of a vector into cells results in the expression ofthesiNA.

00 Figure 9D: Cells are sorted based on phenotypic change that is associated with Smodulation of the target nucleic acid sequence.

Figure 9E: The siNA is isolated from the sorted cells and is sequenced to identify i efficacious target sites within the target nucleic acid sequence.

Figure 10 shows non-limiting examples of different stabilization chemistries (1that can be used, for example, to stabilize the 3'-end of siNA sequences of the invention, including [3-3']-inverted deoxyribose; deoxyribonucleotide; 3'-deoxyribonucleotide; [5'-3']-ribonucleotide; [5'-3']-3'-O-methyl ribonucleotide; 3'-glyceryl; [3'-5']-3'-deoxyribonucleotide; [3'-3']-deoxyribonucleotide; 2']-deoxyribonucleotide; and (10) [5-3']-dideoxyribonucleotide. In addition to modified and unmodified backbone chemistries indicated in the figure, these chemistries can be combined with different backbone modifications as described herein, for example, backbone modifications having Formula I. In addition, the 2'-deoxy nucleotide shown to the terminal modifications shown can be another modified or unmodified nucleotide or non-nucleotide described herein, for example modifications having any of Formulae I- VII or any combination thereof.

Figure 11 shows a non-limiting example of a strategy used to identify chemically modified siNA constructs of the invention that are nuclease resistance while preserving the ability to mediate RNAi activity. Chemical modifications are introduced into the siNA construct based on educated design parameters introducing 2'-mofications, base modifications, backbone modifications, terminal cap modifications etc). The modified construct in tested in an appropriate system human serum for nuclease resistance, shown, or an animal model for PK/delivery parameters). In parallel, the siNA construct is tested for RNAi activity, for example in a cell culture system such as a luciferase reporter assay). Lead siNA constructs are then identified which possess a 118 particular characteristic while maintaining RNAi activity, and can be further modified and assayed once again. This same approach can be used to identify siNA-conjugate 2 molecules with improved pharmacokinetic profiles, delivery, and RNAi activity.

Figure 12 shows non-limiting examples of phosphorylated siNA molecules of the invention, including linear and duplex constructs and asymmetric derivatives thereof.

0 Figure 13 shows non-limiting examples of chemically modified terminal phosphate groups of the invention.

SFigure 14A shows a non-limiting example of methodology used to design self complementary DFO constructs utilizing palindrome and/or repeat nucleic acid sequences that are identified in a target nucleic acid sequence. A palindrome or repeat sequence is identified in a nucleic acid target sequence. (ii) A sequence is designed that is complementary to the target nucleic acid sequence and the palindrome sequence. (iii) An inverse repeat sequence of the non-palindrome/repeat portion of the complementary sequence is appended to the 3'-end of the complementary sequence to generate a self complementary DFO molecule comprising sequence complementary to the nucleic acid target. (iv) The DFO molecule can self-assemble to form a double stranded oligonucleotide. Figure 14B shows a non-limiting representative example of a duplex forming oligonucleotide sequence. Figure 14C shows a non-limiting example of the self assembly schematic of a representative duplex forming oligonucleotide sequence.

Figure 14D shows a non-limiting example of the self assembly schematic of a representative duplex forming oligonucleotide sequence followed by interaction with a target nucleic acid sequence resulting in modulation of gene expression.

Figure 15 shows a non-limiting example of the design of self complementary

DFO

constructs utilizing palindrome and/or repeat nucleic acid sequences that are incorporated into the DFO constructs that have sequence complementary to any target nucleic acid sequence of interest. Incorporation of these palindrome/repeat sequences allow the design of DFO constructs that form duplexes in which each strand is capable of mediating modulation of target gene expression, for example by RNAi. First, the target sequence is identified. A complementary sequence is then generated in which nucleotide or non-nucleotide modifications (shown as X or Y) are introduced into the complementary sequence that generate an artificial palindrome (shown as XYXYXY in 119 the Figure). An inverse repeat of the non-palindrome/repeat complementary sequence is N appended to the 3'-end of the complementary sequence to generate a self complementary DFO comprising sequence complementary to the nucleic acid target. The DFO can selfassemble to form a double stranded oligonucleotide.

Figure 16 shows non-limiting examples of multifunctional siNA molecules of the invention comprising two separate polynucleotide sequences that are each capable of M mediating RNAi directed cleavage of differing target nucleic acid sequences. Figure S16A shows a non-limiting example of a multifunctional siNA molecule having a first t region that is complementary to a first target nucleic acid sequence (complementary 10 region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region wherein the first and second complementary regions are situated at the 3'-ends of each polynucleotide sequence in the multifunctional siNA.

The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences. Figure 16B shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region wherein the first and second complementary regions are situated at the 5'-ends of each polynucleotide sequence in the multifunctional siNA.

The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences.

Figure 17 shows non-limiting examples of multifunctional siNA molecules of the invention comprising a single polynucleotide sequence comprising distinct regions that are each capable of mediating RNAi directed cleavage of differing target nucleic acid sequences. Figure 17A shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region wherein the second complementary region is situated at the 3'-end of the polynucleotide sequence in the multifunctional siNA. The dashed portions of each polynucleotide sequence of the 120 Smultifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid Ssequences. Figure 17B shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region wherein the first 00 complementary region is situated at the 5'-end of the polynucleotide sequence in the Smultifunctional siNA. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid N sequences. In one embodiment, these multifunctional siNA constructs are processed in vivo or in vitro to generate multifunctional siNA constructs as shown in Figure 16.

Figure 18 shows non-limiting examples of multifunctional siNA molecules of the invention comprising two separate polynucleotide sequences that are each capable of mediating RNAi directed cleavage of differing target nucleic acid sequences and wherein the multifunctional siNA construct further comprises a self complementary, palindrome, or repeat region, thus enabling shorter bifuctional siNA constructs that can mediate RNA interference against differing target nucleic acid sequences. Figure 18A shows a nonlimiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region wherein the first and second complementary regions are situated at the 3'-ends of each polynucleotide sequence in the multifunctional siNA, and wherein the first and second complementary regions further comprise a self complementary, palindrome, or repeat region. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences. Figure 18B shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the first and second complementary regions are situated at the 5'-ends of each polynucleotide sequence in the multifunctional siNA, and wherein the first and second complementary regions further comprise a self complementary, palindrome, or repeat Sregion. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA O 5 duplex, but do not have complementarity to the target nucleic acid sequences.

Figure 19 shows non-limiting examples of multifunctional siNA molecules of the M invention comprising a single polynucleotide sequence comprising distinct regions that Sare each capable of mediating RNAi directed cleavage of differing target nucleic acid it sequences and wherein the multifunctional siNA construct further comprises a self O 10 complementary, palindrome, or repeat region, thus enabling shorter bifuctional siNA constructs that can mediate RNA interference against differing target nucleic acid sequences. Figure 19A shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region wherein the second complementary region is situated at the 3'-end of the polynucleotide sequence in the multifunctional siNA, and wherein the first and second complementary regions further comprise a self complementary, palindrome, or repeat region. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences. Figure 19B shows a nonlimiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region wherein the first complementary region is situated at the end of the polynucleotide sequence in the multifunctional siNA, and wherein the first and second complementary regions further comprise a self complementary, palindrome, or repeat region. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences. In one embodiment, these multifunctional siNA constructs are processed in vivo or in vitro to generate multifunctional siNA constructs as shown in Figure 18.

Figure 20 shows a non-limiting example of how multifunctional siNA molecules of the invention can target two separate target nucleic acid molecules, such as separate RNA molecules encoding differing proteins, for example, a cytokine and its corresponding receptor, differing viral strains, a virus and a cellular protein involved in viral infection or replication, or differing proteins involved in a common or divergent biologic pathway that is implicated in the maintenance of progression of disease. Each strand of the multifunctional siNA construct comprises a region having complementarity to separate target nucleic acid molecules. The multifunctional siNA molecule is designed such that each strand of the siNA can be utilized by the RISC complex to initiate RNA interference mediated cleavage of its corresponding target. These design parameters can include destabilization of each end of the siNA construct (see for example Schwarz et al., 2003, Cell, 115, 199-208). Such destabilization can be accomplished for example by using guanosine-cytidine base pairs, alternate base pairs wobbles), or destabilizing chemically modified nucleotides at terminal nucleotide positions as is known in the art.

Figure 21 shows a non-limiting example of how multifunctional siNA molecules of the invention can target two separate target nucleic acid sequences within the same target nucleic acid molecule, such as alternate coding regions of a RNA, coding and noncoding regions of a RNA, or alternate splice variant regions of a RNA. Each strand of the multifunctional siNA construct comprises a region having complementarity to the separate regions of the target nucleic acid molecule. The multifunctional siNA molecule is designed such that each strand of the siNA can be utilized by the RISC complex to initiate RNA interference mediated cleavage of its corresponding target region. These design parameters can include destabilization of each end of the siNA construct (see for example Schwarz et al., 2003, Cell, 115, 199-208). Such destabilization can be accomplished for example by using guanosine-cytidine base pairs, alternate base pairs wobbles), or destabilizing chemically modified nucleotides at terminal nucleotide positions as is known in the art.

Figure 22(A-H) shows non-limiting examples of tethered multifunctional siNA constructs of the invention. In the examples shown, a linker nucleotide or nonnucleotide linker) connects two siNA regions two sense, two antisense, or alternately a sense and an antisense region together. Separate sense (or sense and 123

I

t antisense) sequences corresponding to a first target sequence and second target sequence are hybridized to their corresponding sense and/or antisense sequences in the Smultifunctional siNA. In addition, various conjugates, ligands, aptamers, polymers or reporter molecules can be attached to the linker region for selective or improved delivery and/or pharmacokinetic properties.

Figure 23 shows a non-limiting example of various dendrimer based 00 Mn multifunctional siNA designs.

Figure 24 shows a non-limiting example of various supramolecular 0multifunctional siNA designs.

Figure 25 shows a non-limiting example of a dicer enabled multifunctional siNA design using a 30 nucleotide precursor siNA construct. A 30 base pair duplex is cleaved by Dicer into 22 and 8 base pair products from either end (8 b.p. fragments not shown).

For ease of presentation the overhangs generated by dicer are not shown but can be compensated for. Three targeting sequences are shown. The required sequence identity overlapped is indicated by grey boxes. The N's of the parent 30 b.p. siNA are suggested sites of 2'-OH positions to enable Dicer cleavage if this is tested in stabilized chemistries. Note that processing of a 30mer duplex by Dicer RNase III does not give a precise 22+8 cleavage, but rather produces a series of closely related products (with 22+8 being the primary site). Therefore, processing by Dicer will yield a series of active siNAs.

Figure 26 shows a non-limiting example of a dicer enabled multifunctional siNA design using a 40 nucleotide precursor siNA construct. A 40 base pair duplex is cleaved by Dicer into 20 base pair products from either end. For ease of presentation the overhangs generated by dicer are not shown but can be compensated for. Four targeting sequences are shown. The target sequences having homology are enclosed by boxes. This design format can be extended to larger RNAs. If chemically stabilized siNAs are bound by Dicer, then strategically located ribonucleotide linkages can enable designer cleavage products that permit our more extensive repertoire of multiifunctional designs. For example cleavage products not limited to the Dicer standard of approximately 22-nucleotides can allow multifunctional siNA constructs with a target sequence identity overlap ranging from, for example, about 3 to about 15 nucleotides.

124 Figure 27 shows a non-limiting example of additional multifunctional siNA Sconstruct designs of the invention. In one example, a conjugate, ligand, aptamer, label, 2 or other moiety is attached to a region of the multifunctional siNA to enable improved delivery or pharmacokinetic profiling.

Figure 28 shows a non-limiting example of additional multifunctional siNA 0 construct designs of the invention. In one example, a conjugate, ligand, aptamer, label, M or other moiety is attached to a region of the multifunctional siNA to enable improved Sdelivery or pharmacokinetic profiling.

Figure 29 shows a non-limiting example of a cholesterol linked phosphoramidite that can be used to synthesize cholesterol conjugated siNA molecules of the invention.

An example is shown with the cholesterol moiety linked to the 5'-end of the sense strand of a siNA molecule.

Figure 30 shows a non-limiting example of siNA mediated inhibition of expression of myc-tagged human HD protein in HEK-293 cells transfected with active and inverted control siNA constructs along with untreated and transfection controls.

DETAILED DESCRIPTION OF THE INVENTION Mechanism of Action of Nucleic Acid Molecules of the Invention The discussion that follows discusses the proposed mechanism of RNA interference mediated by short interfering RNA as is presently known, and is not meant to be limiting and is not an admission of prior art. Applicant demonstrates herein that chemically-modified short interfering nucleic acids possess similar or improved 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. By "improved capacity to mediate RNAi" or "improved RNAi activity" is meant to include RNAi activity measured in vitro and/or in vivo where the RNAi activity is a reflection of both the ability of the siNA to mediate RNAi and the stability of the siNAs of the invention. In this invention, the product of t these activities can be increased in vitro and/or in vivo compared to an all RNA siRNA or a siNA containing a plurality of ribonucleotides. In some cases, the activity or stability Sof the siNA molecule can be decreased less than ten-fold), but the overall activity of the siNA molecule is enhanced in vitro and/or in vivo.

RNA interference refers to the process of sequence specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs) (Fire et al., 00 M 1998, Nature, 391, 806). The corresponding process in plants is commonly referred to as 0 post-transcriptional gene silencing or RNA silencing and is also referred to as quelling in i fungi. The process of post-transcriptional gene silencing is thought to be an O 10 evolutionarily-conserved cellular defense mechanism used to prevent the expression of foreign genes which is commonly shared by diverse flora and phyla (Fire et al., 1999, Trends Genet., 15, 358). Such protection from foreign gene expression may have evolved in response to the production of double-stranded RNAs (dsRNAs) 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-mediated activation of protein kinase PKR and 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.

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 containing a siRNA, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single-stranded RNA having sequence homologous to the siRNA. Cleavage of the target RNA takes place in the middle of the region complementary to the guide sequence of the siRNA 126 t duplex (Elbashir et al., 2001, Genes Dev., 15, 188). In addition, RNA interference can I also involve small RNA micro-RNA or miRNA) mediated gene silencing, Spresumably though cellular mechanisms that regulate chromatin structure and thereby prevent transcription of target gene sequences (see for example Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237). As such, siNA 00 molecules of the invention can be used to mediate gene silencing via interaction with SRNA transcripts or alternately by interaction with particular gene sequences, wherein I such interaction results in gene silencing either at the transcriptional level or post- 0 10 transcriptional level.

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 has revealed certain requirements for siRNA length, structure, chemical composition, and sequence that are essential to mediate efficient RNAi activity. These studies have shown that 21 nucleotide siRNA duplexes are most active when containing two 2-nucleotide 3'terminal nucleotide overhangs. Furthermore, substitution of one or both siRNA strands with 2'-deoxy or 2'-O-methyl nucleotides abolishes RNAi activity, whereas substitution of 3'-terminal siRNA nucleotides with deoxy nucleotides was shown to be tolerated.

Mismatch sequences in the center of the siRNA duplex were also shown to abolish RNAi activity. In addition, these studies also indicate that the position of the cleavage site in the target RNA is defined by the 5'-end of the siRNA guide sequence rather than the 3'end (Elbashir et al., 2001, EMBO 20, 6877). Other studies have indicated that a 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); however, siRNA molecules lacking a phosphate are active when introduced exogenously, suggesting that 5'-phosphorylation of siRNA constructs may occur in vivo.

Duplex Forming Oligonucleotides (DFO) of the Invention In one embodiment, the invention features siNA molecules comprising duplex forming oligonucleotides (DFO) that can self-assemble into double stranded oligonucleotides. The duplex forming oligonucleotides of the invention can be chemically synthesized or expressed from transcription units and/or vectors. The DFO molecules of the instant invention provide useful reagents and methods for a variety of M therapeutic, diagnostic, agricultural, veterinary, target validation, genomic discovery, O genetic engineering and pharmacogenomic applications.

SApplicant demonstrates herein that certain oligonucleotides, refered to herein for

C

10 convenience but not limitation as duplex forming oligonucleotides or DFO molecules, are potent mediators of sequence specific regulation of gene expression. The oligonucleotides of the invention are distinct from other nucleic acid sequences known in the art siRNA, miRNA, stRNA, shRNA, antisense oligonucleotides etc.) in that they represent a class of linear polynucleotide sequences that are designed to selfassemble into double stranded oligonucleotides, where each strand in the double stranded oligonucleotides comprises a nucleotide sequence that is complementary to a target nucleic acid molecule. Nucleic acid molecules of the invention can thus self assemble into functional duplexes in which each strand of the duplex comprises the same polynucleotide sequence and each strand comprises a nucleotide sequence that is complementary to a target nucleic acid molecule.

Generally, double stranded oligonucleotides are formed by the assembly of two distinct oligonucleotide sequences where the oligonucleotide sequence of one strand is complementary to the oligonucleotide sequence of the second strand; such double stranded oligonucleotides are assembled from two separate oligonucleotides, or from a single molecule that folds on itself to form a double stranded structure, often referred to in the field as hairpin stem-loop structure shRNA or short hairpin RNA). These double stranded oligonucleotides known in the art all have a common feature in that each strand of the duplex has a distict nucleotide sequence.

Distinct from the double stranded nucleic acid molecules known in the art, the applicants have developed a novel, potentially cost effective and simplified method of forming a double stranded nucleic acid molecule starting from a single stranded or linear 128 oligonucleotide. The two strands of the double stranded oligonucleotide formed according to the instant invention have the same nucleotide sequence and are not covalently linked to each other. Such double-stranded oligonucleotides molecules can be readily linked post-synthetically by methods and reagents known in the art and are within the scope of the invention. In one embodiment, the single stranded oligonucleotide of the invention (the duplex forming oligonucleotide) that forms a double stranded 0 oligonucleotide comprises a first region and a second region, where the second region Sincludes a nucleotide sequence that is an inverted repeat of the nucleotide sequence in the first region, or a portion thereof, such that the single stranded oligonucleotide self assembles to form a duplex oligonucleotide in which the nucleotide sequence of one strand of the duplex is the same as the nucleotide sequence of the second strand. Nonlimiting examples of such duplex forming oligonucleotides are illustrated in Figures 14 and 15. These duplex forming oligonucleotides (DFOs) can optionally include certain palindrome or repeat sequences where such palindrome or repeat sequences are present in between the first region and the second region of the DFO.

In one embodiment, the invention features a duplex forming oligonucleotide (DFO) molecule, wherein the DFO comprises a duplex forming self complementary nucleic acid sequence that has nucleotide sequence complementary to a repeat expansion (RE) target nucleic acid sequence. The DFO molecule can comprise a single self complementary sequence or a duplex resulting from assembly of such self complementary sequences.

In one embodiment, a duplex forming oligonucleotide (DFO) of the invention comprises a first region and a second region, wherein the second region comprises a nucleotide sequence comprising an inverted repeat of nucleotide sequence of the first region such that the DFO molecule can assemble into a double stranded oligonucleotide.

Such double stranded oligonucleotides can act as a short interfering nucleic acid (siNA) to modulate gene expression. Each strand of the double stranded oligonucleotide duplex formed by DFO molecules of the invention can comprise a nucleotide sequence region that is complementary to the same nucleotide sequence in a target nucleic acid molecule target repeat expansion (RE) RNA).

0 In one embodiment, the invention features a single stranded DFO that can 1 assemble into a double stranded oligonucleotide. The applicant has surprisingly found a that a single stranded oligonucleotide with nucleotide regions of self complementarity can readily assemble into duplex oligonucleotide constructs. Such DFOs can assemble into duplexes that can inhibit gene expression in a sequence specific manner. The DFO moleucles of the invention comprise a first region with nucleotide sequence that is 0 complementary to the nucleotide sequence of a second region and where the sequence of Sthe first region is complementary to a target nucleic acid RNA). The DFO can Sform a double stranded oligonucleotide wherein a portion of each strand of the double stranded oligonucleotide comprises a sequence complementary to a target nucleic acid sequence.

In one embodiment, the invention features a double stranded oligonucleotide, wherein the two strands of the double stranded oligonucleotide are not covalently linked to each other, and wherein each strand of the double stranded oligonucleotide comprises a nucleotide sequence that is complementary to the same nucleotide sequence in a target nucleic acid molecule or a portion thereof repeat expansion (RE) RNA target). In another embodiment, the two strands of the double stranded oligonucleotide share an identical nucleotide sequence of at least about 15, preferably at least about 16, 17, 18, 19, or 21 nucleotides.

In one embodiment, a DFO molecule of the invention comprises a structure having Formula DFO-I: Z X'-3' wherein Z comprises a palindromic or repeat nucleic acid sequence optionally with one or more modified nucleotides nucleotide with a modified base, such as 2-amino purine, 2-amino-1,6-dihydro purine or a universal base), for example of length about 2 to about 24 nucleotides in even numbers about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 22 or 24 nucleotides), X represents a nucleic acid sequence, for example of length of about 1 to about 21 nucleotides about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides), X' comprises a nucleic acid sequence, for example of length about 1 and about 21 nucleotides about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, t 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides) having nucleotide sequence complementarity N to sequence X or a portion thereof, p comprises a terminal phosphate group that can be 2 present or absent, and wherein sequence X and Z, either independently or together, comprise nucleotide sequence that is complementary to a target nucleic acid sequence or a portion thereof and is of length sufficient to interact base pair) with the target nucleic acid sequence or a portion thereof repeat expansion (RE) RNA target). For 00 example, X independently can comprise a sequence from about 12 to about 21 or more about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more) nucleotides in length that is complementary to nucleotide sequence in a target repeat expansion (RE) RNA or a portion thereof. In another non-limiting example, the length of the nucleotide sequence of X and Z together, when X is present, that is complementary to the target RNA or a portion thereof repeat expansion (RE) RNA target) is from about 12 to about 21 or more nucleotides about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more). In yet another non-limiting example, when X is absent, the length of the nucleotide sequence of Z that is complementary to the target repeat expansion (RE) RNA or a portion thereof is from about 12 to about 24 or more nucleotides about 12, 14, 16, 18, 20, 22, 24, or more). In one embodiment X, Z and X' are independently oligonucleotides, where X and/or Z comprises a nucleotide sequence of length sufficient to interact base pair) with a nucleotide sequence in the target RNA or a portion thereof repeat expansion (RE) RNA target). In one embodiment, the lengths of oligonucleotides X and X' are identical. In another embodiment, the lengths of oligonucleotides X and X' are not identical. In another embodiment, the lengths of oligonucleotides X and Z, or Z and X', or X, Z and X' are either identical or different.

When a sequence is described in this specification as being of"sufficient" length to interact base pair) with another sequence, it is meant that the the length is such that the number of bonds hydrogen bonds) formed between the two sequences is enough to enable the two sequence to form a duplex under the conditions of interest.

Such conditions can be in vitro for diagnostic or assay purposes) or in vivo for therapeutic purposes). It is a simple and routine matter to determine such lengths.

In one embodiment, the invention features a double stranded oligonucleotide construct having Formula DFO-I(a): Z X'-3' Z wherein Z comprises a palindromic or repeat nucleic acid sequence or palindromic or repeat-like nucleic acid sequence with one or more modified nucleotides nucleotides with a modified base, such as 2-amino purine, 2-amino-1,6-dihydro purine or a universal base), for example of length about 2 to about 24 nucleotides in even numbers about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 nucleotides), X represents a nucleic acid sequence, for example of length about 1 to about 21 nucleotides about 1, 2, 3, S4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides), X' comprises N a nucleic acid sequence, for example of length about 1 to about 21 nucleotides about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides) having nucleotide sequence complementarity to sequence X or a portion thereof, p comprises a terminal phosphate group that can be present or absent, and wherein each X and Z independently comprises a nucleotide sequence that is complementary to a target nucleic acid sequence or a portion thereof repeat expansion (RE) RNA target) and is of length sufficient to interact with the target nucleic acid sequence of a portion thereof repeat expansion (RE) RNA target). For example, sequence X independently can comprise a sequence from about 12 to about 21 or more nucleotides about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more) in length that is complementary to a nucleotide sequence in a target RNA or a portion thereof repeat expansion (RE) RNA target).

In another non-limiting example, the length of the nucleotide sequence of X and Z together (when X is present) that is complementary to the target repeat expansion (RE) RNA or a portion thereof is from about 12 to about 21 or more nucleotides about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more). In yet another non-limiting example, when X is absent, the length of the nucleotide sequence of Z that is complementary to the target repeat expansion (RE) RNA or a portion thereof is from about 12 to about 24 or more nucleotides about 12, 14, 16, 18, 20, 22, 24 or more). In one embodiment X, Z and X' are independently oligonucleotides, where X and/or Z comprises a nucleotide sequence of length sufficient to interact base pair) with nucleotide sequence in the target RNA or a portion thereof repeat expansion (RE) RNA target). In one embodiment, the lengths of oligonucleotides X and X' are identical. In another embodiment, the lengths of oligonucleotides X and X' are not identical. In another 132 embodiment, the lengths of oligonucleotides X and Z or Z and X' or X, Z and X' are 2, either identical or different. In one embodiment, the double stranded oligonucleotide Sconstruct of Formula I(a) includes one or more, specifically 1, 2, 3 or 4, mismatches, to the extent such mismatches do not significantly diminish the ability of the double stranded oligonucleotide to inhibit target gene expression.

o In one embodiment, a DFO molecule of the invention comprises structure having M Formula DFO-II: X'-3' N, wherein each X and X' are independently oligonucleotides of length about 12 nucleotides to about 21 nucleotides, wherein X comprises, for example, a nucleic acid sequence of length about 12 to about 21 nucleotides about 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides), X' comprises a nucleic acid sequence, for example of length about 12 to about 21 nucleotides about 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides) having nucleotide sequence complementarity to sequence X or a portion thereof, p comprises a terminal phosphate group that can be present or absent, and wherein X comprises a nucleotide sequence that is complementary to a target nucleic acid sequence repeat expansion (RE) RNA) or a portion thereof and is of length sufficient to interact base pair) with the target nucleic acid sequence of a portion thereof. In one embodiment, the length of oligonucleotides X and X' are identical. In another embodiment the length of oligonucleotides X and X' are not identical. In one embodiment, length of the oligonucleotides X and X' are sufficint to form a relatively stable double stranded oligonucleotide.

In one embodiment, the invention features a double stranded oligonucleotide construct having Formula DFO-II(a): X'-3' wherein each X and X' are independently oligonucleotides of length about 12 nucleotides to about 21 nucleotides, wherein X comprises a nucleic acid sequence, for example of length about 12 to about 21 nucleotides about 12, 13, 14, 15, 16, 17, 18, 133 0 19, 20 or 21 nucleotides), X' comprises a nucleic acid sequence, for example of length i about 12 to about 21 nucleotides about 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 C nucleotides) having nucleotide sequence complementarity to sequence X or a portion thereof, p comprises a terminal phosphate group that can be present or absent, and wherein X comprises nucleotide sequence that is complementary to a target nucleic acid sequence or a portion thereof repeat expansion (RE) RNA target) and is of length 0 sufficient to interact base pair) with the target nucleic acid sequence repeat Sexpansion (RE) RNA) or a portion thereof. In one embodiment, the lengths of oligonucleotides X and X' are identical. In another embodiment, the lengths of oligonucleotides X and X' are not identical. In one embodiment, the lengths of the Soligonucleotides X and X' are sufficint to form a relatively stable double stranded oligonucleotide. In one embodiment, the double stranded oligonucleotide construct of Formula II(a) includes one or more, specifically 1, 2, 3 or 4 mismatches, to the extent such mismatches do not significantly diminish the ability of the double stranded oligonucleotide to inhibit target gene expression.

In one embodiment, the invention features a DFO molecule having Formula

DFO-

I(b): 5'-p-Z-3' where Z comprises a palindromic or repeat nucleic acid sequence optionally including one or more non-standard or modified nucleotides nucleotide with a modified base, such as 2-amino purine or a universal base) that can facilitate base-pairing with other nucleotides. Z can be, for example, of length sufficient to interact base pair) with nucleotide sequence of a target nucleic acid repeat expansion (RE) RNA) molecule, preferably of length of at least 12 nucleotides, specifically about 12 to about 24 nucleotides about 12, 14, 16, 18, 20, 22 or 24 nucleotides). p represents a terminal phosphate group that can be present or absent.

In one embodiment, a DFO molecule having any of Formula DFO-I, DFO-I(a), DFO-I(b), DFO-II(a) or DFO-II can comprise chemical modifications as described herein without limitation, such as, for example, nucleotides having any of Formulae

I-

VII, stabilization chemistries as described in Table IV, or any other combination of t modified nucleotides and non-nucleotides as described in the various embodiments herein.

a In one embodiment, the palidrome or repeat sequence or modified nucleotide nucleotide with a modified base, such as 2-amino purine or a universal base) in Z of DFO constructs having Formula DFO-I, DFO-I(a) and DFO-I(b), comprises chemically C, modified nucleotides that are able to interact with a portion of the target nucleic acid 00 M€ sequence modified base analogs that can form Watson Crick base pairs or non- Watson Crick base pairs).

SIn one embodiment, a DFO molecule of the invention, for example a DFO having Formula DFO-I or DFO-II, comprises about 15 to about 40 nucleotides about 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides). In one embodiment, a DFO molecule of the invention comprises one or more chemical modifications. In a non-limiting example, the introduction of chemically modified nucleotides and/or non-nucleotides into nucleic acid molecules of the invention provides a powerful tool in overcoming potential limitations of in vivo stability and bioavailability inherent to unmodified 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 or in cells or tissues. Furthermore, certain chemical modifications can improve the bioavailability and/or potency of nucleic acid molecules by not only enhancing half-life but also facilitating the targeting of nucleic acid molecules to particular organs, cells or tissues and/or improving cellular uptake of the nucleic acid molecules. Therefore, even if the activity of a chemically modified nucleic acid molecule is reduced in vitro as compared to a native/unmodified nucleic acid molecule, for example when compared to an unmodified RNA molecule, the overall activity of the modified nucleic acid molecule can be greater than the native or unmodified nucleic acid molecule due to improved stability, potency, duration of effect, bioavailability and/or delivery of the molecule.

Multifunctional or Multi-targeted siNA molecules of the Invention In one embodiment, the invention features siNA molecules comprising multifunctional short interfering nucleic acid (multifunctional siNA) molecules that 135 modulate the expression of one or more genes in a biologic system, such as a cell, tissue, or organism. The multifunctional short interfering nucleic acid (multifunctional siNA) 2 molecules of the invention can target more than one region a repeat expansion (RE) target nucleic acid sequence or can target sequences of more than one distinct target nucleic acid molecules. The multifunctional siNA molecules of the invention can be chemically synthesized or expressed from transcription units and/or vectors. The 00 multifunctional siNA molecules of the instant invention provide useful reagents and Smethods for a variety of human applications, therapeutic, cosmetic, diagnostic, agricultural, veterinary, target validation, genomic discovery, genetic engineering and pharmacogenomic applications.

Applicant demonstrates herein that certain oligonucleotides, refered to herein for convenience but not limitation as multifunctional short interfering nucleic acid or multifunctional siNA molecules, are potent mediators of sequence specific regulation of gene expression. The multifunctional siNA molecules of the invention are distinct from other nucleic acid sequences known in the art siRNA, miRNA, stRNA, shRNA, antisense oligonucleotides, etc.) in that they represent a class of polynucleotide molecules that are designed such that each strand in the multifunctional siNA construct comprises a nucleotide sequence that is complementary to a distinct nucleic acid sequence in one or more target nucleic acid molecules. A single multifunctional siNA molecule (generally a double-stranded molecule) of the invention can thus target more than one 2, 3, 4, 5, or more) differing target nucleic acid target molecules. Nucleic acid molecules of the invention can also target more than one 2, 3, 4, 5, or more) region of the same target nucleic acid sequence. As such multifunctional siNA molecules of the invention are useful in down regulating or inhibiting the expression of one or more target nucleic acid molecules. By reducing or inhibiting expression of more than one target nucleic acid molecule with one multifunctional siNA construct, multifunctional siNA molecules of the invention represent a class of potent therapeutic agents that can provide simultaneous inhibition of multiple targets within a disease or pathogen related pathway. Such simultaneous inhibition can provide synergistic therapeutic treatment strategies without the need for separate preclinical and clinical development efforts or complex regulatory approval process.

O Use of multifunctional siNA molecules that target more then one region of a target nucleic acid molecule messenger RNA) is expected to provide potent inhibition of gene expression. For example, a single multifunctional siNA construct of the invention can target both conserved and variable regions of a target nucleic acid molecule, such as repeat expansion (RE) target RNA or DNA, thereby allowing down regulation or inhibition of different splice variants encoded by a single gene, or allowing for targeting 00 of both coding and non-coding regions of a target nucleic acid molecule.

(Generally, double stranded oligonucleotides are formed by the assembly of two Sdistinct oligonucleotides where the oligonucleotide sequence of one strand is complementary to the oligonucleotide sequence of the second strand; such double stranded oligonucleotides are generally assembled from two separate oligonucleotides siRNA). Alternately, a duplex can be formed from a single molecule that folds on itself shRNA or short hairpin RNA). These double stranded oligonucleotides are known in the art to mediate RNA interference and all have a common feature wherein only one nucleotide sequence region (guide sequence or the antisense sequence) has complementarity to a target nucleic acid sequence, such as repeat expansion (RE) targets, and the other strand (sense sequence) comprises nucleotide sequence that is homologous to the target nucleic acid sequence. Generally, the antisense sequence is retained in the active RISC complex and guides the RISC to the target nucleotide sequence by means of complementary base-pairing of the antisense sequence with the target seqeunce for mediating sequence-specific RNA interference. It is known in the art that in some cell culture systems, certain types of unmodified siRNAs can exhibit "off target" effects. It is hypothesized that this off-target effect involves the participation of the sense sequence instead of the antisense sequence of the siRNA in the RISC complex (see for example Schwarz et al., 2003, Cell, 115, 199-208). In this instance the sense sequence is believed to direct the RISC complex to a sequence (off-target sequence) that is distinct from the intended target sequence, resulting in the inhibition of the off-target sequence. In these double stranded nucleic acid molecules, each strand is complementary to a distinct target nucleic acid sequence. However, the off-targets that are affected by these dsRNAs are not entirely predictable and are non-specific.

Distinct from the double stranded nucleic acid molecules known in the art, the applicants have developed a novel, potentially cost effective and simplified method of 137 t down regulating or inhibiting the expression of more than one target nucleic acid 0 sequence using a single multifunctional siNA construct. The multifunctional siNA amolecules of the invention are designed to be double-stranded or partially double stranded, such that a portion of each strand or region of the multifunctional siNA is complementary to a target nucleic acid sequence of choice. As such, the multifunctional siNA molecules of the invention are not limited to targeting sequences that are 00 complementary to each other, but rather to any two differing target nucleic acid Ssequences. Multifunctional siNA molecules of the invention are designed such that each (strand or region of the multifunctional siNA molecule, that is complementary to a given target nucleic acid sequence, is of suitable length from about 16 to about 28 nucleotides in length, preferably from about 18 to about 28 nucleotides in length) for mediating RNA interference against the target nucleic acid sequence. The complementarity between the target nucleic acid sequence and a strand or region of the multifunctional siNA must be sufficient (at least about 8 base pairs) for cleavage of the target nucleic acid sequence by RNA interference. Multifunctional siNA of the invention is expected to minimize off-target effects seen with certain siRNA sequences, such as those described in (Schwarz et al., supra).

It has been reported that dsRNAs of length between 29 base pairs and 36 base pairs (Tuschl et at., International PCT Publication No. WO 02/44321) do not mediate RNAi.

One reason these dsRNAs are inactive may be the lack of turnover or dissociation of the strand that interacts with the target RNA sequence, such that the RISC complex is not able to efficiently interact with multiple copies of the target RNA resulting in a significant decrease in the potency and efficiency of the RNAi process. Applicant has surprisingly found that the multifunctional siNAs of the invention can overcome this hurdle and are capable of enhancing the efficiency and potency of RNAi process. As such, in certain embodiments of the invention, multifunctional siNAs of length of about 29 to about 36 base pairs can be designed such that, a portion of each strand of the multifunctional siNA molecule comprises a nucleotide sequence region that is complementary to a target nucleic acid of length sufficient to mediate RNAi efficiently about 15 to about 23 base pairs) and a nucleotide sequence region that is not complementary to the target nucleic acid. By having both complementary and noncomplementary portions in each strand of the multifunctional siNA, the multifunctional t siNA can mediate RNA interference against a target nucleic acid sequence without being prohibitive to turnover or dissociation where the length of each strand is too long to 2 mediate RNAi against the respective target nucleic acid sequence). Furthermore, design of multifunctional siNA molecules of the invention with internal overlapping regions allows the multifunctional siNA molecules to be of favorable (decreased) size for mediating RNA interference and of size that is well suited for use as a therapeutic agent 00 wherein each strand is independently from about 18 to about 28 nucleotides in 3 length). Non-limiting examples are illustrated in Figures 16-28.

t In one embodiment, a multifunctional siNA molecule of the invention comprises a first region and a second region, where the first region of the multifunctional siNA comprises a nucleotide sequence complementary to a nucleic acid sequence of a first target nucleic acid molecule, and the second region of the multifunctional siNA comprises nucleic acid sequence complementary to a nucleic acid sequence of a second target nucleic acid molecule. In one embodiment, a multifunctional siNA molecule of the invention comprises a first region and a second region, where the first region of the multifunctional siNA comprises nucleotide sequence complementary to a nucleic acid sequence of the first region of a target nucleic acid molecule, and the second region of the multifunctional siNA comprises nucleotide sequence complementary to a nucleic acid sequence of a second region of a the target nucleic acid molecule. In another embodiment, the first region and second region of the multifunctional siNA can comprise separate nucleic acid sequences that share some degree of complementarity from about 1 to about 10 complementary nucleotides). In certain embodiments, multifunctional siNA constructs comprising separate nucleic acid seqeunces can be readily linked post-synthetically by methods and reagents known in the art and such linked constructs are within the scope of the invention. Alternately, the first region and second region of the multifunctional siNA can comprise a single nucleic acid sequence having some degree of self complementarity, such as in a hairpin or stem-loop structure.

Non-limiting examples of such double stranded and hairpin multifunctional short interfering nucleic acids are illustrated in Figures 16 and 17 respectively. These multifunctional short interfering nucleic acids (multifunctional siNAs) can optionally include certain overlapping nucleotide sequence where such overlapping nucleotide

I

sequence is present in between the first region and the second region of the Nmultifunctional siNA (see for example Figures 18 and 19).

In one embodiment, the invention features a multifunctional short interfering nucleic acid (multifunctional siNA) molecule, wherein each strand of the the multifunctional siNA independently comprises a first region of nucleic acid sequence C* that is complementary to a distinct target nucleic acid sequence and the second region of 00 M' nucleotide sequence that is not complementary to the target sequence. The target nucleic acid sequence of each strand is in the same target nucleic acid molecule or different target nucleic acid molecules.

S 10 In another embodiment, the multifunctional siNA comprises two strands, where: the first strand comprises a region having sequence complementarity to a target nucleic acid sequence (complementary region 1) and a region having no sequence complementarity to the target nucleotide sequence (non-complementary region the second strand of the multifunction siNA comprises a region having sequence complementarity to a target nucleic acid sequence that is distinct from the target nucleotide sequence complementary to the first strand nucleotide sequence (complementary region and a region having no sequence complementarity to the target nucleotide sequence of complementary region 2 (non-complementary region 2); the complementary region 1 of the first strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in the non-complementary region 2 of the second strand and the complementary region 2 of the second strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in the noncomplementary region 1 of the first strand. The target nucleic acid sequence of complementary region 1 and complementary region 2 is in the same target nucleic acid molecule or different target nucleic acid molecules.

In another embodiment, the multifunctional siNA comprises two strands, where: the first strand comprises a region having sequence complementarity to a target nucleic acid sequence derived from a gene, such as repeat expansion (RE) (complementary region 1) and a region having no sequence complementarity to the target nucleotide sequence of complementary region 1 (non-complementary region the second strand of the multifunction siNA comprises a region having sequence complementarity to a target nucleic acid sequence derived from a gene that is distinct (from the gene of complementary region 1 (complementary region and a region having 2 no sequence complementarity to the target nucleotide sequence of complementary region 2 (non-complementary region the complementary region 1 of the first strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in the non-complementary region 2 of the second strand and the complementary region 2 of the 00 second strand comprises a nucleotide sequence that is complementary to a nucleotide Ssequence in the non-complementary region 1 of the first strand.

In another embodiment, the multifunctional siNA comprises two strands, where: the first strand comprises a region having sequence complementarity to a target nucleic acid sequence derived from a gene, such as repeat expansion (RE), (complementary region 1) and a region having no sequence complementarity to the target nucleotide sequence of complementary region 1 (non-complementary region the second strand of the multifunction siNA comprises a region having sequence complementarity to a target nucleic acid sequence distinct from the target nucleic acid sequence of complementary region 1 (complementary region provided, however, that the target nucleic acid sequence for complementary region 1 and target nucleic acid sequence for complementary region 2 are both derived from the same gene, and a region having no sequence complementarity to the target nucleotide sequence of complementary region 2 (non-complementary region the complementary region 1 of the first strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in the non-complementary region 2 of the second strand and the complementary region 2 of the second strand comprises a nucleotide sequence that is complementary to nucleotide sequence in the non-complementary region 1 of the first strand.

In one embodiment, the invention features a multifunctional short interfering nucleic acid (multifunctional siNA) molecule, wherein the multifunctional siNA comprises two complementary nucleic acid sequences in which the first sequence comprises a first region having nucleotide sequence complementary to nucleotide sequence within a target nucleic acid molecule, and in which the second seqeunce comprises a first region having nucleotide sequence complementary to a distinct nucleotide sequence within the same target nucleic acid molecule. Preferably, the first region of the first sequence is also complementary to the nucleotide sequence of the 141 t second region of the second sequence, and where the first region of the second sequence is complementary to the nucleotide sequence of the second region of the first sequence.

a In one embodiment, the invention features a multifunctional short interfering nucleic acid (multifunctional siNA) molecule, wherein the multifunctional siNA comprises two complementary nucleic acid sequences in which the first sequence C* comprises a first region having a nucleotide sequence complementary to a nucleotide 00 M€ sequence within a first target nucleic acid molecule, and in which the second seqeunce Scomprises a first region having a nucleotide sequence complementary to a distinct Snucleotide sequence within a second target nucleic acid molecule. Preferably, the first region of the first sequence is also complementary to the nucleotide sequence of the second region of the second sequence, and where the first region of the second sequence is complementary to the nucleotide sequence of the second region of the first sequence.

In one embodiment, the invention features a multifunctional siNA molecule comprising a first region and a second region, where the first region comprises a nucleic acid sequence having about 18 to about 28 nucleotides complementary to a nucleic acid sequence within a first target nucleic acid molecule, and the second region comprises nucleotide sequence having about 18 to about 28 nucleotides complementary to a distinct nucleic acid sequence within a second target nucleic acid molecule.

In one embodiment, the invention features a multifunctional siNA molecule comprising a first region and a second region, where the first region comprises nucleic acid sequence having about 18 to about 28 nucleotides complementary to a nucleic acid sequence within a target nucleic acid molecule, and the second region comprises nucleotide sequence having about 18 to about 28 nucleotides complementary to a distinct nucleic acid sequence within the same target nucleic acid molecule.

In one embodiment, the invention features a double stranded multifunctional short interfering nucleic acid (multifunctional siNA) molecule, wherein one strand of the multifunctional siNA comprises a first region having nucleotide sequence complementary to a first target nucleic acid sequence, and the second strand comprises a first region having a nucleotide sequence complementary to a second target nucleic acid sequence. The first and second target nucleic acid sequences can be present in separate target nucleic acid molecules or can be different regions within the same target nucleic 142 t acid molecule. As such, multifunctional siNA molecules of the invention can be used to I target the expression of different genes, splice variants of the same gene, both mutant Sand conserved regions of one or more gene transcripts, or both coding and non-coding sequences of the same or differeing genes or gene transcripts.

In one embodiment, a target nucleic acid molecule of the invention encodes a single protein. In another embodiment, a target nucleic acid molecule encodes more than 00oO M one protein 1, 2, 3, 4, 5 or more proteins). As such, a multifunctional siNA 0 construct of the invention can be used to down regulate or inhibit the expression of t several proteins. For example, a multifunctional siNA molecule comprising a region in O 10 one strand having nucleotide sequence complementarity to a first target nucleic acid sequence derived from a gene encoding one protein and the second strand comprising a region with nucleotide sequence complementarity to a second target nucleic acid sequence present in target nucleic acid molecules derived from genes encoding two or more proteins two or more differing repeat expansion (RE) target sequences) can be used to down regulate, inhibit, or shut down a particular biologic pathway.by targeting, for example, two or more targets involved in a biologic pathway.

In one embodiment the invention takes advantage of conserved nucleotide sequences present in different isoforms of cytokines or ligands and receptors for the cytokines or ligands. By designing multifunctional siNAs in a manner where one strand includes a sequence that is complementary to a target nucleic acid sequence conserved among various isoforms of a cytokine and the other strand includes sequence that is complementary to a target nucleic acid sequence conserved among the receptors for the cytokine, it is possible to selectively and effectively modulate or inhibit a biological pathway or multiple genes in a biological pathway using a single multifunctional siNA.

In one embodiment, a double stranded multifunctional siNA molecule of the invention comprises a structure having Formula MF-I: Z X'-3' Z wherein each 5'-p-XZX'-3' and 5'-p-YZY'-3' are independently an oligonucleotide of length of about 20 nucleotides to about 300 nucleotides, preferably of about 20 to about 143 200 nucleotides, about 20 to about 100 nucleotides, about 20 to about 40 nucleotides, about 20 to about 40 nucleotides, about 24 to about 38 nucleotides, or about 26 to about 2 38 nucleotides; XZ comprises a nucleic acid sequence that is complementary to a first target nucleic acid sequence; YZ is an oligonucleotide comprising nucleic acid sequence that is complementary to a second target nucleic acid sequence; Z comprises nucleotide sequence of length about 1 to about 24 nucleotides about 1, 2, 3, 4, 5, 6, 7, 8, 9, 00 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides) that is self ¢3complimentary; X comprises nucleotide sequence of length about 1 to about 100 N nucleotides, preferably about 1 to about 21 nucleotides about 1, 2, 3, 4, 5, 6, 7, 8, 9, 3 10 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides) that is complementary to nucleotide sequence present in region Y comprises nucleotide sequence of length about 1 to about 100 nucleotides, prefereably about 1- about 21 nucleotides about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides) that is complementary to nucleotide sequence present in region each p comprises a terminal phosphate group that is independently present or absent; each XZ and YZ is independently of length sufficient to stably interact base pair) with the first and second target nucleic acid sequence, respectively, or a portion thereof. For example, each sequence X and Y can independently comprise sequence from about 12 to about 21 or more nucleotides in length about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more) that is complementary to a target nucleotide sequence in different target nucleic acid molecules, such as target RNAs or a portion thereof. In another non-limiting example, the length of the nucleotide sequence of X and Z together that is complementary to the first target nucleic acid sequence or a portion thereof is from about 12 to about 21 or more nucleotides about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more). In another non-limiting example, the length of the nucleotide sequence of Y and Z together, that is complementary to the second target nucleic acid sequence or a portion thereof is from about 12 to about 21 or more nucleotides about 12, 13, 14, 15, 16, 17, 18, 19, 21, or more). In one embodiment, the first target nucleic acid sequence and the second target nucleic acid sequence are present in the same target nucleic acid molecule repeat expansion (RE) RNA). In another embodiment, the first target nucleic acid sequence and the second target nucleic acid sequence are present in different target nucleic acid molecules repeat expansion (RE) targets). In one embodiment,

Z

comprises a palindrome or a repeat sequence. In one embodiment, the lengths of 144 oligonucleotides X and X' are identical. In another embodiment, the lengths of N oligonucleotides X and X' are not identical. In one embodiment, the lengths of Soligonucleotides Y and Y' are identical. In another embodiment, the lengths of oligonucleotides Y and Y' are not identical. In one embodiment, the double stranded oligonucleotide construct of Formula I(a) includes one or more, specifically 1, 2, 3 or 4, mismatches, to the extent such mismatches do not significantly diminish the ability of 0 the double stranded oligonucleotide to inhibit target gene expression.

O In one embodiment, a multifunctional siNA molecule of the invention comprises a t structure having Formula MF-II: X'-3' wherein each and are independently an oligonucleotide of length of about 20 nucleotides to about 300 nucleotides, preferably about 20 to about 200 nucleotides, about 20 to about 100 nucleotides, about 20 to about 40 nucleotides, about to about 40 nucleotides, about 24 to about 38 nucleotides, or about 26 to about 38 nucleotides; X comprises a nucleic acid sequence that is complementary to a first target nucleic acid sequence; Y is an oligonucleotide comprising nucleic acid sequence that is complementary to a second target nucleic acid sequence; X comprises a nucleotide sequence of length about 1 to about 100 nucleotides, preferably about 1 to about 21 nucleotides about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 21 nucleotides) that is complementary to nucleotide sequence present in region Y comprises nucleotide sequence of length about 1 to about 100 nucleotides, prefereably about 1 to about 21 nucleotides about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20 or 21 nucleotides) that is complementary to nucleotide sequence present in region each p comprises a terminal phosphate group that is independently present or absent; each X and Y independently is of length sufficient to stably interact base pair) with the first and second target nucleic acid sequence, respectively, or a portion thereof. For example, each sequence X and Y can independently comprise sequence from about 12 to about 21 or more nucleotides in length about 12, 13, 14, 16, 17, 18, 19, 20, 21, or more) that is complementary to a target nucleotide sequence in different target nucleic acid molecules, such as repeat expansion, RBL1, and RBL2, tt target sequences or a portion thereof. In one embodiment, the first target nucleic acid N sequence and the second target nucleic acid sequence are present in the same target nucleic acid molecule repeat expansion (RE) RNA or DNA). In another embodiment, the first target nucleic acid sequence and the second target nucleic acid sequence are present in different target nucleic acid molecules, such as repeat expansion, RBL1, and RBL2, target sequences or a portion thereof. In one embodiment, Z 00 comprises a palindrome or a repeat sequence. In one embodiment, the lengths of oligonucleotides X and X' are identical. In another embodiment, the lengths of oligonucleotides X and X' are not identical. In one embodiment, the lengths of oligonucleotides Y and Y' are identical. In another embodiment, the lengths of oligonucleotides Y and Y' are not identical. In one embodiment, the double stranded oligonucleotide construct of Formula I(a) includes one or more, specifically 1, 2, 3 or 4, mismatches, to the extent such mismatches do not significantly diminish the ability of the double stranded oligonucleotide to inhibit target gene expression.

In one embodiment, a multifunctional siNA molecule of the invention comprises a structure having Formula MF-III: X X'

Y,-W-Y

wherein each X, Y, and Y' is independently an oligonucleotide of length of about nucleotides to about 50 nucleotides, preferably about 18 to about 40 nucleotides, or about 19 to about 23 nucleotides; X comprises nucleotide sequence that is complementary to nucleotide sequence present in region X' comprises nucleotide sequence that is complementary to nucleotide sequence present in region Y; each X and X' is independently of length sufficient to stably interact base pair) with a first and a second target nucleic acid sequence, respectively, or a portion thereof; W represents a nucleotide or non-nucleotide linker that connects sequences Y' and Y; and the multifunctional siNA directs cleavage of the first and second target sequence via RNA interference. In one embodiment, the first target nucleic acid sequence and the second target nucleic acid sequence are present in the same target nucleic acid molecule repeat expansion (RE) RNA). In another embodiment, the first target nucleic acid sequence and the second target nucleic acid sequence are present in different target 3 nucleic acid molecules such as repeat expansion, RBL1, and RBL2, target sequences or a portion thereof. In one embodiment, region W connects the 3'-end of sequence Y' with the 3'-end of sequence Y. In one embodiment, region W connects the 3'-end of sequence Y' with the 5'-end of sequence Y. In one embodiment, region W connects the 5'-end of sequence Y' with the 5'-end of sequence Y. In one embodiment, region W connects the 5'-end of sequence Y' with the 3'-end of sequence Y. In one embodiment, 0 a terminal phosphate group is present at the 5'-end of sequence X. In one embodiment, a Sterminal phosphate group is present at the 5'-end of sequence In one embodiment, a I terminal phosphate group is present at the 5'-end of sequence Y. In one embodiment, a terminal phosphate group is present at the 5'-end of sequence In one embodiment, W connects sequences Y and Y' via a biodegradable linker. In one embodiment,

W

further comprises a conjugate, label, aptamer, ligand, lipid, or polymer.

In one embodiment, a multifunctional siNA molecule of the invention comprises a structure having Formula MF-IV: X X'

SY'-W-Y

wherein each X, Y, and Y' is independently an oligonucleotide of length of about nucleotides to about 50 nucleotides, preferably about 18 to about 40 nucleotides, or about 19 to about 23 nucleotides; X comprises nucleotide sequence that is complementary to nucleotide sequence present in region X' comprises nucleotide sequence that is complementary to nucleotide sequence present in region Y; each Y and Y' is independently of length sufficient to stably interact base pair) with a first and a second target nucleic acid sequence, respectively, or a portion thereof; W represents a nucleotide or non-nucleotide linker that connects sequences Y' and Y; and the multifunctional siNA directs cleavage of the first and second target sequence via RNA interference. In one embodiment, the first target nucleic acid sequence and the second target nucleic acid sequence are present in the same target nucleic acid molecule repeat expansion (RE) RNA). In another embodiment, the first target nucleic acid sequence and the second target nucleic acid sequence are present in different target nucleic acid molecules, such as repeat expansion, RBL1, and RBL2, target sequences or a portion thereof. In one embodiment, region W connects the 3'-end of sequence Y' t with the 3'-end of sequence Y. In one embodiment, region W connects the 3'-end of sequence Y' with the 5'-end of sequence Y. In one embodiment, region W connects the 5'-end of sequence Y' with the 5'-end of sequence Y. In one embodiment, region W connects the 5'-end of sequence Y' with the 3'-end of sequence Y. In one embodiment, a terminal phosphate group is present at the 5'-end of sequence X. In one embodiment, a terminal phosphate group is present at the 5'-end of sequence In one embodiment, a 00 terminal phosphate group is present at the 5'-end of sequence Y. In one embodiment, a Sterminal phosphate group is present at the 5'-end of sequence In one embodiment, W connects sequences Y and Y' via a biodegradable linker. In one embodiment, W 0 10 further comprises a conjugate, label, aptamer, ligand, lipid, or polymer.

In one embodiment, a multifunctional siNA molecule of the invention comprises a structure having Formula MF-V: X X'

Y'-W-Y

wherein each X, Y, and Y' is independently an oligonucleotide of length of about nucleotides to about 50 nucleotides, preferably about 18 to about 40 nucleotides, or about 19 to about 23 nucleotides; X comprises nucleotide sequence that is complementary to nucleotide sequence present in region X' comprises nucleotide sequence that is complementary to nucleotide sequence present in region Y; each X, Y, or Y' is independently of length sufficient to stably interact base pair) with a first, second, third, or fourth target nucleic acid sequence, respectively, or a portion thereof; W represents a nucleotide or non-nucleotide linker that connects sequences Y' and Y; and the multifunctional siNA directs cleavage of the first, second, third, and/or fourth target sequence via RNA interference. In one embodiment, the first, second, third and fourth target nucleic acid sequence are all present in the same target nucleic acid molecule repeat expansion (RE) RNA). In another embodiment, the first, second, third and fourth target nucleic acid sequence are independently present in different target nucleic acid molecules, such as repeat expansion, RBL1, and RBL2, target sequences or a portion thereof. In one embodiment, region W connects the 3'-end of sequence Y' with the 3'end of sequence Y. In one embodiment, region W connects the 3'-end of sequence Y' with the 5'-end of sequence Y. In one embodiment, region W connects the 5'-end of 148 O sequence Y' with the 5'-end of sequence Y. In one embodiment, region W connects the of sequence Y' with the 3'-end of sequence Y. In one embodiment, a terminal phosphate group is present at the 5'-end of sequence X. In one embodiment, a terminal phosphate group is present at the 5'-end of sequence In one embodiment, a terminal phosphate group is present at the 5'-end of sequence Y. In one embodiment, a terminal phosphate group is present at the 5'-end of sequence In one embodiment,

W

00 connects sequences Y and Y' via a biodegradable linker. In one embodiment, W further Scomprises a conjugate, label, aptamer, ligand, lipid, or polymer.

In one embodiment, regions X and Y of multifunctional siNA molecule of the invention having any of Formula MF-I MF-V), are complementary to different i target nucleic acid sequences that are portions of the same target nucleic acid molecule.

In one embodiment, such target nucleic acid sequences are at different locations within the coding region of a RNA transcript. In one embodiment, such target nucleic acid sequences comprise coding and non-coding regions of the same RNA transcript. In one embodiment, such target nucleic acid sequences comprise regions of alternately spliced transcripts or precursors of such alternately spliced transcripts.

In one embodiment, a multifunctional siNA molecule having any of Formula

MF-I

MF-V can comprise chemical modifications as described herein without limitation, such as, for example, nucleotides having any of Formulae I-VII described herein, stabilization chemistries as described in Table IV, or any other combination of modified nucleotides and non-nucleotides as described in the various embodiments herein.

In one embodiment, the palidrome or repeat sequence or modified nucleotide nucleotide with a modified base, such as 2-amino purine or a universal base) in Z of multifunctional siNA constructs having Formula MF-I or MF-II comprises chemically modified nucleotides that are able to interact with a portion of the target nucleic acid sequence modified base analogs that can form Watson Crick base pairs or non- Watson Crick base pairs).

In one embodiment, a multifunctional siNA molecule of the invention, for example each strand of a multifunctional siNA having MF-I MF-V, independently comprises about 15 to about 40 nucleotides about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides). In one embodiment, 149 O a multifunctional siNA molecule of the invention comprises one or more chemical modifications. In a non-limiting example, the introduction of chemically modified nucleotides and/or non-nucleotides into nucleic acid molecules of the invention provides a powerful tool in overcoming potential limitations of in vivo stability and bioavailability inherent to unmodified RNA molecules that are delivered exogenously. For example, the use of chemically modified nucleic acid molecules can enable a lower dose of a 0 particular nucleic acid molecule for a given therapeutic effect since chemically modified Snucleic acid molecules tend to have a longer half-life in serum or in cells or tissues.

C. Furthermore, certain chemical modifications can improve the bioavailability and/or potency of nucleic acid molecules by not only enhancing half-life but also facilitating the targeting of nucleic acid molecules to particular organs, cells or tissues and/or improving cellular uptake of the nucleic acid molecules. Therefore, even if the activity of a chemically modified nucleic acid molecule is reduced in vitro as compared to a native/unmodified nucleic acid molecule, for example when compared to an unmodified RNA molecule, the overall activity of the modified nucleic acid molecule can be greater than the native or unmodified nucleic acid molecule due to improved stability, potency, duration of effect, bioavailability and/or delivery of the molecule.

In another embodiment, the invention features multifunctional siNAs, wherein the multifunctional siNAs are assembled from two separate double-stranded siNAs, with one of the ends of each sense strand is tethered to the end of the sense strand of the other siNA molecule, such that the two antisense siNA strands are annealed to their corresponding sense strand that are tethered to each other at one end (see Figure 22).

The tethers or linkers can be nucleotide-based linkers or non-nucleotide based linkers as generally known in the art and as described herein.

In one embodiment, the invention features a multifunctional siNA, wherein the multifunctional siNA is assembled from two separate double-stranded siNAs, with the of one sense strand of the siNA is tethered to the end of the sense strand of the other siNA molecule, such that the 5'-ends of the two antisense siNA strands, annealed to their corresponding sense strand that are tethered to each other at one end, point away (in the opposite direction) from each other (see Figure 22 The tethers or linkers can be nucleotide-based linkers or non-nucleotide based linkers as generally known in the art and as described herein.

3 In one embodiment, the invention features a multifunctional siNA, wherein the Smultifunctional siNA is assembled from two separate double-stranded siNAs, with the 3'-end of one sense strand of the siNA is tethered to the end of the sense strand of the other siNA molecule, such that the 5'-ends of the two antisense siNA strands, annealed to their corresponding sense strand that are tethered to each other at one end, face each other (see Figure 22 The tethers or linkers can be nucleotide-based linkers or non- Snucleotide based linkers as generally known in the art and as described herein.

O In one embodiment, the invention features a multifunctional siNA, wherein the Smultifunctional siNA is assembled from two separate double-stranded siNAs, with the 5'-end of one sense strand of the siNA is tethered to the end of the sense strand of the other siNA molecule, such that the 5'-end of the one of the antisense siNA strands annealed to their corresponding sense strand that are tethered to each other at one end, faces the 3'-end of the other antisense strand (see Figure 22 The tethers or linkers can be nucleotide-based linkers or non-nucleotide based linkers as generally known in the art and as described herein.

In one embodiment, the invention features a multifunctional siNA, wherein the multifunctional siNA is assembled from two separate double-stranded siNAs, with the of one antisense strand of the siNA is tethered to the end of the antisense strand of the other siNA molecule, such that the 5'-end of the one of the sense siNA strands annealed to their corresponding antisense sense strand that are tethered to each other at one end, faces the 3'-end of the other sense strand (see Figure 22 In one embodiment, the linkage between the 5'-end of the first antisense strand and the 3'end of the second antisense strand is designed in such a way as to be readily cleavable biodegradable linker) such that the 5'end of each antisense strand of the multifunctional siNA has a free 5'-end suitable to mediate RNA interefence-based cleavage of the target RNA. The tethers or linkers can be nucleotide-based linkers or non-nucleotide based linkers as generally known in the art and as described herein.

In one embodiment, the invention features a multifunctional siNA, wherein the multifunctional siNA is assembled from two separate double-stranded siNAs, with the 5'-end of one antisense strand of the siNA is tethered to the end of the antisense strand of the other siNA molecule, such that the 3'-end of the one of the sense siNA 3 strands annealed to their corresponding antisense sense strand that are tethered to each N other at one end, faces the 3'-end of the other sense strand (see Figure 22 In one Sembodiment, the linkage between the 5'-end of the first antisense strand and the of the second antisense strand is designed in such a way as to be readily cleavable biodegradable linker) such that the 5'end of each antisense strand of the multifunctional siNA has a free 5'-end suitable to mediate RNA interefence-based cleavage of the target 0 RNA. The tethers or linkers can be nucleotide-based linkers or non-nucleotide based Slinkers as generally known in the art and as described herein.

In one embodiment, the invention features a multifunctional siNA, wherein the multifunctional siNA is assembled from two separate double-stranded siNAs, with the 3'-end of one antisense strand of the siNA is tethered to the end of the antisense strand of the other siNA molecule, such that the 5'-end of the one of the sense siNA strands annealed to their corresponding antisense sense strand that are tethered to each other at one end, faces the 3'-end of the other sense strand (see Figure 22 In one embodiment, the linkage between the 5'-end of the first antisense strand and the of the second antisense strand is designed in such a way as to be readily cleavable biodegradable linker) such that the 5'end of each antisense strand of the multifunctional siNA has a free 5'-end suitable to mediate RNA interefence-based cleavage of the target RNA. The tethers or linkers can be nucleotide-based linkers or non-nucleotide based linkers as generally known in the art and as described herein.

In any of the above embodiments, a first target nucleic acid sequence or second target nucleic acid sequence can independently comprise repeat expansion (RE) RNA, DNA or a portion thereof. In one embodiment, the first target nucleic acid sequence is a repeat expansion (RE) RNA, DNA or a portion thereof and the second target nucleic acid sequence is a repeat expansion (RE) RNA, DNA of a portion thereof. In one embodiment, the first target nucleic acid sequence is a repeat expansion (RE) RNA, DNA or a portion thereof and the second target nucleic acid sequence is a another RNA, DNA of a portion thereof.

Synthesis of Nucleic Acid Molecules Synthesis of nucleic acids greater than 100 nucleotides in length is difficult using automated methods, and the therapeutic cost of such molecules is prohibitive. In this 152 3 invention, small nucleic acid motifs ("small" refers to nucleic acid motifs no more than N 100 nucleotides in length, preferably no more than 80 nucleotides in length, and most Spreferably no more than 50 nucleotides in length; individual siNA oligonucleotide sequences or siNA sequences synthesized in tandem) are preferably used for exogenous delivery. The simple structure of these molecules increases the ability of the nucleic acid to invade targeted regions of protein and/or RNA structure. Exemplary molecules of the 00 instant invention are chemically synthesized, and others can similarly be synthesized.

SOligonucleotides certain modified oligonucleotides or portions of Soligonucleotides lacking ribonucleotides) are synthesized using protocols known in the art, for example as described in Caruthers et al., 1992, Methods in Enzymology 211, 3- 19, Thompson et al., International PCT Publication No. WO 99/54459, Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, Methods Mol. Bio., 74, 59, Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45, and Brennan, U.S. Pat. No.

6,001,311. All of these references are incorporated herein by reference. The synthesis of oligonucleotides makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end. In a nonlimiting example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc.

synthesizer using a 0.2 tmol scale protocol with a 2.5 min coupling step for methylated nucleotides and a 45 second coupling step for 2'-deoxy nucleotides or 2'deoxy-2'-fluoro nucleotides. Table V outlines the amounts and the contact times of the reagents used in the synthesis cycle. Alternatively, syntheses at the 0.2 Pmol scale can be performed on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, CA) with minimal modification to the cycle. A 33-fold excess pL of 0.11 M 6.6 tmol) of 2'-O-methyl phosphoramidite and a 105-fold excess of Sethyl tetrazole (60 uL of 0.25 M 15 tmol) can be used in each coupling cycle of methyl residues relative to polymer-bound 5'-hydroxyl. A 22-fold excess (40 IL of 0.11 M 4.4 tmol) of deoxy phosphoramidite and a 70-fold excess of S-ethyl tetrazole tL of 0.25 M 10 umol) can be used in each coupling cycle of deoxy residues relative to polymer-bound 5'-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, are typically 97.5-99%. Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer include the following: detritylation solution is 3% TCA in 0 methylene chloride (ABI); capping is performed with 16% N-methyl imidazole in THF C, (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); and oxidation solution is 16.9 mM 12, 49 mM pyridine, 9% water in THF (PerSeptive Biosystems, Inc.).

Burdick Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle.

S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc. Alternately, for the introduction of 0 phosphorothioate linkages, Beaucage reagent (3H-l,2-Benzodithiol-3-one 1,1-dioxide, 0.05 M in acetonitrile) is used.

Deprotection of the DNA-based oligonucleotides is performed as follows: the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 40% aqueous methylamine (1 mL) at 65 °C for minutes. After cooling to -20 the supernatant is removed from the polymer support.

The support is washed three times with 1.0 mL of EtOH:MeCN:H20/3:1:1, vortexed and the supernatant is then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, are dried to a white powder.

The method of synthesis used for RNA including certain siNA molecules of the invention follows the procedure as described in Usman et al., 1987, J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res., 18, 5433; and Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684 Wincott et al., 1997, Methods Mol. Bio., 74, 59, and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end. In a non-limiting example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc.

synthesizer using a 0.2 jimol scale protocol with a 7.5 min coupling step for alkylsilyl protected nucleotides and a 2.5 min coupling step for 2'-O-methylated nucleotides.

Table V outlines the amounts and the contact times of the reagents used in the synthesis cycle. Alternatively, syntheses at the 0.2 imol scale can be done on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, CA) with minimal modification to the cycle. A 33-fold excess (60 pL of 0.11 M 6.6 jimol) of methyl phosphoramidite and a 75-fold excess of S-ethyl tetrazole (60 jtL of 0.25 M .mol) can be used in each coupling cycle of 2'-O-methyl residues relative to polymerbound 5'-hydroxyl. A 66-fold excess (120 uL of 0.11 M 13.2 [tmol) of alkylsilyl (ribo) protected phosphoramidite and a 150-fold excess of S-ethyl tetrazole (120 uL of 0.25 M 154 30 umol) can be used in each coupling cycle of ribo residues relative to polymer- Sbound 5'-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc.

2 synthesizer, determined by colorimetric quantitation of the trityl fractions, are typically 97.5-99%. Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer include the following: detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methyl imidazole in THF (ABI) and 0 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); oxidation solution is 16.9 mM 12, 49 mM pyridine, 9% water in THF (PerSeptive Biosystems, Inc.). Burdick Jackson CN Synthesis Grade acetonitrile is used directly from the reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American C International Chemical, Inc. Alternately, for the introduction of phosphorothioate linkages, Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,l-dioxide0.05 M in acetonitrile) is used.

Deprotection of the RNA is performed using either a two-pot or one-pot protocol.

For the two-pot protocol, the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 40% aq. methylamine (1 mL) at 65 "C for 10 min. After cooling to -20 the supernatant is removed from the polymer support. The support is washed three times with 1.0 mL of EtOH:MeCN:H20/3:1:1, vortexed and the supernatant is then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, are dried to a white powder. The base deprotected oligoribonucleotide is resuspended in anhydrous TEA/HF/NMP solution (300 jtL of a solution of 1.5 mL N-methylpyrrolidinone, 750 pL TEA and 1 mL TEA-3HF to provide a 1.4 M HF concentration) and heated to 65 "C.

After 1.5 h, the oligomer is quenched with 1.5 M NH 4

HCO

3 Alternatively, for the one-pot protocol, the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 33% ethanolic methylamine/DMSO: 1/1 (0.8 mL) at 65 "C for 15 minutes.

The vial is brought to room temperature TEA-3HF (0.1 mL) is added and the vial is heated at 65 "C for 15 minutes. The sample is cooled at -20 OC and then quenched with 1.5 MNH 4

HCO

3 t For purification of the trityl-on oligomers, the quenched NH 4

HCO

3 solution is C1 loaded onto a C-18 containing cartridge that had been prewashed with acetonitrile followed by 50 mM TEAA. After washing the loaded cartridge with water, the RNA is detritylated with 0.5% TFA for 13 minutes. The cartridge is then washed again with water, salt exchanged with 1 M NaCl and washed with water again. The oligonucleotide is then eluted with 30% acetonitrile.

00 r The average stepwise coupling yields are typically >98% (Wincott et al., 1995 0Nucleic Acids Res. 23, 2677-2684). Those of ordinary skill in the art will recognize that Sthe scale of synthesis can be adapted to be larger or smaller than the example described 10 above including but not limited to 96-well format.

Alternatively, the nucleic acid molecules of the present invention can be synthesized separately and joined together post-synthetically, for example, by ligation (Moore et al., 1992, Science 256, 9923; Draper et al., International PCT publication No.

WO 93/23569; Shabarova et al., 1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204), or by hybridization following synthesis and/or deprotection.

The siNA molecules of the invention can also be synthesized via a tandem synthesis methodology as described in Example 1 herein, wherein both siNA strands are synthesized as a single contiguous oligonucleotide fragment or strand separated by a cleavable linker which is subsequently cleaved to provide separate siNA fragments or strands that hybridize and permit purification of the siNA duplex. The linker can be a polynucleotide linker or a non-nucleotide linker. The tandem synthesis of siNA as described herein can be readily adapted to both multiwell/multiplate synthesis platforms such as 96 well or similarly larger multi-well platforms. The tandem synthesis of siNA as described herein can also be readily adapted to large scale synthesis platforms employing batch reactors, synthesis columns and the like.

A siNA molecule can also be assembled from two distinct nucleic acid strands or fragments wherein one fragment includes the sense region and the second fragment includes the antisense region of the RNA molecule.

I

t The nucleic acid molecules of the present invention can be modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-fluoro, 2'-O-methyl, 2'-H (for a review see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163). siNA constructs can be purified by gel electrophoresis using general methods or can be purified by high pressure liquid chromatography (HPLC; see Wincott et al., supra, the totality of which is 0 hereby incorporated herein by reference) and re-suspended in water.

SIn another aspect of the invention, siNA molecules of the invention are expressed t from transcription units inserted into DNA or RNA vectors. The recombinant vectors can O 10 be DNA plasmids or viral vectors. siNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus.

The recombinant vectors capable of expressing the siNA molecules can be delivered as described herein, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of siNA molecules.

Optimizing Activity of the nucleic acid molecule of the invention.

Chemically synthesizing nucleic acid molecules with modifications (base, sugar and/or phosphate) can prevent their degradation by serum ribonucleases, which can increase their potency (see Eckstein et al., International Publication No. WO 92/07065; Perrault et al., 1990 Nature 344, 565; Pieken et al., 1991, Science 253, 314; Usman and Cedergren, 1992, Trends in Biochem. Sci. 17, 334; Usman et al., International Publication No. WO 93/15187; and Rossi et al., International Publication No. WO 91/03162; Sproat, U.S. Pat. No. 5,334,711; Gold et al., U.S. Pat. No. 6,300,074; and Burgin et al., supra; all of which are incorporated by reference herein). All of the above references describe various chemical modifications that can be made to the base, phosphate and/or sugar moieties of the nucleic acid molecules described herein.

Modifications that enhance their efficacy in cells, and removal of bases from nucleic acid molecules to shorten oligonucleotide synthesis times and reduce chemical requirements are desired.

There are several examples in the art describing sugar, base and phosphate modifications that can be introduced into nucleic acid molecules with significant enhancement in their nuclease stability and efficacy. For example, oligonucleotides are 157

I

t modified to enhance stability and/or enhance biological activity by modification with I nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-fluoro, 2'-O-methyl, Sallyl, nucleotide base modifications (for a review see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163; Burgin et al., 1996, Biochemistry, 35, 14090). Sugar modification of nucleic acid molecules have been extensively described in the art (see Eckstein et al., International Publication PCT No.

00 WO 92/07065; Perrault et al. Nature, 1990, 344, 565-568; Pieken et al. Science, 1991, S253, 314-317; Usman and Cedergren, Trends in Biochem. Sci., 1992, 17, 334-339; N Usman et al. International Publication PCT No. WO 93/15187; Sproat, U.S. Pat. No.

5,334,711 and Beigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman et al., International PCT publication No. WO 97/26270; Beigelman et al., U.S. Pat. No.

5,716,824; Usman et al., U.S. Pat. No. 5,627,053; Woolf et al., International PCT Publication No. WO 98/13526; Thompson et al., USSN 60/082,404 which was filed on April 20, 1998; Karpeisky et al., 1998, Tetrahedron Lett., 39, 1131; Earnshaw and Gait, 1998, Biopolymers (Nucleic Acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu.

Rev. Biochem., 67, 99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5, 1999-2010; all of the references are hereby incorporated in their totality by reference herein). Such publications describe general methods and strategies to determine the location of incorporation of sugar, base and/or phosphate modifications and the like into nucleic acid molecules without modulating catalysis, and are incorporated by reference herein. In view of such teachings, similar modifications can be used as described herein to modify the siNA nucleic acid molecules of the instant invention so long as the ability of siNA to promote RNAi is cells is not significantly inhibited.

While chemical modification of oligonucleotide internucleotide linkages with phosphorothioate, phosphorodithioate, and/or 5'-methylphosphonate linkages improves stability, excessive modifications can cause some toxicity or decreased activity.

Therefore, when designing nucleic acid molecules, the amount of these internucleotide linkages should be minimized. The reduction in the concentration of these linkages should lower toxicity, resulting in increased efficacy and higher specificity of these molecules.

Short interfering nucleic acid (siNA) molecules having chemical modifications that maintain or enhance activity are provided. Such a nucleic acid is also generally more 158 t resistant to nucleases than an unmodified nucleic acid. Accordingly, the in vitro and/or in vivo activity should not be significantly lowered. In cases in which modulation is the goal, therapeutic nucleic acid molecules delivered exogenously should optimally be stable within cells until translation of the target RNA has been modulated long enough to reduce the levels of the undesirable protein. This period of time varies between hours to days depending upon the disease state. Improvements in the chemical synthesis of RNA 00 and DNA (Wincott et al., 1995, Nucleic Acids Res. 23, 2677; Caruthers et al., 1992, SMethods in Enzymology 211, 3-19 (incorporated by reference herein)) have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability, as described above.

In one embodiment, nucleic acid molecules of the invention include one or more about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) G-clamp nucleotides. A G-clamp nucleotide is a modified cytosine analog wherein the modifications confer the ability to hydrogen bond both Watson-Crick and Hoogsteen faces of a complementary guanine within a duplex, see for example Lin and Matteucci, 1998, Am. Chem. Soc., 120, 8531- 8532. A single G-clamp analog substitution within an oligonucleotide can result in substantially enhanced helical thermal stability and mismatch discrimination when hybridized to complementary oligonucleotides. The inclusion of such nucleotides in nucleic acid molecules of the invention results in both enhanced affinity and specificity to nucleic acid targets, complementary sequences, or template strands. In another embodiment, nucleic acid molecules of the invention include one or more about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) LNA "locked nucleic acid" nucleotides such as a 4'- C methylene bicyclo nucleotide (see for example Wengel et al., International PCT Publication No. WO 00/66604 and WO 99/14226).

In another embodiment, the invention features conjugates and/or complexes of siNA molecules of the invention. Such conjugates and/or complexes can be used to facilitate delivery of siNA molecules into a biological system, such as a cell. The conjugates and complexes provided by the instant invention can impart therapeutic activity by transferring therapeutic compounds across cellular membranes, altering the pharmacokinetics, and/or modulating the localization of nucleic acid molecules of the invention. The present invention encompasses the design and synthesis of novel conjugates and complexes for the delivery of molecules, including, but not limited to, 159 small molecules, lipids, cholesterol, phospholipids, nucleosides, nucleotides, nucleic N acids, antibodies, toxins, negatively charged polymers and other polymers, for example Sproteins, peptides, hormones, carbohydrates, polyethylene glycols, or polyamines, across cellular membranes. In general, the transporters described are designed to be used either individually or as part of a multi-component system, with or without degradable linkers.

These compounds are expected to improve delivery and/or localization of nucleic acid 00 molecules of the invention into a number of cell types originating from different tissues, Sin the presence or absence of serum (see Sullenger and Cech, U.S. Pat. No. 5,854,038).

I Conjugates of the molecules described herein can be attached to biologically active molecules via linkers that are biodegradable, such as biodegradable nucleic acid linker C, molecules.

The term "biodegradable linker" as used herein, refers to a nucleic acid or nonnucleic acid linker molecule that is designed as a biodegradable linker to connect one molecule to another molecule, for example, a biologically active molecule to a siNA molecule of the invention or the sense and antisense strands of a siNA molecule of the invention. The biodegradable linker is designed such that its stability can be modulated for a particular purpose, such as delivery to a particular tissue or cell type. The stability of a nucleic acid-based biodegradable linker molecule can be modulated by using various chemistries, for example combinations of ribonucleotides, deoxyribonucleotides, and chemically-modified nucleotides, such as 2'-O-methyl, 2'-fluoro, 2'-amino, 2'-O-amino, 2'-C-allyl, 2'-O-allyl, and other 2'-modified or base modified nucleotides. The biodegradable nucleic acid linker molecule can be a dimer, trimer, tetramer or longer nucleic acid molecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length, or can comprise a single nucleotide with a phosphorus-based linkage, for example, a phosphoramidate or phosphodiester linkage. The biodegradable nucleic acid linker molecule can also comprise nucleic acid backbone, nucleic acid sugar, or nucleic acid base modifications.

The term "biodegradable" as used herein, refers to degradation in a biological system, for example, enzymatic degradation or chemical degradation.

The term "biologically active molecule" as used herein refers to compounds or molecules that are capable of eliciting or modifying a biological response in a system.

SNon-limiting examples of biologically active siNA molecules either alone or in 2Ni combination with other molecules contemplated by the instant invention include Stherapeutically active molecules such as antibodies, cholesterol, hormones, antivirals, peptides, proteins, chemotherapeutics, small molecules, vitamins, co-factors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex forming oligonucleotides, 2,5-A chimeras, siNA, dsRNA, allozymes, 00 aptamers, decoys and analogs thereof. Biologically active molecules of the invention Salso include molecules capable of modulating the pharmacokinetics and/or I pharmacodynamics of other biologically active molecules, for example, lipids and polymers such as polyamines, polyamides, polyethylene glycol and other polyethers.

The term "phospholipid" as used herein, refers to a hydrophobic molecule comprising at least one phosphorus group. For example, a phospholipid can comprise a phosphorus-containing group and saturated or unsaturated alkyl group, optionally substituted with OH, COOH, oxo, amine, or substituted or unsubstituted aryl groups.

Therapeutic nucleic acid molecules siNA molecules) delivered exogenously optimally are stable within cells until reverse transcription of the RNA has been modulated long enough to reduce the levels of the RNA transcript. The nucleic acid molecules are resistant to nucleases in order to function as effective intracellular therapeutic agents. Improvements in the chemical synthesis of nucleic acid molecules described in the instant invention and in the art have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability as described above.

In yet another embodiment, siNA molecules having chemical modifications that maintain or enhance enzymatic activity of proteins involved in RNAi are provided. Such nucleic acids are also generally more resistant to nucleases than unmodified nucleic acids. Thus, in vitro and/or in vivo the activity should not be significantly lowered.

Use of the nucleic acid-based molecules of the invention will lead to better treatments by affording the possibility of combination therapies multiple siNA molecules targeted to different genes; nucleic acid molecules coupled with known small molecule modulators; or intermittent treatment with combinations of molecules, including different motifs and/or other chemical or biological molecules). The treatment 161 of subjects with siNA molecules can also include combinations of different types of i nucleic acid molecules, such as enzymatic nucleic acid molecules (ribozymes), Sallozymes, antisense, 2,5-A oligoadenylate, decoys, and aptamers.

In another aspect a siNA molecule of the invention comprises one or more and/or a cap structure, for example, on only the sense siNA strand, the antisense siNA strand, or both siNA strands.

00 By "cap structure" is meant chemical modifications, which have been incorporated at either terminus of the oligonucleotide (see, for example, Adamic et al., U.S. Pat. No.

S5,998,203, incorporated by reference herein). These terminal modifications protect the nucleic acid molecule from exonuclease degradation, and may help in delivery and/or localization within a cell. The cap may be present at the 5'-terminus (5'-cap) or at the 3'terminal (3'-cap) or may be present on both termini. In non-limiting examples, the includes, but is not limited to, glyceryl, inverted deoxy abasic residue (moiety); methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide, 4'-thio nucleotide; carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic dihydroxypentyl nucleotide, 3'-3'-inverted nucleotide moiety; 3'-3'-inverted abasic moiety; 3'-2'-inverted nucleotide moiety; 3'-2'-inverted abasic moiety; 1,4-butanediol phosphate; 3'-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3'-phosphate; 3'-phosphorothioate; phosphorodithioate; or bridging or non-bridging methylphosphonate moiety. Non-limiting examples of cap moieties are shown in Figure Non-limiting examples of the 3'-cap include, but are not limited to, glyceryl, inverted deoxy abasic residue (moiety), 5'-methylene nucleotide; 1-(beta-Derythrofuranosyl) nucleotide; 4'-thio nucleotide, carbocyclic nucleotide; phosphate; 1,3-diamino-2-propyl phosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate; nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; 3,4dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5'-5'-inverted nucleotide 3 moiety; 5'-5'-inverted abasic moiety; 5'-phosphoramidate; 5'-phosphorothioate; 1,4- Sbutanediol phosphate; 5'-amino; bridging and/or non-bridging Sphosphorothioate and/or phosphorodithioate, bridging or non bridging methylphosphonate and 5'-mercapto moieties (for more details see Beaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporated by reference herein).

By the term "non-nucleotide" is meant any group or compound which can be M incorporated into a nucleic acid chain in the place of one or more nucleotide units, 0 including either sugar and/or phosphate substitutions, and allows the remaining bases to ¢n exhibit their enzymatic activity. The group or compound is abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine and therefore lacks a base at the l'-position.

An "alkyl" group refers to a saturated aliphatic hydrocarbon, including straightchain, branched-chain, and cyclic alkyl groups. Preferably, the alkyl group has 1 to 12 carbons. More preferably, it is a lower alkyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkyl group can be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, N02 or N(CH3)2, amino, or SH. The term also includes alkenyl groups that are unsaturated hydrocarbon groups containing at least one carbon-carbon double bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkenyl group has 1 to 12 carbons.

More preferably, it is a lower alkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkenyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, N02, halogen, N(CH3)2, amino, or SH. The term "alkyl" also includes alkynyl groups that have an unsaturated hydrocarbon group containing at least one carbon-carbon triple bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkynyl group has 1 to 12 carbons. More preferably, it is a lower alkynyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkynyl group may be substituted or unsubstituted.

When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, =0, N02 or N(CH3)2, amino or SH.

Such alkyl groups can also include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups. An "aryl" group refers to an aromatic group that has at 163 3 least one ring having a conjugated pi electron system and includes carbocyclic aryl, N heterocyclic aryl and biaryl groups, all of which may be optionally substituted. The preferred substituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An "alkylaryl" group refers to an alkyl group (as described above) covalently joined to an aryl group (as described above). Carbocyclic aryl groups are groups wherein the ring atoms on the aromatic ring 00 are all carbon atoms. The carbon atoms are optionally substituted. Heterocyclic aryl Sgroups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and N the remainder of the ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted. An "amide" refers to an where R is either alkyl, aryl, alkylaryl or hydrogen.

An "ester" refers to an where R is either alkyl, aryl, alkylaryl or hydrogen.

By "nucleotide" as used herein is as recognized in the art to include natural bases (standard), and modified bases well known in the art. Such bases are generally located at the 1' position of a nucleotide sugar moiety. Nucleotides generally comprise a base, sugar and a phosphate group. The nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and other; see, for example, Usman and McSwiggen, supra; Eckstein et al., International PCT Publication No. WO 92/07065; Usman et al., International PCT Publication No.

WO 93/15187; Uhlman Peyman, supra, all are hereby incorporated by reference herein). There are several examples of modified nucleic acid bases known in the art as summarized by Limbach et al., 1994, Nucleic Acids Res. 22, 2183. Some of the nonlimiting examples of base modifications that can be introduced into nucleic acid molecules include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-methylcytidine), 5-alkyluridines ribothymidine), 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines 6methyluridine), propyne, and others (Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman Peyman, supra). By "modified bases" in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at 1' position or their equivalents.

In one embodiment, the invention features modified siNA molecules, with NI phosphate backbone modifications comprising one or more phosphorothioate, Sphosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl, substitutions. For a review of oligonucleotide backbone modifications, see Hunziker and Leumann, 1995, Nucleic Acid 00 Analogues: Synthesis and Properties, in Modern Synthetic Methods, VCH, 331-417, and SMesmaeker et al., 1994, Novel Backbone Replacements for Oligonucleotides, in Carbohydrate Modifications in Antisense Research, ACS, 24-39.

Ni3 By "abasic" is meant sugar moieties lacking a base or having other chemical groups in place of a base at the 1' position, see for example Adamic et al., U.S. Pat. No.

5,998,203.

By "unmodified nucleoside" is meant one of the bases adenine, cytosine, guanine, thymine, or uracil joined to the 1' carbon of 13-D-ribo-furanose.

By "modified nucleoside" is meant any nucleotide base which contains a modification in the chemical structure of an unmodified nucleotide base, sugar and/or phosphate. Non-limiting examples of modified nucleotides are shown by Formulae I-VII and/or other modifications described herein.

In connection with 2'-modified nucleotides as described for the present invention, by "amino" is meant 2'-NH 2 or NH 2 which can be modified or unmodified. Such modified groups are described, for example, in Eckstein et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic et al., U.S. Pat. No. 6,248,878, which are both incorporated by reference in their entireties.

Various modifications to nucleic acid siNA structure can be made to enhance the utility of these molecules. Such modifications will enhance shelf-life, half-life in vitro, stability, and ease of introduction of such oligonucleotides to the target site, to enhance penetration of cellular membranes, and confer the ability to recognize and bind to targeted cells.

Administration of Nucleic Acid Molecules A siNA molecule of the invention can be adapted for use to treat, for example, Huntinton disease and related conditions such as progressive chorea, rigidity, dementia, Sand seizures, spinocerebellar ataxia, spinal and bulbar muscular dystrophy (SBMA), dentatorubropallidoluysian atrophy (DRPLA) and any other diseases or conditions that are related to or will respond to the levels of a repeat expansion (repeat expansion (RE)) gene in a cell or tissue, alone or in combination with other therapies. For example, a 00 siNA molecule can comprise a delivery vehicle, including liposomes, for administration Sto a subject, carriers and diluents and their salts, and/or can be present in N pharmaceutically acceptable formulations. Methods for the delivery of nucleic acid 8 10 molecules are described in Akhtar et al., 1992, Trends Cell Bio., 2, 139; Delivery (N Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995, Maurer et al., 1999, Mol. Membr. Biol., 16, 129-140; Hofland and Huang, 1999, Handb. Exp.

Pharmacol., 137, 165-192; and Lee et al., 2000, ACS Symp. Ser., 752, 184-192, all of which are incorporated herein by reference. Beigelman et al., U.S. Pat. No. 6,395,713 and Sullivan et al., PCT WO 94/02595 further describe the general methods for delivery of nucleic acid molecules. These protocols can be utilized for the delivery of virtually any nucleic acid molecule. Nucleic acid molecules can be administered to cells by a variety of methods known to those of skill in the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as biodegradable polymers, hydrogels, cyclodextrins (see for example Gonzalez et al., 1999, Bioconjugate Chem., 10, 1068-1074; Wang et al., International

PCT

publication Nos. WO 03/47518 and WO 03/46185), poly(lactic-co-glycolic)acid

(PLGA)

and PLCA microspheres (see for example US Patent 6,447,796 and US Patent Application Publication No. US 2002130430), biodegradable nanocapsules, and bioadhesive microspheres, or by proteinaceous vectors (O'Hare and Normand, International PCT Publication No. WO 00/53722). In another embodiment, the nucleic acid molecules of the invention can also be formulated or complexed with polyethyleneimine and derivatives thereof, such as polyethyleneiminepolyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL) or polyethyleneiminepolyethyleneglycol-tri-N-acetylgalactosamine (PEI-PEG-triGAL) derivatives.

Alternatively, the nucleic acid/vehicle combination is locally delivered by direct injection or by use of an infusion pump. Many examples in the art describe CNS delivery methods of oligonucleotides by osmotic pump, (see Chun et al., 1998,

I

3 Neuroscience Letters, 257, 135-138, D'Aldin et al., 1998, Mol. Brain Research, 55, 151- 164, Dryden et al., 1998, J. Endocrinol., 157, 169-175, Ghimikar et al., 1998, SNeuroscience Letters, 247, 21-24) or direct infusion (Broaddus et al., 1997, Neurosurg.

Focus, 3, article Various devices as are known in the art can be utilized to deliver nucleic acid molecules of the invention (see for example Turner, 2003, Acta Neurochir Suppl., 87, 29-35). Other routes of delivery include, but are not limited to oral (tablet or 00 pill form) and/or intrathecal delivery (Gold, 1997, Neuroscience, 76, 1153-1158). For a Scomprehensive review on drug delivery strategies including broad coverage of CNS delivery, see Ho et al., 1999, Curr. Opin. Mol. Ther., 1, 336-343 and Jain, Drug Delivery Systems: Technologies and Commercial Opportunities, Decision Resources, 1998 and Groothuis et al., 1997, J. NeuroVirol., 3, 387-400. Direct injection of the nucleic acid molecules of the invention, whether subcutaneous, intramuscular, or intradermal, can take place using standard needle and syringe methodologies, or by needle-free technologies such as those described in Conry et al., 1999, Clin. Cancer Res., 5, 2330- 2337 and Barry et al., International PCT Publication No. WO 99/31262. The molecules of the instant invention can be used as pharmaceutical agents. Pharmaceutical agents prevent, modulate the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state in a subject.

In one embodiment, a siNA molecule of the invention is administered to a subject or organism via local administration to relevant tissues or cells, such as brain cells and tissues basal ganglia, striatum, or cortex), for example, by administration of siNA, vectors or expression cassettes of the invention to relevant cells basal ganglia, striatum, cortex, cerebellum, motor neurons etc.). In one embodiment, the siNA, vector, or expression cassette is administered to the subject or organism by stereotactic or convection enhanced delivery to the brain. For example, US Patent No. 5,720,720 provides methods and devices useful for stereotactic and convection enhanced delivery of reagents to the brain. Such methods and devices can be readily used for the delivery of siNAs, vectors, or expression cassettes of the invention to a subject or organism, and is incorporated by reference herein in its entirety. US Patent Application Nos.

2002/0141980; 2002/0114780; and 2002/0187127 all provide methods and devices useful for stereotactic and convection enhanced delivery of reagents that can be readily adapted for delivery of siNAs, vectors, or expression cassettes of the invention to a subject or organism, and are incorporated by reference herein in their entirety. Particular devices that may be useful in delivering siNAs, vectors, or expression cassettes of the invention to a subject or organism are for example described in US Patent Application No. 2004/0162255, which is incorporated by reference herein in its entirety. The siNA molecule of the invention can be chemically synthesized or expressed from vectors as described herein or otherwise known in the art to target appropriate tissues or cells in the 00 subject or organism.

SExperiments have demonstrated the efficient in vivo uptake of nucleic acids by neurons. As an example of local administration of nucleic acids to nerve cells, Sommer O 10 et al., 1998, Antisense Nuc. Acid Drug Dev., 8, 75, describe a study in which a phosphorothioate antisense nucleic acid molecule to c-fos is administered to rats via microinjection into the brain. Antisense molecules labeled with tetramethylrhodamineisothiocyanate (TRITC) or fluorescein isothiocyanate (FITC) were taken up by exclusively by neurons thirty minutes post-injection. A diffuse cytoplasmic staining and nuclear staining was observed in these cells. As an example of systemic administration of nucleic acid to nerve cells, Epa et al., 2000, Antisense Nuc. Acid Drug Dev., 10, 469, describe an in vivo mouse study in which beta-cyclodextrin-adamantane-oligonucleotide conjugates were used to target the p75 neurotrophin receptor in neuronally differentiated PC12 cells. Following a two week course of IP administration, pronounced uptake of p 7 5 neurotrophin receptor antisense was observed in dorsal root ganglion (DRG) cells.

In addition, a marked and consistent down-regulation of p75 was observed in DRG neurons. Additional approaches to the targeting of nucleic acid to neurons are described in Broaddus et al., 1998, J. Neurosurg., 88(4), 734; Karle et al., 1997, Eur. J.

Pharmocol., 340(2/3), 153; Bannai et al., 1998, Brain Research, 784(1,2), 304; Rajakumar et al., 1997, Synapse, 26(3), 199; Wu-pong et al., 1999, BioPharm, 12(1), 32; Bannai et al., 1998, Brain Res. Protoc., 83; Simantov et al., 1996, Neuroscience, 74(1), 39. Nucleic acid molecules of the invention are therefore amenable to delivery to and uptake by cells that express repeat expansion allelic variants for modulation of repeat expansion (RE) gene expression.

The delivery of nucleic acid molecules of the invention, targeting repeat expansion (RE) is provided by a variety of different strategies. Traditional approaches to CNS delivery that can be used include, but are not limited to, intrathecal and 168

I

t intracerebroventricular administration, implantation of catheters and pumps, direct injection or perfusion at the site of injury or lesion, injection into the brain arterial 2 system, or by chemical or osmotic opening of the blood-brain barrier. Other approaches can include the use of various transport and carrier systems, for example though the use of conjugates and biodegradable polymers. Furthermore, gene therapy approaches, for example as described in Kaplitt et al., US 6,180,613 and Davidson, WO 04/013280, can 0 be used to express nucleic acid molecules in the CNS.

SIn one embodiment, a siNA composition of the invention can comprise a delivery t vehicle, including liposomes, for administration to a subject, carriers and diluents and 10 their salts, and/or can be present in pharmaceutically acceptable formulations. Methods for the delivery of nucleic acid molecules are described in Akhtar et al., 1992, Trends Cell Bio., 2, 139; Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed.

Akhtar, 1995, Maurer et al., 1999, Mol. Membr. Biol., 16, 129-140; Hofland and Huang, 1999, Handb. Exp. Pharmacol., 137, 165-192; and Lee et al., 2000, ACS Symp. Ser., 752, 184-192, all of which are incorporated herein by reference. Beigelman et al., U.S. Pat.

No. 6,395,713 and Sullivan et al., PCT WO 94/02595 further describe the general methods for delivery of nucleic acid molecules. These protocols can be utilized for the delivery of virtually any nucleic acid molecule. Nucleic acid molecules can be administered to cells by a variety of methods known to those of skill in the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as biodegradable polymers, hydrogels, cyclodextrins (see for example Gonzalez et al., 1999, Bioconjugate Chem., 10, 1068-1074; Wang et al., International PCT publication Nos. WO 03/47518 and WO 03/46185), poly(lactic-coglycolic)acid (PLGA) and PLCA microspheres (see for example US Patent 6,447,796 and US Patent Application Publication No. US 2002130430), biodegradable nanocapsules, and bioadhesive microspheres, or by proteinaceous vectors (O'Hare and Normand, International PCT Publication No. WO 00/53722). In another embodiment, the nucleic acid molecules of the invention can also be formulated or complexed with polyethyleneimine and derivatives thereof, such as polyethyleneiminepolyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL) or polyethyleneiminepolyethyleneglycol-tri-N-acetylgalactosamine (PEI-PEG-triGAL) derivatives. In one embodiment, the nucleic acid molecules of the invention are formulated as described in

I

0 United States Patent Application Publication No. 20030077829, incorporated by reference herein in its entirety.

a In one embodiment, a siNA molecule of the invention is complexed with membrane disruptive agents such as those described in U.S. Patent Application Publication No. 20010007666, incorporated by reference herein in its entirety including 0 the drawings. In another embodiment, the membrane disruptive agent or agents and the M siNA molecule are also complexed with a cationic lipid or helper lipid molecule, such as O those lipids described in U.S. Patent No. 6,235,310, incorporated by reference herein in Sits entirety including the drawings.

In one embodiment, a siNA molecule of the invention is complexed with delivery systems as described in U.S. Patent Application Publication No. 2003077829 and International PCT Publication Nos. WO 00/03683 and WO 02/087541, all incorporated by reference herein in their entirety including the drawings.

In one embodiment, delivery systems of the invention include, for example, aqueous and nonaqueous gels, creams, multiple emulsions, microemulsions, liposomes, ointments, aqueous and nonaqueous solutions, lotions, aerosols, hydrocarbon bases and powders, and can contain excipients such as solubilizers, permeation enhancers fatty acids, fatty acid esters, fatty alcohols and amino acids), and hydrophilic polymers polycarbophil and polyvinylpyrolidone). In one embodiment, the pharmaceutically acceptable carrier is a liposome or a transdermal enhancer. Examples of liposomes which can be used in this invention include the following: CellFectin, 1:1.5 liposome formulation of the cationic lipid N,NI,NII,NIII-tetramethyl-N,NI,NII,NIII-tetrapalmit-yspermine and dioleoyl phosphatidylethanolamine (DOPE) (GIBCO BRL); Cytofectin GSV, 2:1 liposome formulation of a cationic lipid and DOPE (Glen Research); DOTAP (N-[1-(2,3-dioleoyloxy)-N,N,N-tri-methyl-ammoniummethylsulfate) (Boehringer Manheim); and Lipofectamine, 3:1 liposome formulation of the polycationic lipid DOSPA and the neutral lipid DOPE (GIBCO BRL).

In one embodiment, delivery systems of the invention include patches, tablets, suppositories, pessaries, gels and creams, and can contain excipients such as solubilizers and enhancers propylene glycol, bile salts and amino acids), and other vehicles 170 t polyethylene glycol, fatty acid esters and derivatives, and hydrophilic polymers N such as hydroxypropylmethylcellulose and hyaluronic acid).

SIn one embodiment, a siNA molecule of the invention is administered iontophoretically, for example to the dermis or to other relevant tissues such as the inner ear/cochlea. Non-limiting examples of iontophoretic delivery are described in, for example, WO 03/043689 and WO 03/030989, which are incorporated by reference in 00 Cr their entireties herein.

cNi In one embodiment, siNA molecules of the invention are formulated or complexed Swith polyethylenimine linear or branched PEI) and/or polyethylenimine 10 derivatives, including for example grafted PEIs such as galactose PEI, cholesterol PEI, antibody derivatized PEI, and polyethylene glycol PEI (PEG-PEI) derivatives thereof (see for example Ogris et al., 2001, AAPA PharmSci, 3, 1-11; Furgeson et al., 2003, Bioconjugate Chem., 14, 840-847; Kunath et al., 2002, Phramaceutical Research, 19, 810-817; Choi et al., 2001, Bull. Korean Chem. Soc., 22, 46-52; Bettinger et al., 1999, Bioconjugate Chem., 10, 558-561; Peterson et al., 2002, Bioconjugate Chem., 13, 845- 854; Erbacher et al., 1999, Journal of Gene Medicine Preprint, 1, 1-18; Godbey et al., 1999., PNAS USA, 96, 5177-5181; Godbey et al., 1999, Journal of Controlled Release, 149-160; Diebold et al., 1999, Journal of Biological Chemistry, 274, 19087-19094; Thomas and Klibanov, 2002, PNAS USA, 99, 14640-14645; and Sagara, US 6,586,524, incorporated by reference herein.

In one embodiment, a siNA molecule of the invention comprises a bioconjugate, for example a nucleic acid conjugate as described in Vargeese et al., USSN 10/427,160, filed April 30, 2003; US 6,528,631; US 6,335,434; US 6, 235,886; US 6,153,737; US 5,214,136; US 5,138,045, all incorporated by reference herein.

Thus, the invention features a pharmaceutical composition comprising one or more nucleic acid(s) of the invention in an acceptable carrier, such as a stabilizer, buffer, and the like. The polynucleotides of the invention can be administered RNA, DNA or protein) and introduced to a subject by any standard means, with or without stabilizers, buffers, and the like, to form a pharmaceutical composition. When it is desired to use a liposome delivery mechanism, standard protocols for formation of liposomes can be followed. The compositions of the present invention can also be formulated and used as 171 3 creams, gels, sprays, oils and other suitable compositions for topical, dermal, or transdermal administration as is known in the art.

a The present invention also includes pharmaceutically acceptable formulations of the compounds described. These formulations include salts of the above compounds, acid addition salts, for example, salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid.

00oO A pharmacological composition or formulation refers to a composition or N formulation in a form suitable for administration, systemic or local administration, into a cell or subject, including for example a human. Suitable forms, in part, depend S 10 upon the use or the route of entry, for example oral, transdermal, or by injection. Such forms should not prevent the composition or formulation from reaching a target cell a cell to which the negatively charged nucleic acid is desirable for delivery). For example, pharmacological compositions injected into the blood stream should be soluble.

Other factors are known in the art, and include considerations such as toxicity and forms that prevent the composition or formulation from exerting its effect.

In one embodiment, siNA molecules of the invention are administered to a subject by systemic administration in a pharmaceutically acceptable composition or formulation.

By "systemic administration" is meant in vivo systemic absorption or accumulation of drugs in the blood stream followed by distribution throughout the entire body.

Administration routes that lead to systemic absorption include, without limitation: intravenous, subcutaneous, portal vein, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular. Each of these administration routes exposes the siNA molecules of the invention to an accessible diseased tissue. The rate of entry of a drug into the circulation has been shown to be a function of molecular weight or size. The use of a liposome or other drug carrier comprising the compounds of the instant invention can potentially localize the drug, for example, in certain tissue types, such as the tissues of the reticular endothelial system (RES). A liposome formulation that can facilitate the association of drug with the surface of cells, such as, lymphocytes and macrophages is also useful. This approach can provide enhanced delivery of the drug to target cells by taking advantage of the specificity of macrophage and lymphocyte immune recognition of abnormal cells.

3 By "pharmaceutically acceptable formulation" or "pharmaceutically acceptable i composition" is meant, a composition or formulation that allows for the effective Sdistribution of the nucleic acid molecules of the instant invention in the physical location most suitable for their desired activity. Non-limiting examples of agents suitable for formulation with the nucleic acid molecules of the instant invention include: Pglycoprotein inhibitors (such as Pluronic P85),; biodegradable polymers, such as poly 0 (DL-lactide-coglycolide) microspheres for sustained release delivery (Emerich, DF et al, S1999, Cell Transplant, 8, 47-58); and loaded nanoparticles, such as those made of N polybutylcyanoacrylate. Other non-limiting examples of delivery strategies for the 0 10 nucleic acid molecules of the instant invention include material described in Boado et al., ,I 1998, J. Pharm. Sci., 87, 1308-1315; Tyler et al., 1999, FEBS Lett., 421, 280-284; Pardridge et al., 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv. Drug Delivery Rev., 15, 73-107; Aldrian-Herrada et al., 1998, Nucleic Acids Res., 26, 4910-4916; and Tyler et al., 1999, PNAS USA., 96, 7053-7058.

The invention also features the use of a composition comprising surface-modified liposomes containing poly (ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes) and nucleic acid molecules of the invention. These formulations offer a method for increasing the accumulation of drugs siNA) in target tissues. This class of drug carriers resists opsonization and elimination by the mononuclear phagocytic system (MPS or RES), thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated drug (Lasic et al.

Chem. Rev. 1995, 95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005- 1011). Such liposomes have been shown to accumulate selectively in tumors, presumably by extravasation and capture in the neovascularized target tissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et al., 1995, Biochim. Biophys. Acta, 1238, 86- The long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA, particularly compared to conventional cationic liposomes which are known to accumulate in tissues of the MPS (Liu et al., J. Biol.

Chem. 1995, 42, 24864-24870; Choi et al., International PCT Publication No. WO 96/10391; Ansell et al., International PCT Publication No. WO 96/10390; Holland et al., International PCT Publication No. WO 96/10392). Long-circulating liposomes are also likely to protect drugs from nuclease degradation to a greater extent compared to cationic t liposomes, based on their ability to avoid accumulation in metabolically aggressive MPS tissues such as the liver and spleen.

The present invention also includes compositions prepared for storage or administration that include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, 00 for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R.

Gennaro edit. 1985), hereby incorporated by reference herein. For example, r preservatives, stabilizers, dyes and flavoring agents can be provided. These include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. In addition, antioxidants and suspending agents can be used.

A pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state. The pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors that those skilled in the medical arts will recognize.

Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients is administered dependent upon potency of the negatively charged polymer.

The nucleic acid molecules of the invention and formulations thereof can be administered orally, topically, parenterally, by inhalation or spray, or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and/or vehicles. The term parenteral as used herein includes percutaneous, subcutaneous, intravascular intravenous), intramuscular, or intrathecal injection or infusion techniques and the like. In addition, there is provided a pharmaceutical formulation comprising a nucleic acid molecule of the invention and a pharmaceutically acceptable carrier. One or more nucleic acid molecules of the invention can be present in association with one or more non-toxic pharmaceutically acceptable carriers and/or diluents and/or adjuvants, and if desired other active ingredients. The pharmaceutical compositions containing nucleic acid molecules of the invention can be in a form suitable for oral use, for example, as tablets, troches, 3 lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs.

9 Compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more such sweetening agents, flavoring agents, coloring agents or preservative agents in order to provide pharmaceutically elegant and palatable 00 M preparations. Tablets contain the active ingredient in admixture with non-toxic Spharmaceutically acceptable excipients that are suitable for the manufacture of tablets.

These excipients can be, for example, inert diluents; such as calcium carbonate, sodium 0 10 carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets can be uncoated or they can be coated by known techniques. In some cases such coatings can be prepared by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monosterate or glyceryl distearate can be employed.

Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in a mixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents can be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions can also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

Oily suspensions can be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions can contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents and flavoring agents can be added to provide palatable oral preparations. These compositions can be preserved by the addition of an anti-oxidant such as ascorbic acid Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents or suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, can also be present.

Pharmaceutical compositions of the invention can also be in the form of oil-inwater emulsions. The oily phase can be a vegetable oil or a mineral oil or mixtures of these. Suitable emulsifying agents can be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions can also contain sweetening and flavoring agents.

Syrups and elixirs can be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol, glucose or sucrose. Such formulations can also contain a demulcent, a preservative and flavoring and coloring agents. The pharmaceutical compositions can be in the form of a sterile injectable aqueous or oleaginous suspension.

This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above.

176 The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3- Sbutanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono-or diglycerides. In addition, 00 fatty acids such as oleic acid find use in the preparation of injectables.

The nucleic acid molecules of the invention can also be administered in the form of t suppositories, for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter and polyethylene glycols.

Nucleic acid molecules of the invention can be administered parenterally in a sterile medium. The drug, depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle. Advantageously, adjuvants such as local anesthetics, preservatives and buffering agents can be dissolved in the vehicle.

Dosage levels of the order of from about 0.1 mg to about 140 mg per kilogram of body weight per day are useful in the treatment of the above-indicated conditions (about 0.5 mg to about 7 g per subject per day). The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the host treated and the particular mode of administration. Dosage unit forms generally contain between from about 1 mg to about 500 mg of an active ingredient.

It is understood that the specific dose level for any particular subject depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy.

For administration to non-human animals, the composition can also be added to the animal feed or drinking water. It can be convenient to formulate the animal feed and drinking water compositions so that the animal takes in a therapeutically appropriate I quantity of the composition along with its diet. It can also be convenient to present the Scomposition as a premix for addition to the feed or drinking water.

The nucleic acid molecules of the present invention can also be administered to a subject in combination with other therapeutic compounds to increase the overall therapeutic effect. The use of multiple compounds to treat an indication can increase the 00 M beneficial effects while reducing the presence of side effects.

In one embodiment, the invention comprises compositions suitable for 0 administering nucleic acid molecules of the invention to specific cell types. For example, the asialoglycoprotein receptor (ASGPr) (Wu and Wu, 1987, J. Biol. Chem.

262, 4429-4432) is unique to hepatocytes and binds branched galactose-terminal glycoproteins, such as asialoorosomucoid (ASOR). In another example, the folate receptor is overexpressed in many cancer cells. Binding of such glycoproteins, synthetic glycoconjugates, or folates to the receptor takes place with an affinity that strongly depends on the degree of branching of the oligosaccharide chain, for example, triatennary structures are bound with greater affinity than biatenarry or monoatennary chains (Baenziger and Fiete, 1980, Cell, 22, 611-620; Connolly et al., 1982, J. Biol.

Chem., 257, 939-945). Lee and Lee, 1987, Glycoconjugate 4, 317-328, obtained this high specificity through the use ofN-acetyl-D-galactosamine as the carbohydrate moiety, which has higher affinity for the receptor, compared to galactose. This "clustering effect" has also been described for the binding and uptake of mannosyl-terminating glycoproteins or glycoconjugates (Ponpipom et al., 1981, J. Med. Chem., 24, 1388- 1395). The use of galactose, galactosamine, or folate based conjugates to transport exogenous compounds across cell membranes can provide a targeted delivery approach to, for example, the treatment of liver disease, cancers of the liver, or other cancers. The use of bioconjugates can also provide a reduction in the required dose of therapeutic compounds required for treatment. Furthermore, therapeutic bioavailability, pharmacodynamics, and pharmacokinetic parameters can be modulated through the use of nucleic acid bioconjugates of the invention. Non-limiting examples of such bioconjugates are described in Vargeese et al., USSN 10/201,394, filed August 13, 2001; and Matulic-Adamic et al., USSN 60/362,016, filed March 6, 2002.

I

3 Alternatively, certain siNA molecules of the instant invention can be expressed within cells from eukaryotic promoters Izant and Weintraub, 1985, Science, 229, 345; McGarry and Lindquist, 1986, Proc. Natl. Acad. Sci., USA 83, 399; Scanlon et al., 1991, Proc. Natl. Acad. Sci. USA, 88, 10591-5; Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Dropulic et al., 1992, J. Virol., 66, 1432-41; Weerasinghe et al., 1991, J. Virol., 65, 5531-4; Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802- S6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Sarver et al., 1990 Science, 247, S1222-1225; Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Good et al., 1997, SGene Therapy, 4, 45. Those skilled in the art realize that any nucleic acid can be expressed in eukaryotic cells from the appropriate DNA/RNA vector. The activity of such nucleic acids can be augmented by their release from the primary transcript by a enzymatic nucleic acid (Draper et al., PCT WO 93/23569, and Sullivan et al., PCT WO 94/02595; Ohkawa et al., 1992, Nucleic Acids Symp. Ser., 27, 15-6; Taira et al., 1991, Nucleic Acids Res., 19, 5125-30; Ventura et al., 1993, Nucleic Acids Res., 21, 3249-55; Chowrira et al., 1994, J. Biol. Chem., 269, 25856.

In another aspect of the invention, RNA molecules of the present invention can be expressed from transcription units (see for example Couture et al., 1996, TIG., 12, 510) inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. siNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. In another embodiment, pol III based constructs are used to express nucleic acid molecules of the invention (see for example Thompson, U.S. Pats. Nos. 5,902,880 and 6,146,886). The recombinant vectors capable of expressing the siNA molecules can be delivered as described above, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of nucleic acid molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the siNA molecule interacts with the target mRNA and generates an RNAi response. Delivery of siNA molecule expressing vectors can be systemic, such as by intravenous or intra-muscular administration, by administration to target cells ex-planted from a subject followed by reintroduction into the subject, or by any other means that would allow for introduction into the desired target cell (for a review see Couture et al., 1996, TIG., 12, 510).

In one aspect the invention features an expression vector comprising a nucleic acid Ssequence encoding at least one siNA molecule of the instant invention. The expression vector can encode one or both strands of a siNA duplex, or a single self-complementary strand that self hybridizes into a siNA duplex. The nucleic acid sequences encoding the siNA molecules of the instant invention can be operably linked in a manner that allows expression of the siNA molecule (see for example Paul et al., 2002, Nature 00 Biotechnology, 19, 505; Miyagishi and Taira, 2002, Nature Biotechnology, 19, 497; Lee Set al., 2002, Nature Biotechnology, 19, 500; and Novina et al., 2002, Nature Medicine, Sadvance online publication doi:10.1038/nm7 2 0 10 In another aspect, the invention features an expression vector comprising: a) a transcription initiation region eukaryotic pol I, II or III initiation region); b) a transcription termination region eukaryotic pol I, II or III termination region); and c) a nucleic acid sequence encoding at least one of the siNA molecules of the instant invention, wherein said sequence is operably linked to said initiation region and said termination region in a manner that allows expression and/or delivery of the siNA molecule. The vector can optionally include an open reading frame (ORF) for a protein operably linked on the 5' side or the 3'-side of the sequence encoding the siNA of the invention; and/or an intron (intervening sequences).

Transcription of the siNA molecule sequences can be driven from a promoter for eukaryotic RNA polymerase I (pol RNA polymerase II (pol II), or RNA polymerase III (pol III). Transcripts from pol II or pol III promoters are expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type depends on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby. Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic

RNA

polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci. USA, 87, 6743-7; Gao and Huang 1993, Nucleic Acids Res., 21, 2867-72; Lieber et al., 1993, Methods Enzymol., 217, 47-66; Zhou et al., 1990, Mol.

Cell. Biol., 10, 4529-37). Several investigators have demonstrated that nucleic acid molecules expressed from such promoters can function in mammalian cells (e.g.

Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Ojwang et al., 1992, Proc.

Natl. Acad. Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Yu et al., 1993, Proc. Natl. Acad. Sci. USA, 90, 6340-4; L'Huillier et al., 1992, EMBO 180 3 11, 4411-8; Lisziewicz et al., 1993, Proc. Natl. Acad. Sci. U. S. A, 90, 8000-4; Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Sullenger Cech, 1993, Science, Q 262, 1566). More specifically, transcription units such as the ones derived from genes encoding U6 small nuclear (snRNA), transfer RNA (tRNA) and adenovirus VA RNA are useful in generating high concentrations of desired RNA molecules such as siNA in cells (Thompson et al., supra; Couture and Stinchcomb, 1996, supra; Noonberg et al., 1994, 0 Nucleic Acid Res., 22, 2830; Noonberg et al., U.S. Pat. No. 5,624,803; Good et al., 1997, SGene Ther., 4, 45; Beigelman et al., International PCT Publication No. WO 96/18736.

N The above siNA transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated virus vectors), or viral RNA vectors (such as retroviral or alphavirus vectors) (for a review see Couture and Stinchcomb, 1996, supra).

In another aspect the invention features an expression vector comprising a nucleic acid sequence encoding at least one of the siNA molecules of the invention in a manner that allows expression of that siNA molecule. The expression vector comprises in one embodiment; a) a transcription initiation region; b) a transcription termination region; and c) a nucleic acid sequence encoding at least one strand of the siNA molecule, wherein the sequence is operably linked to the initiation region and the termination region in a manner that allows expression and/or delivery of the siNA molecule.

In another embodiment the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an open reading frame; and d) a nucleic acid sequence encoding at least one strand of a siNA molecule, wherein the sequence is operably linked to the 3'-end of the open reading frame and wherein the sequence is operably linked to the initiation region, the open reading frame and the termination region in a manner that allows expression and/or delivery of the siNA molecule. In yet another embodiment, the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; and d) a nucleic acid sequence encoding at least one siNA molecule, wherein the sequence is operably linked to the initiation region, the intron and the termination region in a manner which allows expression and/or delivery of the nucleic acid molecule.

t In another embodiment, the expression vector comprises: a) a transcription N initiation region; b) a transcription termination region; c) an intron; d) an open reading Sframe; and e) a nucleic acid sequence encoding at least one strand of a siNA molecule, wherein the sequence is operably linked to the 3'-end of the open reading frame and wherein the sequence is operably linked to the initiation region, the intron, the open reading frame and the termination region in a manner which allows expression and/or 0 delivery of the siNA molecule.

0 Huntingtin biology and biochemistry

(N

SThe following discussion is adapted from the Revilla et al., 2002, Huntington

C

I 10 Disease, Copyright 2004, eMedicine.com, Inc. and the OMIM database entry for Huntington disease, Copyright 1966-2004 Johns Hopkins University. Huntington disease (HD) is an incurable, adult-onset, autosomal dominant inherited disorder associated with cell loss within a specific subset of neurons in the basal ganglia and cortex. HD is named after George Huntington, the physician who described it as hereditary chorea in 1872. Characteristic features of HD include involuntary movements, dementia, and behavioral changes. Huntington disease (HD) is inherited as an autosomal dominant disease that gives rise to progressive, selective or localized neural cell death associated with choreic movements and dementia. The classic signs of Huntington disease are progressive chorea, rigidity, and dementia, oftem associated with seizures. A characteristic atrophy of the caudate nucleus is seen in radiographic images. The most striking neuropathology in HD occurs within the neostriatum, in which gross atrophy of the caudate nucleus and putamen is accompanied by selective neuronal loss and astrogliosis. Other regions, including the globus pallidus, thalamus, subthalamic nucleus, substantia nigra, and cerebellum, show varying degrees of atrophy depending on the pathologic grade. The extent of gross striatal pathology, neuronal loss, and gliosis provides a basis for grading the severity of HD pathology (grades Typically, there is a prodromal phase of mild psychotic and behavioral symptoms which precedes frank Huntington chorea by up to 10 years.

The disease is associated with increases in the length of a polyglutamine or CAG triplet repeat present in the Huntingtin gene located on chromosome 4pl6.3. The function of huntingtin is not known. Normally, it is located in the cytoplasm. The 3 association of huntingtin with the cytoplasmic surface of a variety of organelles, I including transport vesicles, synaptic vesicles, microtubules, and mitochondria, raises the Spossibility of the occurrence of normal cellular interactions that might be relevant to neurodegeneration. Although the variation in age at onset of HD is partly explained by the size of the expanded CAG repeat, it is strongly heritable, which suggests that other genes modify the age at onset.

00 M Studies have shown that mutant huntingtin protein from human brain, transgenic Sanimals, and cells is more resistant to proteolysis than normal huntingtin. The Nit terminal cleavage fragments that arise from the processing of normal huntingtin are O 10 sequestered by full-length huntingtin. One model has been proposed in which inhibition of proteolysis of mutant huntingtin leads to aggregation and neurotoxicity through the sequestration of important targets, including normal huntingtin. The presence of neuronal intranuclear inclusions (NIIs) initially led to the view that they are toxic and, hence, pathogenic. More recent data from striatal neuronal cultures transfected with mutant huntingtin and transgenic mice carrying the spinocerebellar ataxia-1 (SCA-1) gene (another CAG repeat disorder) suggest that NIIs may not be necessary or sufficient to cause neuronal cell death, but translocation into the nucleus is sufficient to cause neuronal cell death. Caspase inhibition in clonal striatal cells showed no correlation between the reduction of aggregates in the cells and increased survival.

Cytoplasmic protein extracts from several rat brain regions, including striatum and cortex (sites of neuronal degeneration in HD), contain a 63 kD RNA-binding protein that interacts specifically with CAG repeat sequences. It has been noted that the protein RNA interactions are dependent upon the length of the CAG repeat, and that longer repeats bind substantially more protein. Two CAG binding proteins have been identified in human cortex and striatum, one of 63 kD and another of 49 kD. These data suggest mechanisms by which RNA binding proteins may be involved in the pathological course of trinucleotide-associated neurologic diseases (see for example McLaughlin et al., 1996, Hum. Genet. 59, 561-569.

The Huntington's Disease Collaborative Research Group (1993, Cell, 72, 971-983) found a gene, designated IT15 (important transcript 15) and later called huntingtin, which was isolated using cloned trapped exons and which contains a polymorphic t trinucleotide repeat that is expanded and unstable on HD chromosomes. A (CAG)n repeat longer than the normal range was observed on HD chromosomes from all disease Sfamilies examined. The families came from a variety of ethnic backgrounds and demonstrated a variety of 4p16.3 haplotypes. The (CAG)n repeat appeared to be located within the coding sequence of a predicted protein of about 348 kD that is widely expressed but unrelated to any known gene. Thus, the HD mutation involves an unstable 00 DNA segment similar to those previously observed in several disorders, including the Sfragile X syndrome, Kennedy syndrome, and myotonic dystrophy. The fact that the phenotype of HD is completely dominant suggests that the disorder results from a gainof-function mutation in which either the mRNA product or the protein product of the disease allele has some new property or is expressed inappropriately (see for example, Myers et al., 1989, Am. J. Hum. Genet., 34, 481-488).

The use of small interfering nucleic acid molecules targeting HD, for example mutant alleles associated with Huntington disease, or alternately bot mutant and wild type HD alleles, provides a class of novel therapeutic agents that can be used in the the treatment of Huntington Disease and any other disease or condition that responds to modulation of HD genes.

Examples: The following are non-limiting examples showing the selection, isolation, synthesis and activity of nucleic acids of the instant invention.

Example 1: Tandem synthesis of siNA constructs Exemplary siNA molecules of the invention are synthesized in tandem using a cleavable linker, for example, a succinyl-based linker. Tandem synthesis as described herein is followed by a one-step purification process that provides RNAi molecules in high yield. This approach is highly amenable to siNA synthesis in support of high throughput RNAi screening, and can be readily adapted to multi-column or multi-well synthesis platforms.

After completing a tandem synthesis of a siNA oligo and its complement in which the 5'-terminal dimethoxytrityl (5'-O-DMT) group remains intact (trityl on synthesis), the O oligonucleotides are deprotected as described above. Following deprotection, the siNA I sequence strands are allowed to spontaneously hybridize. This hybridization yields a duplex in which one strand has retained the 5'-O-DMT group while the complementary strand comprises a terminal 5'-hydroxyl. The newly formed duplex behaves as a single molecule during routine solid-phase extraction purification (Trityl-On purification) even though only one molecule has a dimethoxytrityl group. Because the strands form a 00 stable duplex, this dimethoxytrityl group (or an equivalent group, such as other trityl Sgroups or other hydrophobic moieties) is all that is required to purify the pair of oligos, for example, by using a C 18 cartridge.

Standard phosphoramidite synthesis chemistry is used up to the point of introducing a tandem linker, such as an inverted deoxy abasic succinate or glyceryl succinate linker (see Figure 1) or an equivalent cleavable linker. A non-limiting example of linker coupling conditions that can be used includes a hindered base such as diisopropylethylamine (DIPA) and/or DMAP in the presence of an activator reagent such as Bromotripyrrolidinophosphoniumhexaflurorophosphate (PyBrOP). After the linker is coupled, standard synthesis chemistry is utilized to complete synthesis of the second sequence leaving the terminal the 5'-O-DMT intact. Following synthesis, the resulting oligonucleotide is deprotected according to the procedures described herein and quenched with a suitable buffer, for example with 50mM NaOAc or 1.5M NH 4

H

2 CO3.

Purification of the siNA duplex can be readily accomplished using solid phase extraction, for example, using a Waters C18 SepPak 1g cartridge conditioned with 1 column volume (CV) of acetonitrile, 2 CV H20, and 2 CV 50mM NaOAc. The sample is loaded and then washed with 1 CV H20 or 50mM NaOAc. Failure sequences are eluted with 1 CV 14% ACN (Aqueous with 50mM NaOAc and 50mM NaCI). The column is then washed, for example with 1 CV H20 followed by on-column detritylation, for example by passing 1 CV of 1% aqueous trifluoroacetic acid (TFA) over the column, then adding a second CV of 1% aqueous TFA to the column and allowing to stand for approximately 10 minutes. The remaining TFA solution is removed and the column washed with H20 followed by 1 CV 1M NaCl and additional H20. The siNA duplex product is then eluted, for example, using 1 CV 20% aqueous

CAN.

3 Figure 2 provides an example of MALDI-TOF mass spectrometry analysis of a Npurified siNA construct in which each peak corresponds to the calculated mass of an 2 individual siNA strand of the siNA duplex. The same purified siNA provides three peaks when analyzed by capillary gel electrophoresis (CGE), one peak presumably corresponding to the duplex siNA, and two peaks presumably corresponding to the separate siNA sequence strands. Ion exchange HPLC analysis of the same siNA contract 00 only shows a single peak. Testing of the purified siNA construct using a luciferase reporter assay described below demonstrated the same RNAi activity compared to siNA constructs generated from separately synthesized oligonucleotide sequence strands.

Example 2: Identification of potential siNA target sites in any RNA sequence The sequence of an RNA target of interest, such as a viral or human mRNA transcript, is screened for target sites, for example by using a computer folding algorithm. In a non-limiting example, the sequence of a gene or RNA gene transcript derived from a database, such as Genbank, is used to generate siNA targets having complementarity to the target. Such sequences can be obtained from a database, or can be determined experimentally as known in the art. Target sites that are known, for example, those target sites determined to be effective target sites based on studies with other nucleic acid molecules, for example ribozymes or antisense, or those targets known to be associated with a disease, trait, or condition such as those sites containing mutations or deletions, can be used to design siNA molecules targeting those sites.

Various parameters can be used to determine which sites are the most suitable target sites within the target RNA sequence. These parameters include but are not limited to secondary or tertiary RNA structure, the nucleotide base composition of the target sequence, the degree of homology between various regions of the target sequence, or the relative position of the target sequence within the RNA transcript. Based on these determinations, any number of target sites within the RNA transcript can be chosen to screen siNA molecules for efficacy, for example by using in vitro RNA cleavage assays, cell culture, or animal models. In a non-limiting example, anywhere from 1 to 1000 target sites are chosen within the transcript based on the size of the siNA construct to be used. High throughput screening assays can be developed for screening siNA molecules using methods known in the art, such as with multi-well or multi-plate assays to determine efficient reduction in target gene expression.

186 Example 3: Selection of siNA molecule target sites in a RNA The following non-limiting steps can be used to carry out the selection of siNAs a targeting a given gene sequence or transcript.

O 1. The target sequence is parsed in silico into a list of all fragments or subsequences of a particular length, for example 23 nucleotide fragments, contained within the target 0sequence. This step is typically carried out using a custom Perl script, but commercial Ssequence analysis programs such as Oligo, MacVector, or the GCG Wisconsin CPackage can be employed as well.

2. In some instances the siNAs correspond to more than one target sequence; such would be the case for example in targeting different transcripts of the same gene, targeting different transcripts of more than one gene, or for targeting both the human gene and an animal homolog. In this case, a subsequence list of a particular length is generated for each of the targets, and then the lists are compared to find matching sequences in each list. The subsequences are then ranked according to the number of target sequences that contain the given subsequence; the goal is to find subsequences that are present in most or all of the target sequences. Alternately, the ranking can identify subsequences that are unique to a target sequence, such as a mutant target sequence. Such an approach would enable the use of siNA to target specifically the mutant sequence and not effect the expression of the normal sequence.

3. In some instances the siNA subsequences are absent in one or more sequences while present in the desired target sequence; such would be the case if the siNA targets a gene with a paralogous family member that is to remain untargeted. As in case 2 above, a subsequence list of a particular length is generated for each of the targets, and then the lists are compared to find sequences that are present in the target gene but are absent in the untargeted paralog.

4. The ranked siNA subsequences can be further analyzed and ranked according to GC content. A preference can be given to sites containing 30-70% GC, with a further preference to sites containing 40-60% GC.

t 5. The ranked siNA subsequences can be further analyzed and ranked according to selffolding and internal hairpins. Weaker internal folds are preferred; strong hairpin k structures are to be avoided.

6. The ranked siNA subsequences can be further analyzed and ranked according to whether they have runs of GGG or CCC in the sequence. GGG (or even more Gs) in 0 either strand can make oligonucleotide synthesis problematic and can potentially M interfere with RNAi activity, so it is avoided whenever better sequences are available.

SCCC is searched in the target strand because that will place GGG in the antisense n strand.

C, 10 7. The ranked siNA subsequences can be further analyzed and ranked according to whether they have the dinucleotide UU (uridine dinucleotide) on the 3'-end of the sequence, and/or AA on the 5'-end of the sequence (to yield 3' UU on the antisense sequence). These sequences allow one to design siNA molecules with terminal TT thymidine dinucleotides.

8. Four or five target sites are chosen from the ranked list of subsequences as described above. For example, in subsequences having 23 nucleotides, the right 21 nucleotides of each chosen 23-mer subsequence are then designed and synthesized for the upper (sense) strand of the siNA duplex, while the reverse complement of the left 21 nucleotides of each chosen 23-mer subsequence are then designed and synthesized for the lower (antisense) strand of the siNA duplex (see Tables II and III). If terminal TT residues are desired for the sequence (as described in paragraph then the two 3' terminal nucleotides of both the sense and antisense strands are replaced by TT prior to synthesizing the oligos.

9. The siNA molecules are screened in an in vitro, cell culture or animal model system to identify the most active siNA molecule or the most preferred target site within the target RNA sequence.

Other design considerations can be used when selecting target nucleic acid sequences, see, for example, Reynolds et al., 2004, Nature Biotechnology Advanced Online Publication, 1 February 2004, doi:10.1038/nbt936 and Ui-Tei et al., 2004, Nucleic Acids Research, 32, doi: 10.1093/nar/gkh247.

188 In an alternate approach, a pool of siNA constructs specific to a repeat expansion target sequence is used to screen for target sites in cells expressing repeat expansion S(RE) RNA, such as cultured Jurkat, HeLa, A549, 293T such as COS-1 cells (see for example Sittler et al., 2001, Human Molecular Genetics, 10, 1307-1315). The general strategy used in this approach is shown in Figure 9. A non-limiting example of such is a pool comprising sequences having any of SEQ ID NOS 1-3575. Cells expressing repeat 00 expansion (RE) are transfected with the pool of siNA constructs and cells that Sdemonstrate a phenotype associated with repeat expansion (RE) inhibition are sorted.

The pool of siNA constructs can be expressed from transcription cassettes inserted into 0 10 appropriate vectors (see for example Figure 7 and Figure The siNA from cells demonstrating a positive phenotypic change decreased proliferation, decreased repeat expansion (RE) mRNA levels or decreased repeat expansion (RE) protein expression), are sequenced to determine the most suitable target site(s) within the target repeat expansion (RE) RNA sequence.

Example 4: Repeat expansion (RE) targeted siNA design siNA target sites were chosen by analyzing sequences of the repeat expansion (RE) RNA target and optionally prioritizing the target sites on the basis of folding (structure of any given sequence analyzed to determine siNA accessibility to the target), by using a library of siNA molecules as described in Example 3, or alternately by using an in vitro siNA system as described in Example 6 herein, siNA molecules were designed that could bind each target and are optionally individually analyzed by computer folding to assess whether the siNA molecule can interact with the target sequence. Varying the length of the siNA molecules can be chosen to optimize activity. Generally, a sufficient number of complementary nucleotide bases are chosen to bind to, or otherwise interact with, the target RNA, but the degree of complementarity can be modulated to accommodate siNA duplexes or varying length or base composition. By using such methodologies, siNA molecules can be designed to target sites within any known RNA sequence, for example those RNA sequences corresponding to the any gene transcript.

Chemically modified siNA constructs are designed to provide nuclease stability for systemic administration in vivo and/or improved pharmacokinetic, localization, and delivery properties while preserving the ability to mediate RNAi activity. Chemical 3 modifications as described herein are introduced synthetically using synthetic methods described herein and those generally known in the art. The synthetic siNA constructs are Sthen assayed for nuclease stability in serum and/or cellular/tissue extracts liver extracts). The synthetic siNA constructs are also tested in parallel for RNAi activity using an appropriate assay, such as a luciferase reporter assay as described herein or another suitable assay that can quantity RNAi activity. Synthetic siNA constructs that 0 possess both nuclease stability and RNAi activity can be further modified and re- Sevaluated in stability and activity assays. The chemical modifications of the stabilized active siNA constructs can then be applied to any siNA sequence targeting any chosen RNA and used, for example, in target screening assays to pick lead siNA compounds for Cl therapeutic development (see for example Figure 11).

Example 5: Chemical Synthesis and Purification of siNA siNA molecules can be designed to interact with various sites in the RNA message, for example, target sequences within the RNA sequences described herein. The sequence of one strand of the siNA molecule(s) is complementary to the target site sequences described above. The siNA molecules can be chemically synthesized using methods described herein. Inactive siNA molecules that are used as control sequences can be synthesized by scrambling the sequence of the siNA molecules such that it is not complementary to the target sequence. Generally, siNA constructs can by synthesized using solid phase oligonucleotide synthesis methods as described herein (see for example Usman et al., US Patent Nos. 5,804,683; 5,831,071; 5,998,203; 6,117,657; 6,353,098; 6,362,323; 6,437,117; 6,469,158; Scaringe et al., US Patent Nos. 6,111,086; 6,008,400; 6,111,086 all incorporated by reference herein in their entirety).

In a non-limiting example, RNA oligonucleotides are synthesized in a stepwise fashion using the phosphoramidite chemistry as is known in the art. Standard phosphoramidite chemistry involves the use of nucleosides comprising any of dimethoxytrityl, 2'-O-tert-butyldimethylsilyl, 3'-O-2-Cyanoethyl N,N-diisopropylphosphoroamidite groups, and exocyclic amine protecting groups N6-benzoyl adenosine, N4 acetyl cytidine, and N2-isobutyryl guanosine). Alternately, 2'-O-Silyl Ethers can be used in conjunction with acid-labile 2'-O-orthoester protecting groups in the synthesis of RNA as described by Scaringe supra. Differing 2' chemistries can require different protecting groups, for example 2'-deoxy-2'-amino nucleosides can utilize N-phthaloyl i protection as described by Usman et al., US Patent 5,631,360, incorporated by reference Sherein in its entirety).

During solid phase synthesis, each nucleotide is added sequentially to direction) to the solid support-bound oligonucleotide. The first nucleoside at the 3'-end of the chain is covalently attached to a solid support controlled pore glass or 00 M polystyrene) using various linkers. The nucleotide precursor, a ribonucleoside Sphosphoramidite, and activator are combined resulting in the coupling of the second nucleoside phosphoramidite onto the 5'-end of the first nucleoside. The support is then washed and any unreacted 5'-hydroxyl groups are capped with a capping reagent such as acetic anhydride to yield inactive 5'-acetyl moieties. The trivalent phosphorus linkage is then oxidized to a more stable phosphate linkage. At the end of the nucleotide addition cycle, the 5'-O-protecting group is cleaved under suitable conditions acidic conditions for trityl-based groups and Fluoride for silyl-based groups). The cycle is repeated for each subsequent nucleotide.

Modification of synthesis conditions can be used to optimize coupling efficiency, for example by using differing coupling times, differing reagent/phosphoramidite concentrations, differing contact times, differing solid supports and solid support linker chemistries depending on the particular chemical composition of the siNA to be synthesized. Deprotection and purification of the siNA can be performed as is generally described in Usman et al., US 5,831,071, US 6,353,098, US 6,437,117, and Bellon et al., US 6,054,576, US 6,162,909, US 6,303,773, or Scaringe supra, incorporated by reference herein in their entireties. Additionally, deprotection conditions can be modified to provide the best possible yield and purity of siNA constructs. For example, applicant has observed that oligonucleotides comprising 2'-deoxy-2'-fluoro nucleotides can degrade under inappropriate deprotection conditions. Such oligonucleotides are deprotected using aqueous methylamine at about 35 0 C for 30 minutes. If the 2'-deoxy- 2'-fluoro containing oligonucleotide also comprises ribonucleotides, after deprotection with aqueous methylamine at about 35 0 C for 30 minutes, TEA-HF is added and the reaction maintained at about 65°C for an additional 15 minutes.

Example 6: RNAi in vitro assay to assess siNA activity An in vitro assay that recapitulates RNAi in a cell-free system is used to evaluate siNA constructs targeting repeat expansion (RE) RNA targets. The assay comprises the system described by Tuschl et al., 1999, Genes and Development, 13, 3191-3197 and Zamore et al., 2000, Cell, 101, 25-33 adapted for use with repeat expansion (RE) target RNA. A Drosophila extract derived from syncytial blastoderm is used to reconstitute RNAi activity in vitro. Target RNA is generated via in vitro transcription from an appropriate repeat expansion (RE) expressing plasmid using T7 RNA polymerase or via chemical synthesis as described herein. Sense and antisense siNA strands (for example 20 uM each) are annealed by incubation in buffer (such as 100 mM potassium acetate, mM HEPES-KOH, pH 7.4, 2 mM magnesium acetate) for 1 minute at 90 0 C followed by 1 hour at 37°C then diluted in lysis buffer (for example 100 mM potassium acetate, mM HEPES-KOH at pH 7.4, 2mM magnesium acetate). Annealing can be monitored by gel electrophoresis on an agarose gel in TBE buffer and stained with ethidium bromide.

The Drosophila lysate is prepared using zero to two-hour-old embryos from Oregon R flies collected on yeasted molasses agar that are dechorionated and lysed. The lysate is centrifuged and the supernatant isolated. The assay comprises a reaction mixture containing 50% lysate [vol/vol], RNA (10-50 pM final concentration), and 10% [vol/vol] lysis buffer containing siNA (10 nM final concentration). The reaction mixture also contains 10 mM creatine phosphate, 10 ug/ml creatine phosphokinase, 100 um GTP, 100 uM UTP, 100 uM CTP, 500 uM ATP, 5 mM DTT, 0.1 U/uL RNasin (Promega), and 100 uM of each amino acid. The final concentration of potassium acetate is adjusted to 100 mM. The reactions are pre-assembled on ice and preincubated at 250 C for 10 minutes before adding RNA, then incubated at 250 C for an additional 60 minutes. Reactions are quenched with 4 volumes of 1.25 x Passive Lysis Buffer (Promega). Target RNA cleavage is assayed by RT-PCR analysis or other methods known in the art and are compared to control reactions in which siNA is omitted from the reaction.

Alternately, internally-labeled target RNA for the assay is prepared by in vitro transcription in the presence of [alpha- 32 p] CTP, passed over a G50 Sephadex column by spin chromatography and used as target RNA without further purification. Optionally, target RNA is 5'-32P-end labeled using T4 polynucleotide kinase enzyme. Assays are performed as described above and target RNA and the specific RNA cleavage products 192 t generated by RNAi are visualized on an autoradiograph of a gel. The percentage of ,I cleavage is determined by PHOSPHOR IMAGER® (autoradiography) quantitation of bands representing intact control RNA or RNA from control reactions without siNA and the cleavage products generated by the assay.

In one embodiment, this assay is used to determine target sites in the repeat 0 expansion (RE) RNA target for siNA mediated RNAi cleavage, wherein a plurality of 00 C siNA constructs are screened for RNAi mediated cleavage of the repeat expansion (RE) RNA target, for example, by analyzing the assay reaction by electrophoresis of labeled target RNA, or by northern blotting, as well as by other methodology well known in the 10 art.

Example 7: Nucleic acid inhibition of repeat expansion (RE) target RNA in vivo siNA molecules targeted to the huma repeat expansion (RE) RNA are designed and synthesized as described above. These nucleic acid molecules can be tested for cleavage activity in vivo, for example, using the following procedure. The target sequences and the nucleotide location within the repeat expansion (RE) RNA are given in Table II and

III.

Two formats are used to test the efficacy of siNAs targeting repeat expansion (RE).

First, the reagents are tested in cell culture using, for example, Jurkat, HeLa, A549, COS-1 or 293T cells, to determine the extent of RNA and protein inhibition. siNA reagents see Tables II and III) are selected against the repeat expansion (RE) target as described herein. RNA inhibition is measured after delivery of these reagents by a suitable transfection agent to, for example, Jurkat, HeLa, A549 or 293T cells.

Relative amounts of target RNA are measured versus actin using real-time PCR monitoring of amplification ABI 7700 TAQMAN®). A comparison is made to a mixture of oligonucleotide sequences made to unrelated targets or to a randomized siNA control with the same overall length and chemistry, but randomly substituted at each position. Primary and secondary lead reagents are chosen for the target and optimization performed. After an optimal transfection agent concentration is chosen, a RNA timecourse of inhibition is performed with the lead siNA molecule. In addition, a cell-plating format can be used to determine RNA inhibition.

Delivery of siNA to Cells SCells Jurkat, HeLa, A549 or 293T cells) are seeded, for example, at 1x10 8 cells per well of a six-well dish in EGM-2 (BioWhittaker) the day before transfection.

siNA (final concentration, for example 20nM) and cationic lipid final concentration 2tg/ml) are complexed in EGM basal media (Biowhittaker) at 37 0 C for 30 minutes in 00 polystyrene tubes. Following vortexing, the complexed siNA is added to each well and c incubated for the times indicated. For initial optimization experiments, cells are seeded, c for example, at lx103 in 96 well plates and siNA complex added as described. Efficiency Sof delivery of siNA to cells is determined using a fluorescent siNA complexed with lipid.

Cells in 6-well dishes are incubated with siNA for 24 hours, rinsed with PBS and fixed in 2% paraformaldehyde for 15 minutes at room temperature. Uptake of siNA is visualized using a fluorescent microscope.

TAOMAN® (real-time PCR monitoring of amplification) and Lightcycler quantification of mRNA Total RNA is prepared from cells following siNA delivery, for example, using Qiagen RNA purification kits for 6-well or Rneasy extraction kits for 96-well assays. For TAQMAN® analysis (real-time PCR monitoring of amplification), dual-labeled probes are synthesized with the reporter dye, FAM or JOE, covalently linked at the 5'-end and the quencher dye TAMRA conjugated to the 3'-end. One-step RT-PCR amplifications are performed on, for example, an ABI PRISM 7700 Sequence Detector using 50 pl reactions consisting of 10 tl total RNA, 100 nM forward primer, 900 nM reverse primer, 100 nM probe, IX TaqMan PCR reaction buffer (PE-Applied Biosystems), 5.5 mM MgCl2, 300 pM each dATP, dCTP, dGTP, and dTTP, 10U RNase Inhibitor (Promega), 1.25U AMPLITAQ GOLD® (DNA polymerase) (PE-Applied Biosystems) and 10U M- MLV Reverse Transcriptase (Promega). The thermal cycling conditions can consist of minutes at 48 0 C, 10 minutes at 95 0 C, followed by 40 cycles of 15 seconds at 95 0

C

and 1 minute at 60 0 C. Quantitation of mRNA levels is determined relative to standards generated from serially diluted total cellular RNA (300, 100, 33, 11 ng/reaction) and normalizing to B-actin or GAPDH mRNA in parallel TAQMAN® reactions (real-time PCR monitoring of amplification). For each gene of interest an upper and lower primer and a fluorescently labeled probe are designed. Real time incorporation of SYBR Green 194 I dye into a specific PCR product can be measured in glass capillary tubes using a Slightcyler. A standard curve is generated for each primer pair using control cRNA.

SValues are represented as relative expression to GAPDH in each sample.

Western blotting Nuclear extracts can be prepared using a standard micro preparation technique (see 00 for example Andrews and Faller, 1991, Nucleic Acids Research, 19, 2499). Protein Sextracts from supernatants are prepared, for example using TCA precipitation. An equal NI volume of 20% TCA is added to the cell supernatant, incubated on ice for 1 hour and Spelleted by centrifugation for 5 minutes. Pellets are washed in acetone, dried and 10 resuspended in water. Cellular protein extracts are run on a 10% Bis-Tris NuPage (nuclear extracts) or 4-12% Tris-Glycine (supernatant extracts) polyacrylamide gel and transferred onto nitro-cellulose membranes. Non-specific binding can be blocked by incubation, for example, with 5% non-fat milk for 1 hour followed by primary antibody for 16 hour at 4 0 C. Following washes, the secondary antibody is applied, for example (1:10,000 dilution) for 1 hour at room temperature and the signal detected with SuperSignal reagent (Pierce).

Example 8: Animal Models useful to evaluate the down-regulation of HD gene expression Evaluating the efficacy of anti-HD agents in animal models is an important prerequisite to human clinical trials. Although the HD mRNA and protein product (huntingtin) show widespread distribution, the progressive neurodegeneration is selective in location, with regional neuron loss and gliosis in striatum, cerebral cortex, thalamus, subthalamus, and hippocampus. An experimental transgenic mouse model has utilized widespread expression of full-length human HD cDNA in mice with either 16, 48, or 89 CAG repeats. Only mice with 48 or 89 CAG repeats manifested progressive behavioral and motor dysfunction with neuron loss and gliosis in striatum, cerebral cortex, thalamus, and hippocampus (Reddy et al., 1998, Nature Genet. 20, 198-202). These animals represent a clinically relevant model for HD pathogenesis and can provide insight into the underlying pathophysiologic mechanisms of other triplet repeat disorders.

Other neurodegenerative animal models as are known in the art can similarly be utilized to evaluate siNA molecules of the invention, for example models that utilize systemic or 195 localized delivery direct injection, intrathecal delivery, osmotic pump etc.) of therapeutic compounds to the CNS, (see for example Ryu et al., 2003, Exp Neurol., 183, 700-4). As such, this model provides an animal model for testing therapeutic drugs, including siNA constructs of the instant invention.

Example 9: RNAi mediated inhibition of repeat expansion (RE) expression In vitro siNA mediated inhibition of repeat expansion (RE) RNA siNA constructs (Table III) are tested for efficacy in reducing repeat expansion (RE) RNA expression in, for example, COS-1 or Hela cells. Cells are plated approximately 24 hours before transfection in 96-well plates at 5,000-7,500 cells/well, 100 pl/well, such that at the time of transfection cells are 70-90% confluent. For transfection, annealed siNAs are mixed with the transfection reagent (Lipofectamine 2000, Invitrogen) in a volume of 50 tl/well and incubated for 20 minutes at room temperature. The siNA transfection mixtures are added to cells to give a final siNA concentration of 25 nM in a volume of 150 tl. Each siNA transfection mixture is added to 3 wells for triplicate siNA treatments. Cells are incubated at 370 for 24 hours in the continued presence of the siNA transfection mixture. At 24 hours, RNA is prepared from each well of treated cells. The supernatants with the transfection mixtures are first removed and discarded, then the cells are lysed and RNA prepared from each well.

Target gene expression following treatment is evaluated by RT-PCR for the target gene and for a control gene (36B4, an RNA polymerase subunit) for normalization. The triplicate data is averaged and the standard deviations determined for each treatment.

Normalized data are graphed and the percent reduction of target mRNA by active siNAs in comparison to their respective inverted control siNAs is determined.

In a non-limiting example, siNA molecules targeting human huntingtin (HD) were evaluated in cell culture using the transgenic allele (HD82Q) used to make the HD model N171-82Q. A myc tag to the HD protein was utilized for western blot analysis. HEK- 293 cells were transfected with HD82Q-myc construct alone or with active siNA constructs 1, 2, and 3 (Sirna Compound Nos. 31993/31994, 31995/31996, 31997/31998 respectively, Table III) or matched chemistry inverted control constructs 4, 5, and 6 (Sirna Compound Nos. 31999/32000, 32001/32002, 32003/32004 respectively, Table III) at two concentrations (0.5 ng and 5 ng) using lipofectamine 2000. Cells were harvested 196 48 hours later and protein extracts run on SDS-PAGE, blotted to nitrocellulose, and probed with anti-myc antibodies. Neomycin phosphotransferase is expressed on the same plasmid as the myc-tagged construct, allowing for a transfection control. The experiment was run in duplicate. As shown in Figure 30, the active siNA constructs (Sirna Compound Nos. 31993/31994, 31995/31996, 31997/31998) all demonstrate inhibition of HD82Q-myc compared with the inverted matched chemistry siNA 00 constructs. Furthermore, the active siNA constructs show selectivity for inhibiting the Smyc tagged HD82Q compared to c-myc and the necomycin transfection control.

N Additional experiments are utilized to evaluate silencing of the full-length HD construct by western blot and QPCR. This rapid in vitro screen is useful for identifying effective C, siNA constructs prior to in vivo studies, utilizing for example N171-82Q mice.

Example 10: Indications The present body of knowledge in HD research indicates the need for methods to assay HD activity and for compounds that can regulate HD expression for research, diagnostic, and therapeutic use. As described herein, the nucleic acid molecules of the present invention can be used in assays to diagnose disease state related of HD levels. In addition, the nucleic acid molecules can be used to treat disease state related to HD levels.

Particular conditions and disease states that can be associated with HD expression modulation include, but are not limited to Huntinton disease and related conditions such as progressive chorea, rigidity, dementia, and seizures, spinocerebellar ataxia, spinal and bulbar muscular dystrophy (SBMA), dentatorubropallidoluysian atrophy (DRPLA), and any other diseases or conditions that are related to or will respond to the levels of a repeat expansion (RE) protein in a cell or tissue, alone or in combination with other therapies.

The use of caspase inhibitors, agents that disrupt RE protein aggregation, and neuroprotective agents pryridoxine) are non-limiting examples of chemotherapeutic agents that can be combined with or used in conjunction with the nucleic acid molecules siNA molecules) of the instant invention. Those skilled in the art will recognize that other anti-cancer compounds and therapies can similarly be readily combined with t the nucleic acid molecules of the instant invention siNA molecules) and are hence within the scope of the instant invention.

Example 11: Multifunctional siNA Inhibition of repeat expansion (RE) RNA expression SMultifunctional siNA design 0 Once target sites have been identified for multifunctional siNA constructs, each 00 strand of the siNA is designed with a complementary region of length, for example, of (about 18 to about 28 nucleotides, that is complementary to a different target nucleic acid Ssequence. Each complementary region is designed with an adjacent flanking region of Sabout 4 to about 22 nucleotides that is not complementary to the target sequence, but which comprises complementarity to the complementary region of the other sequence (see for example Figure 16). Hairpin constructs can likewise be designed (see for example Figure 17). Identification of complementary, palindrome or repeat sequences that are shared between the different target nucleic acid sequences can be used to shorten the overall length of the multifunctional siNA constructs (see for example Figures 18 and 19).

In a non-limiting example, three additional categories of additional multifunctional siNA designs are presented that allow a single siNA molecule to silence multiple targets.

The first method utilizes linkers to join siNAs (or multiunctional siNAs) in a direct manner. This can allow the most potent siNAs to be joined without creating a long, continuous stretch of RNA that has potential to trigger an interferon response. The second method is a dendrimeric extension of the overlapping or the linked multifunctional design; or alternatively the organization of siNA in a supramolecular format. The third method uses helix lengths greater than 30 base pairs. Processing of these siNAs by Dicer will reveal new, active 5' antisense ends. Therefore, the long siNAs can target the sites defined by the original 5' ends and those defined by the new ends that are created by Dicer processing. When used in combination with traditional multifunctional siNAs (where the sense and antisense strands each define a target) the approach can be used for example to target 4 or more sites.

I. Tethered Bifunctional siNAs t The basic idea is a novel approach to the design of multifunctional siNAs in which I two antisense siNA strands are annealed to a single sense strand. The sense strand 2 oligonucleotide contains a linker non-nulcoetide linker as described herein) and two segments that anneal to the antisense siNA strands (see Figure 22). The linkers can also optionally comprise nucleotide-based linkers. Several potential advantages and variations to this approach include, but are not limited to: 00 M 1. The two antisense siNAs are independent. Therefore, the choice of target sites is Snot constrained by a requirement for sequence conservation between two sites.

Any two highly active siNAs can be combined to form a multifunctional siNA.

2. When used in combination with target sites having homology, siNAs that target a sequence present in two genes different repeat expansion (RE) isoforms), the design can be used to target more than two sites. A single multifunctional siNA can be for example, used to target RNA of two different repeat expansion (RE) RNAs.

3. Multifunctional siNAs that use both the sense and antisense strands to target a gene can also be incorporated into a tethered multifuctional design. This leaves open the possibility of targeting 6 or more sites with a single complex.

4. It can be possible to anneal more than two antisense strand siNAs to a single tethered sense strand.

5. The design avoids long continuous stretches of dsRNA. Therefore, it is less likely to initiate an interferon response.

6. The linker (or modifications attached to it, such as conjugates described herein) can improve the pharmacokinetic properties of the complex or improve its incorporation into liposomes. Modifications introduced to the linker should not impact siNA activity to the same extent that they would if directly attached to the siNA (see for example Figures 27 and 28).

7. The sense strand can extend beyond the annealed antisense strands to provide additional sites for the attachment of conjugates.

3 8. The polarity of the complex can be switched such that both of the antisense 3' ends are adjacent to the linker and the 5' ends are distal to the linker or combination thereof.

Dendrimer and supramolecular siNAs a In the dendrimer siNA approach, the synthesis of siNA is initiated by first 00 3 synthesizing the dendrimer template followed by attaching various functional siNAs.

Various constructs are depicted in Figure 23. The number of functional siNAs that can 3 be attached is only limited by the dimensions of the dendrimer used.

Supramolecular approach to multifunctional siNA The supramolecular format simplifies the challenges of dendrimer synthesis. In this format, the siNA strands are synthesized by standard RNA chemistry, followed by annealing of various complementary strands. The individual strand synthesis contains an antisense sense sequence of one siNA at the 5'-end followed by a nucleic acid or synthetic linker, such as hexaethyleneglyol, which in turn is followed by sense strand of another siNA in 5' to 3' direction. Thus, the synthesis of siNA strands can be carried out in a standard 3' to 5' direction. Representative examples of trifunctional and tetrafunctional siNAs are depicted in Figure 24. Based on a similar principle, higher functionality siNA constucts can be designed as long as efficient annealing of various strands is achieved.

Dicer enabled multifunctional siNA Using bioinformatic analysis of multiple targets, stretches of identical sequences shared between differeing target sequences can be identified ranging from about two to about fourteen nucleotides in length. These identical regions can be designed into extended siNA helixes >30 base pairs) such that the processing by Dicer reveals a secondary functional 5'-antisense site (see for example Figure 25). For example, when the first 17 nucleotides of a siNA antisense strand 21 nucleotide strands in a duplex with 3'-TT overhangs) are complementary to a target RNA, robust silencing was observed at 25 nM. 80% silencing was observed with only 16 nucleotide complementarity in the same format.

a Incorporation of this property into the designs of siNAs of about 30 to 40 or more base pairs results in additional multifunctional siNA constructs. The example in Figure 25 illustrates how a 30 base-pair duplex can target three distinct sequences after processing by Dicer-RNaseIIl; these sequences can be on the same mRNA or separate 00 RNAs, such as viral and host factor messages, or multiple points along a given pathway inflammatory cascades). Furthermore, a 40 base-pair duplex can combine a t bifunctional design in tandem, to provide a single duplex targeting four target sequences.

An even more extensive approach can include use of homologous sequences to enable five or six targets silenced for one multifunctional duplex. The example in Figure demonstrates how this can be achieved. A 30 base pair duplex is cleaved by Dicer into 22 and 8 base pair products from either end (8 b.p. fragments not shown). For ease of presentation the overhangs generated by dicer are not shown but can be compensated for. Three targeting sequences are shown. The required sequence identity overlapped is indicated by grey boxes. The N's of the parent 30 b.p. siNA are suggested sites of 2'- OH positions to enable Dicer cleavage if this is tested in stabilized chemistries. Note that processing of a 30mer duplex by Dicer RNase III does not give a precise 22+8 cleavage, but rather produces a series of closely related products (with 22+8 being the primary site). Therefore, processing by Dicer will yield a series of active siNAs.

Another non-limiting example is shown in Figure 26. A 40 base pair duplex is cleaved by Dicer into 20 base pair products from either end. For ease of presentation the overhangs generated by dicer are not shown but can be compensated for. Four targeting sequences are shown in four colors, blue, light-blue and red and orange. The required sequence identity overlapped is indicated by grey boxes. This design format can be extended to larger RNAs. If chemically stabilized siNAs are bound by Dicer, then strategically located ribonucleotide linkages can enable designer cleavage products that permit our more extensive repertoire of multiifunctional designs. For example cleavage products not limited to the Dicer standard of approximately 22-nucleotides can allow multifunctional siNA constructs with a target sequence identity overlap ranging from, for example, about 3 to about 15 nucleotides.

Example 12: Diagnostic uses The siNA molecules of the invention can be used in a variety of diagnostic applications, such as in the identification of molecular targets RNA) in a variety of applications, for example, in clinical, industrial, environmental, agricultural and/or research settings. Such diagnostic use of siNA molecules involves utilizing reconstituted RNAi systems, for example, using cellular lysates or partially purified cellular lysates.

siNA molecules of this invention can be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of endogenous or exogenous, for example viral, RNA in a cell. The close relationship between siNA activity and the structure of the target RNA allows the detection of mutations in any region of the molecule, which alters the base-pairing and three-dimensional structure of the target RNA. By using multiple siNA molecules described in this invention, one can map nucleotide changes, which are important to RNA structure and function in vitro, as well as in cells and tissues. Cleavage of target RNAs with siNA molecules can be used to inhibit gene expression and define the role of specified gene products in the progression of disease or infection. In this manner, other genetic targets can be defined as important mediators of the disease. These experiments will lead to better treatment of the disease progression by affording the possibility of combination therapies multiple siNA molecules targeted to different genes, siNA molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations siNA molecules and/or other chemical or biological molecules). Other in vitro uses of siNA molecules of this invention are well known in the art, and include detection of the presence of mRNAs associated with a disease, infection, or related condition. Such RNA is detected by determining the presence of a cleavage product after treatment with a siNA using standard methodologies, for example, fluorescence resonance emission transfer (FRET).

In a specific example, siNA molecules that cleave only wild-type or mutant forms of the target RNA are used for the assay. The first siNA molecules those that cleave only wild-type forms of target RNA) are used to identify wild-type RNA present in the sample and the second siNA molecules those that cleave only mutant forms of target RNA) are used to identify mutant RNA in the sample. As reaction controls, synthetic substrates of both wild-type and mutant RNA are cleaved by both siNA 202 molecules to demonstrate the relative siNA efficiencies in the reactions and the absence of cleavage of the "non-targeted" RNA species. The cleavage products from the synthetic substrates also serve to generate size markers for the analysis of wild-type and mutant RNAs in the sample population. Thus, each analysis requires two siNA molecules, two substrates and one unknown sample, which is combined into six reactions. The presence of cleavage products is determined using an RNase protection 00 assay so that full-length and cleavage fragments of each RNA can be analyzed in one lane of a polyacrylamide gel. It is not absolutely required to quantify the results to gain ,I insight into the expression of mutant RNAs and putative risk of the desired phenotypic changes in target cells. The expression of mRNA whose protein product is implicated in the development of the phenotype disease related or infection related) is adequate to establish risk. If probes of comparable specific activity are used for both transcripts, then a qualitative comparison of RNA levels is adequate and decreases the cost of the initial diagnosis. Higher mutant form to wild-type ratios are correlated with higher risk whether RNA levels are compared qualitatively or quantitatively.

All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually.

One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The methods and compositions described herein as presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art, which are encompassed within the spirit of the invention, are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varying substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. Thus, such additional embodiments are within the scope of the present invention and the following claims. The present invention teaches one skilled in the art to test various combinations and/or substitutions of chemical 203 t modifications described herein toward generating nucleic acid constructs with improved (Ni activity for mediating RNAi activity. Such improved activity can comprise improved Sstability, improved bioavailability, and/or improved activation of cellular responses mediating RNAi. Therefore, the specific embodiments described herein are not limiting and one skilled in the art can readily appreciate that specific combinations of the modifications described herein can be tested without undue experimentation toward 0 identifying siNA molecules with improved RNAi activity.

00 The invention illustratively described herein suitably can be practiced in the t absence of any element or elements, limitation or limitations that are not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising", "consisting essentially of', and "consisting of' may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the description and the appended claims.

In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.

204 Table I: POLYQ repeat Accession Numbers S 5 NM_002111 Homo sapiens huntingtin (Huntington disease) mRNA CD gi1387884041refjNM_002111.4 [387884041 00 10 AB01679 4

C

c Homo sapiens mRNA for huntingtin, complete cds gil4126798ldbjlAB016794.1|[4126798] L12392 15 omo sapiens Huntington's Disease (HD) mRNA, complete cds gi1l70999l1gbIL1239 2 .11HUMHDA[170 999 1] AC0055 16 Homo sapiens Chromosome 4p16.

3 BAC clone 399e10 containing Huntington's Disease gene; exons 1-67, complete sequence gij3900835|gbjAC005516.1AC00551 6 [3900835] AL39005 9 Human DNA sequence from clone RP11-399E10 on chromosome 4, complete sequence gi|26984367|embAL390059.9 [26984367] Z69837 Human DNA sequence from clone LA04NC01-113B 6 on chromosome 4, complete sequence gi|12129491emblZ69837.1|HSL113B6[1212949] L20431 Homo sapiens Huntington disease-associated protein

(HD)

mRNA, complete cds gi|3980 28 gbLL20431.1HUMHUNTDIS[398028] NM_000332 Homo sapiens spinocerebellar ataxia 1 (olivopontocerebellar ataxia 1, autosomal dominant, ataxin 1) (SCAl), mRNA gi|4506792jrefiNM_000332.1 [4506792] X7 9204 H.sapiens SCAl mRNA for ataxin gil 5296611 embIX79204 .1 IHSSCA1 [5296611 AL 009031 Human DNA sequence from clone RP3-467D16 on chromosome 6p22.3-24.1 Contains the 5' end of the SCAl gene for spinocerebellar ataxia 1 (olivopontocerebellar ataxia 1, autosomal dominant, ataxin 1) with a polyglutamine (CAG repeat) polymorphism and the 3' part of the GMPR gene for GMP reductase, Guanosine -monophosphate oxidoreductase, complete sequence gil 28084221 embIAL00903l .1 1HS467D16 [28084221 S64648 SCAl {CAG repeat} [human, Genomic Mutant, 506 nt] gil407593 1bbml3l63931bbsII 36468 1gbIS64648 .lIS64648 [407593] BC047894 Homo sapiens spinocerebellar ataxia 1 ataxia 1, autosomal dominant, ataxin mRNA (cDNA clone partial cds gil288390521gbIBC047894.1I [28839052] NM_002973 Homo sapiens spinocerebellar ataxia 2 ataxia 2, autosomal dominant, ataxin 2) (SCA2), mRNA gil45067941refINKL002973.ll [4506794] (olivopontocerebellar IMAGE:4472404), (olivopontocerebellar U70323 Human ataxin-2 (SCA2) mRNA, complete cds gil16796831gbIU70323.11HSU703 23 [1 679 68 3 1 Y08262 H.sapiens mRNA for SCA2 protein gill770389lemblY08262.11HSDANSCA2[177O389] AK095017 206 Homo sapiens cDNA FLJ37698 f is, clone BRH1P2015679, highly similar to Human ataxin-2 (SCA2) mRNA gil2l754l98dbj lAK095017.ll [21754198] BC033711 Homo sapiens Machado-Joseph disease (spinocerebellar ataxia 3, olivopontocerebellar ataxia 3, autosomal dominant, ataxin mRNA (cDNA clone MGC:44934 IMAGE:4393766), complete cds gil2l70805llgbIBC03371l.1I [217080511 U64822 Homo sapiens josephin MJD1 mR'A, partial cds gij 22621981 gbjU64822 .1 HSU64822 [2262198] S75313 MJD1=MJD1 protein (CAG repeats) [human, brain, mRNA, 1776 nt] gil 833927 Ibbml 3603251bbsl160590 gbjS75313 .11S75313 [833927] NM_004993 Homo sapiens Machado-Joseph disease (spinocerebellar ataxia 3, olivopontocerebellar ataxia 3, autosomal dominant, ataxin 3) (MJD), transcript variant 1, mR\A gil135180l81refINYI904993.2I [13518018] U64821 Homo sapiens josephin MJD1 mRNA, cds gil 22621961 gbIU64821 .1 1HSU64821 [2262196] U64820 Homo sapiens josephin MJD1 mRNA, complete cds gil 22621941 gbIU64820 .1 1HSU64820 [2262194] AB 050194 Homo sapiens mRNSA for ataxin-3, complete cds gilll559485ldbjIAB050194.lI [11559485] NM_030660 0 Homo sapiens Machado-Joseph disease (spinocerebellar ataxia CM 3, Solivopontocerebellar ataxia 3, autosomal dominant, ataxin 3) (MJD), transcript variant 2, mRNA gil13518012|refINM_030660.1|[13518012] S 10 BC022245 00 Homo sapiens Machado-Joseph disease (spinocerebellar ataxia 3, Solivopontocerebellar ataxia 3, autosomal dominant, ataxin I mRNA (cDNA clone IMAGE:4717161), containing frame-shift errors gi|18490814|gbIBC022245.1| [18490814] AB038653 Homo sapiens genomic DNA, chromosome 14q32.1, BAC clone:B445M7 gi|14149091jdbjjAB038653.1 [14149091] AJ000501 Homo sapiens DNA for CAG/CTG repeat region gi|2274960|emb|AJ000501.1|HSCAGCTG[2274960] NM_000068 Homo sapiens calcium channel, voltage-dependent, P/Q type, alpha 1A subunit (CACNA1A), transcript variant 1, mRNA gi113386499|refINM_000068.2| [13386499 NM_023035 Homo sapiens calcium channel, voltage-dependent, P/Q type, alpha 1A subunit (CACNA1A), transcript variant 2, mRNA gi|13386497|ref|NM_023035.1 [13386497] U79666 Homo sapiens alphalA-voltage-dependent calcium channel mRNA, splice form BI-1-Vi-GGCAG, complete cds gi|22817511gb|U79666.1|HSU79666[2281751] 208 X99897 H.sapiens mRNA for P/Q-type calcium channel aiphal subunit gil 1657332 erbjX99897 .1 IHSPQCCAl [1657332] AB 035726 Homo sapiens CAGNAlA mRNA for aiphalA-voltage-dependent calcium channel, partial cds, gil7630l80ldbilABO35726.ll [7630180] AF 004883 Homo sapiens neuronal calcium channel alpha isoform 1A-2 MRNA, complete cds gil 22139101 gblAFOO4883 .1 1AF004883 [2213910] AF004884 Homo sapiens neuronal calcium channel alpha isoform. A-i mRNA, complete cds gij 22139121 gbjAFOO4884 .11AF004884 [2213912] 1A subunit 1A subunit AB 035727 Homo sapiens CAGNAlA mRNA for alphalA-Voltage-dependent calcium channel, complete cds, isolate:TNDN-CNTO00l gil97ll928ldbjlAB035727.2l [9711928] U0 67 02 Human clone CCA54 mRNA containing CCA trinucleotide repeat gil4762661gbIU06702.lIHSUO 67

O

2 [476266] NMYI000333 Homo sapiens spinocerebellar ataxia 7 (olivopontocerebellar atrophy with retinal degeneration) (SCA7), mRNA gil45067961refINML000333.lI [4506796] AJ 000517 Homo Sapiens mRNA for spinocerebellar ataxia 7 gil2370154lemblAJ000517.11HSSCA7[ 237 0lS 4 209 AF032105 Homo sapiens ataxin-7 (SCA7) mRNA, complete cds gi13192953 gb|AF032105.1|AF032105[319 29 53] AF032103 Homo sapiens ataxin-7 (SCA7) mRNA, 3' end, partial cds gij3192949|gbAF03211A03.1AF3210 3 [31 9 2 9 4 9 oO M AK125125 Homo sapiens cDNA FLJ43135 fis, clone CTONG3006629 Sgi 34531113 dbj AK125125.1 [34531113] (i S AF020275 Homo sapiens expanded SCA7 CAG repeat gij25019551gbjAF020275.1|AF020275[2501955] NM_004576 Homo sapiens protein phosphatase 2 (formerly 2A), regulatory subunit B (PR 52), beta isoform (PPP2R2B), transcript variant 1, mRNA gij32307122|ref|NM_004576.21[32307122] M64930 Human protein phosphatase 2A beta subunit mRNA, complete cds gi|1904231gbIM64930.1|HUMPROP2AB[190 4 2 3 NM_181675 Homo sapiens protein phosphatase 2 (formerly 2A), regulatory subunit B (PR 52), beta isoform (PPP2R2B), transcript variant 3, mRNA gi|323071141refNM_181675.1 [32307114] NM_181674 Homo sapiens protein phosphatase 2 (formerly 2A), regulatory subunit B (PR 52), beta isoform (PPP2R2B), transcript variant 2, mRNA gi|32307112|refINM_181674.1 [32307112] BC031790 Homo sapiens protein phosphatase 2 (formerly 2A), regulatory subunit B (PR 52),

I

beta isoform, transcript variant 2, mRNA (cDNA clone MGC:24888 IMAGE:493998l), complete cds gi 216l93 041gbiBC031790.11[216193041 AK056192 Homo sapiens cDNA FLJ31630 fis, clone NT2RI200336 1 highly similar to PROTEIN PHOSPHATASE PP2A, 55 KD REGULATORY SUBUNIT,

NEURONAL

ISOFORM

gijl6551529ldbilAK05692.11[16551529 NM 000044 Homo sapiens androgen receptor (dihydrotestosterone receptor; testicular feminization; spinal and bulbar muscular atrophy; Kennedy disease) mRNA gil213222511refiNMK-000044.2l [21322251] M20132 Human androgen receptor (AR) mRNA, complete cds gi1786271gbIM20132.1 HUMANDREC[1 7 862 7 M21748 Human androgen receptor mRNA, complete cds, clones Al and J8 gil178871 gbIM21748.lIHUMARA[178871] M73069 Human androgen receptor mutant gene, mRNA, complete cds gil178655jgbIM73O69 .1HUMANRE[178655] BC051795 Homo sapiens dentatorubral-pallidoluYsian atrophy (atrophin-1), mRNA (cDNA clone MGC:57647 IMAGE:418159 2 complete cds gil34193087gbIBC051795.21 [34193087] NM_001940 Homo sapiens dentatorubral-pallidoluysian atrophy (atrophin-l) (DRPLA), mRNA gil60059981refINN00194021 [60059981 U23 851 Human atrophin-1 mRNA, complete cds gil9l53251gbIU23851.lIHSU23B51[9l5 32 D38529 Homo sapiens mRNA for DRPLA protein, complete cds gill732443ldbjID38529.lIHUMDRPLA[17324431 D31840 Homo sapiens DRPLA mRNA, complete cds gilB62329ldbjID3184O.11HUMDRPLA1[8623291 AC 006512 Homo sapiens 12 PAC RP3-461F17 (Roswell Park Cancer Institute Human PAC Library) complete sequence gil294694881gbIACOO6512.13I [29469488] 2005201389 01 Apr 2005 Table 11: HD siNA and Target Sequences dbSNP ID Pos Target Seq Seq ID UPos Upper seq SeqiD LPos Lower seq Seq ID rs396875 85 CAAUCAUGGUGGCGGCGU 1 85 CAAUCAUGCUGGCCGGCGU 1 103 ACGCCGGCCAGCAUGAUUG 1753 rs396875 86 AAUCAUGCUGGCCGGCGUG 2 86 AAUCAUGCUGGCCGGCGUG 2 104 CACGCCGGCCAGCAUGAUU. 1754 rs396875 87 AUCAUGCUGGCCGGCGUGG 3 87 AUCAUGCUGGCCGGCGUGG 3 105 CCACGCCGGCCAGCAUGAU 1755 rs396875 88 UCAUGCUGGCCGGCGUGGC 4 88 UCAUGCUGGCCGGCGUG.GC 4 106 -GCCACGCCGGCCAGCAUGA 1756 rs396875 89 CAUGCUGGCCGGCGUGGCC 5 89 CAUGCUGGCCGGCGUGGCC 5 107 GGCCACGCCGGCCAGCAUG 1757 rs396875 90 AUGCUGGCCGGCGUGGCCC 6 90 AUGCUGGCCGGCGUGGCCC 6 108 GGGCCACGCCGGCCAGCAU 1758 rs396875 91 UGCUGGCCGGCGUGGCCCC 7 91 UGCUGGCCGGCGUGGCCCC 7 109 GGGGCCACGCCGGCCAGCA 1759 rs396875 92 GCUGGCCGGCGUGGCCCCG -8 92 GCUGGCCGGCGUGGCCCCG 8 110 CGGGGCOACGCCGGCCAGC 1760 rs396875 93 CUGGCCGGCGUGGCCCCGC 9 93 CUGGCCGGCGUGGCCCCGC 9 i11 GC GGGGCCACGCCGGCCAG 1761 rs396875 94 UGGCCGGCGUGGCCCCGCC -10 94 UGGCCGGCGUGGCCCCGCC 10 112 IGGCGGGGCCACGCCGGCCA 1762 rs396875 95 GGCC GGCGUGGCCCCGCCU 11 95 GGCCGGCGUGGCCCCGCCU 11 113 JAGGCGGGGCCACGCCGGCC 1763 rs396875 96 GCCGGCGUGGCCCCGCCUC 12 96 GCCGGCGUGGCCCCGCCUC 12 114 GAGGCGGGGCCACGCCGGC 1764 rs396875 97 CCGGCGUGGCCCCGCCUCC 13 97 CCGGCGUGGCCCCGCCUCC 13 115 GGAGGCGGGGCCACGCCGG 1765 rs396875 98 CGGCGUGGCCCCGCCUCCG 14 98 CGGCGUGGCCCCGCGUCCG 14 116 CGGAGGCGGGGCCACGCCG 1766 rs396875 99 GGCGUGGCCCCGCCUCCGC 15 99 GGCGUGGCCCCGCCUCCGC 15 117 GCGGAGGCGGGGCCACGCC 1767 rs396875 1100 GCGUGGCCCCGCCUCCGCC 16 100 GCGUGGCCCCGCCUCCGCC 16 118 GGCGGAGGCGGGGCCACGC 1768 rs396875 1101 CGUGGCCCCGCCUCCGCCG 17 101 CGUGGCCCCGCCUCCGCCG 17 119 CGGCGGAGGCGGGGCCACG 1769 rs396875 102 GUGGCCCCGCCUCCGCCGG 18 102 GUGGCCCCGCCUCCGCCGG 18 120 CCGGCGGAGGCGGGGCCAC 1770 rs396875 103 UGGCCCCGCCUCCGCCGGC 19 103 UGGCCCCGCCUCCGCCGGC- 19 121 GCCGGCGGAGGCGGGGCCA 1771 rs396875 85 CAAUCAUGCUGGCCGGCGC 20 85 CAAUCAUGCUGGCCGGCGC 20 103 GCGCCGGCCAGCAUGAUUG 1772 rs396875 86 AAUCAUGCUGGCCGGCGCG 21 86 AAUCAUGCUGGCCGGCGCG 21 104 CGCGCCGGCCAGCAUGAUU 1773 rs396875 87 AUCAUGCUGGCCGGCGCGG 22 87 AUCAUGCUGGCCGGCGCGG 22 105 CCGCGCCGGCCAGCAUGAU 1774 rs396875 88 UCAUGCUGGCCGGCGCGGC 23 88 UCAUGCUGGCCGGCGCGGC 23 106 GCCGCGCCGGCCAGCAUGA 1775 rs396875 89 CAUGCUGGCCGGCGCGGCC 24 89 CAUGCUGGCCGGCGCGGC 24 107 GGCCGCGCCGGCCAGCAUG. 1776 rs396875 90 AUGCUGGCCGGCGCGGCCC 25 90 AUGCUGGCCGGCGCGGCCC 25 108 GGGCCGCGCCGGCCAGCAU. 1777 rs396875 91 UGCUGGCCGGCGCGGCCCC 26 91 UGCUGGCCGGCGCGGCCCC 26 109 GGGGCCGCGCCGGCCAGCA 1778 rs396875 92 GCUGGCCGGCGCGGCCCCG 27 92 GCUGGCCGGCGCGGCCCCG 27 110 CGGGGCCGCGCCGGCCAGC 1779 rs396875 93 CUGGCCGGCGCGGCCCCGC 28 93 CUGGCCGGCGCGGCCCCGC 28 111 GCGGGGCCGCGCCGGCCAG 1780 rs396875 94 UGGCCGGCGCGGCCCCGCC 29 94 UGGCCGGCGCGGCCCCGCC 29 112 GGCGGGGCCGCGCCGGCCA 1781 rs396875 95 GGCCGGCGCGGCCCCGCCU 30 95 GGCCGGCGCGGCCCCGCCU 30 113 AGGCGGGGCCGCGCCGGCC 1782 rs396875 96 GCCGGCGCGGCCCCGCCUC 31 96 GCCGGCGCGGCCCCGCCUC 31 114 GAGGCGGGGCCGCGCCGGC 1783 rs396875 97 CCGGCGCGGCCCCGCCUCC 32 97 CCGGCGCGGCCCCGCCUCC 32 115 GGAGGCGGGGCCGCGCCGG 1784 rs396875 98 CGGCGCGGCCCCGCCUCCG 33 98 CGGCGCGGCCCCGCCUCCG 33 116 CGGAGGCGGGGCCGCGCCG 1785 rs396875 99 GGCGCGGCCCCGCCUCCGC 34 99 GGCGCGGCCCCGCCUCCGC 34 117 GCGGAGGCGGGGCCGCGCC 1786 rs396875 1100 GCGCGGCCCCGCCUCCGCC 35 100 GCGCGGCCCCGCCUCCGCC 35 -118 GGCGGAGGCGGGGCCGCGC 1787 2005201389 01 Apr 2005 rs396875 101 CGCGGCCCCGCCUCCGCCG__ 36__1 101 jCGCGGCCCCGCCUCCGCCG 136 T119 I f t 7 1 120 CGGCGGAGGCGGGGCCGCG 1788 179 I 'Aflo75y I n13 nrnn(lrlPflnr rHrrnrrnn .it

I

10JU~. 1'J L I rs396875 103 CGGCCCCGCCUCCGCCGGC 138 103 jCGGCCCCGCCUCCGCCGGC [38 E121 A~ n~r T20 f-A~P~-AnAG 39 328 IGAAAAGCUGAUGAAGGCCU I39 346

CCGGC

AGGCC

rs10701858 329 -AAAAGCUGAUGAAGGCCUU -40 329 -AAAAGCUGAUGAAGGCCUU 40 347 AAGGC rsl 0701858 330 AAAGCUGAUGAAGGCCUUC -~41 330 MAGCUGAUGAAGGCCUUC 41 [348 GA rsl0701858 331 AAGCUGAUGAAGGCCUUCG 42 331 -AAGCUGAUGAAGGCCUUCG 42 349 CGAAC rs1 0701858 332 AGCUGAUGAAGGCCUUCGA 43 32 AGCUGAUGAAGGCCUUCGA 43 350 UCGAI rsl 0701858 333 1GCUGAUGAAGGCCUUCGAG 44-33 GCUGAUGAAGGCCUUCGAG 44-351 CUCGA AI I) A rrII C A(I- ffAfI I A r A( CC

GGAGGCGGGGCCGC

UCAUCGCUC

UUCAUCAGCUUUUC

;CUUCAUCAGCUUUU

~GCUCAUCAGCUUU

GCCUUCAUCAGCUU

GGCCUUCAUCAGCU

3AGGCCUUCAUCAGG 3AAGGCCUUCAUCA 1791 1792 1793 1794 1795 1796 1797 1798 fl7flA 0CC rs lutu 10001 1 i i rsl0701858 335 UGAUGAAGGCCUUC3A(GUC 4b J.JZ I 179 rsl0701858 336 GAUGMAGGCCUUCGAGUCC 47 336 GAUGAAGGCCUUCGAGUCC 47 354 GGACUCGAAGGCCUUCAUC 79 rsl0701858 337 AUGAAGGCCUUCGAGUCCC 48 337 AUGAAGGCCUUCGAGUCCC 48 355 GGGACUCGAAGGCCUUCAU 1800 rsl0701858 338 UGAAGGCCUUCGAGUCCCU 49 338 UGAAGGCCUUCGAGUCCCU 49 356 AGGGACUCGAAGGCCUUCA 1801 rsl0701858, 339 GAAGGCCUUCGAGUCCCUC 50 339 GAAGGCCUUCGAGUCCCUC 50 357 jGAGGGACUCGAAGGCCUUC 1802 rsl 07018581 340 AAGGCCUUCGAGUCCCUCA 51 340 AAGGCCUUCGAGUCCCUCA 51 358 UGAGGGACUCGAAGGCCUU 1803 rsl07018581 341 AGGCCUUCGAGUCCCUCAA 52 341 AGGCCUUCGAGUCCCUCAA 52 359 IUUGAGGGACUCGAAGGCCU 1804 rsl 07018581 342 GGCCUUCGAGUCCCUCAAG 53 342 GGCCUUCGAGUCCCUCAAG 53 360 ICUUGAGGGACUCGAAGGCC 1805 rsl07018581 343 GCCUUCGAGUCCCUCAAGU 54 343 GCCUUCGAGUCCCUCAAGU 54 361 ACUUGAGGGACUCGAAGGC 1806 rsl 07018581 344 CCUUCGAGUCCCUCAAGU 55.9 344 CCUUCGAGUCCCUCAAGU 55 362 ACUUGAGGGACUCGAAGG 1807 rsl 07018581 328 GAAAAGCUGAUGAAGGCCG 56 328 GAAAAGCUGAUGAAGGCCG 56 346 CGGCCUUCAUCAGCUUUUC 1808 rsl07018581 329 AAAAGCUGAUGAAGGCCGC 57 329 AAAAGCUGAUGAAGGCCGC 57 347 GCGGCCUUCAUCAGCUUUU 1809 rsl 07018581 330 AAAGCUGAUGAAGGCCGCC 58 330 MAGCUGAUGAAGGCCGCC 58 348 GGCGGCCUUCAUCAGCUUU 1810 rs1 07018581 331 AAGCUGAUGAAGGCCGCCU 59 331 AAGCUGAUGAAGGCCGCCU 59 349 JAGGCGGCCUUCAUCAGCUU 1811 rsl07018581 332 AGCUGAUGAAGGCCGCCUU 60 332 AGCUGAUGAAGGCCGCCUU 60 350 IAAGGCGGCCUUCAUCAGCU 1812 rs1 07018581 333 GCUGAUGAAGGCCGCCUUC 61 333 GCUGAUGAAGGCCGCCUUC 61 351 IGAAGGCGGCCUUCAUCAGC 1813 rs1 07018581 334 CUGAUGAAGGCCGCCUUCG 62 334 CUGAUGAAGGCCGCCUUCG 62 352 ICGAAGGCGGCCUUCAUCAG 1814 rsl07018581 335 UGAUGAAGGCCGCCUUCGA 63 335 UGAUGAAGGCCGCCUUCGA 63 353 UCGAAGGCGGCCUUCAUCA 1815 rs1 07018581 336 GAUGAAGGCCGCCUUCGAG 64 336 GAUGAAGGCCGCCUUCGAG 64 354 CUCGAAGGCGGCCUUCAUC 1816 rs1 07018581 337 AUGAAGGCCGCCUUCGAGU 65 337 AUGAAGGCCGCCUUCGAGU 65 355 ACUCGAAGGCGGCCUUCAU 1817 rsl07018581 338 UGAAGGCCGCCUUCGAGUC 66 338 UGAAGGCCGCCUUCGAGUC 66 3-5-6 -GACUCGAAGGCGGCCUUCA 1818 rsl07018581 339 GAAGGCCGCCUUCGAGUCC -67 339 GAAGGCCGCCUUCGAGUCC 67 357 GGACUCGMAGGCGGCCUUC 1819 rsl07018581 340 AAGGCCGCCUUCGAGUCCC 68 340 AAGGCCGCCUUCGAGUCCC 68 358 GGGACUCGAAGGCGGCCUU 1820 rsl07018581 341 AGGCCGCCUUCGAGUCCCU 69 341 AGGCCGCCUUCGAGUCCCU 69 359 AGGGACUCGAAGGCGGC0CU 1821 rs107018581 342 GGCCGCCUUCGAGUCCCUC 70 342 GGCCGCCUUCGAGUCCCUC 70 360 GAGGGACUCGAAGGCGGCC 1822 rsl0701858 343 GCCGCCUUCGAGUCCCUCA 71 343 GCCGCCUUCGAGUCCCUCA 71 361 -UGAGGGACUCGAAGGCGG3C 1823 rsl0701858 344 CCGCCUUCGAGUCCCUCAA 72 344 CCGCCUUCGAGUCCCUCAA 72 362 _UUGAGGGACUCGAAGGCGG 1824 rs10701858 345 CGCCUUCGAGUCCCUCAAG 73 345- C-GCCUUCGAGUCCCUCAAG 73 363 CUUGAGGGACUCGAAGGCG 1825 rs1936033 1070T UUUUGUUAAAGGCCUUCAU 74 1070 UUUUGUUAAAGGCCUUCAU 174 1088 AUGAAGGCCUUUAACAAAA 1826 214 2005201389 01 Apr 2005 rs1936033 1071 UUUGUUAAAGGCCUUCAUA 75 1071 UUUGUUAAAGGCCUUCAUA 75 1089 UAUGAAGGCCUUUAACAAA 1827 rs1936033 1072 UUGUUAAAGGCCUUCAUAG 76 1072 UUGUUAAAGGCCUUCAUAG 76 1090 CUAUGAAGGCCUUUAACAA 1828 rs1936033 1073 UGUUAAAGGCCUUCAUAGC 77 1073 UGUUAAAGGCCUUCAUAGC 77 1091 GCUAUGAAGGCCUUUAACA 1829 rs1936033 1074 GUUAAAGGCCUUCAUAGCG 78 1074 GUUAAAGGCCUUCAUAGCG 78 1092 CGCUAUGAAGGCCUUUAAC 1830 rs1936033 1075 UUAAAGGCCUUCAUAGCGA 79 1075 UUAAAGGCCUUCAUAGCGA 79 1093 UCGCUAUGAAGGCCUUUAA 1831 rs1936033 11076 UAAAGGCCUUCAUAGCGAA 80 1076 UAAAGGCCUUCAUAGCGAA 80 1094 UUCGCUAUGAAGGCCUUUA 1832 rs1936033 1077 AAAGGCCUUCAUAGCGAAC 81 1077 AAAGGCCUUCAUAGCGAAC 81 1095 GUUCGCUAUGAAGGCCUUU 1833 rs1936033 1078 AAGGCCUUCAUAGCGAACC 82 1078 AAGGCCUUCAUAGCGAACC 82 1096 GGUUCGCUAUGAAGGCCUU 1834 rs1936033 1079 AGGCCUUCAUAGCGAACCU 83 1079 AGGCCUUCAUAGCGAACCU 83 1097 AGGUUCGCUAUGAAGGCCU 1835 rs1936033 1080 GGCCUUCAUAGCGAACCUG 84 1080 GGCCUUCAUAGCGAACCUG 84 1098 CAGGUUCGCUAUGAAGGCC 1836 rs1936033 1081 GCCUUCAUAGCGAACCUGA 85 1081 GCCUUCAUAGCGAACCUGA 85 1099 UCAGGUUCGCUAUGAAGGC 1837 rs1936033 1082 CCUUCAUAGCGAACCUGAA 86 1082 CCUUCAUAGCGAACCUGAA 86 1100 UUCAGGUUCGCUAUGAAGG 1838 rs1936033 1083 CUUCAUAGCGAACCUGAAG 87 1083 CUUCAUAGCGAACCUGAAG 87 1101 CUUCAGGUUCGCUAUGAAG 1839 rs1936033 1084 UUCAUAGCGAACCUGAAGU 88 1084 UUCAUAGCGAACCUGAAGU 88 1102 ACUUCAGGUUCGCUAUGAA 1840 rs1936033 1085 UCAUAGCGAACCUGAAGUC 89 1085 UCAUAGCGAACCUGAAGUC 89 1103 IGACUUCAGGUUCGCUAUGA 1841 rs1936033 1086 CAUAGCGAACCUGAAGUCA 90 1086 CAUAGCGAACCUGAAGUCA 90 1104 JUGACUUCAGGUUCGCUAUG 18421 rs1936033 1087 AUAGCGAACCUGAAGUCAA 91 1087 AUAGCGAACCUGAAGUCAA 91 1105 UUGACUUCAGGUUCGCUAU 1843 rs1936033 1088 UAGCGAACCUGAAGUCAAG 92 1088 UAGCGAACCUGAAGUCAAG 92 1106 CUUGACUUCAGGUUCGCUA 1844 rs1936033 1070 UUUUGUUAAAGGCCUUCAC 93 1070 UUUUGUUAAAGGCCUUCAC 93 1088 GUGAAGGCCUUUAACAAAA 1845 rs1936033 1071 UUUGUUAAAGGCCUUCACA 94 1071 UUUGUUAAAGGCCUUCACA 94 1089 UGUGAAGGCCUUUAACAAA 1846 rs1936033 1072 UUGUUAAAGGCCUUCACAG 95 1072 UUGUUAAAGGCCUUCACAG 95 1090 CUGUGAAGGCCUUUAACAA 1847 rs1936033 1073 UGUUAAAGGCCUUCACAGC 96 1073 UGUUAAAGGCCUUCACAGC 96 1091 1GCUGUGAAGGCCUUUAACA 1848 rs1936033 1074 GUUAAAGGCCUUCACAGCG 97 1074 GUUAAAGGCCUUCACAGCG 97 1092 ICGCUGUGAAGGCCUUUAAC 1849 rs1936033 1075 UUAAAGGCCUUCACAGCGA 98 1075 UUAAAGGCCUUCACAGCGA 98 1093 UCGCUGUGAAGGCCUUUAA 1850 rs1936033 1076 UAAAGGCCUUCACAGCGAA 99 1076 UAAAGGCCUUCACAGCGAA 99 1094 UUCGCUGUGAAGGCCUUUA 1851 rs1936033 1077 AAAGGCCUUCACAGCGAAC 100 1077 AAAGGCCUUCACAGCGAAC 100 1095 GUUCGCUGUGAAGGCCUUU 1852 rs1936033 1078 AAGGCCUUCACAGCGAACC 101 1078 AAGGCCUUCACAGCGAACC 101 1096 GGUUCGCUGUGAAGGCCUU 1853 rs1936033 1079 AGGCCUUCACAGCGAACCU 102 1079 AGGCCUUCACAGCGMACCU 102 1097 AGGUUCGCUGUGAAGGCCU 1854 rs1936033 1080 GGCCUUCACAGCGAACCUG 103 1080 GGCCUUCACAGCGMACCUG 103 1098 CAGGUUCGCUGUGAAGGCC 1855 rs1936033 1081 GCCUUCACAGCGAACCUGA 104 1081 GCCUUCACAGCGAACCUGA '104 1099 JUCAGGUUCGCUGUGAAGGC 1856 rs1936033 1082 CCUUCACAGCGAACCUGMA 105 1082 CCUUCACAGCGAACCUGAA 105 1100 UUCAGGUUCGCUGUGAAGG 1857 rs1936033 1083 CUUCACAGCGAACCUGAAG 106 1083 CUUCACAGCGAACCUGAAG 106 1101 CUUCAGGUUCGCUGUGAAG 1858 rs1936033 1084 UUCACAGCGAACCUGAAGU 107 1084 UUCACAGCGAACCUGAAGU 107 1102 ACUUCAGGUUCGCUGUGAA 1859 rs1936033 1085 UCACAGCGAACCUGAAGUC 108 1085 UCACAGCGAACCUGAAGUG 108 1103 GACUUCAGGUUCGCUGUGA 1860 rs1936033 1086 CACAGCGAACCUGAAGUCA 109 1086 CACAGCGAACCUGAAGUCA 109 1104 UGACUUCAGGUUCGCUGUG 1861 rs1936033 1087 ACAGCGAACCUGAAGUCAA 110 1087 ACAGCGAACCUGAAGUCAA 110 1105 UUGACUUCAGGUUCGCUGU 1862 rs1 936033 1088 CAGCGAACCUGAAGUCAAG 111 1088 CAGCGAACCUGAAGUCAAG 111 1106 CUUGACUUCAGGUUCGCUG 1863 rs1936032 1188 UUGGCUACUAAAUGUGCUC 112 1188 UUGGCUACUAAAUGUGCUC 112 1206 GAGCACAUUUAGUAGCCAA 1864 rs1936032 1189 UGGCUACUAAAUGUGCUCU 113 1189 1UGGCUr-ACUAAAUGUGCUCU 1113 1207 AGAGCACAUUUAGUAGCCA 1865 2005201389 01 Apr 2005 rs1936032 1190 GGCUACUMAAUGUGCUCUU 114 1190 GGCUACUAAAUGUGCUCUU 114 1208 AAGAGCACAUUUAGUAGCC 1866 rs1936032 1191 GCUACUAAAUGUGCUCUUA 115 1191 GCUACUAAAUGUGCUCUUA 115 1209 UAAGAGCACAUUUAGUAGC 1867 rs1 936032 1192 CUACUAAAUGUGCUCUUAG 116 1192 CUACUAAAUGUGCUCUUAG 116 1210 CUAAGAGCACAUUUAGUAG 1868 rs1 936032 1193 UACUAAAUGUGCUCUUAGG 117 1193 UACUAAAUGUGCUCUUAGG 117 1211 CCUAAGAGCACAUUUAGUA 1869 rs1936032 1194 ACUAAAUGUGCUCUUAGGC 118 1194 ,ACUAAAUGUGCUCUUAGGC 118 1212 GCCUAAGAGCACAUUUAGU 1870 rs1936032 1195 CUAAAUGUGCUCUUAGGCU 119 1195 CUAAAUGUGCUCUUAGGCU 119 1213 AGCCUAAGAGCACAUUUAG 1871 rs1936032 1196 UAAAUGUGCUCUUAGGCUU 120 1196 UAAAUGUGCUCUUAGGCUU 120 1214 AAGCCUAAGAGCACAUUUA 1872 rs1 936032 1197 AAAUGUGCUCUUAGGCUUA 121 1197 AAAUGUGCUCUUAGGCUUA 121 1215 UAAGCCUAAGAGCACAUUU 1873 rs1936032 1198 AAUGUGCUCUUAGGCUUAC 122 1198 AAUGUGCUCUUAGGCUUAC 122 1216 GUAAGCCUAAGAGCACAUU 1874 rs1 936032 1199 AUGUGCUCUUAGGCUUACU 123 1199 AUGUGCUCUUAGGCUUACU 123 1217 AGUAAGCCUAAGAGCACAU 1875 rs1 936032 1200 UGUGCUCUUAGGCUUACUC 124 1200 UGUGCUCUUAGGCUUACUC 124 1218 GAGUAAGCCUAAGAGCACA 1876 rs1936032 1201 GUGCUCUUAGGCUUACUCG 125 1201 GUGCUCUUAGGCUUACUCG 125 1219 CGAGUAAGCCUAAGAGCAC. 1877 rs1936032 1202 UGCUCUUAGGCUUACUCGU 126 1202 UGCUCUUAGGCUUACUCGU 126 1220 ACGAGUAAGCCUAAGAGCA 1878 rs1936032 1203 GCUCUUAGGCUUACUCGUU 127 1203 GCUCUUAGGCUUACUCGUU 127 1221 AACGAGUAAGCCUAAGAGC 1879 rs1936032 1204 CUCUUAGGCUUACUCGUUC 128 1204 CUCUUAGGCUUACUCGUUC 128 1222 GAACGAGUAAGCCUAAGAG 1880 rs1936032 1205 UCUUAGGCUUACUCGUUCC 129 1205 UCUUAGGCUUACUCGUUCC 129 1223 GGAACGAGUAAGCCUAAGA 1881 rs1936032 1206 CUUAGGCUUACUCGUUCCU 130 1206 CUUAGGCUUACUCGUUCCU 130 1224 AGGAACGAGUAAGCCUAAG 1882 rs1936032 1188 UUGGCUACUAAAUGUGCUG 131 1188 UUGGCUACUAAAUGUGCUG 131 1206 CAGCACAUUUAGUAGCCAA 1883 rs1936032 1189 UGGCUACUAAAUGUGCUGU 132 1189 UGGCUACUAAAUGUGCUGU 132 1207 ACAGCACAUUUAGUAGCCA 1884 rs1936032 1190 GGCUACUAAAUGUGCUGUU 133 1190 GGCUACUAAAUGUGCUGUU 133 1208 AACAGCACAUUUAGUAGCC 1885 rs1936032 1191 GCUACUAAAUGUGCUGUUA 134 1191 GCUACUAAAUGUGCUGUUA 134 1209 UAACAGCACAUUUAGUAGC 1886 rs1936032 1192 CUACUAAAUGUGCUGUUAG 135 1192 CUACUAAAUGUGCUGUUAG 135 1210 CUAACAGCACAUUUAGUAG 1887 rs1936032 1193 UACUMAAUGUGCUGUUAGG 136 1193 UACUAAAUGUGCUGUUAGG 136 1211 CCUAACAGCACAUUUAGUA 1888 rs1936032 1194 ACUAAAUGUGCUGUUAGGC 137 1194 ACUAAAUGUGCUGUUAGGC 137 1212 -GCCUAACAGCACAUUUAGU 1889 rs1936032 1195 CUAAAUGUGCUGUUAGGCU 138 1195 CUAAAUGUGCUGUUAGGCU 138 1213 AGCCUAACAGCACAUUUAG 1890 rs1936032 1196 UAAAUGUGCUGUUAGGCUU 139 1196 UAAAUGUGCUGUUAGGCUU 139 1214 AAGCCUAACAGCACAUUUA 1891 rs1936032 1197 AAAUGUGCUGUUAGGCUUA 140 1197 AAAUGUGCUGUUAGGCUUA 140 1215 UAAGCCUAACAGCACAUUU 1892 rs1936032 1198 MAUGUGCUGUUAGGCUUAC 141 1198 AAUGUGCUGUUAGGCUUAC 141 1216 GUAAGCCUAACAGCACAUU 1893 rs1936032 1199 AUGUGCUGUUAGGCUUACU 142 1199 AUGUGCUGUUAGGCUUACU 142 1217 AGUAAGCCUAACAGCACAU 1894 rs1936032 1200 UGUGCUGUUAGGCUUACUC 143 1200 UGUGCUGUUAGGCUUACUC 143 1218 GAGUAAGCCUAACAGCACA 1895 rs1936032 1201 GUGCUGUUAGGCUUACUCG 144 1201 GUGCUGUUAGGCUUACUCG 144 1219 CGAGUAAGCCUAACAGCAC 1896 rs1936032 1202 UGCUGUUAGGCUUACUCGU -45 1202 -UGCUGUUAGGCUUACUCGU 145 1220 ACGAGUAAGCCUAACAGCA 1897 rs1936032 1203 GCUGUUAGGCUUACUCGUU 146 1203 GCUGUUAGGCUUACUCGUU 146 1221 AACGAGUAAGCCUAACAGC 1898 rs1 936032 1204 CUGUUAGGCUUACUCGUUC 147 1204 CUGUUAGGCUUACUCGUUC 147 1222 GAACGAGUAAGCCUAACAG 1899 rs1936032 1205 UGUUAGGCUUACUCGUUCC 148 1205 .UGUUAGGCUUACUCGUUCC 148 1223 GGAACGAGUAAGCCUAACA 1900 rs1936032 1206 GUUAGGCUUACUCGUUCCU 149 1206 GUUAGGCUUACUCGUUCCU 149 1224 AGGAACGAGUAAGCCUAAC 1901 rs1 065745 1491 GCUUCUGCAAACCCUGACC 150 1491 GCUUCUGCAAACCCUGACC 150 1509 GGUCAGGGUUUGCAGAAGC 1902 rs1 065745 1492 CUUCUGCAAACCCUGACCG 151 1492 CUUCUGCAAACCCUGACCG j151 1510 iCGGUCAGGGUUUGCAGAAG, 1903, rs1 065745 1493 UUCUGCAAACCCUACC 12 49 UCGAACUACGC 15 1511 GCGGUCAGGGUUUGCAGAA 1904 2005201389 01 Apr 2005 rs1 065745 1494 UCUGCAAACCCUGAcCGCA 153 1494 UCUGCAAACCCUGACCGCA 153 1512 UGCGGUCAGGGUUUGCAGA 1905 rs1065745 1495 CUGCAAACCCUGACCGCAG 154 1495 CUGCAAACCCUGACCGCAG 154 1513 CUGCGGUCAGGGUUUGCAG 1906 rs1 065745 1496 UGCAAACCCUGACCGCAGU 155 1496 UGCAAACCCUGACCGCAGU 155 1514 ACUGCGGUCAGGGUUUGCA 1907 rs1065745 1497 GCAAACCCUGACCGCAGUC 156 1497 GCAAACCCUGACCGCAGUC 156 1515 GACUGCGGUCAGGGUUUGC 1908 rsl065745 1498 CAAACCCUGACCGCAGUCG 157 1498 CAAACCCUGACCGCAGUCG 157 1516 CGACUGCGGUCAGGGUUUG 1909 rs1 065745 1499 AAACCCUGACCGCAGUCGG 158 1499 AAACCCUGACCGCAGUCGG 158 1517 CCGACUGCGGUCAGGGUUU 1910 rs1065745 1500 AACCCUGACCGCAGUCGGG 159 1500 AACCCUGACCGCAGUCGGG 159 1518 CCCGACUGCGGUCAGGGUU 1911 rsl065745 1501 ACCCUGACCGCAGUCGGGG 160 1501 ACCCUGACCGCAGUCGGGG 160 1519 CCCCGACUGCGGUCAGGGU- 1912 rs1 065745 1502 CCCUGACCGCAGUCGGGGG 161 1502 CCCUGACCGCAGUCGGGGG 161 1520 CCCCCGACUGCGGUCAGGG 1913 rsl065745 1503 CCUGACCGCAGUCGGGGGC 162 1503 CCUGACCGCAGUCGGGGGC 162 1521 IGCCCCCGACUGCGGUCAGG 1914 rsl065745 1504 CUGACCGCAGUCGGGGGCA 163 1504 CUGACCGCAGUCGGGGGCA 163 1522 UGCCCCCGACUGCGGUCAG- 1915 rsl065745 1505 UGACCGCAGUCGGGGGCAU 164 1505 UGACCGCAGUCGGGGGCAU 164 1523 AUGCCCCCGACUGCGGUCA 1916 rsl065745 1506 GACCGCAGUCGGGGGCAUU 165 1506 GACCGCAGUCGGGGGCAUU 165 1524 AAUGCCCCCGACUGCGGUC 1917 rsl065745 1507 ACCGCAGUCGGGGGCAUUG 166 1507 ACCGCAGUCGGGGGCAUUG 166 1525 CAAUGCCCCCGACUGCGGU 1918 rsl065745 1508 CCGCAGUCGGGGGCAUUGG 167 1508 CCGCAGUCGGGGGCAUUGG 167 1526 CCAAUGCCCCCGACUGCGG 1919 rsl065745 1509 CGCAGUCGGGGGCAUUGGG 168 1509 CGCAGUCGGGGGCAUUGGG 168 1527 CCCAAUGCCCCCGACUGCG 1920 rsl065745 1491 GCUUCUGCAAACCCUGACU 169 1491 GCUUCUGCAAACCCUGACU 169 1509 AGUCAGGGUUUGCAGAAGC 1921 rsl065745 1492 CUUCUGCAAACCCUGACUG 170 1492 CUUCUGCAAACCCUGACUG 170 1510 CAGUCAGGGUUUGCAGAAG 1922 rsl065745 1493 UUCUGCAAACCCUGACUGC 171 1493 UUCUGCAAACCCUGACUGC 171 1511 GCAGUCAGGGUUUGCAGAA 1923 rsl065745 1494 UCUGCAAACCCUGAcUGCA 172 1494 UCUGCAAACCCUGACUGCA 172 1512 UGCAGUCAGGGUUUGCAGA 1924 rsl065745 1495 CUGCAAACCCUGACUGCAG 173 1495 CUGCAAACCCUGACUGCAG 173 1513 CUGCAGUCAGGGUUUGCAG 1925 rsl065745 1496 UGCAAACCCUGACUGCAGU 174 1496 UGCAAACCCUGACUGCAGU 174 1514 IACUGCAGUCAGGGUUUGCA 1926 rsl065745 1497 GCAAACCCUGACUGCAGUC 175 1497 GCAAACCCUGACUGCAGUC 175 1515 GACUGCAGUCAGGGUUUGC 1927 rsl065745 1498 CAAACCCUGACUGCAGUCG 176 1498 CAAACCCUGACUGCAGUCG 176 1516 CGACUGCAGUCAGGGUUUG 1928 rsl065745 1499 AAACCCUGACUGCAGUCGG 177 1499 AAACCCUGACUGCAGUCGG 177 1517 CCGACUGCAGUCAGGGUUU 1929 rsl065745 1500 AACCCUGACUGCAGUCGGG 178 1500 AACCCUGACUGCAGUCGGG 178 1518 CCCGACUGCAGUCAGGGUU- 1930 rsl065745 1501 ACCCUGACUGCAGUCGGGG 179 1501 ACCCUGACUGCAGUCGGGG 179 1519 CCCCGACUGCAGUCAGGGU 1931 rsl065745 1502 CCCUGACUGCAGUCGGGGG 180 1502 CCCUGACUGCAGUCGGGGG 180 1520 ICCCCCGACUGCAGUCAGGG 1932 rsl065745 1503 CCUGACUGCAGUCGGGGGC 181 1503 CCUGACUGCAGUCGGGGGC 181 1521 GCCCCCGACUGCAGUCAGG 1933 rsl065745 1504 CUGACUGCAGUCGGGGGCA 182 1504 CUGACUGCAGUCGGGGGCA 182 1522 UGCCCGCGACUGCAGUCAG 1934 rsl065745 1505 UGACUGCAGUCGGGGGCAU 183 1505 UGACUGCAGUCGGGGGCAU 183 1523 AUGCCCCCGACUGCAGUCA 1935 rsl065745 1506 GACUGCAGUCGGGGGCAUU 184 1506 GACUGCAGUCGGGGGCAUU 184 1524 AAUGCCCCCGACUGCAGUC 1936 rsl065745 1507 ACUGCAGUCGGGGGcAUUG 185 1507 ACUGCAGUCGGGGGCAUUG 185 1525 CAAUGCCCCCGACUGCAGU 1937 rsl065745 1508 CUGCAGUCGGGGGCAUUGG 186 1508 CUGCAGUCGGGGGCAUUGG 186 1526 CCAAUGCCCCCGACUGCAG 1938 rsl065745 1509 UGCAGUCGGGGGCAUUGGG 187 1509 UGCAGUCGGGGGCAUUGGG 187 1527 CCCAAUGCCCCCGACUGCA- 1939 rs2301367 1839 GGCGGACUCAGUGGAUCUG 188 1839 GGCGGACUCAGUGGAUCUG 188 1857 CAGAUCCACUGAGUCCGCC 1940 rs2301367 1840 GCGGACUCAGUGGAUCUGG 189 1840 GCGGACUCAGUGGAUCUGG 189 1858 CCAGAUCCACUGAGUCCGC 1941 rs2301367 1841 CGGACUCAGUGGAUCUGGC 190 1841 CGGACUCAGUGGAUCUGGC 190 1859 GCCAGAUCCACUGAGUCCG 1942 rs2301367 1842 GGACUCAGUGGAUCUGGCC 191 1842 GGACUCAGUGGAUCUGGCC 191 1860 1GGCCAGAUCCACUGAGUCC 1943 217 2005201389 01 Apr 2005 rs2301367 1843 GACUCAGUGGAUCUGGGCA 192 1843 GACUCAGUGGAUCUGGCCA 192 1861 UGGCCAGAUCCACUGAGUC 1944 rs2301367 1844 ACUCAGUGGAUCUGGCCAG 193 1844 ACUCAGUGGAUCUGGCCAG 193 1862 CUGGCCAGAUCCACUGAGU 1945 rs2301367 1845 CUCAGUGGAUCUGGCCAGC 194 1845 CUCAGUGGAUCUGGCCAGC 194 1863 GCUGGCCAGAUCCACUGAG 1946 rs2301367 1846 UCAGUGGAUCUGGCCAGCU 195 1846 UCAGUGGAUCUGGCCAGCU 195 1864 AGCUGGCCAGAUCCACUGA 1947 rs2301367 1847 CAGUGGAUCUGGCCAGCUG 196 1847 CAGUGGAUCUGGCCAGCUG 196 1865 CAGCUGGCCAGAUCCACUG 1948 rs2301367 1848 AGUGGAUCUGGCCAGCUGU 197 1848 AGUGGAUCUGGCCAGCUGU 197 1866 ACAGCUGGCCAGAUCCACU 1949 rs2301367 1849 GUGGAUCUGGCCAGCUGUG 198 1849 GUGGAUCUGGCCAGCUGUG 198 1867 CACAGCUGGCCAGAUCCAC 1950 rs2301367 1850 UGGAUCUGGCCAGCUGUGA 199 1850 UGGAUCUGGCCAGCUGUGA 199 1868 UCACAGCUGGCCAGAUCCA 1951 rs2301367 1851 GGAUCUGGCCAGCUGUGAC 200 1851 GGAUCUGGCCAGCUGUGAC 200 1869 GUCACAGCUGGCCAGAUCC 1952 rs2301367 1852 GAUCUGGCCAGCUGUGACU 201 1852 GAUCUGGCCAGCUGUGACU 201 1870 AGUCACAGCUGGCCAGAUC- 1953 rs2301367 1853 AUCUGGCCAGCUGUGACUU 202 1853 AUCUGGCCAGCUGUGACUU 202 1871 AAGUCACAGCUGGCCAGAU 1954 rs2301367 1854 UCUGGCCAGCUGUGACUUG 203 1854 UCUGGCCAGCUGUGACUUG 203 1872 CAAGUCACAGCUGGCCAGA 1955 rs2301 367 1855 CUGGCCAGCUGUGACUUGA 204 1855 CUGGCCAGCUGUGACUUGA 204 1873 UCAAGUCACAGCUGGCCAG 1956 rs2301367 1856 UGGCCAGCUGUGACUUGAC 205 1856 UGGCCAGCUGUGACUUGAC 205 1874 GUCAAGUCACAGCUGGCCA 1957 rs2301367 1857 GGCCAGCUGUGACUUGACA 206 1857 GGCCAGCUGUGACUUGACA 206 1875 UGUCAAGUCACAGCUGGCC 1958 rs2301367 1839 GGCGGACUCAGUGGAUCUA 207 1839 GGCGGACUCAGUGGAUCUA 207 1857 UAGAUCCACUGAGUCCGCC- 1959 rs2301 367 1840 GCGGACUCAGUGGAUCUAG 208 1840 GCGGACUCAGUGGAUCUAG 208 1858 CUAGAUCCACUGAGUCCGC 1960 rs2301367 1841 CGGACUCAGUGGAUCUAGC 209 1841 CGGACUCAGUGGAUCUAGC 209 1859 GCUAGAUCCACUGAGUCCG- 1961 rs2301367 1842 GGACUCAGUGGAUCUAGCC 210 1842 GGACUCAGUGGAUCUAGCC 210 1860 GGCUAGAUCCACUGAGUCC 1962 rs2301367 1843 GACUCAGUGGAUCUAGCCA 211 1843 GACUCAGUGGAUCUAGCCA 211 1861 UGGCUAGAUCCACUGAGUC 1963 rs2301367 1844 ACUCAGUGGAUCUAGCCAG 212 1844 ACUCAGUGGAUCUAGCCAG 212 1862 CUGGCUAGAUCCACUGAGU 1964 rs2301367 1845 CUCAGUGGAUCUAGCCAGC 213 1845 CUCAGUGGAUCUAGCCAGC 213 1863 GCUGGCUAGAUCCACUGAG- 1965 rs2301367 1846 UCAGUGGAUCUAGCCAGCU 214 1846 -UCAGUGGAUCUAGCCAGCU 214 1864 AGCUGGCUAGAUCCACUGA 1966 rs2301367 1847 CAGUGGAUCUAGCCAGCUG 215 1847 CAGUGGAUCUAGCCAGCUG 215 1865 CAGCUGGCUAGAUCCACUG- 1967 rs2301367 1848 AGUGGAUCUAGCCAGCUGU 216 1848 AGUGGAUCUAGCCAGCUGU 216 1866 ACAGCUGGCUAGAUCCACU 1968 rs2301367 1849 GUGGAUCUAGCCAGCUGUG 217 1849 GUGGAUCUAGCCAGCUGUG 217 1867 CACAGCUGGCUAGAUCCAC 1969 rs2301367 1850 UGGAUCUAGCCAGCUGUGA 218 1850 UGGAUCUAGCCAGCUGUGA 218 1868 UCACAGCUGGCUAGAUCCA 1970 rs2301367 1851 GGAUCUAGCCAGCUGUGAC 219 1851 GGAUCUAGCCAGCUGUGAC 219 1869 GUCACAGCUGGCUAGAUCC 1971 rs2301367 1852 GAUCUAGCCAGCUGUGACU 220 1852 GAUCUAGCCAGCUGUGACU 220 1870 AGUCACAGCUGGCUAGAUC- 1972 rs2301367 1853 AUCUAGCCAGCUGUGACUU 221 1853 AUCUAGCCAGCUGUGACUU 221 1871 _AAGUCACAGCUGGCUAGAU 1973 rs2301367 1854 UCUAGCCAGCUGUGACUUG 222 1854 -UCUAGCCAGCUGUGACUUG 222 1872 CAAGUCACAGCUGGCUAGA- 1974 rs2301367 1855 CUAGCCAGCUGUGACUUGA 223 1855 CUAGCCAGCUGUGACUUGA 223 1873 IUCAAGUCACAGCUGGCUAG 1975 rs2301367 1856 UAGCCAGCUGUGACUUGAC 224 1856 UAGCCAGCUGUGACUUGAC 224 1874 GUCAAGUCACAGCUGGCUA 1976 rs2301367 1857 AGCCAGCUGUGACUUGACA 225 1857 AGCCAGCUGUGACUUGACA 225 1875 UGUCAAGUCACAGCUGGCU 1977 rs363075 2980 GCAGAAAACUUACACAGAG 226 2980 GCAGAAAACUUACACAGAG 226 2998 ICUCUGUGUAAGUUUUCUGC 17 rs363075 2981 CAGAAAACUUACACAGAGG 227 2981 CAGAAAACUUACACAGAGG 227 2999 ICCUCUGUGUAAGUUUUCUG 1979 rs363075 2982 AGAAAACUUACACAGAGGG 228 2982 AGAAAACUUACACAGAGGG 228 3000 CCCUCUGUGUAAGUUUUCU 1980 rs363075 2983 GAAAACUUACACAGAGGGG 229 2983 GAAAACUUACACAGAGGGG 229 3001 CCCCUCUGUGUAAGUUUUC 1981 rs363075 2984 AAAACUUACACAGAGGGGC -r23-0 2984 AAAACUUACACAGAGGGGC 230 3002 GCCCCUCUGUGUAAGUUUU 18 2005201389 01 Apr 2005 rs363075 2985 AAACUUACACAGAGGGGCU 231 2985 AAACUUACACAGAGGGGCU 231 3003 AGCCCCUCUGUGUAAGUUU 1983 rs363075 2986 AACUUACACAGAGGGGCUC 232 2986 AACUUACACAGAGGGGCUC 232 3004 GAGCCCCUCUGUGUAAGUU 1984 rs363075 2987 ACUUACACAGAGGGGCUCA 233 2987 ACUUACACAGAGGGGCUCA 233 3005 UGAGCCCCUCUGUGUAAGU- 1985 rs363075 2988 CUUACACAGAGGGGCUCAU 234 2988 CUUACACAGAGGGGCUCAU 234 3006 AUGAGCCCCUCUGUGUAAG 1986 rs363075 2989 UUACACAGAGGGGCUCAUC 235 2989 UUACACAGAGGGGCUCAUC 235 3007 GAUGAGCCCCUCUGUGUAA 1987 rs363075 2990 UACACAGAGGGGCUCAUCA 236 2990 1UACACAGAGGGGCUCAUCA 236 3008 UGAUGAGCCCCUCUGUGUA 1988 rs363075 2991 ACACAGAGGGGCUCAUCAU 237 2991 ACACAGAGGGGCUCAUCAU 237 3009 AUGAUGAGCCCCUCUGUGU 1989 rs363075 2992 CACAGAGGGGCUCAUCAUU 238 2992 CACAGAGGGGCUCAUCAUU 238 3010 AAUGAUGAGCCCCUCUGUG 1990 rs363075 2993 ACAGAGGGGCUCAUCAUUA 239 2993 ACAGAGGGGCUCAUCAUUA 239 3011 UAAUGAUGAGCCCCUCUGU 1991 rs363075 2994 CAGAGGGGCUCAUCAUUAU 240 2994 CAGAGGGGCUCAUCAUUAU 240 3012 AUAAUGAUGAGCCCCUCUG 1992 rs363075 2995 AGAGGGGCUCAUCAUUAUA 241 2995 AGAGGGGCUCAUCAUUAUA 241 3013 UAUAAUGAUGAGCCCCUCU 1993 rs363075 2996 GAGGGGCUCAUCAUUAUAC 242 2996 GAGGGGCUCAUCAUUAUAC 242 3014 GUAUAAUGAUGAGCCCCUC 1994 rs363075 2997 AGGGGCUCAUCAUUAUACA 243 2997 AGGGGCUCAUCAUUAUACA 243 3015 UGUAUAAUGAUGAGCCCCU 1995 rs363075 2998 GGGGCUCAUCAUUAUACAG 244 2998 GGGGCUCAUCAUUAUACAG 244 3016 CUGUAUAAUGAUGAGCCCC 1996 rs363075 2980 GCAGAAAACUUACACAGAA 245 2980 GCAGAAAACUUACACAGAA 245 2998 UUCUGUGUAAGUUUUCUGC 1997 rs363075 2981 CAGAAAACUUACACAGAAG 246 2981 CAGAAAACUUACACAGAAG 246 2999 ICUUCUGUGUAAGUUUUCUG 1998 rs363075 2982 AGAAAACUUACACAGAAGG 247 2982 AGAAAACUUACACAGAAGG 247 3000 CCUUCUGUGUAAGUUUUCU 1999 rs363075 2983 GAAAACUUACACAGAAGGG 248 2983 GAAAACUUACACAGAAGGG 248 3001 CCCUUCUGUGUAAGUUUUC 2000 rs363075 2984 AAAACUUACACAGAAGGGC 249 2984 AAAACUUACACAGAAGGGC 249 3002 GCCCUUCUGUGUAAGUUUU 2001 rs363075 2985 AAACUUACACAGAAGGGCU 250 2985 AAACUUACACAGAAGGGCU 250 3003 AGCCCUUCUGUGUAAGUUU 2002 rs363075 2986 AACUUACACAGAAGGGCUC 251 2986 AACUUACACAGAAGGGCUC 251 3004 GAGCCCUUCUGUGUAAGUU 2003 rs363075 2987 ACUUACACAGAAGGGCUCA 252 2987 ACUUACACAGAAGGGCUCA 252 3005 UGAGCCCUUCUGUGUAAGU- 2004 rs363075 2988 CUUACACAGAAGGGCUCAU 253 2988 CUUACACAGAAGGGCUCAU 253 3006 AUGAGCCCUUCUGUGUAAG 2005 rs363075 2989 UUACACAGAAGGGCUCAUC 254 2989 UUACACAGAAGGGCUCAUC 254 3007 GAUGAGCCCUUCUGUGUAA- 2006 rs363075 2990 UACACAGAAGGGCUCAUCA 255 2990 UACACAGAAGGGCUCAUCA 255 3008 UGAUGAGCCCUUCUGUGUA 2007 rs363075 2991 ACACAGAAGGGCUCAUCAU 256 2991 ACACAGAAGGGCUCAUCAU 256 3009 AUGAUGAGCCCUUCUGUGU 2008 rs363075 2992 CACAGAAGGGCUCAUCAUU 257 2992 CACAGAAGGGCUCAUCAUU 257 3010 AAUGAUGAGCCCUUCUGUG 2009 rs363075 2993 ACAGAAGGGCUCAUCAUUA 258 2993 ACAGAAGGGCUCAUCAUUA 258 3011 UAAUGAUGAGCCCUUCUGU 2010 rs363075 2994 CAGAAGGGCUCAUCAUUAU 259 2994 CAGMAGGGCUCAUCAUUAU 259 3012 AUAAUGAUGAGCCCUUCUG 2011 rs363075 2995 AGAAGGGCUCAUCAUUAUA 260 2995 AGAAGGGCUCAUCAUUAUA 260 3013 UAUAAUGAUGAGCCCUUCU 2012 rs363075 2996 GAAGGGCUCAUCAUUAUAC 261 2996 GAAGGGCUCAUCAUUAUAC 261 3014 GUAUAAUGAUGAGCCCUUC 2013 rs363075 2997 AAGGGCUCAUCAUUAUACA 262 2997 AAGGGCUCAUCAUUAUACA 262 3015 UGUAUAAUGAUGAGCCCUU 2014 rs363075 2998 AGGGCUCAUCAUUAUACAG 263 2998 AGGGCUCAUCAUUAUACAG 263 3016 CUGUAUAAUGAUGAGCCCU 2015 rsl065746 3547 UCAGCUUGGUUCCCAUUGG 264 3547 UCAGCUUGGUUCCCAUUGG 264 3565 CCAAUGGGAACCAAGCUGA 2016 rsl065746 3548 CAGCUUGGUUCCCAUUGGA 265 3548 CAGCUUGGUUCCCAUUGGA 265 3566 UCCAAUGGGAACCAAGCUG 2017 rs1065746 3549 AGCUUGGUUCCCAUUGGAU 266 3549 AGCUUGGUUCCCAUUGGAU 266 3567 AUCCAAUGGGAACCAAGCU 2018 rs1 065746 3550 GCUUGGUUCCCAUUGGAUC 267 3550 GCUUGGUUCCCAUUGGAUC 267 3568 GAUCCAAUGGGAACCAAGC 2019 rsl 065746 3551 CUUGGUUCCCAUUGGAUCU 268 3551 CUUGGUUCCCAUUGGAUCU 268 3569 AGAUCCAAUGGGAACCAAG 2020 rsl 065746 3552 UUGGUUCCCAUUGGAUCUC 269 3552 UUGGUUCCCAUUGGAUCUC 269 350 GAGAUCCAAUGGGAACCAA 12021 219 2005201389 01 Apr 2005 rsl 065746 13553 rsl 065746 3554 rsl 065746 3555 rsl 065746 3556 rsl 065746 3557 rsl 065746 3558 rsl065746 3559 rsl065746 3560 rsl065746 3561 rsl 065746 3562 rs1065746 3563 rsl065746 3564 rsl 065746 3565 rsl 065746 3547 rrsl 065746 3548 rs1065746 3549 rs1065746 3 550 rs1065746 3551 rs10546 35 rsl 065746 3553 rs:l 065746 3554 rsl 065746 3 555 rsl 065746 3556 rsl 065746 3557 rsl 065746 3 558 rsl 065746 3559 rs1065746 3560 rsl 065746 3561 rsl 065746 3562 rsl 065746 3563 r sl 065746 3:564 rsl065746 3565 rs1065746 3 547 rsl 065746 35487 rsl 065746 3549 rsl06574 6 3550 rsl 065746 3551 rsl 065746 3552 rsl 065746 3553

UGGUUCCCAUUGGACU

GGUUCCCAUUGGAUCUCUC

GUUCCCAUUGGAUCUCUCA

UUCCCAUUGGAUCUCUCAG

UCCCAUUGGAUCUCUCAGC

CCCAUUGGAUCUCUCAGCC

CCAUUGGAUCUCUCAGCCC

CAUUGGAUCUCUCAGCCCA

AUUGGAUCUCUCAGCCCAU

UUGGAUCUCUCAGCCCAUC

-UGGAUCUCUCAGOCCAUCA

GGAUCUCUCAGCCCAUCAA

GAUCUCUCAGCCCAUCAAG

UCAGCUUGGUUCCCAUUGA

CAGCUUGGUUCCCAUUGAA

AGCUUGGUUCCCAUUGAAU

GCUUGGUUCCCAUUGAAUC

CUUGGUUCCCAUUGAAUCU

UUGGUUCCCAUUGAAUCUC

-UGGUUCCCAUUGAAUCUCU

GGUUCCCAUUGAAUCUCUC

GUUCCCAUUGAAUCUCUCA

UUCCCAUUGAAUCUCUCAG

UCCCAUUGAAUCUCUCAGC

CCCAUUGAAUCUCUCAGCC

CCAUUGAAUCUCUCAGCCC

CAUUGMAUCUCUCAGCCCA

AUUGMAUCUCUCAGCCCAU

UUGAAUCUCUCAGCCCAUC

UGAAUCUCUCAGCCCAUCA

GAAUCUCUCAGCCCAUCAA

AAUCUCUCAGCCCAUCAAG

UCAGCUUGGUUCCCAUUGC

CAGCUUGGUUCCCAUUGCA

AGCUUGGUUCCCAUUGCAU

GCUUGGUUCCCAUUGCAUC

CUUGGUUCCCAUUGCAUCU

UUGGUUCCCAUUGCAUCUC

F UGGUUCCCAUUGCAUCUCU 2 70 272 273 274 275 276 2 77 278 279 280 281 282 283 284 285 286 287 28 8 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303- 304 305 30 6 307 308 35 UGGUUCAUUGGAUCC 3-554 LGUUCCCAUUGGAUCUCU 3555 GGUUCCCAUUGGAUCUUC 3557UCCCUUGGUCUAG 3558 GUCCCAUUGGAUCUCUCA 3559 CCA

UGGAUCUCUCC

3556 UCAUUGGGAUCUCUCAGC 351 UUAC

UCUCCA

3562 UGGAUC

CUCGCU

3563 UCUGGAUCUCUCAGCA 3564 CUGGAUCUCUCAGCCA 355 CAGAUCUCUCAGACA 3j5470 CAUUGGUUCCAGA 3548 CAUGGUUCCAGAA 3549 AUUGGUUCCAGAU 3550 GGCUUUUCCCAUAA 355 GCUUUUCCCAUAAC 3552 UCUUGGUUCCCAUUGAU 3553 CAUGGUUCCCAUUGAAC 354%GGGUUCCCAUUGAAUU 3555 GCGUUCCCAUUGAAUCC 3556 CUUUCCCAUUGAAUCU 3557 UUUCCCAUUGAAUCUC 3558 UGCCCAUUGAAUCUCUC 3559 GGCCAUUGAAUCUCUCC 3560 GUCAUUGAAUCUCUCAC 356 UUAUUGAAUCUCUCAGA 3562 UCUUGAAUCUCUCAGCU 3563 CUGAAUCUCUCAGCCC 3564 CGAAUCUCUCAGCCCA 3565 AAAUCUCUCAGCCCAUA 3547 UCACUGGUCCCAUGC 3548 CUACUUGCCCAUCA 3549 AGUCUUGCCCAUCAUG 3550 AGCUUGGUUCCCAUUGCAU 3551 GCUUGGUUCCCAUUGCAUC -3552 CUUGGUUCCCAUUGAUCU 3553 UUGUCCCAUUGCAUCUC 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 3571 AGAGAUCCAAUGGGAACCA 3572 -GAGAGAUCCAAUGGGAACC 3573 UGAGAGAUCCAAUGGGAAC 3574 CUGAGAGAUCCAAUGGGAA 3575 GCUGAGAGAUCCAAUG3GGA 3576 -GGCUGAGAGAUCCAAUGGG 3577 GGGCUGAGAGAUCCAAUGG 3578 UGGGCUGAGAGAUCCAAUG 3579 AUGGGCUGAGAGAUCCAAU 3580 GAUGGGCUGAGAGAUCCAA 3581 UGAUGGGCUGAGAGAUCCA 3582 UUGAUGGGCUGAGAGAUCC 3583 CUUGAUGGGCUGAGAGAUC 3565 UCAAUGGGAACCAAGCUGA 3566 UUCAAUGGGAACCAAGCUG 3567 AUUCAAUGGGAACCAAGCU 3568 GAUUCAAUGGGAACCAAGC 3569 AGAUUCAAUGGGAACC;AAG 3570 GAGAUUCAAUGGGAACCAA 3571 AGAGAUUCAAUGGGAACCA 3572 GAGAGAUUCAAUGGGAACC 3573 UGAGAGAUUCAAUGGGAAC 3574 -CUGAGAGAUUCAAUGGGAA 3575 GCUGAGAGAUUCAAUGGGA 3576 GGCUGAGAGAUUCAAUGGG 3577 GGGCUGAGAGAUUCAAUGG 3578 UGGGCUGAGAGAUUCAAUG 3579 AUGGGCUGAGAGAUUCAAU 3580 GAUIGGGCUGAGAGAUUCAA 3581 UGAUGGGCUGAGAGAUUCA 3582 UUGAUGGGCUGAGAGAUUC 3583 CUUGAUGGGCUGAGAGAUU 3565 GCAAUGGGAACCAAGCUGA 3566 UGCAAUGGGAACCAAGCUG 3567 AUGCAAUGGGAACCAAGCU 3568 GAUGCAAUGGGAACCAAGC 3569 AGAUGCAAUGGGAACCAAG 3570 GAGAUGCAAUGGGAACCAA 3571 LAGAGAUGCAAUGGGAACCA 2022 2023 2024 2025 2026 2027 2028 2029 2030 2031 2032 2033 2034 2035 2036 2037 2038 2039 2040 2041 2042 2043 2044 2045 2046 2047 2048 2049 2050 2051_ 2052 2053 2054 2055 2056 2057 2058 2059 2060 220 2005201389 01 Apr 2005 rsl 065746 3554 GGUUCCCAUUGCAUCUCUC 309 3554 GGUUCCCAUUGCAUCUCUC 309 3572 GAGAGAUGCAAUGGGAACC 2061 rsl065746 3555 GUUCCCAUUGCAUCUCUCA 310 3555 GUUCCCAUUGCAUCUCUCA 310 3573 UGAGAGAUGCAAUGGGAAC 2062 rsl065746 3556 UUCCCAUUGCAUCUCUCAG 311 3556 UUCCCAUUGCAUCUCUCAG 311 3574 CUGAGAGAUGCAAUGGGAA 2063 rsl 065746 3557 UCCCAUUGCAUCUCUCAGC 312 3557 UCCCAUUGCAUCUCUCAGC 312 3575 GCUGAGAGAUGCAAUGGGA- 2064 rsl 065746 3558 CCCAUUGCAUCUCUCAGCC 313 3558 CCCAUUGCAUCUCUCAGCC 313 3576 GGCUGAGAGAUGCAAUGGG- 2065 rsl 065746 3559 CCAUUGCAUCUCUCAGCCC 314 3559 CCAUUGCAUCUCUCAGCCC 314 3577 GGGCUGAGAGAUGCAAUGG 2066 rsl065746 3560 CAUUGCAUCUCUCAGCCCA 315 3560 CAUUGCAUCUCUCAGCCCA 315 3578 UGGGCUGAGAGAUGCAAUG 2067 rsl065746 3561 AUUGCAUCUCUCAGCCCAU 316 3561 AUUGCAUCUCUCAGCCCAU 316 3579 AUGGGCUGAGAGAUGCAAU 2068 rsl065746 3562 UUGCAUCUCUCAGCCCAUC 317 3562 UUGCAUCUCUCAGCCCAUC 317 3580 GAUGGGCUGAGAGAUGCAA 2069 rsl065746 3563 UGCAUCUCUCAGCCCAUCA 318 3563 UGCAUCUCUCAGCCCAUCA 318 3581 IUGAUGGGCUGAGAGAUGCA 2070 rsl 065746 3564 GCAUCUCUCAGCCCAUCAA 319 3564 GCAUCUCUCAGCCCAUCAA 319 3582 UUGAUGGGCUGAGAGAUGC 2071 rsl 065746 3565 CAUCUCUCAGCCCAUCAAG 320 3565 CAUCUCUCAGCCCAUCAAG 320 3583 CUUGAUGGGCUGAGAGAUG 2072 rsl065747 3647 GGGCCUCUGAAGAAGAAGC 321 3647 GGGCCUCUGAAGAAGAAGC 321 3665 GCUUCUUCUUCAGAGGCCC- 2073 rsl065747 3648 GGCCUCUGAAGAAGAAGCC 322 3648 GGCCUCUGAAGAAGAAGCC 322 3666 GGCUUCUUCUUCAGAGGCC 2074 rsl065747 3649 GCCUCUGAAGAAGAAGCCA 323 3649 GCCUCUGAAGAAGAAGCCA 323 3667 UGGCUUCUUCUUCAGAGGC 2075 rsl065747 3650 CCUCUGAAGAAGAAGCCAA 324 3650 CCUCUGAAGAAGAAGCCAA 324 3668 UUGGCUUCUUCUUCAGAGG 2076 rsl 065747 3651 CUCUGAAGAAGAAGCCAAC 325 3651 CUCUGAAGAAGAAGCCAAC 325 3669 GUUGGCUUCUUCUUCAGAG 2077 rsl 065747 3652 UCUGAAGAAGAAGCCAACC 326 3652 UCUGAAGAAGAAGCCAACC 326 3670 GGUUGGCUUCUUCUUCAGA 2078 rsl 065747 3653 CUGAAGAAGMAGCCAACCC 327 3653 CUGAAGAAGAAGCCAACCC 327 3671 IGGGUUGGCUUCUUCUUCAG 2079 rsl 065747 3654 UGAAGAAGAAGCCAACCCA 328 3654 UGAAGAAGAAGCCAACCCA 328 3672 JUGGGUUGGCUUCUUCUUCA 2080 rsl 065747 3655 GAAGAAGAAGCCAACCCAG 329 3655 GAAGAAGAAGCCAACCCAG 329 3673 ICUGGGUUGGCUUCUUCUUC 2081 rsl065747 3656 AAGAAGAAGCCAACCCAGC 330 3656 AAGAAGAAGCCAACCCAGC 330 3674 GCUGGGUUGGCUUCUUCUU 2082 rsl 065747 3657 AGAAGAAGCCAACCCAGCA 331 3657 AGAAGAAGCCAACCCAGCA 331 3675 UGCUGGGUUGGCUUCUUCU 2083 rsl 065747 3658 GAAGAAGCCMACCCAGCAG 332 3658 GAAGAAGCCAACCCAGCAG 332 3676 CUGCUGGGUUGGCUUCUUC 2084 rs1065747 3659 AAGAAGCCAACCCAGCAGC 333 3659 AAGAAGCCAACCCAGCAGC 333 3677 GCUGCUGGGUUGGCUUCUU 2085 rsl 065747 3660 AGAAGCCAACCCAGCAGCC 334 3660 AGAAGCCAACCCAGCAGCC 334 3678 GGCUGCUGGGUUGGCUUCU 2086 rsl 065747 3661 GAAGCCAACCCAGCAGCCA 335 3661 GAAGCCAACCCAGCAGCCA 335 3679 UGGCUGCUGGGUUGGCUUC 2087 rsl065747 3662 AAGCCAACCCAGCAGCCAC 336 3662 AAGCCAACCCAGCAGCCAC 336 3680 GUGGCUGCUGGGUUGGCUU 2088 rsl 065747 3663 AGCCAACCCAGCAGCCACC 337 3663 AGCCAACCCAGCAGCCACC 337 3681 GGUGGCUGCUGGGUUGGCU 2089 rsl 065747 3664 GCCAACCCAGCAGCCACCA 338 3664 GCCAACCCAGCAGCCACCA 338 3682 UGGUGGCUGCUGGGUUGGC 2090 rsl 065747 3665 CCAACCCAGCAGCCACCAA 339 3665 CCAACCCAGCAGCCACCAA 339 3683 IUUGGUGGCUGCUGGGUUGG 2091 rsl 065747 3647 GGGCCUCUGAAGAAGAAGG 340 3647 GGGCCUCUGAAGAAGAAGG 340 3665 CCUUCUUCUUCAGAGGCCC 2092 rsl 065747 3648 GGCCUCUGAAGAAGAAGGC 341 3648 GGCCUCUGAAGAAGAAGGC 341 3666 GCCUUCUUCUUCAGAGGCC 2093 rsl 065747 3649 GCCUCUGAAGAAGAAGGCA 342 3649 GCCUCUGAAGAAGAAGGCA 342 3667 UGCCUUCUUCUUCAGAGGC 2094 rsl 065747 3650 CCUCUGAAGAAGAAGGCAA 343 3650 CCUCUGAAGAAGAAGGCAA 343 3668 UUGCCUUCUUCUUCAGAGG 2095 rsl 065747 3651 CUCUGAAGAAGAAGGCAAC 344 3651 -CUCUGAAGAAGAAGGCAAC 344 3669 -GUUGCCUUCUUCUUCAGAG 2096 rsl 065747 3652 UCUGAAGAAGAAGGCAACC 345 3652 UCUGAAGAAGAAGGCAACC 345 3670 GGUUGCCUUCUUCUUCAGA 2097 rsl 065747 3653 CUGAAGAAGAAGGCAACCC 346 3653 -CUGAAGAAGAAGGCAACCC 346 3671 GGGUUGCCUUCUUCUUCAG 2098 rsl065747 3654 ,UGAAGAAGAAGGCAACCCA 347 3654 1UGAAGAAGAAGGCAACCCA ,347 3-6-72 UGGGUUGCCUUCUUCUUCA 2099 2005201389 01 Apr 2005 rsl065747 3655 GAAGAAGAAGGCAACCCAG 348 3655 GAAGAAGAAGGCAACCCAG 348 3673 CUGGGUUGCCUUCUUCUUC 2100 rsl065747 3656 AAGAAGAAGGCMACCCAGC I349 3656 AAGAAGAAGGCMACCCAGC 349 3674 GCUGGGUUGCCUUCUUCUU 2101 rsl065747 3657 AGAAGAAGGCAACCCAGCA 350 3657 AGAAGAAGGCAACCCAGCA 350 3675 UGCUGGGUUGCCUUCUUCU 2102 rsl065747 3658 GMAGAAGGCAACCCAGCAG 351 3658 GAAGAAGGCAACCCAGCAG 351 3676 CUGCUGGGUUGCCUUCUUC 2103 rsl065747 3659 AAGAAGGCAACCCAGCAGC 352 3659 AAGAAGGCAACCCAGCAGC 352 3677 GCUGCUGGGUUGCCUUCUU 2104 rsl065747 3660 AGAAGGCAACCCAGCAGCC 353 3660 AGAAGGCAACCCAGCAGCC 353 3678 GGCUGCUGGGUUGCCUUCU 2105 rsl065747 3661 GAAGGCAACCCAGCAGCCA 354 3661 GAAGGCAACCCAGCAGCCA 354 3679 UGGCUGCUGGGUUGCCUUC 2106 rsl065747 3662 AAGGCAACCCAGCAGCCAC 355 3662 AAGGCAACCCAGCAGCCAC 355 3680 GUGGCUGCUGGGUUGCCUU 2107 rsl065747 3663 AGGCAACCCAGCAGCCACC 356 3663 AGGCAACCCAGCAGCCACC 356 3681 GGUGGCUGCUGGGUUGCCU 2108 rsl065747 3664 GGCAACCCAGCAGCCACCA 357 3664 GGCAACCCAGCAGCCACCA 357 3682 UGGUGGCUGCUGGGUUGCC 2109 rsl 065747 3665 GCAACCCAGCAGCCACCAA 358 3665 GCAACCCAGCAGCCACCAA 358 3683 JUU GGUGGCUGCUGGGUUGC 2110 rs2530588 3803 CUGGACCCGCAAUAAAGGC 359 3803 CUGGACCCGCAAUAAAGGC 359 3821 GCCUUUAUUGCGGGUCCAG 2111 rs2530588 3804 UGGACCCGCAAUAAAGGCA 360 3804 UGGACCCGCAAUAAAGGCA 360 3822 UGCCUUUAUUGCGGGUCCA 2112 rs2530588 3805 GGACCCGCAAUAAAGGCAG 361 3805 GGACCCGCAAUAAAGGCAG 361 3823 CUGCCUUUAUUGCGGGUCC 2113 rs2530588 3806 GACCCGCAAUAAAGGCAGC 362 3806 GACCCGCAAUAAAGGCAGC 362 3824 GCUGCCUUUAUUGCGGGUC 2114 rs2530588 3807 ACCCGCAAUAAAGGCAGCC 36-3 3807 ACCCGCAAUAAAGGCAGCC 363 3825 GGCUGCCUUUAUUGCGGGU 2115 rs2530588 3808 CCCGCAAUAAAGGCAGCCU 364 3808 CCCGCAAUAAAGGCAGCCU 364 3826 AGGCUGCCUUUAUUGCGGG 2116 rs2530588 3809 CCGCAAUAAAGGCAGCCUU 365 3809 CCGCAAUAAAGGCAGCCUU 365 3827 -AAGGCUGCCUUUAUUGCGG 2117 rs2530588 3810 CGCAAUAAAGGCAGCCUUG 366 3810 CGCAAUAAAGGCAGCCUUG 366 3828 CAAGGCUGCCUUUAUUGCG 2118 rs2530588 3811 GCAAUAAAGGCAGCCUUGC 367 3811 GCAAUAAAGGCAGCCUUGC 367 3829 GCAAGGCUGCCUUUAUUGC 2119 rs2530588 3812 CAAUAAAGGCAGCCUUGCC 368 3812 CAAUAAAGGCAGCCUUG3CC 368 3830 GGCAAGGCUGCCUUUAUUG 2120 rs2530588 3813 AAUAAAGGCAGCCUUGCCU 369 3813 MAUAAAGGCAGCCUUGCCU 369 3831 AGGCAAGGCUGCCUUUAUU 2121 rs2530588 3814 AUAAAGGCAGCCUUGCCUU 370 3814 AUAAAGGCAGCCUUGCC UU 370 3832 AAGGCAAGGCUGCCUUUAU 2122 rs2530588 3815 UAAAGGCAGCCUUGCCUUC 371 3815 UAAAGGCAGCCUUGCCUUC 371 3833 GAAGGCAAGGCUGCCUUUA 2123 rs2530588 3816 AAAG;GCAGCCUUGCCUUCU 37 2 381 6 AAAGGCAGCCUUGCCUUCU 372 3834 AGAAGGCAAGGCUGCCUUU 2124 rs2530588 3817 AAGGCAGCCUUGCCUUCUC 373 3817 AAGGCAGCCUUGCCUUCUC 373 3835 IGAGAAGGCAAGGCUGCCUU 2125 rs2530588 3818 AGGCAGCCUUGCCUUCUCU 374 3818 AGGCAGCCUUGCCUUCUCU 374 3836 AGAGAAGGCAAGGCUGCCU 2126 rs2530588 3819 GGCAGCCUUGCCUUCUCUA 375 3819 GGCAGCCUUGCCUUCUCUA 375 3837 UAGAGAAGGCAAGGCUGCC 2127 rs2530588 3820 GCAGCCUUGCCUUCUCUAA 376 3820 GCAGCCUUGCCUUCUCUAA 376 3838 UUAGAGAAGGCAAGGCUGC 2128 rs2530588 3821 CAGCCUUGCCUUCUCUAAC 377 3821 CAGCCUUGCCUUCUCUAAC 377 3839 GUUAGAGAAGGCAAGGCUG 2129 rs25305B8 3803 CUGGACCCGCAAUAAAGGA 378 3803 CUGGACCCGCAAUAAAGGA 378 381 UCCUUUAUUGCGGGUCCAG- 2130 rs2530588 3804 UGGACCCGCAAUAAAGGAA 379 3804 UGGACCCGCAAUAAAGGAA 379 3822 IUUCCUUUAUUGCGGGUCCA- 2131 rs2530588 3805 GGACCCGCAAUAAAGGAAG 380 3805 GGACCCGCAAUAAAGGAAG 380 3823 ICUUCCUUUAUUGCGGGUCC 2132 rs2530588 3806 GACCCGCAAUAAAGGAAGC 381 3806 GACCCGCAAUAAAGGAAGC 381 3824 GCUUCCUUUAUUGCGGGUC 2133 rs2530588 3807 ACCCGCAAUAAAGGAAGCC 382 3807 ACCCGCAAUAAAGGAAGCC 382 3825 GGCUUCCUUUAUUGCGGGU 2134 rs2530588 3808 CCCGCAAUAAAGGAAGCCU 383 3808 CCCGCAAUAAAGGAAGCCU 383 3826 JAGGCUUCCUUUAUUGCGGG 2135 rs2530588 3809 CCGCAAUAAAGGAAGCCUU 384 3809 CCGCAAUAAAGGAAGCCUU 384 3827 IAAGGCUUCCUUUAUUGCGG 2136 rs2530588 3810 CGCAAUAAAGGAAGCCUUG 385 3810 CGCAAUAAAGGAAGCCUUG 385 3828 CAAGGCUUCCUUUAUUGCG 12137 rs2530588 81 1 GCAAUAAAGGAAGCCUUGC 36 3811 GCAAUAAAGGAAGCCUUGC 386 3829 IGCAAGGCUUCCUUUAUUGC 2138 222 2005201389 01 Apr 2005 rs2530588 3812 CAAUAAAGGAAGCCUUGCC 387 3812 CAAUAAAGGAAGCCUUGCC 387 3830 GGCAAGGCUUCCUUUAUUG 2139 rs2530588 3813 AAUAAAGGAAGCCUUGCCU 388 3813 AAUAAAGGAAGCCUUGCCU 388 3831 AGGCAAGGCUUCCUUUAUU 2140 rs2530588 3814 AUAAAGGAAGCCUUGCCUU 389 3814 AUAAAGGAAGCCUUGCCUU 389 3832 AAGGCAAGGCUUCCUUUAU 2141 rs2530588 3815 UAAAGGAAGCCUUGCCUUC 390 3815 UAAAGGAAGCCUUGCCUUC 390 3833 GAAGGCAAGGCUUCCUUUA 2142 rs2530588 3816 AAAGGAAGCCUUGCCUUCU 391 3816 AAAGGAAGCCUUGCCUUCU 391 3834 AGAAGGCAAGGCUUCCUUU 2143 rs2530588 3817 AAGGAAGCCUUGCCUUCUC 392 3817 AAGGAAGCCUUGCCUUCUC 392 3835 GAGAAGGCAAGGCUUCCUU 2144 rs2530588 3818 AGGAAGCCUUGCCUUCUCU 393 3818 AGGAAGCCUUGCCUUCUCU 393 3836 AGAGAAGGCAAGGCUUCCU 2145rs2530588 3819 GGAAGCCUUGCCUUCUCUA 394 3819 GGAAGCCUUGCCUUCUCUA 394 3837 UAGAGAAGGCAAGGCUUCC 2146 rs2530588 3820 GAAGCCUUGCCUUCUCUAA 395 3820 GAAGCCUUGCCUUCUCUAA 395 3838 UUAGAGAAGGCMAGGCUUC 2147 rs2530588 3821 AAGCCUUGCCUUCUCUAAC 396 3821 AAGCCUUGCCUUCUCUAAC 396 3839 GUUAGAGAAGGCAAGGCUU 2148 rs3025843 3822 AGCCUUGCCUUCUCUAACA 397 3822 AGCCUUGCCUUCUCUAACA 397 3840 UGUUAGAGAAGGCAAGGCU 2149 rs3025843 3823 GCCUUGCCUUCUCUAACAA 398 3823 GCCUUGCCUUCUCUAACAA 398 3841 UUGUUAGAGAAGGCAAGGC 2150 rs3025843 3824 CCUUGCCUUCUCUAACAAA 399 3824 CCUUGCCUUCUCUAACAAA 399 3842 UUUGUUAGAGAAGGCAAGG 2151 rs3025843 3825 CUUGCCUUCUCUAACAAAC 400 3825 CUUGCCUUCUCUAACAAAC 400 3843 GUUUGUUAGAGAAGGCAAG 2152 rs3025843 3826 UUGCCUUCUCUAACAAACC 401 3826 UUGCCUUCUCUAACAAACC 401 3844 GGUUUGUUAGAGAAGGCAA 2153 rs3025843 3827 UGCCUUCUCUAACAAACCC 402 3827 UGCCUUCUCUAACAAACCC 402 3845 GGGUUUGUUAGAGAAGGCA 2154 rs3025843 3828 GCCUUCUCUAACAAACCCC 403 3828 GCCUUCUCUAACAAACCCC 403 3846 GGGGUUUGUUAGAGAAGGC 2155 rs3025843 3829 CCUUCUCUAACAAACCCCC 404 3829 CCUUCUCUAACAAACCCCC 404 3847 GGGGGUUUGUUAGAGAAGG 2156 rs3025843 3830 CUUCUCUAACAAACCCCCC 405 3830 CUUCUCUAACAAACCCCCC 405 3848 GGGGGGUUUGUUAGAGAAG 2157 rs3025843 3831 UUCUCUAACAAACCCCCCU 406 3831 UUCUCUAACAAACCCCCCU 406 3849 AGGGGGGUUUGUUAGAGAA 2158 rs3025843 3832 UCUCUAACAAACCCCCCUU 407 3832 UCUCUAACAAACCCCCCUU 407 3850 AAGGGGGGUUUGUUAGAGA 2159 rs3025843 3833 CUCUMACAAACCCCCCUUC 408 3833 CUCUAACAAACCCCCCUUC 408 3851 GAAGGGGGGUUUGUUAGAG 2160 rs3025843 3834 UCUAACAAACCCCCCUUCU 409 3834 UCUAACAAACCCCCCUUCU 409 3852 AGAAGGGGGGUUUGUUAGA 2161 rs3025843 3835 CUAACAAACCCCCCUUCUC 410 3835 CUAACAAACCCCCCUUCUC 410 3853 GAGAAGGGGGGUUUGUUAG 2162 rs3025843 3836 UAACAAACCCCCCUUCUCU 411 3836 UAACAAACCCCCCUUCUCU 411 3854 AGAGMAGGGGGGUUUGUUA 2163 rs3025843 3837 AACAAACCCCCCUUCUCUA 412 3837 AACAAACCCCCCUUCUCUA 412 3855 JUAGAGAAGGGGGGUUUGUU 2164 rs3025843 3838 ACAAACCCCCCUUCUCUAA 413 3838 ACAAACCCCCCUUCUCUAA 413 3856 UUAGAGAAGGGGGGUUUGU 2165 rs3025843 3820 GCAGCCUUGCCUUCUCUAG 414 3820 GCAGCCUUGCCUUCUCUAG 414 3838 CUAGAGMAGGCAAGGCUGC 2166 rs3025843 3821 CAGCCUUGCCUUCUCUAGC 415 3821 CAGCCUUGCCUUCUCUAGC 415 3839 GCUAGAGAAGGCAAGGCUG 2167 rs3025843 3822 AGCCUUGCCUUCUCUAGCA 416 3822 AGCCUUGCCUUCUCUAGCA 416 3840 UGCUAGAGAAGGCAAGGCU 2168 rs3025843 3823 GCCUUGCCUUCUCUAGCAA 417 3823 GCCUUGCCUUCUCUAGCAA 417 3841 UUGCUAGAGAAGGCAAGGC 2169 rs3025843 3824 CCUUGCCUUCUCUAGCAAA 418 3824 CCUUGCCUUCUCUAGCAAA 418 3842 UUUGCUAGAGAAGGCAAGG 2170 rs3025843 3825 CUUGCCUUCUCUAGCAAAC 419 3825 CUUGCCUUCUCUAGCAAAC 419 3843 GUUUGCUAGAGAAGGCAAG 2171 rs3025843 3826 UUGCCUUCUCUAGCAAACC 420 3826 UUGCCUUCUCUAGCAAACC 420 3844 GGUUUGCUAGAGAAGGCAA 2172 rs3025843 3827 UGCCUUCUCUAGCAAACCC 421 3827 UGCCUUCUCUAGCAAACCC 421 3845 GGG UUUGCUAGAGAAGGCA 2173 rs3025843 3828 GCCUUCUCUAGCAAACCCC 422 3828 GCCUUCUCUAGCAAACCCC 422 3846 GGGGUUUGCUAGAGAAGGC 2174 rs3025843 3829 CCUUCUCUAGCAAACCCCC 423 3829 CCUUCUCUAGCAAACCCCC 423 3847 GGGGGUUUGCUAGAGAAGG 2175 rs3025843 3830 CUUCUCUAGCAAACCCCCC 424 3830 CUUCUCUAGCAAACCCCCC 424 3848 GGGGGGUUUGCUAGAGAAG 2176 rs3025843 3831 UUCUCUAGCMAACCCCCCU 425 3831 UUCUCUAGCAAACCCCCCUI 42-5 3849 AGGGGGGUUUGCUAGAGAA 2177 2005201389 01 Apr 2005 rs3025843 3832 UCUCUAGCAAACCCCCCUU 426 3832 UCUCUAGCAAACCCCCCUU 426 3850 AAGGGGGGUUUGCUAGAGA 2178 rs3025843 3833 CUCUAGCAAACCCCCCUUC 427 3833 CUCUAGCAAACCCCCCUUC 427 3851 GAAGGGGGGUUUGCUAGAG 2179 rs3025843 3834 UCUAGCAAACCCCCCUUCU 428 3834 UCUAGCAAACCCCCCUUCU 428 3852 AGAAGGGGGGUUUGCUAGA 2180 rs3025843 3835 CUAGGAAACCCCCCUUCUC 429 3835 CUAGCAAACCCCCCUUCUC 429 3853 GAGAAGGGGGGUUUGCUAG 2181 rs3025843 3836 UAGCMAACCCCCCUUCUCU 430 3836 UAGCAAACCCCCCUUCUCU 430 3854 AGAGAAGGGGGGUUUGCUA 2182 rs3025843 3837 AGCMAACCCCCCUUCUCUA 431 3837 AGCAAACCCCCCUUCUCUA 431 3855 UAGAGAAGGGGGGUUUGCU 2183 rs3025843 3838 GCAAACCCCCCUUCUCUAA 432 3838 GCAAACCCCCCUUCUCUAA 432 3856 UUAGAGAAGGGGGGUUUGC 2184 rs4690074 4104 AAAGUUUGGAGGGUUUCUC 433 4104 AAAGUUUGGAGGGUUUCUC 433 4122 GAGAAACCCUCCAAACUUU- 2185 rs4690074 4105 AAGUUUGGAGGGUUUCUCC 434 4105 AAGUUUGGAGGGUUUCUCC 434 4123 GGAGAAACCCUCCAAACUU 2186 rs4690074 4106 AGUUUGGAGGGUUUCUCCG 435 4106 AGUUUGGAGGGUUUCUCCG 435 4124 CGGAGAAACCCUCCAAACU 2187 rs4690074 4107 GUUUGGAGGGUUUCUCCGC 436 4107 GUUUGGAGGGUUUCUCCGC 436 4125 GCGGAGAAACCCUCCAAAC 2188 rs4690074 4108 UUUGGAGGGUUUCUCCGCU 437 4108 UUUGGAGGGUUUCUCCGCU 437 4126 AGCGGAGAAACCCUCCAAA 2189 rs4690074 4109 UUGGAGGGUUUCUCCGCUC 438 4109 UUGGAGGGUUUCUCCGCUC 438 4127 GAGCGGAGAAACCCUCCAA 2190 rs4690074 4110 UGGAGGGUUUCUCCGCUCA 439 4110 UGGAGGGUUUCUCCGCUCA 439 4128 UGAGCGGAGAAACCCUCCA 2191 rs4690074 4111 GGAGGGUUUCUCCGCUCAG 440 4111 GGAGGGUUUCUCCGCUCAG 440 4129 CUGAGCGGAGAAACCCUCC 2192 rs4690074 4112 GAGGGUUUCUCCGCUCAGC 441 4112 GAGGGUUUCUCCGCUCAGC 441 4130 GCUGAGCGGAGAAACCCUC 2193 rs46007 413 AGGUUCUCGCUAGC 44 411 AGGUUCUCGCUCGCC 442 413 GGUGAGGGAAAACCU 219 rs4690074 4114 AGGGUUUCUCCGCUCAGCC 443 4114 GGGUUUCUCCGCUCAGCC 443 4132 GGCUGAGCGGAGAAACCCU 2195 rs4690074~~ 4153UUUCCCGCU 44 41 GUCCGUACU 4 1332 AGUACGAAAC 29 rs4690074 4116 GGUUUCUCCGCUCAGCCU 445 4116 GGUUUCUCCGCUCAGCCU 445 4134 CAGGCUGAGCGGAGAAAC 2197 rs469074 417 UUUUCCGCCAGCCUGG 44 4117 UUUCUCGCUCGCCUUG 446 41353 CAGUACGAAA 29 rs4690074 4118 GGUUCUCCGCUCAGCCUU 447 4118 GGUUCUCCGCUCAGCCUU 447 4136 UAAGGCUGAGCGGAGAA 2199 rs4690074 4119 GUUCUCCGCUCAGCCUUG 448 4119 GUUCUCCGCUCAGCCUUG 448 4137 ACAAGGCUGAGCGGAGA 2200 rs4690074 4120 UUCUCCGCUCAGCCUUGG 449 4120 UUCUCCGCUCAGCCUUGG 449 4138 CCCAAGGCUGAGCGGAGA 221 rs4690074 4118 UUCCGCUCAGCCUUGGA 450 4121 UCCGCUCAGCCUUGGA 450 4139 AAUCCAAGGCUGAGCGGGA 2202 rs4690074 4122 UCCGCUCAGCCUUGGAU 451 4122 UCCCGCUCAGCCUUGGAU 451 4140 AAUCCAAGGCUGAGCGGG 2203 rs4690074 4120 AAGUCUUGGAUU 452 4120 AACGUCUUGGAUU 452 4122 AAGuCAACCCUCAU 2204 rs4690074 4105 AGUCCUUGGAUU 453 4105 AACGUCAUUGGAUU 453 4139 GAAC CUCCGUAAACUU 2205 rs4690074 4106 AGUUGAGGUUGUUU 454 4106 AGUUGAGGUUGUUU 454 414 CAAAAUCCUAAACU 2206 rs4690074 4107 AGUUUGGAGGGUUUCUU 455 4107 AGUUUGGAGGGUUUCUUG 455 4125 GAAGAAACCCUCCAAAC 2207 rs4690074 4108 AAUUUGGAGGGUUUCUUC 456 4108 AAUUUGGAGGGUUUCUUC 456 4126 AGAAGAAACCCUCCAAA 2208 rs4690074 4109 AGUUGGAGGGUUUCUUCG 457 4109 AGUUGGAGGGUUUCUUCG 457 4127 GCGAAGAAACCCUCCAA 2209 rs4690074 410 GUGGAGGGUUUCUUCGC 458 410 GUGGAGGGUUUCUUCGC 458 4128 UGCGAAGAAACCCUCCAC 2207 rs4690074 4111 UUGGAGGGUUUCUCGCU 459 4111 UUGGAGGGUUUCUUCGCU 459 4129 CAGCGAAGAAACCCUCC 2211 rs4690074 4109 UUGAGGGUUUCUUCGCUC 460 4112 UUGAGGGUUUCUUCGCUC 460 4130 GGAGCGAAGAAACCCUCA 2212 rs4690074 4113 UGAGGGUUUCUUCGCUCA 461 4113 UGAGGGUUUCUUCGCUCA 461 4131 GUGAGCGAAGAAACCCUC 2213 rs4690074 4114 GGGGUUUCUUCGCUCAG 462 4114 GGGGGUUUCUUCGCUCAG 462 4129 ACUGAGCGAAGAAACCCC 2214 rs4690074 4115 GAGGUUUCUUCGCUCAGC 463 4115 GAGGUUUCUUCGCUCAGC 463 4133 AGCUGAGCGAAGAAACCC 2215 rs4690074 4116 AGGUUUCUUCGCUCAGCC 464 4116 AGGUUUCUUCGCUCAGCC 464 4134 CGGCUGAGCGAAGAAACC 2216 224 2005201389 01 Apr 2005 rs4690074 4117 UUUCUUCGCUCAGCCUUGG 465 4117 UUUCUUCGCUCAGCCUUGG 465 4135 CCAAGGCUGAGCGAAGAAA- 2217 rs4690074 4118 UUCUUCGCUCAGCCUUGGA 466 4118 UUCUUCGCUCAGCCUUGGA 466 4136 UCCAAGGCUGAGCGAAGAA 2218 rs4690074 4119 UCUUCGCUCAGCCUUGGAU 467 4119 UCUUCGCUCAGCCUUGGAU 467 4137 AUCCAAGGCUGAGCGAAGA 2219 rs4690074 4120 CUUCGCUCAGCCUUGGAUG 468 4120 CUUCGCUCAGCCUUGGAUG 468 4138 CAUCCAAGGCUGAGCGAAG_ 2220 rs4690074 4121 UUCGCUCAGCCUUGGAUGU 469 4121 UUCGCUCAGCCUUGGAUGU 469 4139 ACAUCCAAGGCUGAGCG3AA 2221 rs4690074 4122 UCGCUCAGCCUUGGAUGUU 470 4122 UCGCUCAGCCUUGGAUGUU 470 4140 AACAUCCAAGGCUGAGCGA 2222 rs3025837 4456 GUGCAGGCGGAGCAGGAGA 471 4456 GUGCAGGCGGAGCAGGAGA 471 4474 UCUCCUGCUCCGCCUGCAC. 2223 rs3025837 4457 UGCAGGCGGAGCAGGAGAA 472 4457 UGCAGGCGGAGCAGGAGAA 472 4475 UUCUCCUGCUCCGCCUGCA- 2224 rs3025837 4458 GCAGGCGGAGCAGGAGAAC 473 4458 GCAGGCGGAGCAGGAGAAC 473 4476 GUUCUCCUGCUCCGCCUGC 2225 rs3025837 4459 CAGGCGGAGCAGGAGAACG 474 4459 CAGGCGGAGCAGGAGAACG 474 4477 CGUUCUCCUGCUCCGCCUG 2226 rs3025837 4460 AGGCGGAGCAGGAGAACGA 475 4460 AGGCGGAGCAGGAGAACGA 475 4478 UCGUUCUCCUGCUCCGCCU 2227 rs3025837 4461 GGCGGAGCAGGAGAACGAC 476 4461 GGCGGAGCAGGAGAACGAC 476 4479 GUCGUUCUCCUGCUCCGCC 2228 rs3025837 4462 GCGGAGCAGGAGAACGACA 477 4462 GCGGAGCAGGAGAACGACA 477 P4480 UGUCGUUCUCCUGCUCCGC 2229 rs3025837 4463 CGGAGCAGGAGAACGACAC 478 4463 CGGAGCAGGAGAACGACAC 478 4481 GUGUCGUUCUCCUGCUCCG 2230 rs3025837 4464 GGAGCAGGAGAACGACACC 479 4464 GGAGCAGGAGAACGACACC 479 4482 GGUGUCGUUCUCCUGCUCC 2231 rs3025837 4465 GAGCAGGAGAACGACACCU 480 4465 GAGCAGGAGAACGACACCU 480 4483 AGGUGUCGUUCUCCUGCUC 2232 rs3025837 4466 AGCAGGAGAACGACACCUC 481 4466 AGCAGGAGAACGACACCUC 481 4484 GAGGUGUCGUUCUCCUGCU 2233 rs3025837 4467 GCAGGAGAACGACACCUCG 482 4467 GCAGGAGAACGACACCUCG 482 4485 CGAGGUGUCGUUCUCCUGC 2234 rs3025837 4468 CAGGAGAACGACACCUCGG 483 4468 CAGGAGAACGACACCUCGG 483 4486 CCGAGGUGUCGUUCUCCUG 2235 rs3025837 4469 AGGAGAACGACACCUCGGG 484 4469 AGGAGAACGACACCUCGGG 484 4487 CCCGAGGUGUCGUUCUCCU 2236 rs3025837 4470 GGAGAACGACACCUCGGGA 485 4470 GGAGAACGACACCUCGGGA 485 4488 UCCCGAGGUGUCGUUCUCC 2237 rs3025837 4471 GAGAACGACACCUCGGGAU 486 4471 GAGAACGACACCUCGGGAU 486 4489 AUCCCGAGGUGUCGUUCUC 2238 rs3025837 4472 AGAACGACACCUCGGGAUG 487 4472 AGAACGACACCUCGGGAUG 487 4490 CAUCCCGAGGUGUCGUUCU 2239 rs3025837 4473 GAACGACACCUCGGGAUGG 488 4473 GAACGACACCUCGGGAUGG 488 4491 CCAUCCCGAGGUGUCGUUC 2240 rs3025837 4474 AACGACACCUCGGGAUGGU 489 4474 AC GACACCUCGGGAUGGU 489 4492 ACCAUCCCGAGGUGUCGUU 2241 rs3025837 4456 GUGCAGGCGGAGCAGGAGC 490 4456 GUGCAGGCGGAGCAGGAGC 490 4474 GCUCCUGCUGCGCCUGCAC- 2242 rs3025837 4457 UGCAGGCGGAGCAGGAGCA 491 4457 UGCAGGCGGAGCAGGAGCA 491 4475 UGCUCCUGCUCCGCCUGCA 2243 rs3025837 4458 GCAGGCGGAGCAG;GAGCAC 492 4458 GCAGGCGGAGCAGGAGC;AC 492 4476 GUGCUCCUGCUCCGCCUGC 2244 rs3025837 4459 CAGGCGGAGCAGGAGCACG 493 4459 CAGGCGGAGCAGGAGCACG 493 4477 CGUGCUCCUGCUCCGCCUG 2245 rs3025837 4460 AGGCGGAGCAGGAGCACGA 494 4460 AGGCGGAGCAGGAGCACGA 44 4478 UCGUGCUCCUGCUCCGCCU 2246 rs3025837 4461 GGCGGAGCAGGAGGACGAC 495 4461 GGCGGAGCAGGAGCACGAC 495 449 GUCGUGCUCCUGCUCCGCC; 2247 rs3025837 4462 GCGGAGCAGGAGCACGACA 496 4462 GCGGAGCAGGAGCACGACA 496 4480 UGUCGUGCUCCUGCUCCGC 2248 rs3025837 4463 CGGAGCAGGAGCACGACAC 497 4463 CGGAGCAGGAGCACGACAC 497 4481 GUGUCGUGCUCCUGCUCCG 2249 rs3 258 7 4 64 GA CAG AGC CGA AC 49 44 4 G AGC GG GCA GAC CC 984478 2 G U C U C C U C CC rs3025837 4465 GGAGCAGGAGCACGACACC 499 4465 GGAGCAGGAGCACGACACC 499 4483 GGUGUCGUGCUCCUGCUCC 2251 rs302537 446 AGCAGAGCACGCACCU 500 466 AGCGGAGCAGACACCC 500 44841AGGCUCUCGU rs3025837 4467 GGCAGGAGCACGACACCU 501 4467 GGCAGGAGCACGACACCU 501 4485 CAGGUGUCGUGCUCCUGC 2253 rs3025837 4468 ACAGGAGCACGACACCUCG 502 4468 ACAGGAGCACGACACCUCG 502 4486 CGAGGUGUCGUGCUCCUG 2254 rs3025837 4469 GAGGAGCACGACACCUCGG 503 4469 GAGGAGCACGACACCUCGG 503 4487 CGAGGUGUCGUGCUCCU 2255 2005201389 01 Apr 2005 rs3025837 4470 GGAGCACGACACCUCGGGA 504 4470 GGAGCACGACACCUCGGGA 504 4488 UCCCGAGGUGUCGUGCUCC 2256 rs3025837 4471 GAGCACGACACCUCGGGAU 505 4471 GAGCACGACACCUCGGGAU 505 4489 AUCCCGAGGUGUCGUGCUC 2257 rs3025837 4472 AGCACGACACCUCGGGAUG 506 4472 AGCACGACACCUCGGGAUG 506 4490 CAUCCCGAGGUGUCGUGCU 2258 rs3025837 4473 GCACGACACCUCGGGAUGG 507 4473 GCACGACACCUCGGGAUGG 507 4491 CCAUCCCGAGGUGUCGUGC 2259 rs3025837 4474 CACGACACCUCGGGAUGGU 508 4474 CACGACACCUCGGGAUGGU 508 4492 ACCAUCCCGAGGUGUCGUG 2260 rs363129 4967 UCUUUGUAUUAAGAGGAAC 509 4967 UCUUUGUAUUAAGAGGAAC 509 4985 GUUCCUCUUAAUACAAAGA 2261 rs363129 4968 CUUUGUAUUAAGAGGMACA 510 4968 CUUUGUAUUAAGAGGAACA 510 4986 UGUUCCUCUUAAUACAAAG 2262 rs363129 4969 UUUGUAUUAAGAGGAACAA 511 4969 UUUGUAUUAAGAGGAACAA 511 4987 UUGUUCCUCUUAAUACAAA 2263 rs363129 4970 UUGUAUUAAGAGGAACAAA .512 4970 UUGUAUUAAGAGGAACAAA 512 4988 UUUGUUCCUCUUAAUACAA 2264 rs363129 4971 UGUAUUAAGAGGAACAAAU 513 4971 UGUAUUAAGAGGAACAAAU 513 4989 AUUUGUUCCUCUUAAUACA 2265 rs363129 4972 GUAUUAAGAGGAACAAAUA 514 4972 GUAUUAAGAGGAACAAAUA 514 4990 UAUUUGUUCCUCUUAAUAC 2266 rs363129 4973 UAUUAAGAGGAACAAAUAA 515 4973 UAUUAAGAGGAACAAAUAA 515 4991 UUAUUUGUUCCUCUUAAUA 2267 rs363129 4974 AUUAAGAGGAACAAAUAAA 516 4974 AUUAAGAGGAACAAAUAAA 516 4992 UUUAUUUGUUCCUCUUAAU 2268 rs363129 4975 UUAAGAGGAACAAAUAAAG 517 4975 UUAAGAGGAACAAAUAAAG, 517 4993 CUUUAUUUGUUCCUCUUAA 2269 rs363129 4976 UAAGAGGAACAAAUAAAGC 518 4976 UAAGAGGAACAAAUAAAGC 518 4994 GCUUUAUUUGUUCCUCUUA 2270 rs363129 4977 AAGAGGAACAAAUAAAGCU 519 4977 AAGAGGAACAAAUAAAGCU 519 4995 AGCUUUAUUUGUUCCUCUU 2271 rs363129 4978 AGAGGAACAAAUAAAGCUG 520 4978 AGAGGAACAAAUAAAGCUG 520 4996 CAGCUUUAUUUGUUCCUCU 2272 rs363129 4979 GAGGAACAAAUAAAGCUGA 521 4979 GAGGAACAAAUAAAGCUGA 521 4997 UCAGCUUUAUUUGUUCCUC 2273 rs363129 4980 AGGAACAAAUAAAGCUGAU 522 4980 AGGAACMAAUAAAGCUGAU 522 4998 AUCAGCUUUAUUUGUUCCU 2274 rs363129 4981 GGAACAAAUAAAGCUGAUG 523 4981 GGAACAAAUAAAGCUGAUG 523 4999 CAUCAGCUUUAUUUGUUCC 2275 rs363129 4982 GAACAAAUAAAGCUGAUGC 524 4982 GMACAAAUAAAGCUGAUGC 524 5000 GCAUCAGCUUUAUUUGUUC 2276 rs363129 4983 AACAAAUAAAGCUGAUGCA 525 4983 AACAAAUAAAGCUGAUGCA 525 5001 UGCAUCAGCUUUAUUUGUU 2277 rs363129 4984 ACAAAUAAAGCUGAUGCAG 526 4984 ACAAAUAAAGCUGAUGCAG 526 5002 CUGCAUCAGCUUUAUUUGU 2278 rs363129 4985 CAAAUAAAGCUGAUGCAGG 527 4985 CAAAUAAAGCUGAUGCAGG 527 5003 CCUGCAUCAGCUUUAUUUG 2279 rs363129 4967 UCUUUGUAUUAAGAGGAAU 528 4967 UCUUUGUAUUAAGAGGAAU 528 4985 AUUCCUCUUAAUACAAAGA 2280 rs363129 4968 CUUUGUAUUAAGAGGAAUA 529 4968 CUUUGUAUUAAGAGGAAUA 529 4986 UAUUCCUCUUAAUACAAAG 2281 rs363129 4969 UUUGUAUUAAGAGGAAUAA 530 4969 UUUGUAUUAAGAGGAAUAA 530 4987 UUAUUCCUCUUAAUACAAA 2282 rs363129 4970 UUGUAUUAAGAGGAAUAAA 531 4970 UUGUAUUAAGAGGAAUAAA 531 4988 UUUAUUCCUCUUMAUACAA 2283 rs363129 4971 UGUAUUAAGAGGAAUAAAU 532 4971 UGUAUUAAGAGGAAUAAAU 532 4989 AUUUAUUCCUCUUAAUACA 2284 rs363129 4972 GUAUUAAGAGGAAUAAAUA 533 4972 GUAUUAAGAGGAAUAAAUA 533 4990 UAUUUAUUCCUCUUAAUAC 2285 rs363129 4973 UAUUAAGAGGAAUAAAUAA 534 4973 UAUUAAGAGGAAUAAAUAA 534 4991 UUAUUUAUUCCUCUUAAUA 2286 rs363129 4974 AUUAAGAGGAAUAAAUAAA 535 4974 AUUAAGAGGAAUAAAUAAA 535 4992 UUUAUUUAUUCCUCUUAAU 2287 rs363129 4975 UUAAGAGGAAUAAAUAAAG 536 4975 UUAAGAGGAAUAAAUAAAG 536 4993 CUUUAUUUAUUCCUCUUAA 2288 rs363129 4976 UAAGAGGAAUAAAUAAAGC 537 4976 UAAGAGGAAUAAAUAAAGC 537 4994 GCUUUAUUUAUUCCUCUUA 2289 rs363129 4977 AAGAGGAAUAAAUAAAGCU 538 4977 AAGAGGAAUAAAUAAAGCU 538 4995 AGCUUUAUUUAUUCCUCUU 2290 rs363129 4978 AGAGGAAUAAAUAAAGCUG 539 4978 AGAGGAAUAAAUAAAGCUG 539 4996 CAGCUUUAUUUAUUCCUCU- 2291 rs363129 4979 GAGGAAUAAAUAAAGCUGA 540 4979 GAGGAAUAAAUAAAGCUGA 540 4997 UCAGCUUUAUUUAUUCCUC- 2292 rs363129 4980 AGGAAUAAAUAAAGCUGAU 541 4980 AGGAAUAAAUAAAGCUGAU 541 4998 AUCAGCUUUAUUUAUUCCU 2293 rs363129 491 GGAAUAAAUAAAGCUGAUG 54 98 GGAAAUAAAGCUGAUG 542 4999 CAUCAGCUUUAUUUAUUCC 2294 226 2005201389 01 Apr 2005 1 00 [CAUCAGCUUUAU UC 2295 V i r AflO'~ (~AAI IAAAI IAAA(~CI I(~AI I 543 rs612 92 AAAAUjMUAAAAAAACUA(GCA 1 44 1 5001 1 UGCAUCAGCUUUAUUUAUU 12296 363129~ 498 -AAAAACGUC 544-~- 4983 rs363129 4984 IAUAAAUAAAGCUGAUGCAG [545 t4984 AUAAAUAAAGCUGAUGCAG 545 rs363129 I 4985 1UAAAUAAAGCUGAUGCAGG 546 4985 rs363125 j5462 1UAAGAGAUGGGGACAGUAC 547 5462 rs363125 jj5463 AAGAGAUGGGGACAGUACU 548 5463 rs36~3125 I5464 IAGAGAUGGGGACAGUACUU 549 I5464

UAAAUAAAGCUGA~

UAAGAGAUGGGGA

AAGAGAUGGGGAC

AGAGAUGGGGACA

rs363125 5465 GAGAUGGGGACAGUACUUC 550 5465 GAGAUGGGGACAG rs363125 5466 AGAUGGGGACAGUACUUCA -551 5466 AGAUGGGGACAGL rs363125 5467 GAUGGGGACAGUACUUCAA 552 5467 GAUGGGGACAGUA rs363125 5468 AUGGGGACAGUACUUCMAC 553 5468 AUGGGGACAGUAC rs363125 5469 UGGGGACAGUACUUCMACG 554 5469 UGGGGACAGUACL rs363125 5470 GGGGACAGUACUUCAACGC 555 5470 GGGGACAGUACUL rs363125 5471 GGGACAGUACUUCAACGCU 556 5471 GGGACAGUACUUC

U

C

A

G

U

A

r

U

C

A

C

;AGA

GUAC

GUAC UU

UACUU

CUU

CUUCA

JUCAA

UCC

CAACG

ACGC

A.CGCU

CGCUA

(3CUAG

CUAGA

IAGA A 547 548 549 550 551 552 -53 554 555 556 557 558 559 560 5002 5003 5480 5481 5482 1 5483 1 5484 5485 -5486 5487 5488 5489 5490 5491 5492 5493 AAGUACUGUCCCCAUU

C

GAAGUACUGUCCCCAUC

U

UGAAGUACUGUCCCCAUCU

UUGAAGUACUGUCCCCAUC

GUUGAAGUACUGUCCCCA

CGUUGAAGUACUGUCCCCA

GCGUUGAAGUACUGUCCCC

AGCGUUGAAGUACUGUCCC

UAGCGUUGAAGUACUGUCC

CUAGCGUUGAAGUACUGU

UCUAGCGUUGAAGUACUGU

U CUAGCGUUGAAGUACUG 2302 2303 2304 2305 2306 2307 2308 2309 2310 2311 2312 2313

CUGCAUCAGCUUUAUUUAU

CCUG3CAUCAGCUUUAUUUA

GUACUGUCCCCAUCUCUUA

AGUACUGUCCCCAUCUCUU

2298 2299 2300 ,2301 rs363125 I5472 IGGACAGUACUUCAACGCUA 557 5472 558 15473 IGACAGUACUUCAA Ts632 5473 1 (ACAGUACUUCAACGC3LUAGj rs363125 5474 ACAkGUACUUCAACGCUAGA 15591 5474 ACAGUACUUCAACG rs363125 5475 CAGUACUUCMACGCUAGAA 15601 5475 CAGUACUUCAACGC rs363125 5476 AGUACUUCAACGCUA(,AAG 561 5476 AGU U I' 561 5494__ rs363125 5477 GUACUUCAACGCUAGMAGA 5 62 5 477 GUACUUCAACGCUAGAAGA 562 5495 UCUUCUAGCGUUGAAGUAC 2314 rs363125 5 478 -UACUUCMACGCUAGAAGAA 563 ,5478 UACUUCAACGCUAGAAGAA 563 5496 UUCUUCUAGCGUUGAAGUA 2315 rs363125 5479 ACUUCAACGCUAGAAGAAC 564 5479 ACUUCAACGCUAGAAGAAC 564 5497 GUUCUUCUAGCGUU"GAAGU 2316 rs363125 5480 CUUCAACGCUAGAAGAACA 565 5480 CUUCAACGCUAGAAGAACA 565 5498 UGUUCUUCUAGCGUUGAAG 2317 rs363125 5462 UAAGAGAUGGGGACAGUAA 566 5462 UAAGAGAUGGGGACAGUAA 566 5480 UUACUGUCCCCAUCUCUUA 2318 rs363125 5463 AAGAGAUGGGGACAGUAAU 567 5463 AAGAGAUGGGGACAGUAAU 567 5481 AUUACUGUCCCCAUCUCUU 2319 rs363125 5 464 AWGAGAUGGGGACAGUAAUU 568 5464 AGAGAUGGGGACAGUAAUU 568 5482 AAUUACUGUCCCCAUCUCU 2320 rs363125 5465 GAGAUGGGGACAGUAAUUC 569 5465 GAGAUGGGGACAGUAAUUC 569 5483 GAAUUACUGUCCCCAUCUC 2321 rs363125 5466 AGAUGGGGACAGUAAUUCA 570 5466 AGAUGGGGACAGUAAUUCA 570 5484 UGAAUUACUGUCCCCAUCU 2322 rs363125 5467 GAUGGGGACAGUAAUUCAA 571 5467 GAUGGGGACAGUAAUUCAA 571 5485 UUGAAUUACUGUCCCCAUC 2323 rs363125 5468 AUGGGGACAGUAAUUCAAC 572 5468 AUGGGGACAGUAAUUCAAC 572 5486 GUUGAAUUACUGUCCCCAU 2324 rs363125 5469 UGGGGACAGUAAUUCAACG 53 5469 UGGGGACAGUAAUUCAACG 573 5487 CGUUGAAUUACUGUCCCCA 2325 II~fA('~574 5488R ((CIJ1(AAUUACUGUCCCC 2326 rs363125 j5470 GGACAGUAAUUCAACGC rs363125 I5471 IGGGACAGUMAUUCAACGCU 54 5470 575 5471 GGGACAGUAAUUCACC 1I 575 5489 5490

AGCGUUGAAUUACUGUCCC

UAGCGUUGAAUUACUGUCC

2328 2329 rs3631 25 rs3631 25 5472 GGACAGUAAUUCAACGCUA 576 5472 GGACAGUAAUUCAA 5431GACAGUAAUUCAACGCUAG I577 I5473 1GACAGUAAUUCAAC ~CGCUA 1~ 576 CUAGCGUUGAAUUACUGUC 577 rs363125 547 ACAGUAAUUCAACGCUAGA j578 5474 ]ACAGUAAUUCAACGCUAGA I5475 JCAGUAAUUCAACGCUAGAA j579 j5475 jCAGUAAUUCAACGCUAGAA Ib~U. 'S A7rI AM~ AI UUCAAfCCUAGAAG I580 I5476 1AGUAAUUCAACGCUAGAAG 578 5492 CUAGCGUUGAAUUACUGUC I I CU AGCGUUGAAUUACUGU I I J 2330 2331 579 5493 580 5494 CUUCUAGCGUUGAAUUACU j2332 581 5- 4-95 J UCUUCUAGCGUUGAAUUAC J2333 rs3315 47 GUAAUUCAACGCUAGAAGA 581 5477J GPUAAUUCAACGCUAGAAGA 2005201389 01 Apr 2005 rs363125 5478 UAAUUCAACGCUAGAAGAA 582 5478 UAAUUCAACGCUAGAAGAA 582 5496 UUCUUCUAGCGUUGAAUUA 2334 rs363125 5479 AAUUCAACGCUAGAAGAAC 583 5479 AAUUCAACGCUAGAAGAAC 583 5497 GUUCUUCUAGCGUUGAAUU 2335 rs363125 5480 AUUCAACGCUAGAAGAACA 584 5480 AUUCAACGCUAGAAGAACA 584 5498 UGUUCUUCUAGCGUUGAAU 2336 rs4690077 6894 GCCCGAGCUGCCUGCAGAG 585 6894 GCCCGAGCUGCCUGCAGAG 585 6912 CUCUGCAGGCAGCUCGGGC 2337 rs4690077 6895 CCCGAGCUGCCUGCAGAGC 586 6895 CCCGAGCUGCCUGCAGAGC 586 6913 GCUCUGCAGGCAGCUCGGG 2338 rs4690077 6896 CCGAGCUGCCUGCAGAGCC 587 6896 CCGAGCUGCCUGCAGAGCC 587 6914 GGCUCUGCAGGCAGCUCGG3 2339 rs4690077 6897 CGAGCUGCCUGCAGAGCCG 588 6897 CGAGCUGCCUGCAGAGCCG 588 6915 CGGCUCUGCAGGCAGCUCG 2340 rs4690077 6898 GAGCUGCCUGCAGAGCCGG 589 6898 GAGCUGCCUGCAGAGCCGG 589 6916 CCGGCUCUGCAGGCAGCUC 2341 rs4690077 6899 AGCUGCCUGCAGAGCCGGC 590 6899 AGCUGCCUGCAGAGCCGGC 590 6917 GCCGGCUCUGCAGGCAGCU 2342 rs4690077 6900 GCUGCCUGCAGAGCCGGCG 591 6900 GCUGCCUGCAGAGCCGGCG 591 6918 CGCCGGCUCUGCAGGCAGC 2343 rs4690077 6901 CUGCCUGCAGAGCCGGCGG 592 6901 CUGCCUGCAGAGCCGGCGG 592 6919 CCGCCGGCUCUGCAGGCAG 2344 rs4690077 6902 UGCCUGCAGAGCCGGCGGC 593 6902 UGCCUGCAGAGCCGGCGGC_ 593 6920 GCCGCCGGCUCUGCAGGCA 2345 rs4690077 6903 GCCUGCAGAGCCGGCGGCC 594 6903 GCCUGCAGAGCCGGCGGCC 594 6921 GGCCGCCGGCUCUGCAGGC 2346 rs4690077 6904 CCUGCAGAGCCGGCGGCCU 595 6904 CCUGCAGAGCCGGCGGCCU 595 6922 AGGCCGCCGGCUCUGCAGG 2347 rs4690077 6905 CUGCAGAGCCGGCGGCCUA 596 6905 CUGCAGAGCCGGCGGCCUA 596 E6923 UAGGCCGCCGGCUCUGCAG 2348 rs4690077 6906 UGCAGAGCCGGCGGCCUAC 597 6906 UGCAGAGCCGGCGGCCUAC 597 6924 GUAGGCCGCCGGCUCUGCA 2349 rs4690077 6907 GCAGAGCCGGCGGCCUACU 598 6907 GCAGAGCCGGCGGCCUACU 598 6925 AGUAGGCCGCCGGCUCUGC 2350 rs4690077 6908 CAGAGCCGGCGGCCUACUG 599 6908 CAGAGCCGGCGGCCUACUG 599 6926 CAGUAGGCCGCCGGCUCUG 2351 rs4690077 6909 AGAGCCGGCGGCCUACUGG 600 6909 AGAGCCGGCGGCCUACUGG 600 6927 CCAGUAGGCCGCCGGCUCU 2352 rs4690077 6910 GAGCCGGCGGCCUACUGGA 601 6910 GAGCCGGCGGCCUACUGGA 601 6928 UCCAGUAGGCCGCCGGCUC 2353 rs4690077 6911 AGCCGGCGGCCUACUGGAG 602 6911 AGCCGGCGGCCUACUGGAG 602 6929 CUCCAGUAGGCCGCCGGCU 2354 rs4690077 6912 GCCGGCGGCCUACUGGAGC 603 6912 GCCGGCGGCCUACUGGAGC 603 6930 GCUCCAGUAGGCCGCCGGC 2355 rs4690077 6894 GCCCGAGCUGCCUGCAGAA 604 6894 GCCCGAGCUGCCUGCAGAA 604 6912 UUCUGCAGGCAGCUCGGGC 2356 rs4690077 6895 CCCGAGCUGCCUGCAGAAC 605 6895 CCCGAGCUGCCUGCAGAAC 605 6913 GUUCUGCAGGCAGCUCGGG 2357 rs4690077 6896 CCGAGCUGCCUGCAGAACC 606 6896 CCGAGCUGCCUGCAGAACC 606 6914 GGUUCUGCAGGCAGCUCGG 2358 rs4690077 6897 CGAGCUGCCUGCAGAACCG 607 6897 CGAGCUGCCUGCAGAACCG 607 6915 CGGUUCUGCAGGCAGCUCG 2359 rs4690077 6898 GAGCUGCCUGCAGAACCGG 608 6898 GAGCUGCCUGCAGAACCGG 608 6916 CCGGUUCUGCAGGCAGCUC 2360 rs4690077 6899 AGCUGCCUGCAGAACCGGC 609 6899 AGCUGCCUGCAGAACCGGC 609 6917 GCCGGUUCUGCAGGCAGCU 2361 rs4690077 6900 GCUGCCUGCAGAACCGGCG 610 6900 GCUGCCUGCAGAACCGGCG 610 6918 CGCCGGUUCUGCAGGCAGC 2362 rs4690077 6901 CUGCCUGCAGAACCGGCGG 611 6901 CUGCCUGCAGAACCGGCGG 611 6919 -CCGCCGGUUCUGCAGGCAG 2363 rs4690077 6902 UGCCUGCAGAACCGGCGGC 612 6902 UGCCUGCAGAACCGGCGGC 612 6920 GCCGCCGGUUCUGCAGGCA 2364 rs4690077 6903 GCCUGCAGAACCGGCGGCC 6-13 3 GCCUGCAGAACCGGCGGCC 613 6921 GGCCGCCGGUUCUGCAGGC 2365 rs4690077 6904 CCUGCAGAACCGGCGGCCU 614 6904 CCUGCAGAACCGGCGGCCU 614 6922 AGGCCGCCGGUUCUGCAGG 2366 rs4690077 6905 CUGCAGAACCGGCGGCCUA 615 6905 CUGCAGAACCGGCGGCCUA 615 6923 UAGGCCGCCGGUUCUGCAG 2367 rs4690077 6906 UGCAGAACCGGCGGCCUAC 61l 6 6906 UGCAGAACCGGCGGCCUAC 616 6924 GUAGGCCGCCGGUUCUGCA 2368 rs4690077 6907 GCAGAACCGGCGGCCUACU 617 6907 GCAGAACCGGCGGCCUACU 617 6925 AG UAGGCCGCCGGUUCUGC- 2369 rs4690077 6908 CAGAACCGGCGGCCUACUG 618 6908 CAGAACCGGCGGCCUACUG n618 6926 CAGUAGGCCGCCGGUUCUG 2370 rs4690077 60 AGACCGGCGGCCUACUGG 619 6909 AGAACCGGCGGCCUACUGG 619 6927 CCAGUAGGCCGCCGGUUCU 2371 rs4690077 6910 GAACCGGCGGCCUCUG 620 6910 GACGCGCCUACUGGA 620 6928 IUCCAGUAGGCCGCCGGUUCI 2372 228 2005201389 01 Apr 2005 rs4690077 6911 AACCGGCGGCCUACUGGAG 621 6911 rs4690077 6912 ACCGGCGGCCUACUGGAGC 622 6912 rs362331 7228 CACGCCUGCUCCCUCAUCU 623 7228 rs362331 7229 ACGCCUGCUCCCUCAUCUA 624 7229 rs362331 7230 CGCCUGCUCCCUCAUCUAC 1625 7230 rs362331 7231 GCCUGCUCCCUCAUCUACU 1 626 7231 -392111 7232 CCUGCUCCCUCAUCUACUG 627 fzj/rs362331 7233 CUGCUCCCUCAUCUACUGU -628 7233 rs3-62331 72 34 -UGCUCCCUCAUCUACUGUG 629 7234 rs362331 7235 GCUCCCUCAUCUACUGUGU 630 7235 rs3233 7236 CUCCCUCAUCUACUGUGUG 631 7236 rs362331 -7237 UCCCUCAUCUACUGUGUGC 632 7237 qczii'ali 7.)q C(C.LICA1JC1ACUGUGUGCA 633 -7238 rs631731CCUUAUUUCC 64 73 rs362331 7240 CCUCAUCUACUGUGUGCAC 6354 24 rs362331 7241 CUCAUCUACUGUGUGCACU 636 7241 rs362331 7242 UCAUCUACUGUGUGCACUU 637 7242 rs362331 7243 CAUCUACUGUGUGCACUUC 638 7243 rs362331 7244 AUCUACUGUGUGCACUUCA 639 7244 rs362331 7245 UCUACUGUGUGCACUUCAU 640 7245 rs362331 ,7246 1UACUGUGUGCACUUCAUCC 641 7246 AACCGGCGGCCUACUGGAG 621 6929 C CACGCCUGCUCCCUCAUCU 623 7246 ACGCCUGCUCCCUCAUCUA 624 7247 CGCCUGCIUCCCUCAUCUAC 625 7248 GCCUGCUCCCUCAUCUACU 1626 7249 CCUGCUCCCUCAUCUACUG3 627 7250z CUEGCUCCCUCAUCUACUG 628 7251 UGCUCCCUCAUCUACUGUG 629 7252 GCUCCCUCAUCUACUGUGU 630 7253 CUCCCUCAUUCGGG 61 75 UCCCUCAUCUACUGUGUG 632 7255 UCCCUCAUCUACUGUGUGC 633 7256 CCCUCAUCUACUGUGUGCA 634 7257 CCUCAUCUACUGUGUGCAC 635 7258 CUCAUCUACUGUGUGCACU 636 7259 UCAUCUACUGUGUGCACUU 637 7260 CAUCUACUGUGUGACUUC 638 7261 UCUACUGUGUGCACUUA 639 7262 UCUACUGUGUGCACUUAU 640 7263 CUACUGUGUGCACUUCAUC 641 7264 CACUGUGUCCCUCAUCC 642 724 ACGCCUGCUCCCUCAUCC 643 7247 CGCCUGCUCCCUCAUCCA 644 7248 CGCCUGCUCCCUCAUCCAU 645 7249 CCUGCUCCCUCAUCCACU 646 7250 CCUGCUCCCUCAUCCACUG 647 7251 UGCUCCCUCAUCCACUGU 648 7252 UGCUCCCUCAUCCACUGUG 649 7253 GCUCCCUCAUCCACUGUGU 650 -72-54 CUCCCUCAUCCACUGUGUG 651 7255 UCCCUCUCCACUGUGUGA 652 7256 CCUCAUCCACUGUGUGCA 653 7257 CUCAUCCACUGUGUGCAC 654 7258 CUCAUCCACUGUGUGCACU 655 7259 UCAUCCACUGUGUGCACUU 656 7260 AUCCACUGUGUGCACUUC 657 7261 UCCACUGUGUGCACUCA 658 7262 UCCACUGUGUGCACUUAU 659 72632:

~UCCAGUAGGCCGCCGGUU

3CUCCAGUAGGCCGCCGGU

~GAUGAGGGAGCAGGCGUG

JAGAUGAGGGAGCAGGCGU

3UAGAUGAGGGAGCAGG A GUAGAUGAGGGAGCAGGC

CAGUAGAUGAGGGAGCAGG

ACAGUAGAUGAGGGAGGAG

CACAGUAGAUGAGGGAGCA

ACACAGUAGAUGAGGGAGC

CACACAGUAGAUGAGGGAG

GCACACAGUAGAUGAGGGA

UGCACACAGUAGAUGAGGG

GUGCACACAGUAGAUGAGG

AGUGCACACAGUAGAUGAG

AAGUGCACACAGUAGAUGA

GAAGUGCACACAGUAGAUG

UGAAGUGCACACAGUAGAU

AUGAAGUGCACACAGUAGA

GAUGAAGUGCACACAGUAG

GGAUGAAGUGCACACAGUA

GGAUGAGGGAGCAGGCGUG

UGGAUGAGGGAGCAGGCGU

GUGGAUGAGGGAGCAGGCG

AGUGGAUGAGGGAGCAGGC

CAGUGGAUGAGGGAGCAGG

ACAGUGGAUGAGGGAGCAG

CACAGUGGAUGAGGGAGCA

ACACAGUGGAUGAGGGAGC

CACACAGUGGAUGAGGGAG

GCACACAGUGGAUGAGGGA_

UGCACACAGUGGAUGA6G

GUGCACACAGUGGAUGAGG

AGUGCACACAGUGGAUGAG

-AAGUGCACACAGUGGAUGA

GAAGUGCACACAGUGGAUG

UGAAGUGCACACAGUGGAU

AUGAAGUGCACACAGUGGA

GAUGAAGUGCACACAGUGG

2373 2374 2375 2376 2377 2378 2379 2380 2381 2382 2383 2384 2385 2386 2387 2388 2389 2390 2391 2392 2393 2394 2395 2396 2397 2398 2399 2400 2401 2402 2403 2404 2405 2406 2407 2408 2409 2410 rs362331 rs362331 rs362331 rs362331 rs362331 rs362331rs362331rs362331rs362331 rs362331s-362331 rs362331 rs362331 rs362331 rs362331 rs362331 rs362331 rs362331 7228 7229 7230 7231 7234 7235 7236

CACGCCUGCUCCCUCAUCC

ACGCCUGCUCCCUCAUCCA

CGCCUGCUCCCUCAUCCAC

GCCUGCUCCCUCAUCCACU

CCUGCUCCCUCAUCCACUG

CUGCUCCCUCAUCCACUGU

UGCUCCCUCAUCCACUGUG

GCUCCCUCAUCCACUGUGU

CUCCCUCAUCCACUGUGUG

643 644 645 646 647 648 649 65-0 7229 7230 7 i23 1 7232 7233 7234 7235 7236 7237 7238 7239 7240 7241 7242 7243 7244 7245

UCCCUCAUCCACUGUGUG

CCUCAUCCACUGUGUGCA

CCUCAUCCACUGU GU[ GCAC

CUCAUCCACUGUGUGCACU-

UCAUCCACUGUGUGCACUU

CAUCCACUGUGUGCACUUC

AUCCACUGUGUGCACUUCA

UCCACUGUGUGCACUUCAU

CCUGUGUGCACUUCAUC

651 7237 652 7238 F653 7239 654 7240 655 7241 656 7242 6T57 7243 658 7244 659 7245 229 2005201389 01 Apr 2005 s-362331 -7246 rs-3025818 7365 rs3025818 7366 rs3025818 76 rs302581 8 7368 rs3025818 7369 rs:3-025818 7 370 rs3-025818 7-371 rs3025818 7372 rs3025818 737-3 rs3025818 7374 rs3025818 7375 rs3025818 7376 rs3025818 7377 rs3025818 7378 rs3025818 7379 rs3025818 7380 rs3025818 7381 rs3025818 7382 rs3-025818 7383 ri3-02581 8 7365 rs3025818 7366 ii3025818 7367 rs3025818 -7368 rs3025818 7369 rsi3-025818 7 370 7s3025 81 TF7371 rs3025818 7372 rs3025818 7373 rs3025818 7- 374 rs0281 7375rs3025818 7376 rs302581 8 7377 rs3025818 7378 rs30258 18 73798 rs3025818 78 rs302581 8 -7381 rs3025818 73821 rs3025818 7382

CACUGUGUGCACUUCAUCC

AAACACACAGAAUCCUAAG

AACACACAGAAUCCUAAGU

ACACACAGAAUCCUAAGUA

CACACAGAAUCCUAAGUAU

ACACAGAAUCCUAAGUAUA

CACAGAAUCCUAAGUAUAU

ACAGAAUCCUAAGUAUAUC

CAGAAUCCU]AAGUAUAUCA

ATGAAUCCUAAGUAUAUCAC

GAAUCCUAAGUAUAUCACU

AAUCCUAAGUAUAUCACUG

AUCCUAAGUAUAUCACUGc

UCCUMAGUAUAUCACUGCA

CCUMAGUAUAUCACUGCAG

CUAAGUAUAUCACUGCAGC

UAAGUAUAUCACUGCAGCC

AAGUAUAUCACUGCAGCCU

AGUAUAUCACUGCAGCCUG

GUAUAUCACUGCAGCCUGU

AAACACACAGAAUCCUAAA

AACACACAGMAUCCUAAAU

ACACACAGAAUCCUAAAUA

CACACAGAAUCCUAAAUAU

ACACAGAAUCCUAAAUAUA

CACAGAAUCCUAAAUAUAU

ACAGAAUCCUAAAUAUAUC

CAGAAUCCUAAAUAUAUCA

AGAAUCCUAAAUAUAUCAC

GAAUCCUAAAUAUAUCACU

AAUCCUAAAUAUAUCACUG

AUCCLJAAAUAUAUCACUGC

UCCUAAAUAUAUCACUGCA

CCUAAAUAUAUCACUGCAG

CUAAAUAUAUCACUGCAGC

UAAAUAUAUCACUGCAGCC

AAAUAUAUCACUUAGCCU

AAUAUAUCACUGCAGCCUG

AUAUAUCACUGCAGCCUGU

660 7246 CACUGUGUGCACUUCAUCC 661 7365 AAA CACACAGAAUCCUAAG 662 7366 AACACACAGAAUCCUAAGU 663 7367 ACACACAGAAUCCUAAG3UA 664 7368 CACACAGAAUCCUAAGUAU 665 7369 ACACAGAAUCCUAAGUAUA 666 7370 CACAGAAUCCUAAGUAUAU 667 7371 ACAGAAUCCUAAGUAUAUC 668 7372 CAGAAUCCUAAGUAUAUCA 669 7373 AGAAUCCUAAGUAUAUCAC 670 7374 GAAUCCUAAGUAUAUCACU 671 7375 AAUCCUAAGUAUAUCACUG 672 7376 AUCCUAAGUAUAUCACUGC 673 7377 UCCUAAGUAUAUCACUGCA 674 7378 CCUAAGUAUAUCACUGCAG 675 7379 CUAAGUAUAUCACUGC(AGC 676 7380 UAAGUAUAUCACUGCAGCC 677 7381 AAGUAUACACUGCAGCCU 678 7382ts AGUAUAUCACUGCAGCCUG 679 7383 GUAUAUCACUGCAGCCUGU 680 7365 AAACACACAGAAUCC;UAAA 681 7366 AACACACAGAAUCCUAAAU 682 7367 ACACACAGAAUCCUAAAUA 683 7368 CACACAGAAUCCUAAAUAU 64 7369 ACACAGAAUCCUAAAUAUA 685 7370 CACAGMAUCCUAAAUAUAU 686 7371 ACAGMAUCCUAAAUAUAUC 687 7372 CAGAAUCCUAAAUAUAUCA 688 7373 AGAAUCCUAWAUAUAUCAC 689 7374 GOAAU1CCUlAAAUAUAUCACU 690 7375 AAUCCLUAAAUAUAUCACUG 691 73V76 AUCCUAAAUAUAUCACUGC 692 7377 UCCUAAAUAUAUCACUGCA 693 7378 CCUAAAUAUAUCACUGCAG3 694 7379 CUAAAUAUAUCACUG3CAGiC 695 7380 UAAAUAUAUCACUGCAGCC 696 7381 AAAUAUAUCACUGCAGCCU 697 7382 AAUAUAUCACUGCAGCC;UG 698 ,7383 AUAUAUCACUGCAGCCUG 660 661 662 663 664 665 666 667 7264 7383 7384 7385] 7386] 7387 7388 7389

GGAUGAAGUGCACACAGUG

CUUAGGAUUCUGUGUGUUU

ACUUAGGAUUCUGUGUGUU

[UACUUAGGAUUCUGUGUGU

AUACUUAGGAUUCUGUGUG

-UAUACUUAGGAUUCUGUGU

AUAUACUUAGGAUUCUGUG

GAUAUACUUAGGAUUCUGU

2412 2413 2414 2415 2416 2417 2418 2419j 669 670 671 672 673 674 675 676 67-7 678 679 680 681 682 683 684 685 686 687 688s 689 690 691 692 693 694 695 696 697 698 7391 7392 7393 7394 7395 7396 7397 7398 7399 7400 7401 7383 7384 7385 7386 7387 7388 7389 7390 7391 7392 7i393 7394 7395 7396 7397 7398 7399 7400 7401

UGAUAUACUUAGGAUUCUG

GUGAUAUACUUAGGAUUCU

AGUGAUAUACUUAGGAUUC

CAGUGAUAUACUUAGGAUU

GCAGUGAUAUACUUAGGA

UGCAGUGAUAUACUUAGGA

CUGCAGUGAUAUACUUAGG

GCUGCAGUGAUAUACUUA

GGCUGCAGUGAUAUACUUA

AGGCUGCAGUGAUAUACUU

CAGGCUGCAGUGAUAUACU

UUAGGUCGUGUGUUUG

UUUAGGAUUCUGUGUGUUU

AUUUAGGAUUCUGUGUGUU

UAUUUAGGAUUCUGUGUGU

AUAUUUAGGAUUCUGUGUG

UAUAUUUAGGAUUCUGUGU

AUAUAUUUAGGAUUCUGUG

GAUAUAUUUAGGAUUCUGU

UGAUAUAUUUAGGAUUUU

GUGAUAUAUUUAGGAUUCU

AGUGAUAUAUUUAGGAUUC

CAGUGAUAUAUUUAGGAUU

GCAGUGAUAUAUUUAGGA

UGCAGUGAUAUAUU

UAGGA

CUGCAGUGAUAUAUUUAGG

GGCUGCAGUGAUAUAUUUA3 GGCUGCAGUGAUAUAUU

U

AGGCUGCAGUGAUAUAUUU

CAGGCUGCAGUGAUAUAUU

2421 2422 2423 2424 2425 2426 2427 2428 2429 2430 2431 2432 2433 2434 2435 2436 2437 2438 2439 2440 2441 2442 2443 2444 2445 2446 2447 2448 2449 2450 230 2005201389 01 Apr 2005 rs87 9 49 GUCCCCAUGCUC 699 7479 GUUUCUCACGCCAUUGCUC 699 7497 GAG CAAJU'3 3 UGA G 2451 rs2857 790 7480 UUUUCACGj-CCAUUUC 70 7480 UUUCUCACGCC;AUUGCUCA 700 7498 UGAGCAAUGGGUAA 2452 rs28577 90 7481 UUUCACGCCAUUGCUCA 701 7481 UUCUCACGCCAUUGCUCAG 701 7499 CUGAGCMUGCGUGAGM rs285;;:;: 0 748 UCCCGCUGCCG 70i42UCCCCAUGUAG 72 50 CUAGCAAUGGCGUGAGA 2454 rs28577 90 7483 CUACGC'lCAUUGCUCAG 703 743 CUCACGCCAUUUCAGG 70 51 UCGGAAUGGCGUGAG 2455 rs2857 790 7484 cu U AGCU UAGG~ 704 74842 CCCAUCCGM 74 702 UCUACAGCUA 28I:5 7790 7485 C;LAGCUUCCAGGC 70 7485 CACGCCAUUGUuAGC 0 70 GUCUACAUGC 2457 rs257 7 0 792 UGCAAGAACUCACA 31 492 UUGUGACUCAUCAA 731 750 UGCUGACUGUCGUA 28 790 743 UGUAGACACU 3 7493 UCAAGGA CUCAUC- 732 7511 GAGUCUUGC 2484 rs257 90 49 GCAAACAUCAUCAUC 733 7494 CAGCCUAAGCUCAUCAAC 73 752 GGAAUUCUG 48 rs285 7790 749 CAGACUAUAC 3 5 CMUCAUACA 76 7513 UGAUCUGAGCAUGUCUA 2486 rs285 7790 746 UA GGAAUAA 73 746 AGGCAUC ACAU 735 7514 AUAUUAGUUCCU UA GC 2487 rs285 7790 747 GGAACUCACAUCGC 76 49 AA GAACCAUCAG 736 7515 UGAUGAJCUGAGCUU 2488 rs277l 7665 GCUUGCUCAGCAUCC 737 7665 G;UUGCUCAGAUM 3 783 GUGAUG CUGAGCAUGM 2489 490 CAUGCCAGAAAUAU 71 49 AUGCCAGAAAUAU 10uUCUGGCAU231bj 2005201389 01 Apr 2005 rs362321 7666 UUCAUCUACCGCAUCAACA 738 7666 1-UU1 uCCGCAUCMACA 73G64 UGUUGAUGCGGUAAUGM 2490rs36232 1 7667 UCAUCUACCGCAUCMACAC 739 7667 UCAUCUACCGCAUCAACAC 739 7685 GGUAGGUGUA 29 rs3623 21 7668 CAUCUACCGCAUCAACACA 740 7668 CAUCUCGACCC 740 7686 UGUGUGAUGCGGUAGAUG 2492 rs36232I 7669 AUCUACCGCAUCAACACAC 741 7669 AUCUACCGZCAUCMACACAC 741 7687 GUGUGUUGAUGCGGUAG3AU 2493 rs36232l 7670 UCUACCGCAUCAACACACU 742 7670 UCUACCGCAUlLCAACACACU 742 7688 AGUGUGUUGAUG7CGGUAGA 2494 r21 2 7671 CUACCGCAUCAACACACUA 743 7671 CUACCGCAUCAACACACUA 74 69 UGGGUAGGUG 2495 rs36232l 7672 UACCGCAUCAACACACUAG 744 7672 UACCUACCCUAG 744 7690 CUAGUGUGUUGAUGCGGUA 2496 rs3623 21 7678 AC AUCAACACACUAGGG 7450 7678 ACAUCAACACACUAGGG 7450 7696 UCCCUAGUGUGUUGA U 2502U rs36232l 7679 UCAACAACUAGGCGAC 71 69 UACCCAGUGC 751 7697 GUGCCUAGUGUUUGAL, 2503 rs3623 2 l 7666 CCUCAUCUACCCACUA 757 7666 UUCAUCACCAUCU 75 64 UIGUCGAAUG 2509 rs3623 2 l 767 UCAUCUACCCACUAC 758 767 UCAUCAACCACUAC 758 7685 AGCUAUGUGGUGAG 2510 rs3623 21 7672 CGCAUCAAUACACUAGU 763 7672 UGCAUCAAUACACUA;G 763 7690 CCUAGUGUUUGAUGG 2515 rs362 321 7673 AGCAUCAAUACACUAGGG 764 7673 AGAUAAUACACUAGG 74 7691 CCUGAGUGUUUGAUGU 25016 rs3623 2 l 77 CCCAUCACACUAG GC3U;UU 765 7674 CC.AU.ACACUAGGC 75 7692 UGCCUAGUGUUUGAUG 2517 rs3623 2 l 7679 U CAACACUAGGCUGGAC 770 7679 UCAAACACUAG(GUGGAC 77 769 GUCCAGCCUAGUGUAUUGA 2522 rs3623 2 l 7680 AAC AUAGCUGGACC%,' 771 7680 CAAACACUAGGCGGACC 77 7698 GGUCCAGCCAUGUAUUG 22 rs36 232 l 7681s mU,.ACACUAGGCUGGACCA 772 7681 AAACACUAGGCUGGACCA 772 7699 UGGUCCAGCCUAGUGUAUU 22 rs3623 2 l 7682 AACACUAGGCUGGACCAG 773 7682 ACACAGCGACCAG 773 7700 CUGGUCCAGCCUAGUGUAU 22 rs36 232 l 7683 CACUAGGCUGGACCAGU 774 7683 U;AACUAGGCUGGACCAGU 774 7701 ACUGGUCCAGCUAGUGUA 2526 rs322l 775 CUUGUUCCUCGCU 75 775 C UUGGUUC CGACGCU 75 773 AUGGCCC AGGAC 2527 rs322l 776 UUGUUCCUCGCUA 776 776 UUGUUCCGCGCCUA 776 774 UUUGCACGGAACCM 2528 rs3231 6 UAUUACGAUAAAC 75 767 UCUCUCCAC AC 75 785 GUUUAUCGUAA23251 2005201389 01 Apr 2005 rs3025816 7737 UGGUGUCCUGGUGACGCAG 777 7737 UGGUGUCCUGGUGACGCAG 777 7755 CUGCGUCACCAGGACACCA 2529 rs3025816 7738 GGUGUCCUGGUGACGCAGC 778 7738 GGUGUCCUGGUGACGCAGC 778 7756 GCUGCGUCACCAGGACACC 2530 rs3025816 7739 GUGUCCUGGUGACGCAGCC 779 7739 GUGUCCUGGUGACGCAGCC 779 7757 GGCUGCGUCACCAGGACAC 2531 rs3025816 7740 UGUCCUGGUGACGCAGCCC 780 7740 UGUCCUGGUGACGCAGCCC 780 7758 GGGCUGCGUCACCAGGACA 2532 rs3025816 7741 GUCCUGGUGACGCAGCCCC 781 7741 GUCCUGGUGACGCAGCCCC 781 7759 GGGGCUGCGUCACCAGGAC 2533 rs3025816 7742 UCCUGGUGACGCAGCCCCU 782 7742 UCCUGGUGACGCAGCCCCU 782 7760 JAGGGGCUGCGUCACCAGGA 2534 rs3025816 7743 CCUGGUGACGCAGCCCCUC 783 7743 CCUGGUGACGCAGCCCCUC 783 7761 GAGGGGCUGCGUCACCAGG 2535 rs302581 6 7744 CUGGUGACGCAGCCCCUCG 784 7744 CUGGUGACGCAGCCCCUCG 784 7762 CGAGGGGCUGCGUCACCAG 2536 rs302581 6 7745 UGGUGACGCAGCCCCUCGU 785 7745 UGGUGACGCAGCCCCUCGU 785 7763 ACGAGGGGCUGCGUCACCA. 2537 rs3025816 7746 GGUGACGCAGCCCCUCGUG 786 7746 GGUGACGCAGCCCCUCGUG 786 7764 CACGAGGGGCUGCGUCACC 2538 rs3025816 7747 GUGACGCAGCCCCUCGUGA 787 7747 GUGACGCAGCCCCUCGUGA 787 7765 UCACGAGGGGCUGCGUCAC 2539 rs3025816 7748 UGACGCAGCCCCUCGUGAU 788 7748 UGACGCAGCCCCUCGUGAU 788 7766 AUCACGAGGGGCUGCGUCA 2540 rs3025816 7749 GACGCAGCCCCUCGUGAUG 789 7749 GACGCAGCCCCUCGUGAUG 789 7767 CAUCACGAGGGGCUGCGUC 2541 rs302581 6 7750 ACGCAGCCCCUCGUGAUGG 790 7750 ACGCAGCCCCUCGUGAUGG 790 7768 CCAUCACGAGGGGCUGCGU 2542 rs302581 6 7751 CGCAGCCCCUCGUGAUGGA 791 7751 CGCAGCCCCUCGUGAUGGA 791 7769 UCCAUCACGAGGGGCUGCG 2543 rs3025816 7752 GCAGCCCCUCGUGAUGGAG 792 7752 GCAGCCCCUCGUGAUGGAG 792 7770 CUCCAUCACGAGGGGCUGC 2544 rs3025816 7753 CAGCCCCUCGUGAUGGAGC 793 7753 CAGCCCCUCGUGAUGGAGC 793 7771 GCUCCAUCACGAGGGGCUG 2545 rs302581 6 7735 CUUGGUGUCCUGGUGACGU 794 7735 CUUGGUGUCCUGGUGACGU 794 7753 ACGUCACCAGGACACCMAG 2546 rs3025816 7736 UUGGUGUCCUGGUGACGUA 795 7736 UUGGUGUCCUGGUGACGUA 795 7754 UACGUCACCAGGACACCAA 2547 rs3025816 7737 UGGUGUCCUGGUGACGUAG 796 7737 UGGUGUCCUGGUGAGGUAG 796 7755 CUAGGUCACCAGGACACCA 2548 rs3025816 7738 GGUGUCCUGGUGACGUAGC 797 7738 GGUGUCCUGGUGACGUAGC 797 7756 GCUACGUCACCAGGACACC 2549 rs3025816 7739 GUGUCCUGGUGACGUAGCC 798 7739 GUGUCCUGGUGACGUAGCC 798 7757 GGCUACGUCACCAGGACAC 2550 rs3025816 7740 UGUCCUGGUGACGUAGCCC 799 7740 UGUCCUGGUGACGUAGCCC 799 7758 GGGCUACGUCACCAGGACA 2551 rs3025816 7741 GUCCUGGUGACGUAGCCCC 800 7741 GUCCUGGUGACGUAGCCCC 800 7759 GGGGCUACGUCACCAGGAC 2552 rs302581 6 7742 UCCUGGUGACGUAGCCCCU 801 7742 UCCUGGUGACGUAGCCCCU 801 7760 AGGGGCUACGUCACCAGGA 2553 rs302581 6 7743 CCUGGUGACGUAGCCCCUC 802 7743 CCUGGUGACGUAGCCCCUC 802 7761 GAGGGGCUACGUCACCAGG 2554 rs3025816 7744 CUGGUGACGUAGCCCCUCG 803 7744 CUGGUGACGUAGCCCCUCG 803 7762 CGAGGGGCUACGUCACCAG 2555 rs3025816 7745 UGGUGACGUAGCCCCUCGU 804 7745 UGGUGACGUAGCCCCUCGU 804 7763 ACGAGGGGCUACGUCACCA 2556 rs302581 6 7746 GGUGACGUAGCCCCUCGUG 805 7746 GGUGACGUAGCCCCUCGUG 805 7764 CACGAGGGGCUACGUCACC 2557 rs3025816 7747 GUGACGUAGCCCCUCGUGA 806 7747 GUGACGUAGCCCCUCGUGA 806 7765 UCACGAGGGGCUACGUCAC 2558 rs3025816 7748 UGACGUAGCCCCUCGUGAU 807 7748 UGACGUAGCCCCUCGUGAU 807 7766 AUCACGAGGGGCUACGUCA 2559 rs3025816 7749 GACGUAGCCCCUCGUGAUG 808 7749 GACGUAGCCCCUCGUGAUG 808 7767 CAUCACGAGGGGCUACGUC 2560 rs3025816 7750 ACGUAGCCCCUCGUGAUGG 809 7750 ACGUAGCCCCUCGUGAUGG 809 7768 CCAUCACGAGGGGCUACGU 2561 rs3025816 7751 CGUAGCCCCUCGUGAUGGA 810 7751 CGUAGCCCCUCGUGAUGGA 810 7769 UCCAUCACGAGGGGCUACG 2562 rs3025816 7752 GUAGCCCCUCGUGAUGGAG 811 7752 GUAGCCCCUCGUGAUGGAG 811 7770 CUCCAUCACGAGGGGCUAC 2563 rs3025816 7753 UAGCCCCUCGUGAUGGAGC 812 7753 UAGCCCCUCGUGAUGGAGC 812 7771 GCUCCAUCACGAGGGGCUA 2564 rs3025814 7831 CAGGCCAUCACCUCACUGG 813 7831 CAGGCCAUCACCUCACUGG 813 7849 CCAGUGAGGUGAUGGCCUG 2565 rs302581 4 7832 AGGCCAUCACCUCACUGGU 814 7832 AGGCCAUCACCUCACUGGU 814 7850 ACCAGUGAGGUGAUGGCCU- 2566 rs3025814 7833 1GGCCAUCACCUCACUGGUG 815 7833 1GGCCAUCACCUCACUGGUG 815 781 CACCAGUGAGGUGAUGGCC, 2567 233 2005201389 01 Apr 2005 rs3025814 7834 GCGAUCACCUCACUGGUGC 816 784GCCAUCACCUCACUGGUGC 816 7852 GCACCAGUGAGGUGAUGGC 2568 rs3025814 78-35 CCAUCACCUCACUGGUGCU 81l 7 7835 CCAUCACCUCACUGGUGCU 817 -7853 AGCACCAGUGAGGUGAUGG- 2569 rsi3025814 -7836 CAUCACCUCACUGGUGOUC 818 7836 CAUCACCUCACUGGUGCUC 818 7854 GAGCACCAGUGAGGUGAUG 2570 rs3025814 7837 AUCACCUCACUGGUGCUCA 819 7837 AUCACCUCACUGGUGCUCA 819 7855 UGAGCACCAGUGAGGUGAU 2571 rs3025814 7838 UCACCUCACUGGUGCUCAG 820 7838 UCACCUCACUGGUGCUCAG 820 7856 -CUGAGCACCAGUGAGGUGA 2572 rs3025814 78390 CUCACUGGUGCUCAGUG 8g2 73 ACCUCACUGGUGCUCAGUG 821 7857 CUGAGCACCAGUGAGGU 2573 rs302581 4 7840 CACCUCACUGGUGCUCAGU 822 7840 ACCUCACUGGUGCUCAGU 822 7858 ACUGAGCACCAGUGAGGUG 2574 rs3025814 7841 CCUCACUGGUGCUCAGUGC 823 7841 CCUCACUGGUGCUCAGUGC 823 7859 GCACUGAGCACCAGUGAGG 2575 r32847842 CUCACUGGUGCUCAGUGCA 824 7B42 CUCACUGGUGCUCAGUGCA 824 7860 UGCACUGAGCACCAGUGAG- 2576 rs3025814 7843 UCACUGGUGCUCAGUGCAA 825 7843 UCACUGGUGCUCAGUGCAA 825 7861 UUGCACUGAGCACCAGUGA 2577 rs302581 4 7844 CAC UGGUGCUCAGUGCAAU 826 7844 CACUGGUGCUCAGUGCAAU 826 7862 AUUGCACUGAGCACCAGUG 2578 rs3025814 7845 ACUGGUGCUCAGUGCAAUG 827 7845 ACUGGUGCUCAGUGCAAUG 827 7863 CAUUGCACUGAGCACCAGU 2579 rs3025814 7846 CUGGUGCUCAGUGCAAUGA 88 84 CUGGUGCUCAGUGCAAUGA 828 7864 UCAUUGCACUGAGCACCAG 2580 rs302581 4 7847 UGGUGCUCAGUGCAAUGAC 829 7847 UGGUGCUCAGUGCAAUGAC 829 7865 GUCAUUGCACUGAGCACCA 2581 rs3025814 7848 GGGUAUCAGC 3 88GUCCGUGCAAUGACU 830 7866 AGUCAUUGCACUGAGCACC 2582 rs3058147849 GUGCUCAGUGCAAUGACU 831 7849 GUGCUCACAUAU 83 767 AUAUCCGAAC 23 rs3025814 73 CAGCUACCCG 83 781 CGCAACCCUG 832 7849 CAGUAGUGAUGGCC 2584 rs3025814 7832 GGCCUCACUCAUGCU 8331 7832-9 AGUGCCUCACUCAUGCU 3 80 ACGGGUAG U 28 rs3025814 7833 CGGCCAUCACCUCACUGC 834 7833 CGGCCAUCACCUCAUCG 834 7851 CGCAGUGAGGUGAUGGCC 2586 rs3025814 7834 AGCCAUCACCUCACUGCU 835 7834 AGCCAUCACCUCAGUGCU 835 7852 GAGCAGUGAGGUGAUGGC 2587 rs302581 4 7835 GCCAUCACCUCACUGCUG 836 7835 GCCAUCACCUCACUGCUG 836 7853 ACAGCAGUGAGGUGAUGG 2588 rs3025814 7836 GCAUCACCUCACUGCUGC 837 7836 GCAUCACCUCACUGUGC 837 7854 GGCAGCAGUGAGGUGAUG 2589 rs3025814 7837 CAUCACCUCACUGCUGCU 838 7837 CAUCACCUCACUGCUGCU 838 7855 UAGCAGCAGUGAGGUGAU 2590 rs302581 4 78386 -UCACCUCACUGCUGCUC 839 7838 CUCACCUCACUGCUGCUC 839 7856 CGAGCAGCAGUGAGGUGA 259 rs3025814 7839 ACACCUCACUGCUGCAU 840 7839 ACACCUCACUGCUGCUCA 840 7857 AUGAGCAGCAGUGAGGUG 2592 rs3025814 7840 UACCUCACUGCUGCUCAG 841 7840 UACCUCACUGCUGCUCAG 841 7858 CCUGAGCAGCAGUGAGGU 2593 rs3025814 7841 CCCUCACUGCUGC CAGU 842 7841 lCCUCACUGCUGCUCAGU 842 7859 GACUGAGCAGCAGUGAGG 2594 rs302581 4 7842 ACUCACUGCUGCUCAGUG 843 7842 ACUCACUGCUGCUCAUG 843 7860 UCACUGAGCAGCAGUGAG 2595 rs3025814 7843 CUCACUGCUGCUCAGUGC 1844 7843 CUCACUGCUGCUCAGUGC 844 7861 UGCACUGAGCAGCAGUGA 2596 rs3025814 -844 CACUGCUGCUCAGUGCA 845 7844 CCACUGCUGCUCAGUGCAU 845 7862 AUGCACUGAGCAGCAGUG 2597 rs302581 4 7T845 ACUGCUGCUCAGUGAA 846 7845 UACUGCUGCUCAGUGCAA 846 7863 1UUGCACUGAGCAGCAGU 2598 s-3025814 7846 CCUGCUGCUCAGUGCAAU -847 7846 CCUGCUGCUCAGUGCAAUA 847 7864 UAUUGCACUGAGCAGCAG 2599 rs302581 4 7847 AUGCUGCUCAGUGCAAA 848 7847 AUGCUGCUCAGUGCAAUG 848 7865 GCAUUGCACUGAGCAGCA 2600 17- IA'f'-r'A A H(ArH RAQ 7866 AGtJCAUUGCACUGAGCAGC 2601 rs302581 4 rs302581 4 rs3-62273 rs362273 rs362273 rsi3-62273 S78 48 CUGCUCAGUGCOA-U'AIUU 7849 CUGCUCAGUGCAAUGACUG 81F00 -CCACGAGAAGCUGCUGCUA 8101 CACGAGAAGCUGCUGCUAC 8102 -ACGAGAAGCUGCUGCUACA 8103 CGAGAAGCUGCUGCUACAG 850 851 852 853 7849 86100, 8101 8102 8103

CUGCUCAGL

CA-CGA GA A

CACGAGAAG

ACGAGAAGC

CGAGAAGCL

IGCAAUGACUG 850 [7867 jCAGUCAUUGCACUGAGCAG 2602 GCUGCUGCU 851 8118 1UAGCAGCAGCUUCUCGUGG 2603 CUGCUGCUC 852 j8119 GUAGCAGCAGCUUCUCGUG- 2604

:U

3CUGCUACA j853 8120 LUGUAGCAGCAGUUUULU 21505D -UrCAA 5 8121 CUGUAGCAGCAGCUUCUCG j2606 2005201389 01 Apr 2005 rs362273814 AAGUCUUAAA 85 80 GGAGUCGUCG 85 812 UUUGACCUCC 67 rs362273 8105 AGAAGCUGCUGCUACAGAU 856 8105 AGAAGCUGCUGCUACAGAU 856 -8123 AUCUGUAGCAGCAGCUUCU 2608 rs362273 8106 GAAGCUGCUGCUACAGAUC 857 8106 GAAGCUGCUGCUACAGAUC 857 8124 GAUCUGUAGCAGCAGCUUC 2609 rs362273 8107 AAGCUGCUGCUACAGAUCA 858 8107 MAGCUGCUGCUACAGAUCA 858 8125 UGAUCUGUAGCAGCAGCUU -2610 rs362273 8108 AGCUGCUGCUACAGAUCAA 1859 8108 ,AGCUGCUGCUACAGAUCAA 859 8126 UUGAUCUGUAGCAGCAGCU- 2611 1rs362273 810 9 GA GCUGCUGCUACAGA UC A 8 0 810 9 GA GCUGCUGCUACAGA UC A 86 8127 GUGUCUGUAGCAGCAG C C 2612 r362273810 CCUCAAACCC 81 81 CUCGUAAUAC 86 818 GUGUUUCAAG 23 rs362273 8111 UGCUGCUACAGAUCAAC 862 8111 GUGCUGCUACAGAUAAC 862 8129 GGUUGAUCUGUAGCAGCA 2614 rs362273 8112 CGCUGCUACAGAUCAACC 863 8112 CGCUGCUACAGAUCAACCC 863 8130 GGGUUGAUCUGUAGCAGC 2615 rs362273 8113 UCUGCUACAGAUCAACCC 864 8113 1UCUGCUACAGAUCAACCC 864 8131 CGGGUUGAUCUGUAGCAG 2616 rs362273 8114 GUGCUACAGAUCAACCCCA 865 8114 1GUGCUACAGAUCAACCCCA 865 8132 GGGGUUGAUCUGUAGCAC 2617 rs362273 8115 CGCUACAGAUCAACCCCAG 866 8115 CGCUACAGAUCAACCCCAG 866 8133 ,CGGGGUUGAUCUGUAGC 2618 rs362273 8116 UCUACAGAUCAACCCCGA 867 8116 UCUACAGAUCCCCCGA 867 8134 GUCGGGGUUGAUCUGUAGA 2619 rs362273 8117 GUACAGAUCAACCCCGAG 868 8117 GUACAGAUCAACCCCGAG 86 8135 1CUCGGGGUUGAUCUGUA. 2620 rs362273 8118 CACAGAUCAACCCCGAGC 869 1186 CACAGAUCAACCCCGAGC 869 8136 CGCUCGGGGUUGAUCUGU 2619 rs362273 8100 CACAGACAGCCUGUGCG 870 8100 CACAGAACCGUGU 870 8118 CGCACGCUUCUCGUG 2622 rs362273 8118 ACAGACACUCGUGCUGC 871 8118 CAGAGMCUCGUGCGC 871 8119 CGCACGCUUACUGU 2623 rs362273 8102 CACGAGAAGCUGCUGCUGA 872 8102 CACGAGAAGCUGCUGCUGA 872 8120 UCAGCAGCAGCUUCUCGU 2624 rs362273 8103 CCGAGAAGCUGCUGCUGC 873 8C 1 CCGAGAAGCUGCUGCUGC 873 8119 CGCAGCAGCAGCUUCUCG 2625 rs362273 81042 KGAGAAGCUGCUGCUGCA 874 8104 AGAGGCUGCUGCUGCA 874 8122 UUGCAGCAGCAGCUUCUC 2626 rs362273 8105 CAGAAGCUGCUGCUGCAG 875 8105 -CAGAAGCUGCUGCUGCAG 875 8123 ACUGCAGCAGCAGCUUCU 2627 rs362273 8106 GGAAGCUGCUGCUGCAGA 876 81-06 GGAAGCUGCUGCUGCAGA 876 8124 UCUGCAGCAGCAGCUUC 2628 rs362273 8 1075 -K-AAGCUGCUGCUGCAGAU 877 8107 AAAGCUGCUGCUGCAAU 87 8125 UAUCUGCAGCAGCAGCUU 2629 rs362273 8108 GAGCUGCUGCUGCAGAUC 878 8108 GAGCUGCUGCUGCAGAUA 878 8126 UGAUCUGCAGCAGCAGCUC 2630 rs362273 8109 AGCUGCUGCUGCAGAUCA 879 8109 -AGCUGCUGCUGCAGAUCA 879 8127 UGAUCUGCAGCAGCAGC 2631 rs362273 810 ACUGCUGCUGCAGAUCAA 878 810 CUGCUGCUGCAGAUCAAC 88 8128 GUUGAUCUGCAGCAGCAG 2632 rs362273 8111 GUGCUGCUGCAGAUCAAC 881 8111 GUGCUGCUGCAGAUCAAC 881 8129 GGUUGAUCUGCAGCAGCA 2633 rs362273 8112 CGCUGCUGCAGAUCAACCC 882 8112 CGCUGCUGCAGAUCAACCC 882 810 GGGUACGCAGCAG 2634 rs362273 8113 UCUGCUGCAGAUCAACCC 88 113 CUGCUGCAGAUCAACCC 883 8131 CGGGUUGAUCUGCAGCAG 2635 rs362273 8-114 GUGCUGCAGAUCAACCCCA 884 8114 GUGCUGCAGAUCAACCCCA 884 8132 UGGGGUUGAUCUGCAGCA 2636 rs362273 8115 CGCUGCAGAUCAACCCCAG -853 8115 CGCUGCAGAUCAACCCCAG 885 8133 CCGGGGUUGAUCUGCAGC 2637 rs362273 8116 UCUGCAGAUCAACCCCGA 886 8116 -UCUGCAGAUCAACCCCGA 886 8134 UCGGGGUUGAUCUGCAG 2638 rs362273 8117J GUGCAGAUCAACCCCGACG 887 8117 GUGCAGAUCAACCCCGAG 887 8135 CCUCGGGGUUGAUCUGCA 2639 8 136 ICCGCUCGGGGUUGAUCUGC 2640 rs362273 HD-Ex58 HD-ExS8 8118 8231 8232

GCAGAUCAACCCCGAGCGG

ACGAGGAAGAGGAGGAGGA

CGAGGAAGAGGAGGAGGAG

GGAAGAGGAGGAGGAGG

889 890 891 8231 8232 8233 AC AGGAAGAGGAGGAGGA1

CGAGGAAGAGGAGGAGGA

G5AGGAAGAGGAGGAGGAGG,

AGGAAGAGGAGGAGGAGGC

GAAGAGGAGGAGGAGGCC'-

889 890 8249 8250

UCCUCCUCCUCUUCCUCGU

CCCUCCUCCUCUUCCUCG

2641 2642 891 8251 CCUCCUCCUCCUCUUCCUC 2643 892 18252 ]GCCUCCUCCUCCUCUUCCU 2644 HD-Ex58 8234 AGGAAGAGGAGGAGGAGGC [892 18234 8235 jGGMAGAGGAGGAGGAGGCC 893 8235

I

8253 GGCCUCCUCCUCCUCUUCC 2645 235 2005201389 01 Apr 2005 I HD-Ex58 HTD-Ex58 HWD--Ex58 HD-Ex58 fD-Ex58 HD-ExS8 HFD-Ex58 HfD-Ex58 FD-E x5 8 HD-Ex58 HD-Ex58 H-D-Ex58 H D-Ex58 HD-Ex58 s-2276881 rs-22-76881 rs2276881 ri2-27688l rs2276881 rs2-276881 rs2-276881 rs227688l rs2276881 rs227688l rs2276881 Fs2-276881 rs2276881 rs227688& rs227688& rs2276881 rs227688 rs227688 rs227688 rs227688 rs227688 rs227688 rs227688 rs227688 rs227688 8T236 GAAGAGGAGGAGGAGGCCG 8237 MAGAGGAGGAGGAGGCCGA 8238 AAGGAGGAGGAGGCCGAC 8-239 GGGAGGAGGAGGCCGACG 8240 AGGAGGAGGAGGCCGACGC 8241 GGAGGAGGAGGCCG3ACGCC 82-31 AjC-GAGGMAGAGGAGGAGGC 8232 CGAGGAAGAGGAGGAGGCC 8233 GAGGAAGAGGAGGAGGCCG 8234 AGGAAGAGGAGGAGGCCGA 8235 -GAAGAGGAGGAGGCCGAC 8'2 36 GAAGAGGAGGAGGCCGACG 8237 AAGAGGAGGAGGCCGACG3C 8238 AGAGGAGGAGGCCGACG3CC 8460 GCGCAACCAGUUUGAGCUG 8461 CGCAACCAGUUUGAGCUGA 8462 GCAACCAGUUUGAGCUGAU 8463 CAACCAGUUUGAGCUGAUG 8464 AACCAGUUUGAGCUGAUGU 84-65 ACCAGUUUGAGCUGAUGUA 8466 CCAGUUUGAGCUGAUGUAU 8467 CAGUUUGAGCUGAUG3UAUG 8468 AGUUUGAGCUGAUGUAUGU 8-9469 GUUGAGCUGAUGUAUGUC 8470 UUGAGCUGAUGUAUGUGP 8471 UUGAGCUGAUGUAUGUGAC F8472 UGAGCUGAUGUAUGUIGACC 8-473 GGCUGAUGUAUGUGACGC 8-474 A-G-CUGAUGUAUGUGACGC;L 6 475 GCUGAUGUAUGUGACGCUC 1 8476 CUGAUGUAUGUGACGCUGi 1 877 UAUGUAUGUGACGCUGA( 1 8478 GAUGUAUGUGACGCUGAG~ 1 8460 GGCAACCAGUUUGAGCUi 1 8461 CGCAACCAGUUUGAGCUA/ 1 8462 GCAACCAGUUUGAGCUAA 1 8463 CAACCAGUUUGAGCUMUC 1 8464 CCAGUUUGAGCUAAUG, 1 8465 ACCAGUUUGAGCUAAUGU 894 8236 GAAGAGGAGGAGGAGGCCG 894 8254 CGGCCUCCUCCUCCUCUUC 2646 895 8237 MAGAGGAGGAGGAGGCCGA 895 86255 -UC-GGCCUCCUCCUGCUC;UU 2647 896 238 GAGAGGAGCA 9 26 GCGCCUCCUU 24 897 8239 GAGGAGGAGGAGGCCGAG 897 8257 GUCGGCCUCCUCCUCCUCU 2649 898~~ 824 AGGAGGAGCAG 9 28 GGCGCCUCCU 899 841 GAGAGGAGCGCC 9 29 GCUGCUCCUC 8902 8233 GAGGAAGAGGAGGGGCCG 892 8251 CGCGGCCUCCUCCUCCUC 264 903 234GGGAAGGAGGCG 0 22UGGCUUCCUCU 8905 8236 AGAGAGGAGGAGGCGACG 8905 8254 GCGUCGGCCUCCUCCUCU 2657 906 237AGGAGGAGAGCCC 90 825 GGCGCCUUUU 68 8907 8238 AGAGGAGGAGGCCGAG3CC 8907 8256 GGCGUCGGCCUCCUCCUCU 2659 909 8461 ACGCGAAAGUGAGGA 909 82479 UCAGCUCAUCUUCG 2661 910 462 GAGACGUGGUA 1 40 ACGUA UGUC 26 911 843 CGAAAGUGAGCGAUG 911 8481 GCUCCUCAACUUUG 2663 912 8464 GAAAGUAGGUAGU 912 842 CAGUCCUCACUUUC 2664 913 824 ACCGAAAGUUUAGGACUGUA 913 8483 UCAGUCCUCAACUGGCU 265 916 8468 GGAGUUGAGGAGUCAG 916 8486 ACUCAUCAGCCUUCU 2656 917 84369 GUUUAGAGAGGAUGGAGG 917 824 CACUCAUCAGCUCAAC 2669 919 84371 UUAGAGAGUGAUGUGGAC 919 8489 GCUCCUCUCAGCUCAA 2671 9217 843 AGAGCGAGAGGACGC 921 8491 GGCGUCAGCAUCUCCUCU 2673 922 8474 GCGAUCGUUUGAGCU 922 8492 CAGCGUCAAACAGUCGC 2674 923 8475 CGCUAUCGUUGUGAGCUG 923 84793 CAGCGUCAAACUCAUGC 2675 924-- -846 GCUAUGUUGUGAGCUA 924 8494 AUCAGCGUCAAACAGUC 266 925 8477 UAUCGUUUGAGCUGACG 925 8495 GAUCAGCGUCAAACUCAU 2677 9127 8460 GCAACCAGUUUGAGCUA 9127 848 CUAGCUCAAACUGGUUG 2679 928 8461 CACCAGUUUGAGCUAA 928 8479 UCUAGCUCAAACUGGUG 2680 J 929 8462 CCCAGUUUGAGCUAAU 929 684 -PUAUAGCUCAAACUGGC 2681 930 8463 CCAGUUUGAGCUAAUG 930 8481 CAUCUAGCUCAAAGUUG 2682 J 91 84648 CAGUUUGAGCUAAUGU 91 8482 ACAUCUAGCUCAAACGU 26683 932 8465 AGUUUGAGCUAAUGUAG 932 8483 UACAUCUAGCUCAAACU 2684 236 2005201389 01 Apr 2005 rs2768l 866 CCAUUUAGUAAUGUAU 933 8466 CCAGUUUGAGCUMUGUAU 933 8484 AUAUAGCCG 28 rs2276 88 l 8467 CAGUGACU GUAUG 934 8467 CAGUUUGA1GCUAAUGUAUG 934 8485 CAUACAUUAGCUCAACUG 2686 rs22768 8 l 8468 AGUGCAUUUU 93 8468 AGUUUGAGcUAAUGUAUGU 935 8486 ACAUACAUUAGCUCAACU 2687 rs22768 8 l 8469 GUUUGAGUAUGUAUGU 9365 46 GUUUGAGCUAAUGUAUGUG 936 8487 ACAUACAUUAGCUCAAC 2688 rs22768 8 l 8470 UUUGAGCUAAUGUAUGUG 97 847 UUUGAGCUAAUGUAUGUGA 937 8488 U ACUACUUA!1 C 2689 rs22768 8 l 8471 UUGAGCUAAUGUAUGGc 938 8471 UUGAGCUAAUGUAGUGACG 938 84890 GUCCUCUAC 2690 rs22768 8 l 8472 UGAGCUAAUGUAUGUGACG 939 8472 UGAGCUAAUGUAUGAC 99 8490 CGCCUCUACC 2691 rs32 2 7 869 UUGACUGCGACGCA 965 8659 GUCUGGAGCCUGCAGC 965 8740CUGAGCC~ 2717 rs227 2 8660 UUGG CCGA 66 86 UUGGCUCCCU 966 8678 A ,GC6,l!GUACAUUAGCU 21 rs62 27 2 61 74 GAGCCCGUGCGACGCAUC 967 8661 AGGACCGAGCU 96 869 GGC CAGGCC 219 rs622 7 2 86762 GGAGCCCUGCACGCGA 968 662 GAUGCACGCGAUC 968 8680 GCAGCCGUCAUCC 2720 rs362 272 8663 GUGAGCCCUGCACGGCC 969 8663 UUGAGCCCUGCACGGCUCU 969 8681 AGGACCGUGCAI31 ,"GGGCUC 2721 rs3622 72 8664 AGG) CCUGCACGGCUCUC 970 8664 UGAGCCCUGCACGGCUCU 7 8682 GGAGCCGUCAGGGCU 2722 rs362 272 8665 GAGCCUGCACGGCAUCCUCU 951 866 GCCCUGCACGUCCUCU 91 8683 GAGGAGCCGUGCAGGG 2723 rs322Z 864AGC I GCUCCCU 952 863 GAG CCCGGCGG23770 2005201389 01 Apr 2005 rs362272 8666 CCCUGCACGGCAUCCUCUA 972 8666 CCCUGCACGGCAUCCUCUA 972 8684 UAGAGGAUGCCGUGCAGGG 2724 rs362272 8667 CCUGCACGGCAUCCUCUAU 973 8667 CCUGCACGGCAUCCUCUAU 973 8685 AUAGAGGAUGCCGUGCAGG 2725 rs362272 8668 CUGCACGGCAUCCUCUAUG 974 8668 CUGCACGGCAUCCUCUAUG 974 8686 CAUAGAGGAUGCCGUGCAG 2726 rs362272 8669 UGCACGGCAUCCUCUAUGU 975 8669 UGCACGGCAUCCUCUAUGU 975 8687 ACAUAGAGGAUGCCGUGCA 2727 rs362272 8670 GCACGGCAUCCUCUAUGUG 976 8670 GCACGGCAUCCUCUAUGUG 976 8688 CACAUAGAGGAUGCCGUGC 2728 rs362272 8671 CACGGCAUCCUCUAUGUGC 977 8671 CACGGCAUCCUCUAUGUGC 977 8689 GCACAUAGAGGAUGCCGUG 2729 rs362272 8672 ACGGCAUCCUCUAUGUGCU 978 8672 ACGGCAUCCUCUAUGUGCU 978 8690 AGCACAUAGAGGAUGCCGU 2730 rs362272 8673 CGGCAUCCUCUAUGUGCUG 979 8673 CGGCAUCCUCUAUGUGCUG 979 8691 CAGCACAUAGAGGAUGCCG 2731 rs362272 8674 GGCAUCCUCUAUGUGCUGG 980 8674 GGCAUCCUCUAUGUGCUGG 980 8692 CCAGCACAUAGAGGAUGCC 2732 rs362272 8675 GCAUCCUCUAUGUGCUGGA 981 8675 GCAUCCUCUAUGUGCUGGA 981 8693 UCCAGCACAUAGAGGAUGC 2733 rs362272 8676 CAUCCUCUAUGUGCUGGAG 982 8676 CAUCCUCUAUGUGCUGGAG 982 8694 CUCCAGCACAUAGAGGAUG 2734 rs362272 8677 AUCCUCUAUGUGCUGGAGU 983 8677 AUCCUCUAUGUGCUGGAGU 983 8695 ACUCCAGCACAUAGAGGAU 2735 rs3025807 9136 UCAGACCCUMAUCCUGCAG 984 9136 UCAGACCCUAAUCCUGCAG 984 9154 CUGCAGGAUUAGGGUCUGA 2736 rs3025807 9137 CAGACCCUAAUCCUGCAGC 985 9137 CAGACCCUAAUCCUGCAGC 985 9155 GCUGCAGGAUUAGGGUCUG 2737 rs3025807 9138 AGACCCUAAUCCUGCAGCC 986 9138 AGACCCUAAUCCUGCAGCC 986 9156 GGCUGCAGGAUUAGGGUCU 2738 rs3025807 9139 GACCCUAAUCCUGCAGCCC 987 9139 GACCCUAAUCCUGCAGCCC 987 9157 GGGCUGCAGGAUUAGGGUC 2739 rs3025807 9140 ACCCUAAUCCUGCAGCCCC 988 9140 ACCCUAAUCCUGCAGCCCC 988 9158 GGGGCUGCAGGAUUAGGGU 2740 rs3025807 9 141 CCCUAAUCCUGCAGCCCCC 989 9141 CCCUAAUCCUGCAGCCCCC 989 9159 GGGGGCUGCAGGAUUAGGG 2741 rs3025807 9142 CCUAAUCCUGCAGCCCCCG 990 9142 CCUAAUCCUGCAGCCCCCG 990 9160 CGGGGGCUGCAGGAUUAGG 2742 rs3025807 9143 CUAAUCCUGCAGCCCCCGA 991 9143 CUAAUCCUGCAGCCCCCGA 991 9161 UCGGGGGCUGCAGGAUUAG 2743 rs3025807 9144 UAAUCCUGCAGCCCCCGAC 992 9144 UAAUCCUGCAGCCCCCGAC 992 9162 GUCGGGGGCUGCAGGAUUA 2744 rs3025807 9145 AAUCCUGCAGCCCCCGACA 993 9145 AAUCCUGCAGCCCCCGACA 993 9163 UGUCGGGGGCUGCAGGAUU 2745 rs3025807 9146 AUCCUGCAGCCCCCGACAG 994 9146 AUCCUGCAGCCCCCGACAG 994 9164 CUGUCGGGGGCUGCAGGAU 2746 rs3025807 9147 UCCUGCAGCCCCCGACAGC 995 9147 UCCUGCAGCCCCCGACAGC 995 9165 GCUGUCGGGGGCUGCAGGA 2747 rs3025807 9148 CCUGCAGCCCCCGACAGCG 996 9148 CCUGCAGCCCCOGACAGCG 996 9166 CGCUGUCGGGGGCUGCAGG 2748 rs3025807 9149 CUGCAGCCCCCGACAGCGA 997 9149 CUGCAGCCCCCGACAGCGA 997 9167 UCGCUGUCGGGGGCUGCAG 2749 rs3025807 9150 UGCAGCCCCCGACAGCGAG 998 9150 UGCAGCCCCCGACAGCGAG 998 9168 CUCGCUGUCGGGGGCUGCA 2750 rs3025807 9151 GCAGCCCCCGACAGCGAGU 999 9151 GCAGCCCCCGACAGCGAGU 999 9169 ACUCGCUGUCGGGGGCUGC 2751 rs3025807 9152 CAGCCCCCGACAGCGAGUC 1000 9152 CAGCCCCCGACAGCGAGUC 1000 9170 GACUCGCUGUCGGGGGCUG- 2752 rs3025807 9153 AGCCCCCGACAGCGAGUCA 1001 9153 AGCCCCCGACAGCGAGUCA 1001 9171 UGACUCGCUGUCGGGGGCU 2753 rs3025807 9154 GCCCCCGACAGCGAGUCAG 1002 9154 GCCCCCGACAGCGAGUCAG 1002 9172 CUGACUCGCUGUCGGGGGC 2754 rs3025807 9136 UCAGACCCUAAUCCUGCAT 1003 9136 UCAGACCCUAAUCCUGCAT 1003 9154 AUGCAGGAUUAGGGUCUGA 2755 rs3025807 9137 CAGACCCUMAUCCUGCATC 1004 9137 CAGACCCUAAUCCUGCATC 1004 9155 GAUGCAGGAUUAGGGUCUG 2756 rs3025807 9138 AGACCCUAAUCCUGCATCC 1005 9138 AGACCCUAAUCCUGCATCC 1005 9156 GGAUGCAGGAUUAGGGUCU 2757 rs3025807 ,9139 GACCCUAAUCCUGCATCCC 1006 9139 GACCCUAAUCCUGCATCCC 1006 9157 IGGGAUGCAGGAUUAGGGUC 2758 rs3025807 9140 ACCCUAAUCCUGCATCCCC 1007 9140 ACCCUAAUCCUGCATCCCC 1007 9158 GGGGAUGCAGGAU UAGGG U 2759 rs3025807 9141 CCUAAUCCUGCATCCCCC 1008 9141 CCC UAAUCCUGCATCCCCC 1008 .9159 GGGGGAUGCAGGAUUAGGG 2760 rs3025807 9142 CCUAAUCCUGCATCCCCCG 1009 9142 CCUAAUCCUGCATCCCCCG 1009 9160 CGGGGGAUGCAGGAUUAGG 2761 rs30258D7 9143 1CUMAUCCUGCATCCCCCGA 1010 9143 CUAAUCCUGCATCCCCCGA 1010 9161 UCGGGGGAUGCAGGAUUAGJ 2762 238 2005201389 01 Apr 2005 rs3025807 9144 UAAUCCUGCATCCCCCGAC 1011 9144 UAAUCCUGCATCCCCCGAC 1011 9162 GUCGGGGGAUGCAGGAUUA 2763 rs3025807 9145 AAUCCUGCATCCCCCGACA 1012 9145 AAUCCUGCATCCCCCGACA 1012 9163 UGUCGGGGGAUGCAGGAU U 2764 rs3025807 9146 AUCCUGCATCCCCCGACAG 1013 9146 AUCCUGCATCCCCCGACAG 1013 9164 CUGUCGGGGGAUGCAGGAU 2765 rs3025807 9147 UCCUGCATCCCCCGACAGC 1014 9147 UCCUGCATCCCCCGACAGC 1014 9165 GCUGUCGGGGGAUGCAGGA 2766 rs3025807 19148 CCUGCATCCCCCGACAGCG 1015 9148 CCUGCATCCCCCGACAGCG 1015 9166 CGCUGUCGGGGGAUGCAGG 2767 rs3025807 9149 CUGCATCCCCCGACAGCGA 1016 9149 CUGCATCCCCCGACAGCGA 1016_ 9167 UCGCUGUCGGGGGAUGCAG 2768 rs3025807 9150 UGCATCCCCCGACAGCGAG 1017 9150 UGCATCCCCCGACAGCGAG 1017 9168 CUCGCUGUCGGGGGAUGCA 2769 rs3025807 9151 GCATCCCCCGACAGCGAGU 1018 9151 GCATCCCCCGACAGCGAGU 1018 9169 ACUCGCUGUCGGGGGAUGC 2770 rs3025807 9152 CATCCCCCGACAGCGAGUC 1019 9152 CATCCCCCGACAGCGAGUC 1019 9170 GACUCGCUGUCGGGGGAUG 2771 rs3025807 9153 ATCCCCCGACAGCGAGUCA 1020 9153 ATCCCCCGACAGCGAGUCA 1020 9171 UGACUCGCUGUCGGGGGAU 2772 rs3025807 9154 TCCCCCGACAGCGAGUCAG 1021 9154 TCCCCCGACAGCGAGUCAG 1021 9172 CUGACUCGCUGUCGGGGGA 2773 rs362308 9681 AGCCCCAGGAAGCCCAUAU 1022 9681 AGCCCCAGGAAGCCCAUAU 1022 9699 AUAUGGGCUUCCUGGGGCU 2774 rs362308 9682 GCCCCAGGAAGCCCAUAUC 1023 9682 GCCCCAGGAAGCCCAUAUC 1023 9700 GAUAUGGGCUUCCUGGGGC 2775 rs362308 9683 CCCCAGGAAGCCCAUAUCA 1024 9683 CCCCAGGAAGCCCAUAUCA 1024 9701 UGAUAUGGGCUUCCUGGGG 2776 rs362308 9684 CCCAGGAAGCCCAUAUCAC 1025 9684 CCCAGGAAGCCCAUAUCAC 1025 9702 GUGAUAUGGGCUUCCUGGG 2777 rs362308 9685 CCAGGAAGCCCAUAUCACC 1026 9685 CCAGGAAGCCCAUAUCACC 1026 9703 GGUGAUAUGGGCUUCCUGG 2778 rs362308 9686 CAGGAAGCCCAUAUCACCG 1027 9686 CAGGAAGCCCAUAUCACCG 1027 9704 ICGGUGAUAUGGGCUUCCUG 2779 rs362308 9687 AGGAAGCCCAUAUCACCGG 1028 9687 AGGAAGCCCAUAUCACCGG 1028 9705 CCGGUGAUAUGGGCUUCCU 2780 rs362308 9688 GGAAGCCCAUAUCACCGGC 1029 9688 GGAAGCCCAUAUCACCGGC 1029 9706 GCCGGUGAUAUGGGCUUCC- 2781 rs362308 9689 GAAGCCCAUAUCACCGGCU 1030 9689 GAAGCCCAUAUCACCGGCU 1030 9707 AGCCGGUGAUAUGGGCUUC 2782 rs362308 9690 AAGCCCAUAUCACCGGCUG 1031 9690 AAGCCCAUAUCACCGGCUG 1031 9708 CAGCCGGUGAUAUGGGCUU 2783 rs362308 9691 AGCCCAUAUCACCGGCUGC 1032 9691 AGCCCAUAUCACCGGCUGC 1032 9709 GCAGCCGGUGAUAUGGGCU 2784 rs362308 9692 GCCCAUAUCACCGGCUGCU 1033 9692 GCCCAUAUCACCGGCUGCU 1033 9710 AGCAGCCGGUGAUAUGGGC 2785 rs362308 9693 CCCAUAUCACCGGCUGCUG 1034 9693 CCCAUAUCACCGGCUGCUG 1034 9711 CAGCAGCCGGUGAUAUGGG 2786 rs362308 9694 CCAUAUCACCGGCUGCUGA 1035 9694 CCAUAUCACCGGCUGCUGA 1035 9712 UCAGCAGCCGGUGAUAUGG 2787 rs362308 9695 CAUAUCACCGGCUGCUGAC 1036 9695 CAUAUCACCGGCUGCUGAC 1036 9713 GUCAGCAGCCGGUGAUAUG 2788 rs362308 9696 AUAUCACCGGCUGCUGACU 1037 9696 AUAUCACCGGCUGCUGACU 1037 9714 AGUCAGCAGCCGGUGAUAU- 2789 rs362308 9697 UAUCACCGGCUGCUGACUU 1038 9697 UAUCACCGGCUGCUGACUU 1038 9715 AAGUCAGCAGCCGGUGAUA 2790 rs362308 9698 AUCACCGGCUGCUGACUUG 1039 9698 AUCACCGGCUGCUGACUUG 1039 9716 CAAGUCAGCAGCCGGUGAU 2791 rs362308 9699 UCACCGGCUGCUGACUUGU 1040 9699 UCACCGGCUGCUGACUUGU 1040 9717 ACAAGUCAGCAGCCGGUGA 2792 rs362308 9681 AGCCCCAGGAAGCCCAUAC 1041 9681 AGCCCCAGGAAGCCCAUAC 1041 9699 GUAUGGGCUUCCUGGGGCU 2793 rs362308 9682 GCCCCAGGAAGCCCAUACC 1042 9682 GCCCCAGGAAGCCCAUACC 1042 9700 GGUAUGGGCUUCCUGGGGC 2794 rs362308 9683 CCCCAGGAAGCCCAUACCA 1043 9683 CCCCAGGAAGCCCAUACCA 1043- 9701 UGGUAUGGGCUUCCUGGGG 2795 rs362308 9684 CCCAGGAAGCCC5AUACCAC 1044 9684 CCCAGGAAGCCCAUACCAC 1044 9702 GUGGUAUGGGCUUCCUGGG 2796 rs362308 9685 CCAGGAAGCCCAUACCACC 1045 9685 CCAGGAAGCCCAUACCACC 1045 9703 GGUGGUAUGGGCUUCCUGG 2797 rs362308 9686 CAGGAAGCCCAUACCACCG 1046 9686 CAGGAAGCCCAUACCACCG 1046 9704 CGGUGGUAUGGGCUUCCUG 2798 rs362308 9687 AGGAAGCCCAUACCACCGG 1047 9687 AGGAAGCCCAUACCACCGG 1047 9705 CCGGUGGUAUGGGCUUCCU 2799 rs362308 9688 GGAAGCCCAUACCACCGGC 1048 9688 GGAAGCCCAUACCACCGGC 1048 9706 GCCGGUGGUAUGGGCUUCCI 2800 rs362308 9689 1GAAGCCCAUACCACCGGCU 1049 98 GACCUAACGU 109 9707 AGCCGGUGGUAUGGGCUUCI 2801 239 2005201389 01 Apr 2005 rs362308 9690 AAGCCCAUACCACCGGCUG 1050 9690 AAGCCCAUACCACCGGCUG 1050 9708 CAGCCGGUGGUAUGGGCUU 2802 rs362308 9691 AGCCCAUACCACCGGCUGC 1051 9691 AGCCCAUACCACCGGCUGC 1051 9709 GCAGCCGGUGGUAUGGGCU 2803 rs362308 9692 GCCCAUACCACCGGCUGCU 1052 9692 GCCCAUACCACCGGCUGCU 1052 9710 AGCAGCCGGUGGUAUGGGC 2804 rs362308 9693 CCCAUACCACCGGCUGCUG 1053 9693 CCCAUACCACCGGCUGCUG 1053 9711 CAGCAGCCGGUGGUAUGGG 2805 rs362308 9694 CCAUACCACCGGCUGCUGA 1054 9694 CCAUACCACCGGCUGCUGA 1054 9712 UCAGCAGCCGGUGGUAUGG 2806 rs362308 9695 CAUACCACCGGCUGCUGAC 1055 9695 CAUACCACCGGCUGCUGAC 1055 9713 GUCAGCAGCCGGUGGUAUG 2807 rs362308 9696 AUACCACCGGCUGCUGACU 1056 9696 AUACCACCGGCUGCUGACU 1056 9714 AGUCAGCAGCCGGUGGUAU 2808 rs362308 9697 UACCACCGGCUGCUGACUU 1057 9697 UACCACCGGCUGCUGACUU 1057 9715 AAGUCAGCAGCCGGUGGUA 2809 rs362308 9698 ACCACCGGCUGCUGACUUG 1058 9698 ACCACCGGCUGCUGACUUG 1058 9716 CAAGUCAGCAGCCGGUGGU 2810 rs362308 9699 CCACCGGCUGCUGACUUGU 1059 9699 CCACCGGCUGCUGACUUGU 1059 9717 ACAAGUCAGCAGCCGGUGG 2811 rs362307 9791 GGAGCCUUUGGAAGUCUGU 1060 9791 GGAGCCUUUGGAAGUCUGU 1060 9809 ACAGACUUCCAAAGGCUCC 2812 rs362307 9792 GAGCCUUUGGAAGUCUGUG 1061 9792 GAGCCUUUGGMAGUCUGUG 1061 9810 CACAGACUUCCAAAGGCUC 2813 rs362307 9793 AGCCUUUGGAAGUCUGUGC 1062 9793 AGCCUUUGGAAGUCUGUGC 1062 9811 GCACAGACUUCCAAAGGCU 2814 rs362307 9794 GCCUUUGGAAGUCUGUGCC 1063 9794 GCCUUUGGAAGUCUGUGCC 1063 9812 GGCACAGACUUCCAAAGGC 2815 rs362307 9795 CCUUUGGAAGUCUGUGCCC 1064 9795 CCUUUGGAAGUCUGUGCCC 1064 9813 GGGCACAGACUUCCAAAGG 2816 rs362307 9796 CUUUGGAAGUCUGUGCCCU 1065 9796 CUUUGGAAGUCUGUGCCCU 1065 9814 AGGGCACAGACUUCCAAAG 2817 rs362307 9797 UUUGGAAGUCUGUGCCCUU 1066 9797 UUUGGAAGUCUGUGCCCUU 1066 9815 AAGGGCACAGACUUCCAAA 2818 rs362307 9798 UUGGMAGUCUGUGCCCUUG 1067 9798 UUGGAAGUCUGUGCCCUUG 1067 9816 CAAGGGCACAGACUUCCAA- 2819 rs362307 9799 UGGAAGUCUGUGCCCUUGU 1068 9799 UGGAAGUCUGUGCCCUUGU 1068 9817 ACAAGGGCACAGACUUCCA 2820 rs362307 9800 GGAAGUCUGUGCCCUUGUG 1069 9800 GGAAGUCUGUGCCCUUGUG 1069 9818 CACAAGGGCACAGACUUCC 2821 rs362307 9801 GAAGUCUGUGCCCUUGUGC 1070 9801 GAAGUCUGUGCCCUUGUGC 1070 9819 GCACAAGGGCACAGACUUC 2822 rs362307 9802 PAGUCUGUGCCCUUGUGCC 1071 9802 AAGUCUGUGCCCUUGUGCC 1071 9820 GGCACAAGGGCACAGACUU 2823 rs362307 9803 AGUCUGUGCCCUUGUGCCC 1072 9803 AGUCUGUGCCCUUGUGCCC 1072 9821 GGGCACAAGGGCACAGACU 2824 rs362307 9804 GUCUGUGCCCUUGUGCCCU 1073 9804 GUCUGUGCCCUUGUGCCCU 1073 9822 AGGGCACAAGGGCACAGAC 2825 rs362307 9805 UCUGUGCCCUUGUGCCCUG 1074 9805 UCUGUGCCCUUGUGCCCUG 1074 9823 CAGGGCACAAGGGCACAGA 2826 rs362307 9806 CUGUGCCCUUGUGCCCUGC 1075 9806 CUGUGCCCUUGUGCCCUGC 1075 9824 GCAGGGCACAAGGGCACAG 2827 rs362307 9807 UGUGCCCUUGUGCCCUGCC 1076 9807 UGUGCCCUUGUGCCCUGCC 1076 9825 GGCAGGGCACAAGGGCACA- 2828 rs362307 9808 GUGCCCUUGUGCCCUGCCU 1077 9808 GUGCCCUUGUGCCCUGCCU 1077 9826 AGGCAGGGCACAAGGGCAC 2829 rs362307 9809 UGCCCUUGUGCCCUGCCUC 1078 9809 UGCCCUUGUGCCCUGCCUC 1078 9827 GAGGCAGGGCACAAGGGCA 2830 rs362307 9791 GGAGCCUUUGGAAGUCUGC- 1079 9791 GGAGCCUUUGGAAGUCUGC 1079 -9809 GCAGACUUCCAAAGGCUCC 2831 rs362307 9792 GAGCCUUUGGAAGUCUGCG 1080 9792 GAGCCUUUGGMAGUCUGCG 1080 9810 CGCAGACUUCCAAAGGCUC 2832 rs362307 9793 AGCCUUUGGAAGUCUGCGC 1081 9793 AGCCUUUGGAAGUCUGCGC 1081 9811 GCGCAGACUUCCAAAGGCU 2833 rs362307 9794 GCCUUUGGAAGUCUGCGCC 1082 9794 GCCUUUGGAAGUCUGCGCC 1082 9812 GGCGCAGACUUCCAAAGGC 2834 rs362307 9795 CCUUUGGAAGUCUGCGCCC 1083 9795 CCUUUGGAAGUCUGCGCCC 1083 9813 GGGCGCAGACUUCCAAAGG 2835 rs362307 9796 CUUUGGAAGUCUGCGCCCU 1084 9796 CUUUGGAAGUCUGCGCCCU 1084 9814 AGGGCGCAGACUUCCAAAG 2836 rs362307 9797 UUUGGAAGUCUGCGCCCUU 1085 9797 UUUGGAAGUCUGCGCCCUU 1085 9815 AAGGGCGCAGACUUCCAAA 2837 rs362307 9798 UUGGAAGUCUGCGCCCUUG 1086 9798 UUGGMAGUCUGCGCCCUUG 1086 9816 CAAGGGCGCAGACUUCCAA 2838 rs362307 9799 UGGAAGUCUGCGCCCUUGU 1087 9799 UGGAAGUCUGCGCCCUUGU ,1087 9817 ACAAGGGCGCAGACUUCCA 2839 rs362307 9800 GGAAGUCUGCGCCCUUGUG 11088 9800 GGAAGUCUGCC UU 108 H88 CACAAGGGCGCAGACUUCC 12840 240 2005201389 01 Apr 2005 -362307 9 801 GAAGUCUGCGCCCUUGUGC 1089 9801 GAAGUCUGCGCC rs-362307 9-802 AAGUCUGCGCCCU UGUGCC 1090 9802 AAGUCUGCGCCC r-3-62307 9 8-03 AGUCUGCGCCCUUGUGCCC; 1091 9803 AGUCUGCGCCCL r;3-62307 -9804 GUCUGCGCCCUUGUCCU 1092 9804 GUCUGCGCCCUL rs362307 9805 UCUGCGCCCUUGUGCCCUG 1093 9805 UCUGCGCCCUUC rs362307 9806 CUGCGCCCUUGUGCCCUGC 1094 9806 CUGCGCCCUUGL rs362307 9807 UGCGCCCUUGUGCCCUGCC 1095 9807 UGCGCCCUUGU( rs362307 9808 GCGCCCUUGUGCCCUGCCU 1096 9808 GCGCCCUU1 GUG( rs362307 9809 CGCCCUUGUGCCCUGCCUC 1097 9809 CGCCCUGC rs620610046 GCUGGUUGUUGCCAGUUGuu 109 10046 GUGGUUGUUG( rs362306 10047 CUGGUUGUUGCCAGGUUC 1 10047 CUGGUUGUUGCI rs36230 6 10048 UGGUUGUUGCCAGGUUGCA 1100 10048 UGGUUGUUGCCI rs362306 10049 GGUUGUUGCCAGGUUGCAG 1101 10049 GGUUGUUGCCAI rs362306 10050 GUUGUUGCCAGGUUGCAGC 1102 10050 GUUGUUGCCAG, rs362306 10051 UUGUUGCCAGGUUGCAGCU 1103005

UJUGUUGCCAGG

rs362306 10052? IGUCAGUCGU 110 10052M UGUUGCCAGGU rs362306 10053 GUUGCCAGGUUGCAGCUG 11054 05 UGCGU rs362306 10054 GUUGCCAGGUUGCAGCUGC 110 10054 GUUGCCAGGUU s-36-2306 -10055 UUGCCAGGUUGCAGU-CU 1107 10055 UUGCCAGGUUG r7362 -TOO1005 G AGGUUGCAGCUGCUC 1108 10056 UGCCAGGUUGC rs-36230 6 -100576 -ICCAGGUUGCAGCUGCUUU 1109 10057 GCCAGGUUGCA rs362306 10058 CAGGUUGCAGCUGCUCUU 1109 10058 -'CAGGUUGCAG s-36230 6 100-598 AGUUGCAGCUGCUCUUG 1111 10059 CAGGUUGCAGC s-36-2306 100O60 GGUUGCAGCUGCUCUUGCA 12112 106 222 G.%UUGCAGCUC rs36230 6 10061 GUUGCAGCUGCUCUUGCAU 113 10061 GUUGCAGCUGC r 3-6230 6 1f 06 2 UUGCAGCUGCUCUUGCAUC 1114 10062 UUGCAGCUGCL rs3-6230 6 1f00 63 UGCAGCUGCUCUUGCAUCU 1115 10063 UGCAGCUGCUC rsi3-62306 10064 GdCAGCUGCUCUUGCAUCUG 1116 10064 GCAGCUGCUCIL rs362306 10046 GCUGGUUGUUGCCAGGUUA 1117 10046 GCUGGUUGUUC rs362306 J10047 CUGGUUGUUGCCAGGUUAC 1118 10047 CUGGUUGUUG( rs36230 6 10048 UGGUUGUUGCCAGGUUACA 1119 10048 UGGUUGUUGC( rs362306 10049 GGUUGUUGCCAGGUUACAG 11 20 10049 GGUUGUUGCC) rs36230 6 105 GUUGUUGCCAGGUACAGC 1121 10050 GUUGUUGCCA( rs36230 6 10051 UUGUUGCCAGGUUACANGCU 1122 10051 UUGUUGCCAG( rs36230 6 10052 UGUUGCCAGGUUACAGCUG 1123 10052 UGUUGCCAGGI rs-362306 10053 GUUGCCAGGUUACAGCUGC 1124 10053 GUUGCCAGGUI rs36230 6 -10054 UUGCCAGGUUACAGCUGCU 1125 10054 UUGCCAGGUUi s-36-2306 1 0055 UGCCAGGUUACAGCUGCUC 1126 10055 UGCCAGGUUA( rs3-62306 -100 56 GCCAGGUUACAGCUGCUCU 11T2-7 10056 GCCAGGUUAC, 1089 1090 1091 1092 1093 1094 1095 1096 1097 1098 1099 1100 1101 1102 1103 1104 1105 1106 1107 11I0 8 1109 1110 1111 1112 1113 1114 1115 9819 9820 9821 9822 9823 9 8-24 9825 9826 9827 10064 10065 10066 10067 10068 10069 10070 10071 -10072 10073 10074 10075 1i0076 10077 10078

GCACAAGGGCGCAGACUUC

GGCACAAGGGCGCAGACUU

GGGCACAAGGGCGCAGACU

AGGGCACMAGGGCGCAGAC

CAGGGCACAAGGGCGCAGA

GCAGGGCACAAGGGCGGAG

GGCAGGGCACAAGGGGGCA

AGGCAGGGCACAAGGGCGC

GAGGCAGGGCACAAGGGCG

CAACCUGGCAACAACCAGC

GCAACCUGGCAACAACCAG

UGCAACCUGGCAACAACCA

CUGCAACCUGGCAACAACC

GCUGCMACCUGGCAACAAC

AGCUGCAACCUGGCAAGAA

-CAGCUGCAACCUGGCAACA

GCAGCUGCAACCUGGCAAC

AGCAGCUGCAACCUGGCAA

GAGCAGCUGCAACCUGGCA

AGAGCAGCUGCAACCUGGC

-AAGAGCAGCUGCAACCUGG

CMAGAGCAGCUGCMACCUG

GCAAGAGCAGCUGCAACCU

UGCAAGAGCAGCUGCAACC

2841 2842 2843 2844 2845 2846 2847 2848 2849 2850 2851 2852 2853 2854 2855 2856 285-7- 2858 2859 2860 2861 2862 2863 2864 10079 1AUGCAAGAGCAGCUGCAAC] 2865 10081 I GAUGCMAGAGCAGCUGCAA 2867 10O082 AGAUGCAAGAGCAGCUGC 286 Miu 1117 1120 ~1121 1122Y 10064 10065 10066 10067 10068 10069 10070 10071 10072 10073 10074 UAGAUGCAAACACAG

C

UAACCUGGCAACAACCAGC

GUAACCUGGCAACAACCA

UGUAACCUGGCAACAACCA

CUGUAACCUGGCAACAAC

GCUGUAACCUGGCAACAAC

AGCUGUAACCUGGCAACA

CAGCUGUAACCUGGCAAC

GCAGCUGUAACCUGGCAAC

AGCAGCUGUAACCUGGCA

GAGCAGCUGUAACCUGGGC

2869 2870 2871 2872 2873 2874 2875 2876 2877 2878 2879 2005201389 01 Apr 2005 s-36230 6 rs362306 s-362306 rs-362306 rs362306 rs362306 rs36230 6 rs36230 6 rs362268 rs362268 rs362268 rs362268 rs362268 rs36226 8 rs362268 rs36226 8 rs362268 rs36226 8 rs36 226 8 s36226 8 rs362268 rs3622 68 rs36226 8 rs3622 68 rs362268 rs3622 68 rs362268 rs362268 rs362268 rs362268 rs36226f rs36226E rs36226~ rs36226~ rs36226 rs36226 rs362261 rs36226 rs36226 10057 CCAGGUUACAGCUGCUCUU 1128 10057 CCAGGUUACAGCUGCUCUU -1128 10058 CAGGUUACAGCUGCUCUUG 1129 10058 CAGGUUACAGCUGCUCUUG 1129 10059 AGGUUACAGCUGCUCUUGC 1130 10059 AGGUUACAGCUGCUCUUGC 1130 10060 GGUUACAGCUGCUCUUGCA 111100 UAACUGCUCUUGCA 1131 10061 GUUACAGCUGCUCUUGCAU 1132 1006 1 G.UUACAGC,,UGCUJCUUGCAU 1132 10062 UUACAGCUGCUCUUGCAU 1133 10062 UUACAGCUGCUCUUGCAUC 1133 10063 UACAGCUGCUCUUGCAUCU 1134 10063 UACAGCUGCUCUUGCAUCU 1134 10064 ACAGCUGCUCUUGCAUCUG 1135 10064 ACAGCUGCUCUUGCAUCUG 1135 10094 CUCCCUCCUGCAGGCUGGC 1136 109 CUCCrtrrCUCCUGCAGGCUGGC 1136_ 10095 UCCCUCCUGCAGGCUGGCU 1137 10095 uCCCUCCUGCAGGCUGGC'U 1137 10096 CCCUCCUGCAGGCUGGCUG 1138 10096 CCCUCCGAGGCUGGCUG 1138 10097 CCUCCUGCAGGCUGGCUGU 1139 10097 CCUCCUGCAGGCUGGCUGU 1139 10098R CUICCUGCAGGCUGGCUGUU 1140 10098 CUCCUGCAGGCUGGCUGUU 1140 10099 UL;CUGCAGGCUGGCUGUUG 1141 10099 UCCUGCAGGCUGGCUGUUG 1141 10100 CCUGCAGGCUGGCUGUUGG 1 12 10100 CCUGCAGGCUGGCUGUUGG 1142_ 10101 CUGCAGGCUGGCUGUUGGC 1143 10101 CUGCAGGCUGGCUGUUGGC 1143 10102 UGCAGGCUGGCUGUUGGCC 1144 10102 UGCAGGCUGGCUGUUGGCC 1144 10103 GCAGGCUGCUUUGGCCC 1145 10103 GCAGGCUGGCUGUUGGCCC 1145 10104 CAGGCUGGCUGUUGGCCCC 1146 10104 CAGGCUGGCUGUUGGCCCC 1146 10105 AGGCUGGCUGUUGGCCCCU 1147 -110 5 AGGCUGGCUGUUGGCCCCU 1147 10106 GGCUGGCUGUUGGCCCCUC 1148 10106 GGCUGGCUGUUGGCCCCUC 1148 10112 GCCUGUUGGCCCGCUGU 114 10112 GCCUGUUGGCCCUCUGU 114 101094 CUGCCUCCUGCAGGCUGG 1155 101094 CUGCCUCCUGCAGGCUG 1155 101095 UCUGCGGCUG UGG GC 1156 101095 UCCCUGUCCUCUGG 1156 1010 CCCUGUCCCU CUGGU 1157 1010 CCCUGUCCCUCUGGU 1157 10097 CCCUGAGGCCCUGGUG 1158 10097 CCCUGCGGCCUGGUG 1153 10098 CCUGAGGCCCUGGUGU 1159 10098 CUCUGCGGCUGGUGU 1159 310099 CUCCCUGCAGGCUGGUG 1160 10099 CUUCCUGCAGGCUGGUG 1160 310005 UCCCUGCAGGCUG GG 1156 1100 UCCCUGCAGGCUG GG 1156 310017 CCUGCAGGCUGGGUGUG 1162 10017 CCUGCAGGCUGGGUGUC 1162 310028 CUGCAGGCUGGGUGUUC 1163 10028 CUGCAGGCUGGGUGUUC 1163 B 10039 UGCAGGCUGUGUUGC 116 10039 GCAULGGCUGGGUGUUGC 1164 B 10104 CUCAGGCUGGGUGUUGCCCC 1165 -10 10 C CAGGCUGGUUUGGC 1165 8 10105 AGCUGGGUGUUGGCCCC 1166 10105 CAGGCUGGGUGUUGGCCCC 1166 10075 AAGAGCAGCUGUA-ACCUGG 10076 CAAGAGCAGCUGUAACC;UG 10077 GCAAGAGCAGCU-GUAACCU 10078 UGCAAGAGCAGCUGUAACC 10079 AUGCAAGAGCAGCUGUAAC 10080 GAGAGAGCAGCUGUMA 10081 AGAUGCAAGAGCAGCGUUA 10082 CAGAUGCAAGAGCAGCUGU 10112 CCAGCCUGCAGGAGGGAG 10113 AGCCAGCCUGCAGGAGGA 10114 CAGCCAGCCUGCAGGAGGG 10115 ACAGCCAGCCUGCAGGAGG 10116 ACAGCCAGCCUGCAGGAG 10117 CAACAGCCAGCCUGCAGGA- 10118 CC,,AACAGCCAGCCUGCAGG 101 19 GCCAACAGCCAGCCUGCAG 10120 GGCCAACAGCCAGCCUG3CA 10121 GGGCCAACAGCCAGCCUGC 10122 GGGGCCAACAGCCAGCCUG 10123 AGGGGCCAACAGCCAGCCU 10124 GAGGGGCCAACAGCCAGCC 10125 AGAGGGGCCAACAGCCAGC 10126 CAGAGGGGCCAACAGCCAG 101271 GCAGAGGGGCCAACAGCA 10128 AGCAGAGGGGCCAAC;AGCC 10129 CAGCAGAGGGGCCAACAGC 10130 ACAGCAGAGGGGCCAACAG 10112 CCCAGCCUGCAGGAGGGAG- 10113 ACCCAGCCUGCAGGAGG3GA 10114 CACCCAGCCUGAGGAGGG 10115 ACACCCAGCCUGCAGG3AGG 10116 IAACACCCAGCCUGCAGGAG 10117 1CAACACCCAGCCUGCAGGA 10118 CCAACACCCAGCCUGCAGG 10119 GCCAACACCCAGCCUGCAG 10120 GGCCAACACCCAGCCUGCA 10O121 GGGCCAACACCCAGCCUGC 1 0122 GGGGCCAACACCCAGCCUG 10123 AG GG G CCAACAC CCAGCCPU(: 2880 2881 2882 2883 2884 2885 2886 2887 2888 2889 2890 2891 2892 2893 2894 2895 2896 2897 2898 2899 2900 2901 2902 2903 2904 2905 2906 2907 2908 2909 2910U 2911 2912 2913 2914 2915b 242 2005201389 01 Apr 2005 rs3-62305 1-i0113 s3-62305 fO 104 rs-362305 i 15 rs362305 10116] rs362305 10117 rs-362 3-05 101181 rsi3-62305 1 0119 s36 2 305 10-O120 rs362305 10121 rs3-62305 -10122 rs-362305 1-i0123 r 3-62305 -10124 rs362305 10106 rs362305 10107 rs362305 10108 rs362305 10109 rs362305 10110 rs362305 10111 rs362305 101 12 s-362305 -10113 rs3-62305 -10114 rs362305 10115 rs3A62305 10116 rs3-62305 10117 r 3-62305 10O118 rs3-62305 10-119 rs362305 10120 rs362305 10121 rs362305 122 rs362305 1123 ri3-62305 -101-24 rs362304 10218 rs362304 -021 9 rs36230 4 10-22C rs362304 10221 rs36230 4 10222 rs36230 4 1-0222rs63 4 -T10224 rs36230 4 I1022f

UGUUGGCCCCUCUGCUGUC

UUGGCCCCUCUGCUGUCC

7U-UGGCCCCUCUGCUGUCCU

UGGCCCCUCUGCUGUCCUG

GGCCCCUCUGCUGUCCUGC

GCCCCUCUGCUGUCCUGCA

CCCUCUGCUGUCCUGCAG

CCCUCUGCUGUCCUGCAG3U

CCUCUGCUGUCCUGCAGUA

CU CU GC UGUCCU GCAG UAG

UCUGCUGUCCUGCAGUAGA

CUGCUGUCCUGCAGUAGAA

GGCUGGCUGUUGGCCCCUG3

GCUGGCUGUUGGCCCCUGU

CUGGCUGUUGGCCCCUGUG

UGGCUGUUGGCCCCUGUGC

GGCUGUUGGCCCCUGUGCU

GCUGUUGGCCCCUGUGCUG

CZUGUUGGCCCCUGUGCUGU

UGUUGGCCCCUGUGCUGUC

GUUGGCCCCUGUGCUGUCC

UUGGCCCCUGUGCUGUCCU

UGGCCCCUGUGCUGUCCUG

GGCCCCUGUGCUGUCCUGC

GCCCCUGUGCUGUCCUGCA

CCCCUGUGCUGUCCUGCAG

CCCUGUGCUGUCCUGCAGU

CCUGUGCUGUCCUGCAGUA

CU-GUGCUGUCCUGCAGUAG

U-GGUGCUGUCCUGCAGUAGA

GUGCUGUCCUGCAGUAGAA

AUGCACAGAUGCCAUGGCC

UGCACAGAUGCCAUGGCCU

G ;CACAGAUGCCAUGGCCUG

CACAGAUGCCAUGGCCUGU

ACAGAUGCCAUGGCCUGUG

CAGAUGCCAUGGCCUGUG3C

AGAUGCCAUGCCUGUGCU

GAUGCCAUGGCCUGUGCUG

116 1011h 1168 10114 1169 1011 1170 1011 1171 10117 112 10118 1173 1019 1174 10120 1175 10121 1176 10122 1177 10123 1178 10124 1179j~ 10106 118 10107 1181 10108 1182 10109 1183 10110 1184 10111 1185 10112 1186 10113 1187 10114 1188 10115 1189 10116 1190 10117 1191 10118 1192 10119 1193 10120 1194 10121 119-5 10122 1196 10 1 2' 11l9 7 -11 1198 1021~ 11i99 121~ 1200 1022C 1201 1022 1202 1022: 1203 1022: 1204 1022 1205 11022

UGUUGGCCCCUCUGCUGUC

GUUGGCCCCUCUGCU4GUCC UUGGCCCUCUGUGUr

UUGGCCCCUCUGCUUCCU

GGCCCUCUCUGUCUG

UGCCCCUCUGCUGUCCUG

GCCCCUCUGCUGUCCUCAG

GCCCUCUGCUGUCCUGCA

CCCUCUGCUGUCCUGCAG

CCUCUGUGUCCUGAU

CUCUGCUGUCCUGCAGUAA

CCUGCUGUCCUGCAGUAA

GCUGCUGUUGCGUG

CUGCUGUUCCCUGU

GCUGGCUGUUGGCCCCUG

GUGGCUGUUGGCCCCUGU

CGGCUGUUGGCCCCUGUGU

UGCUGUUGGCCCCUGUGC

GCUGUUGGCCCCUGUGCUU

GUGUUGGCCCCUGUGCUG

CGUUGGCCCCUGUGCUGU

UUUGGCCCCUGUGUGUCU

GGCCGUUUCU

UGGCCCCUGUGCUGUCCU

UGCCCCUGUGCUGUCCUG

GCCCCUGUGCUGUCC-GC

R

GUCCCUGUGCUGUCCUCU

GCCCUGUGCUGUGCGUA

UUGCUCCUGAGA

CUGUGCUGUCCUGCAGG

CCGUGCUGUCCUAGUA

3

UGCCAGUGCUG

CCGAUGCCUGGCCUG

1

CACGUGCCUGCG

SCGUGCAUGGCCUGUG

3 CAUGCCAUGCCUGGC 2 AAGAUGCCAUGGCUGUGU 51 GAUGCCAUGGCCUGUGCUG 1167 103 116j8 10132 1169 10133 1170 10134 1171 10135 1172 10136 1173 10137 1174 10138 1175 103 Iii7I- 014 1177~~ 10141 118 10125 118 10127 1183~ 10128 1184~ 10129 118 10130 118 10131 1187- 10132 1188 10133 118 10134 10135 1191 10136 11 92 10137 1193 10138 1194 10139 1195 10140 1196 10141 1197~ 10142 1198~- 10236 1199~ 10237 1200~- 10238 1201 10239 1202 10240 1203 10241 1204 10242 1205 10243 GACAGCAGAGGGGCCAACA 2919 GGACAGCAGAGGGGCCMAC 2920 AGGACAGCAGAGGGGCCAA 2921 CAGGACAGCAGAGGGGCCA 2922 GCAGGACAGCAGAGGGCC 2923 UGCAGGACAGCAGAGGGGC 2924 CUGCAGGACAGCAGAGGGG 2925 ACUGCAGGACAGCAGAGGG 2926 UACU'GCAGGACAGCAGAGG 2927 CUACUGCAGGACAGCAGAG 2928 UCUACUGCAGGACAGCAGA 2929 UUCUACUGCAGGACAGCAG 2930 C'AGGGGCCAACAGCCAGCC 2931 ACAGGGGCCAACAGCCAGC 2932 CACAGGGGCCAACAGCCAG 2933 GCACAGGGGCCAACAGCCA 2934 AGCACAGGGGCCAACAGCC 2935 CAGCACAGGGGCCAACAGC 2936 ACAGCACAGGGGCCAACAG 2937 GACAGCACAGGGGCCAACA 2938 GGACAGCACAGGGGCCAAC 2939 AGGACAGCACAGGGGCCAA 2940 CAGGACAGCACAGGGGCCA 2941 GCAGGACAGCACAGGGGCC 2942 UGCAGGACAGCACAGGGGC 2943 CUGCAGGACAGCACAGGGG 2944 -ACUGCAGGACAGCACAGGG 2945 -UACUGCAGGACAGCACAGG 2946 CUACUGCAGGACAGCACAG 2947 UCUACUGCAGGACAGCACA 2948 UUCUACUGCAGGACAGCAC 2949 GGCCAUGGCAUCUGUGCAU 2950 AGGCCAUGGCAUCUGUGCA 2951 CAGGCCAUGGCAUCUGUGC 2952 AKCAGGCCAUGGCAUCUGUG 2953 CACAGGCCAUGGCAUCUGU 2954 GCACAGGCCAUGGCAUCUG 2955 AGCACAGGCCAUGGCAUCU 2956 CAGCACAGGCCAUGGCAUC 2957 243 rs362304 r 3-623047 rs362304 s-362304 rs362304 rs362304 rs362304 rs3623047 rs362304 rs362304 s3623 rs362304 7s-362304 Rs36 2304 r 3-62304 rs362304 ri3-62304 rs362304 r 3-62304 rs362304 ri3-62304 r;s362304 r 3-6230 4 7s-362304 rs362304 rs362304 rs362304 rs362304 rs362304 r 3-62304 rs3-62303 r 3-62303 rs-36230 3 rs362303 7s-36230 3 rs-362303 rs3-62303 ri3-62303 rs362303 10226 0O227 0O228 FO0229 10230 10231 10232 10233 10234 10235 10236 -0218 10219 10220 10221 10222 10 22 3 -0224 10225 10 22 6 -0227 10228 10-229 10230 10231 10232 TO 2-3 10234 10235 1023( 1025' 1025' 1025~ 1025E 10-25- 10251 10251 10261 -026

AUGCCAUGGCCUGUGCUGG

UGCCAUGGCCUGUGCUGGG

GCAUGGCCUGUGCUGGGC

CAUGGCCUGUGCUGGGCC

CAUGGCCUGUGCUGGGCCA

AUGGCCUGUGCUGGGCCAG

UGGCCUGUGCUGGGCCAGU

GGCCUGUGCUGGGCCAGUGn

GCCUGUGCUGGGCCAGUGG

CCUGUGCUGGGCCA-GUGGC

CUGUGCUGGGCCAGUGGCU

AUGCACAGAUGCCAUGGCA

UGCACAGAUGCCAUGGCAU

GCACAGAUGCCAUGGCAU

CACAGAUGCCAUJGGCA

U

ACAGAUGCCAUGGCAUGU

CAGAUGCCAUGGCAUGUG

AGAUGCCAUGGCAUGUGCU

GAUGCCAUGGCAUGUGCUGi

AUGCCAUGGCAUGUGCUGG

UGCCAUGGCAUGUGCUGGG

GCCAUGGCAUGUGCUGGGC

CCAUGGCAUGUGCUGGGCC

CAUGGCAUGUGCUGGGCCA

AUGCAUUGCGGCAG

UGGCAUGUGCUGGGCCAG

GG-CAUGUGCUGGGCCAGU

GGCAUGUGCUGGGCCAGUG

CAUGUCUGG C U

GAUGUGCUGGGCCAGUGG

CAUGUGGGCAGCC

KUUGGGGGUGCUCCC

5 GGGGGUGCUAGAACC

UGGGGUGCUAGACACCCGC

5GGGGUGCUAGACACCCGGA 3 GGUGCUAGACACCCGGC

-GUGCUAGACACCCGGCA

8 GUGCUAGACACCCGGCACA 9GGCUAGACACCCGGCACC 1206 10226 AUGCCAUGGCCUGUGCUGG 1207 10227 UGCCAUGGCCUGUGCUGG 1208 10228 GCCAUGGCCUGUGCUGGGC 1209 10229 CCAUGGCCUGUGCUGGGCC 1210 10O230 CAUGGCCUGUGCUGGGCCA 1211 10231 AUGGCCUGUGCUGGGCCAG 1212 10232 UGGCCUGUGCUGGGCCAGU 1213 10233 GGCCUGUGCUGGGCCAGUG 1214 10234 'GCCUGUGCUGGGCCA

GUGG

1215 10235 CCUGUGCUGGGCCAGUGG 1216 10236 CUGUGCUGGGCCAGUGGCU 1217 10218 AUGCACAGAUGCCAUGGCA 1218 10219 UGCACAGAUGCCAUGGC3(AU 1219 10220 GCACAGAUGCCAUGGCAUG 1220 10221 ;AC;AGAUGCCAUGGCAUGU 1221 10222 ACAGAUGCCAUGGCAUGUG 1222 10223 CAGAUGCCAUGGCAUGUGC 122 10224 AGUCAGGCAUGUGCL 1224e. V10225 G-UGCC-A-GGCAUGUGCUE 1225 10226 AUGCCAUGGCAUGUGCUGC 1226 10227 UGCCAUGGCAUGUGCUGC 1227 10228 GCCAUGGCAUGUGCUGGG( 1228 10229 CCAUGGCAUGUGCUGGGC( 1229 10230 CAUGGCAUGUGCUGGGCCi 1230 10231 AUGGCAUGUGCUGGGCCLA( 1231 10232 UGGCAUGUGCUGGGCCAGI 1232 10233 CAUG...GCUGGGCCAGU( 1233 10234 GiCAUG..U3CUGGGCCAGUG( 1234 10235 CAUGUGCUGGGCCAGUGGI 1235 10236 AUGUGCUGGGCCAGUGGC' 1236 10253 CUGGGGUGCUAGACA

C

1237 105 GGGUCUAGACACCC 1238 10255 GGGGGU-CUAGACACCCG 1239 10256 GGGGUGCUAGACACCCGG 1240 1 0257- GGUGCUAGACACCCGGC 12-41 10258 GGUGCUAGACACC-CGGCA! 1t24 2 10259 GUCUAGACACCCGGCAC 1243 10260 -~UGCUAGACACCCGGCACC 1 244 121 GCUAGACACCCGGCACCA 2005201389 01 Apr 2005 1206 10244 CCAGCACAGGCCAUGGCAU 2958 1207 10-245 -CCCAGCACAGGCCAUGGC(A 2959 1208 10246 GCCCAGCACAGGCCAUGGC 2960 1209 10247 GGCCCAGCACAGGCCAUGG 2961 1210 10248 UGGCCCAGCACAGGCCAUG 2962 1211 10249 CUGGCCCAGCACAGGCCAU 2963 1212 10250 ACUGGCCCAGCACAGGCCA 2964 1213 10251 CACUGGOCCAGOACAGGOC 296j5 1214 10252 CCACUGGCCCAGCACAGGC 2966 1215 10253 GCCACUGGCCCAGCACAGG 2967 1216 10254 AGCCACUGGCCCAGCACAG 2968 1217 10236 UCCAUGGCAUCUGUGCAU 2969 1218 10237 AUGCCAUGGCAUCUGUGCA 2970 1219 10238 CAUGCCAUGGCAUCUGUGC 2971 1220 10239 ACAUGCCAUGGCAUCGUG 2972 1221 10240 CACAUGCCAUGGCAUCUGU- 2973 1222 1041 GCACAUGCCAUGGCAUCUG 2974 I1223 10242 AGCACAUGCCAUGGCAUCU 2975 1224 10243 -CAGCACAUGCCAUGGCAUC 2976 1225 10244 CCAGCACAUGCCAUGGCAU 2977 3 1226 10245 CCCAGCACAUGCCAUGGCA 2978 S1227 10246 GCCCAGCACAUGCCAUGGC 2979 3 1228 10247 GGCCCAGCACAUGCCAUGG 2980 S1229 10248 UGGCCCAGCACAUGCCAUG 2981 3 1230 10249 CUGGCCCAGCACAUGCCAU 2982 U 1231 10250 ACUGGCCCAGCACAUGCCA 2983 3 1232 10O251 -C ACUGGCCCAGCACAUGCC 2984 G 1233 10252 CCACUGGCCCAGCACAUGC 2985 C 1234 10253 GCCACUGGCCCAGCACAUG 2986 U 1235 10254 AGCCACUGGCCCAGCACAU 2987 C -T 126 121 GGGUGUCUAGCACCCCCAG 2988 G 1237 10272 CGGGUGUCUAGCACCCCCA 2989 G 1238 10273 CGGGUGUCUAGCACCCCC 2990 C 1239 10274 GCCGGGUGUCUAGCACC;CC 2991 A 1240 10275 UGCCGGGUGUCUAGCACCC 2992 C 21 10276 GUGCCGGGUGUCUAGCACC 2993 c 1242 10277 G GUGCCGGGUGUCUAGCAC 2994 A 1243 10278 JUGGUGCCGGGUGUCUAC 2995 U 1244 110279 IAUGGUGCCGGGUGUCUAGCI 29 244 2005201389 01 Apr 2005 rs362303 10262 s-362303 -10263 rs-362 3-03 -10264 Ts-362303 10O265 rs362303 10266 s3-62303 -10267 rs-362303 -10268 7s-362303 -10269 rs362303 10270 rs362303 10271 rs362303 10253 rsj362303 10254 rs-362303 10255 rs362303 10256 rs362303 10257 r362303 10258 rs362303 10259 rs362303 10260 r s36230 3 1i0-261 rs-36230 3 1i026-2 i;s362303 10263 rs362303 10264 r7S362303 10O-265 s-362303 10266 rs36 2 303 10O-267 s-362303 1026E rs362303 10269 rs362303 1 0270 rs362303 10271 rs1557210 10861 rs1 557 2 1 0 1086, rs1 557210 06~ 1155721 0 1086' rs1 557 2 1 0 10861 rs1 55721 0 10861 11557 2 1 0 1086 rs1 55721 1086 rs15.57 2 1 0 1086 r s155721 0 1i087 CUAGACACCCGGCACCAUU 1245 1 UAGACACCCGGCACCAUUC 1246 1 AGACACCCGGCACCAUUCU 1247 1 GACACCCGGCACCAUUCUC 1248 1 ACACCCGGCACCAUUCUCC 1249 1 CACCCGGCACCAUUCUCCC 1250 1 ACCCGGCACCAUUCUCCCU 1251 1 CCCGGCACCAUUCUCCCUU 1252 1 CCGGCACCAUUCUCCCUUC 1253 1 CGGCACCAUUCUCCCUUCU 1254 1 CUGGGGGUGCUAGACACCU 1255 1 UGGGGGUGCUAGACACCUG 12561 GGGGGUGCUAGACACCUGG 125-7 I GGGGUGCUAGACACCUGG 1258 GGGUGCUAGACACCUGGCA 1259 GGUGCUAGACACCUGGCAC 120 GUGCUAGACACCUGGCACC 161 UGCUAGACACCUGGCACCA 162 GCUAGACACCUGCACCAU 126 3 CUAGACACCUGGCACCAUU 1264 UAGACACCUGGCACCAUUC 1265 AGACACCUGGCACCAUUCU 1266 GACACCUGGCACCAUUCUC 1267 ACACCUGGCACCAUUCUCC 1268 CACCUGGCACCAUUCUCCC 1269 ACCUGGCACCAUUCUCCCU 1270 CCUGGCACCAUUCUCCCUU 1271 CUGGCACCAUUCUCCCUUC 1272 UGGCACCAUUCUCCCUUCU 1273 UGUGUUUUGUCUGAGCCUC 1274 GUGUUUUGUCUGAGCCUCU 1275 3 UGUUUUGUCUGAGCCUC;UC 1276 4GUUUUGUCUGAGCCUCUCU 1277 5 UUUUGUCUGAGCCUCUCUC 1278 6 UUUGUCUGAGCCUCUCUCG 1279 7 UUGUCUGAGCCUCUCUCGG 1280 8 UGUCUGAGCCUCUCUCGGU 1281 9 GUCUGAGCCUCUCUCGGUC 1282 0UUGAGCCUCUCUCGGUCA l, 1283 0263 0265 0266 0267 0268 0269 0270 0271 0253 0254 10257 10258 10259 10260n 10261 10262 10263 10264 10265 10266 10267 10268 10269 10270 10271 10861 1086~ 1086 1086' 10861 10861 1086 1086 1086

CUAGACACCCGGCACCAUU

UAGACACCCGGCACCAUUC

AGACACCCGGCACCAUUCU

GACACCCGGCACCAUUCUC

ACACCCGGCACCAUUCUCC

CACCCGGCACCAUUCUCCC

ACCCGGCACCAUUCUCCCU

CCCGGCACCAUUCUCCCUU

CCGGCACCAUUCUCCCUUC

CGGCACCAUUCUCCCUUC

CUGGGGGUGCUAGACAUU

UGGGGGUGCUAGACACCUG

G"GGGUGCUAGACACUGG

GGkGGUG.-CUAGACACCUGGC-

GGGUGCUAGACACCUGGCA

GGUGCUAGACACCUGGCAC

GUGCUAGACACCUGGCACC

UGCUAGACACCUGGCACCA

GCUAGACACCUGGCACCAU

CUAGACACCUGGCACCAUU

UAGACACCUGGCACCAUUC

AGACACCUGGCACCAUUCU

GACACCUGGCACCAU

UCUC

ACACCUGGCACCAUUCUCC

CACCUGGCACCAUUCUCCC

IACCUGGCACAUCUCCCU

ICCUGGCACCAUUCUCCCUU

CUGGCACCAUUCUCCCUUC

UGGCACCAUUCUCCCU

UU

FUGUGUUUUGUCUGAGCCUC

SGUGUUUUGUCUGAGCCUCU

3 UGUUUUGUCUGAGCCUCUC 4 GUUUUGUCUGAGCCUCUCU 5 UUU1UGUCUGSAGCCUICUC

UC

6 UUUGUCUGAGCCUCUCUCG 7 UUGUCUGAGCCUCUCUCGG 8 UGUCUGAGCCUCUCUCGGU 9 GUCUGAGCCUCUCUCGGUC 0UCUGAGCCUCUCUCGG(UCA E1245 10280 1246 -102811 1247 10282] 1248 10283] 1249 10284j 1250 10285 1251 10286 1252 10287 1253 10288 1254 10289 1255 10271 1T256 10272 1257 10273 1258 10274 1259 10O275 1260 10276 1261 10277 1262 10278 1263 10279 1264 10280 1265 10281 1266 10282 1267 10283 1268 10284 1269 10285 1270 10286 1271 10287 12-72 10i288 1273 10289 1274 107 1275 10880 1276 10881 1277 10882 1278 10883 1279 10884 1280 10885 1281 10886

AAUGGUGCCGGGUGUCUAG

GAAUGGUGCCGGGUGUCUA

AGAAUGGUGCCGGGUGUCU

GZAGAAUGGUGCCGGGUGUC

GAGAAUGGUGCCGGGUGU

GGGAGAAUGGUGCCGGGUG

AGGGAGAAUGGUGCCGGGU

AA GGGAGAAUGGUGCCGGG

GAAGGGAGAAUGGUGCCGG

AGAAGGGAGAAUGGUGCCG

AGGUGUCUAGCACCCCCAG

CAGGUGUCUAGCACCCCCA

CCAGGUGUCUAGCACCCCC

GCCAGGUGUCUAGCACCC

UGCCAGGUGUCUAGCACCC

GUGCCAGGUGUCUAGCACC

GGUGCCAGGUGUCUAGCAC

UGGUGCCAGGUGUCUAGCA

AUGGUGCCAGGUGUCUAGC

AAUGGUGCCAGGUGUCUAG

GAAUGGUGCCAGGUGUCUA

AGAAUGGUGCCAGGUGUCU

GAGAAUGGUGCCAGGUGUC

GGAGAAUGGUGCCAGGUGU

GGGAGMAUGGUGCCAGGUG

AGGGAGAAUGGUGCCAGGU

AAGGGAGAAUGGUGCCAGG

2997 2998 2999 3000 3001 3002 3003 3004 3005 3006 3007 3008 3009 3010 3011 3012 3013 3014 3015 3016 3017 3018 3019 3020 3021 3022 3023

GAAGGGAGAAUGGUGCCAG

AGAGGUAGAAAAUCCA

GAGGCUCAGACAAAACACA

AGAGGCUCAGACAAAAC

GAGAGGCUCAGACAAAAC

AGAGAGGCUCAGACAAAAC

GAGAGAGGCUCAGACAAA

CGAGAGAGGCUCAGACAA

ACCGAGAGAGGCUCAGAC

2ACCGAGAGAGGCUCAlA 3025 3026 3027 3028 3029 3030 3031 3032 1283 jF 10888 2005201389 01 Apr 2005 rs5 7 l 07 UACUUUGU~ 1284 10871 CUGAGCCUCUCUCGGUCMA 1284 10889 UUGACCGAGAGllkiAGGCUCAG 3036 rs1557 210 10872 UGAGCCUCUCUCGGUCMC 18 07 UGAGCCUCUCUCGGUCAC 1285 10890 6GUUGACGAGAGAGGCUCA 3037 rs57 2 0 187 AGCCCUGUCAA 1286 10873 GAGCCUCUCUCGGUCCA 1286 10891 tUGUUGACCGAAGAGGCUC 3038 rsl 557210 10874 AGCUUUGUCAG 1287 10874 AGCCUCUCUCGGUCACAG 127 092CGUGCGAGAGAGGCU 3039 rsl 557210 10875 GCCUCUCCGCCGC 18 10875 GCCUCUCUCGGUC'ACAGC 128 183 CGUACCGAGAGAGGC 3040 rsl 557210 10876 CCUCUCUiCljGiGU:liCMCAGC 1289 10876 CCUCUCUCGGUCMCAGCA 1289 1894 UGCUGUUGACCGAGAGAGG 3041 rsl 557210 187CUCUCUCGGUCAACAGCM 1290 10877 CUCUCUCGGUCACAGCAA 1290 1089 UUGCUGUUGACCGAGAGAG 3042 rs1 557 2 1 0 10864 UUCUUGUCGAGCUU 1296 10864 UUUCUG AGCUU 1296 10882 AUGAGUGCCAGA ,C 3048 rs355210 10880 CUCUCGGUCAACAGCAAC 1297 1080UC;UCGGUCAACAGC G 1297 10898 CUUUGCUGUUGACCGAGA 3049 rs36520 10867 UGUCUACUU G 130 23 10867 UUUUGUCUGAGCCUU 1303 108 CAAAGGCUCAGAACM 30455 155721 0 2 10868 GuGUCUGCUCUUUGU 13094 10868 UGUUACUUUGU 1304 10886 AAAAGGCUCAGACAG 3056 rs 5521 0 2 10873 AGUUCGUC; 139 1083 GA UUUG UC 139 10891 GUUAGACUCAGAGGCAC 3061 rs3630 2 1084 ACCUCCUUGUCCAG 131 107 AGCCCUGCU 1310 10892 CUGUGC AAAACU 3062 rs35521 0 2 10875 GUC DUCUGGCCAUC 1311) 107 GCU UU1 UGGCCAG31 183 CGUACGGGC 36 rs3623 02 0880 UCUGGUCAACAGCAGC 1316 1880 UCUGGUCAACAGC GC 1316 10898 GCUUUGCUGUUACCMAGA 3068 rs3623 02 0881 CUGGUCAACAGCAGCU 131 10881 UCUGGUCAAAG CU 1317 0899Y:: AGCUUUGCUGUGACCMAG 3069 rs3623 02 10882 UUGUCAACAGCAAGCUU 131 10882 UGGUCAAAGCAGCUU 1318 10900 AAGCUUUGCUGUUGACCA 3070 rs3623 02 10883 GGUAACAGCAAAGCUUG 131 10883 GGUCAACAGCAAGCUUG 1319 10901 CAAGCUUUGCUGUUGACCAG 3071 rs3O2S8OS 10953 CAG CUGACUCUUCG 130 10953 AUGUCUGCAUCUII[ ,1 H UGCCG 13201 10971 CCGAAGA GUCAGU 3072 rs3O2S 8 OS 10954 G CUGAGCUCUUAGGU 1321 10954 GCUGACUCUUGGU 1321 10972 ACCCAAGAUGCAGCU 3073 rs3025 8 OS 10955 CUGACAUUCUUCACGGUGk 1322t~ 1095 CUGA CUCUUAGGUG 1322 109 CUACCAAGAGGUCAGC 3 074 rs622 082 GACCCUUUGUAA 1081 72UGGCUUCUGUCAC 138 080 UUACAAAGG246 36 246 2005201389 01 Apr 2005 rs3025805 10956 CUGACAUCUUGCACGGUGA 1323 10956 -CUGACAUCUUGCACGGUGA 1323 10974 -UCACCGUGCAAGAUGUCAG 3075 rs3025805 10957 UGACAUCUUGCACGGUGAC 1324 10957 UGACAUCUUGCACGGUGAC 1324 10975 GUCACCGUGCAAGAUGUCA 3076 rs3025805 10958 GACAUCUUGCACGGUGACC -1325 10958 GACAUCUUGCACGGUGACC 1325 10976 GGUCACCGUGCAAGAUGUC 3077 rs3025805 10959 ACAUCUUGCACGGUGACCC 1326 10959 -ACAUCUUGCACGGUGACCC 1326 10977 GGGUCACCGUGCAAGAUGU 3078 rs3025805 10960 CAUCUUGCACGGUGACCCC 1327 10960 CAUCUUGCACGGUGACCCC 1327 10978 GGGGUCACCGUGCAAGAUG 3079 rs3025805 10961 AUCUUGCACGGUGACCCCU 1328 10961 AUCUUGCACGGUGACCCCU 1328 10979 AGGGGUCACCGUGCAAGAU 3080 rs3025805 10962 UCUUGCACGGUGACCCCUU 1329 10962 UCUUGCACGGUGACCCCUU 1329 10980 AAGGGGUCACCGUGCAAGA 3081 rs3025805 10963 CUUGCACGGUGACCCCUUU 1330 1i0963 CUUGCACGGUGACCCCUUU 1330 10981 AAAGGGGUCACCGUGCAAG 3082 rs3025805 10964 UUGCACGGUGACCCCUUUU 1331 10964 UUGCACGGUGACCCCUUUU 1331 10982 AAAAGGGGUCACCGUGCAA 3083 rs3025805 10965 UGCACGGUGACCCCUUUUA 1332 10965 UGCACGGUGACCCCUUUUA 1332 10983 UAAAAGGGGUCACCGUGCA 3084 rs30580 1066 CACGUGACCCUUUG 133 1966GCAGGUGCCCUUUAG 333 098 CUAAAGGGUACCUGC 308 rs3025805 10967 GCACGGUGACCCCUUUUAG 1334 10967 GCACGGUGACCCCUUUAG 1334 10985 CUAAAAGGGGUCACCGUGC 3086 rs3025805 10968 ACGGUGACCCCUUUUAGU 1335 10968 ACGGUGACCCCUUUUAGU 1335 10986 ACUAAAAGGGGUCACCGUG 3087 rs3025805 10969 CGGUGACCCCUUUUAGUC 1336 10969 ACGGUGACCCCUUUUAGUC 1336 10987 GACUAAAAGGGGUCACCGU 3088 rs3025805 10970 CGGUGACCCCUUUUAGUCA 1337 10970 CGGUGACCCCUUUUAGUCA 1337 10988 UGACUAAAAGGGGUCACCG 3089 rs3025805 10971 GGUGACCCCUUUUAGUCAG 1338 10971 GGUGACCCCUUUUAGUCAG 1338 10989 CUGACUAAAAGGGGUCACG 309 rs3025805 10953 CGUGACACUUUGCAGU 1339 10953 GUGACUCUUGUCAGU 1339 10971 CUGACAAGGUCACG 3091 rs3025805 10954 AGCUGACAUCUUGCACGU 1340 10954 CAGCUGACAUCUUGCACGU 1340 10972 ACGUGCAAGAUGUCAGCUG 3092 rs3025805 10955 GCUGACAUCUUGCACGUU 1341 10955 AGCUGACAUCUUGCACGUU 1341 10973 AACGUGCAAGAUGUCAGCU 3093 rs3025805 10956 GCUGACAUCUUGCACGUUG 1342 10956 GCUGACAUCUUGCACGUUG 1342 10974 CAACGUGCMAGAUGUCAGC 3094 rs3025805 10957 CUGACAUCUUGCACGUUGA 1343 10957 CUGACAUCUUGCACGUUGA 1343 10975 UCAACGUGCAAGAUGUCA 3095 rs3025805 10958 UGACAUCUUGCACGUUGAC 1344 10958 UGACAUCUUGCACGUUGAC 1344 10976 GUCMCGUGCAAGAUGUCA. 3096 rs3025805 10959 GACAUCUUGCACGUUGACC 1345 10959 GACAUCUUGCACGUUGACC 1345 10977 GGUCAACGUGCAAGAUGUC 3097 rs3025805 10960 CAUCUUGCACGUUGACCC 1346 10960 CAUCUUGCACGUUGACCC 1346 10978 GGGGUCAACGUGCAAGAUGU 3098 rs3025805 10961 AUCUUGCACGUUGACCCC 1347 10961 CAUCUUGCACGUUGACCCC 1347 10979 GGGGUCAACGUGCAAGAUG 3099 rs3025805 10962 AUCUUGCACGUUGACCCCU 1348 10962 AUCUUGCACGUUGACCCCU 1348 10980 AGGGGUCAACGUGCAAGA 3009 rs3025805 10963 UCUUGCACGUUGACCCCUU 1349 10963 UCUUGCACGUUGACCCCUU 1349 10981 AAGGGGUCAACGUGCAAGA 3101 rs3025805 10964 CUUGCACGUUGACCCCUUU -1350 10964 CUUGCACGUUGACCCCUUU 1350 10982 AAAGGGGUCAACGUGCAAG 3102 rs3025805 10965 UUGCACGUUGACCCCUUUU 1351 10965 UUGCACGUUGACCCCUUUU 1351 10983 AAAAGGGGUCAACGUGCAA 3103 rs3025805 10966 UGCACGUUGACCCCUUUUA 1352 10966 UGCACGUUGACCCCUUUUA 1352 10984 UAAAAGGGGUCAACGUGCA 3104 rs3025805 10967 GCACGUUGACCCCUUUUAG 1353 10967 GCACGUUGACCCCUU UUAG 1353 108 CUAAAAGGGGUCAACGUGC 3105 rs3025805 10968 ACGUUGACCCCUUUUAGU 1354 10968 CACGUUGACCCCUUUUAGU 1354 10986 ACUAAAAGGGGUCAACGUG 3106 rs3025805 10969 CGUUGACCCCUUUUAGUC 1355 10969 ACGUUGACCCCUUUUAGUA 1355 10987 GACUAAAAGGGGUCAACGU 3107 rs3025805 10970 CGUUGACCCCUUUUAGUCA 1356 10970 CGUUGACCCCUUUUAGUCG 1356 10988 UGACUAAAAGGGGUCAACG 3108 rs3025805 10971 GUUGACCCCUUUUAGUCAG 1357 10971 GUUGACCCCUUUUAGUCAG 1357 10989 CUGACUAAAAGGGGUCAA 3109 rs362267 11163, UUUGGGAGCUCUGCUUGCC 1358 11163 UUUGGGAGCUCUGCUUGCC 1358 11181 GGCAAGCAGAGCUCCCAAA 3110 1359 11182 CGGCAAGCAGAGCUCCCAA 3111 I rs362267 11164 11165 11166 UUGGGAGCUCUGUUGCiUU 135 UGGGAGCUCUGCUUGCCGA I 360 GGGAGCUCUGCUUGCCGAC 1361 1165 11166

UGGGACUCGCUUCCG

UGGGAGCUCUGUUCCGA

I ~Rl 1183 UCGGAUUA~A(~UUU I z 113610 11184 I UCGGCAAGCAGAGCUCCC

I-I

1 1361 GUCGGCAAGCAGAGCUCCCI 31 -13 2005201389 01 Apr 2005 CUGCUUGC GACU 1362 11167 GGACf-::iU CU':Ic U UGCCGACU 1362 11 85 AGUCGGCAAGCAGUCC 114 rs362267 1167 GGAGCUCUGCUUGC 1363 11168G GCUG UGGCGCAUG 1363 11186 CAGUCGGCAAGCAGAGCUC 3115 rs362267 I1168 AGCUGCUUGCCGA 136 116UGCUUGCCACUGG 1364 11187 CCAGUCGGCAAGCAGAGCU 3116 s362267 1169 GCUCUGCUUGCCGACUGG 1364 11169 AC UGGCG 1365 11188 GCCAGUCGGCAAGC G 3117 rs362267 1170 GCUCUGCUUGCCGACUGGC 1365 11170 GUUCUGCUUGCCGACUGGC 1366 11189 AGCCAGUCGGCAAGAGAG 3118 rs362267 :1171 UCUGCUUGCCGACCU 1366 117 CU GA UGGCUG 1367 1190 CAGCCAGUCGGCAAGCA 3119 rs362267CUC 1 117 UGCUUGCCGACUGGCUGU 1368 11191 ACAGCCAGUCGGCAAGCAG 3120 rs362267 117 UGCUUGCCGACUGGCUGU 136 117 CCACUGG 1369 192 CACAGCCAGUCGGA 312 rs362267 i1174 UGCUUGCCGACUGGCUGG 1367 11172 CUGUUGCCGACUGGCUGU 1370 11193 UCACAGCCAGUCGGCAAGC 3122 rs362267 11173 GCUUGCCGACUGGCUGUGA 1370 11173 GCCACUGGCUGUG 1371 11194 CUCACAGCCAGUCGAA 3123 rs362267 11174 CUUGCCGACUGGCUGUGA 139 11174 CCG 1372 11195 UCUCACAGCCAGUCGC 3124 rs362267 11175 UGCCGACUGGCUGUGAG 1370 21177 UUGCCGACUGGCUGUGAAC 1373 11196 GUCUCACAGCCAGUGC 3125 rs362267 11178 UGCG(3AUUCUGUGAGAC 1373 11178 UGGAUGCUGUGAGACG 1374 11197 CGUCUCACAGCCAGUCGGC 3126 rs362267 11179 GCCGACUGGCUGUGAGACG 1374 11179 GCCGAC G 1375 11198 UCGUCUCACAGCAGCGG 3127 11626 180 CCGACUGGCUGUGAGACGA 1375 11180 CCGACUGGCUGUGAGACGAG 1375 til99 CUCGUCUCACAGCCLAGUCG 014f rs362267 11181 CGACUGGCUGUGAGACGAG 1376 11181 CUGG GUGAGUCGC G 1376 i111 UCGUCCAAGCGCC 3128 rs362267 11163 UUUGGGAGCUCUGCUUGCU 1377 11163 UUUGGGACUUGCUUGCU 1377 11181 AGCAAGCAGAGCUCCC 3129 rs362267 11164 UUGGGAGCUCUGCUUGCUG 13R3 11-10 IoCU UC 1378 11182 CAGCAAGCAGAGCUCCCA 3130 rs362267 1 165 UGGGAGCUCUGCUUGCUGA 1Ut9 1165 UG C UGCUUGCUGA 1379 11183 UCAGCAAGCAGAGCUCCC 3131 rs362267 11166 GGGAGCUCUGCUUGCUGAC 1380 11166 GGGAGCUCUGCUUGGAC 1380 11184 GUCAGCAAGCAGAGCUCC 3132 rs362267 1 167 GGAGCUCUGCUUGCUGACU 1381 11185 AGUCAGCAAGCAGAGCUC 3133 rs362267 11168 GAGCUCUGCUUGCUGACUG 1382 11186 CAGUCAGCAAGCAGAGCU 3134 rs362267 1 169 AGCUCUGCUUGCUGACUGG 1383 11169 A CUGCUUGCUGACUGG 1383 11187 CCAGUCAGCAAGCAGAGC 3135 rs362267 1i170 GCUCUGCUUGCUGACUGGC 1384 11170 GUUGCUUGCUGACUGGC 1384 il189 GCCAGUCAGCAAGCAGAG 3137 rs362267 11171 CUCUGCUUGCUGACUGGCU 1385 11171 CCUGCUUGCUGACUGGCU 1386 11189 AGCCAGUCAGCAAGCAGAG 3137 rs362267 11172 UCUGCUUGCUGACUG 1386 11172 U G 1386 11190 CAGCCAGUCAGCAAGCAG 3138 rs362267 11173 CUGCUUGCUGACUGGCUGU 1387 11173 UGUCUACU 1387 0191 ACAGCCAGUCAGCAAG 3139 rs362267 11174 UGCU GUG CUGUG 1 88 11174 UGCUUACGCUU 1388 11 122 %,P%%,GCCAGUCG3AGCA 3140 rs362267 11175 GCUUGCUGACUGGCUGUGA 1389 11175 UUGCUGACUGGUUA 1389 11193 UCACAGCCAGUCAGCAAGC 3141 rs362267 11176 UUGUGACUGGGUGUGAG 1390 11176 G UGCUGACUGGUGUGG 1390 11194 CUCACAGCCAGUCGCA G 2 rs362267 11177 UUGCUGACUGGCUGUGAGA 1391 11177 UUGCUGACUGCUUGAA 1392 11195 UCUCACAGCCAGUCAGCA 314 rs362267 11178 UGCUGACUGGCUGUGAGAC 1392 11178 GCUGUCUGUGA 1392 11196 GUCUCACAGCCAGUCAGC 3145 rs362267 11179 GCUGACUGGCUGGAGCAG 1396 11382 CCUGGGAGACG 1393 11197 CGUCUCACCCCACUGCC 314 rs362267 11180 UAUGGGGCA 1394 GG8 FT CGCUGUGA C A 1394 11198 UCGUCUCACAG As 3146 rs362267 11181 UG CUGGCUGUGCA 1395 11181 UGACUGCUGUGAA(;GA 1395 11199 CUCGUCUCACAGCCAGUC 3147 rs362301 11382 UGGCGUGG 1396 11382 UGGCAG GCU~ 1396 11400 AGCUGCUCCCCAGCC 3148 rs362301 11383 GGCAGCUGGGGAA 1397 11383 AGCGACCUG 1397 11401 CAGCUGCUCCCCAGCUGC 3149 rs362301 11384 GCAGCUGGGGAGCAGCUA 1398 11384 GCGCUGGAGCA UGA 1398 11402 UCAGCUGCUCCCCAGC 3150 r,362301 11385 CAGCUGGGGAGCAGCUGAG 1399 6 GCUG C A 1400 11404 CUCAGCUGCUCCCCAGCU 3151 rs362301 11386 AGCUGGGGAGC AG I GC G1UA6 ACUG!113AGCUGAGA 1400 1404 UCUCjGCUGCUCC 3159 248 2005201389 01 Apr 2005 ri3-62301 1i 13 87 GCUGGGGAGCAGCUGAGAU rs-362301 1388 CUGGGGAGCAGCUG

G

r-362301 -11389 UGGGGAGCAGCUGAGAUGU -362301 -11390 GGGGAGCAGCUGAGAUG rs3-62301 11i391 GGGAGCAGCUGAGAUGUGG r 3-62301 11392 GGAGCAGCUGAGAUGUGGA rs3-62301 133GAGCAGCUGAGAUGUGGA s-362301 134AGCAGCUGAGAUGU'GACU.t 7s-362301 11T395 GCAGCUGAGAUGUGGACUU rs362301 11396 CAGCUGAGAUGUGGAC;UUG rs362301 11397 AGCUGAGAUGUGGACUUGU rs36230l 11398 GCUGAGAUGUGGACUUGUA rs362301 11399 CUGAGAUGUGGACUUGUAU rs362301 11400 UGAGAUGUGGAC;UUGUAUG r362301 11388 UGGGCAGCGGAGUG rs362301 11389 GCCUGGGGAGCAGCGGUU rs362301 11390 GGGCAGCGGAGAUGG rs36230l 11391 GGAGCUGGGAGGUGG rs3623 0 l 11392 GAGCGGGAGAGGGA rs36230l 11393 GUGGAGCAGCGGAGAUC rs3623 0 l 11394 CGGAGCAGCGGAGAUGAU rs362301 113895 UGGCAGCGGAGAUGUU rs62148 11441 GCGACAGCGGAGCCCUGC rs364278 11442 CGAAAGCGGAGCCCUGU rs364278 11443 UAAGGGAGCCCUGCUCC rs364278 11444 AGAAGCGGAGCCCUGCUCAU rs364278 11445 AAAGCGGAGCCCUGCUCAAU 1401 11387 GCUGGGGAGCAGCUGAGAU 1402 11388 CUGGGGAGCAGCUGAGAUG 1403 11389 UGGGGAGCAGCUGAGAUGU 1404 11390 GGGGAGCAGCUGAGAUGUG- 1405 111391 1GGI~GAGCAGCUGAGAUGUGG 140 11392 GGAGCAGCUGAGAUGUGGA 1407 11393 GAGCAGCUGAGAUGUGGAC 148119

AGCAGCUGAGAUGUGGACU

1 409 1139

GCAGCUGAGAUGUGGACUU

1410 11396 CAGCUGAGAUGUGGACUUG 1411 11397 AGCUGAGAUGUGGACUUGU 1412 11398 GCUGAGAUGUGGACUUGUA 113 139CGAUUGGACUUGUAU- 1414 11400 UGAGAUGUGGACUUGUAUG 1415I 118 ;UGGCGCUGGGGAGCAGCG 1416 11383 GGCAGCUGGGGAGCAGCGG 1417 11384 GCAGCUGGGGAGCAGCGGA 1418 11385 CAGCUGGGGAGCAGCGGAG 1419 11386 AGCUGGGGAGCAGCGGAGA 14-20 137GCUGGGGAGCAGCGGAGAU 1421 11388 CUGGGGAGCAGCGGAGAUG 1422 11389 UGGGGAGCAGCGGAGAUGU 1423 11390 GGGGAGCAGCGGAGAUGUG 1424 131GGGAGCAGCGGAGAUGUGG 1425 11392 GGAGCAGCGGAGAUGUGGA 1426 11393 GAGCAGCGGAGAUGUGGAC 1427~~ 1194AGAGGAGAUGUGGACU 1428 1139 GCAGCGGGAUGUGGACUU 1429 11396 CAGCGGAGAUGUGGACUUG 1430 11397 AGCGGAGAUGUGGACUUGU 1431 11398 GCGGAGAUGUGGACUUGUA 143 11399 CGGAGAUGUGGACUUGUAU 1433 11400 GGAGAUGUGGACUUGUA;UG 1434 11440 AGCUGAAAGGGAGCCCCUG 1435 11441 GCUGAAAGGGAGCCCCUGC 1436 11442 CUGAAAGGGAGCCCC(UGCU 1437 11443 UGAAAGGGAGCCCCUGCUC 1438 11444 1GAAAGGGAGCCCCUGCUCA 1439 114451 AAAGGGAGCCCCUGCUCAA 1401 11405 AUCUCAGCUGCUCCCCAGC 3153 1402 11406 CAUCUCAGCUGCUCCCCAG 3154 1403 11407 ACAUCUCAGCUGCUCCCCA 3155 1404 11408 CACAUCUCAGCUGCUCCCC 3156 1405 11409 CCACAUCUCAGCUGCUCCC 3157 1i406 1i1410 UCCACAUCUCAGCUGCUCC 3158 1407 11411 GUCCACAUCUCAGCUGCUC 3159 1408 11412 AGUCCACAUCUCAGCUG3CU 3160 1409 11413 AAGUCCACAUCUCAGCUGC 3161 1410 11414 CAAGUCCACAUCUCAGCUG 3162 1411 11415 ACAAGUCCACAUCUCAGCU 3163 1412 11TI416 -UACAAGUCCACAUCUCAGC 3164 -413 -11417 -AUACAAGUCCACAUCUCAG 3165 1414 11418 CAUACAAGUCCACAUCUCA 3166 1415 11400 CGCUGCUCCCCAGCUGCCA 3167 1416 ~11401 CCGCUGCUCCCCAGCUG3CC 3168 1417 11402 UCCGCUGCUCCCCAGCUGC 3169 1418 ~11403 CUCCGCUGCUCCCCAGCUG 3170 1419 11404 UCUCCGCUGCUCCCCAGCU 3171 1T420 11l405 AUCUCCGCUGCUCCCCAGC 3172 1421 11406 CAUCUCCGCUGCUCCCCAG 3173 1422 11407 ACAUCUCCGCUGCUCCCCA- 3174 1423 11408 CACAUCUCCGCUGCUCCCC; 3175 1424 11409 CCACAUCUCCGCUGCUCCC 3176 1425 11410 UCCACAUCUCCGCUGCUC;C; 3177 1426 11411 GUCCACAUCUCCGCUGCUC 3178 1427 11412 AGUCCACAUCUCCGCUGCU 3179 1428 11413 AAGUCCACAUCUCCGCUGC; 3180 17429 144 CAAGUCCACAUCUCCGCUG 3181 1430 11415 ACAAGUCCACAUCUCCGCU 3182 1431 11416 UACAAGUCCACAUCUCCGC 3183 1432 11417 AUACAAGUCCACAUCUCCG 3184 1433 11418 CAUACAAGUCCACAUCUCC 3185 1434 11458 CAGGGGCUCCCUUUCAGCU 3186 1435 11459 GCAGGGGCUCCCUUUCAGC 3187 1436 11460 1AGCAGGGGCUCCCUUUCAG 3188 1437 11 461 GAGCAGGGGCUCCCUUUCA. 3189 1438 11462 UGAGCAGGGGCUCCCUUUCI 3190 1439 116 UGAGCAGGGGCUCCCUUJU 13191 249 2005201389 01 Apr 2005 rs6l 48278 I rs6148278 I rs6148278 I rs6l 48278I rs6148278 1 rsi6l 48278 rs6148278 rs6l 48278 rs6148278 rs6148278 rs6-148278 rs6l 48278 rs6l 48278 rs6l 48278 frs6l 48278 rs6l 48278 rs6l 4827 8 rs6l 48278 rs6l 48278 rs 6l 48278 rs6l 48278 rs5855773 rs5855773 rs5855773 ri5-85577 3 rs5855773 rs585577 3 rs5855773 rs5855773 rs5855773 rs5855773 rs5855773 r 5-855773 rs58557 73 s-5855773 1;5-8557 73 rs585577 3 rs5855773 rs585577 3 1446 144-7 1448 144-9 11450 11451 Fl1452 11453 11454 11455 i11456 11457 11458 i11459 11 46 0 11461 11443 11444 11641 11-642 11 643 11-644 11 645 11 646 11647 11649 11650 11l 651 11 651 11 65' 1165' 11 65 1165~ 1164E 1164 AAGGGAGCCCCUGUCA 1440 AKGGGAGCCCCUGCUCAAAG 1441 GGGAGCCCCUGCUCAAAGG 1442 G GAGCCCCUGCUCAAAGGG 1443 GAGCCCCUGCUCAAAGGGA 1444 AKGCCCCUGCUCAAAGGGAG 14 GCCCCUGCUCAAAGGGAGC 1446 CCCCUGCUCAMAGGGAGCC 1 447 CCCUGCUCAAAGGGAGCCC 1448 CCUGCUCAAAGGGAGCCCC 1449 CUGCUCAAAGGGAGCCCCU 1450 UGCUCAAAGGGAGCCCCUC 1451 GCUCAAAGGGAGCCCCUCC 1452 CUCAAAGGGAGCCCCUCCU 1453 _UCAAAGGGAGCCCCUCCUC 1454 CAAAGGGAGCCCCUCCUCU 145 AGCUGAAAGGGAGCCCCUC 1456 GCUGAAAGGGAGCCCCUCC 1457 CUGAAAGGGAGCCCCUCCU 1458 UGAAAGGGAGCCCCUCCUC 14 59 GAAAGGGAGCCCCUCCUC 1460 GUAAGAAAAUCACCAUUCU 1461 UGAAGAAAAUCACCAUUCUU 1462 AAGAAAAUCACCAUUCUU-- 1463 A-GAAAAUCACCAUUCUUCC 1464 GAAAAUCACCAUUCUUCGG 1 465 -AAAAUCACCAUUCUUCCGU 1466 AAAUCACCAUUCUUCCGUA 14-6 7 AAUCACCAUUCUUCCGUAU 1468 AUCACCAUUCUUCCGUAUU 1469 UCACCAUUCUUCCGUAUUG 1470 CACCAUUCUUCCGUAUUGG 1411 ACCAUUCUUCCGUAUUGGU 147-2 CCAUUCUUCCGUAUUGGUU 1473 CAUUCUUCCGUAUUGGUUG 1474 5 AUUCUUCCGUAUUGGUUGG 1475 6 UUCUUCCGUAUUGGUUGGG 1476 1,GUAAGAAAAUCACCAUUCC 1477 21UAAGAAAAUCACCAUUCC-G 1478 11446 AAGGGAGCCCCUGCUCAAA 11447 AGGGAGCCCCUGCUCAAAG 1148 GGGAGCCCGU

AG

11449 GGAGCCCCUGCUCAAAGG 1450 GGAGCCCUGUCAGA 1145 AGCCCUCUCAAGG

GG

11452 GGGCCCCUGCUCAAAGGG 1145 CCCUCUAGGGACCJ 11454 GACCCUGCUCAAAAAGGGAC 11455 CCCUCAAAGGGAGC 1145 CU CUAAGGGAGCU 11457 UGUAAGAGCCCCU 11458 AGCUCAAAGGGCUCCCUCC33 1145 CU AGGGAGCCU 11440 AG CU G C C

CU

11441 GCCCUGUAAAGGGAGCUC 11442 CCCUG

AAAAGGGAGCCU

114543 CUGGAAAAGGGAGCCCU 11444 CCGAAAGGGACC

CCC

1146 CGUAAAAAUCACCUCU 11427 UAGCAAAACCCUC 11438 GAAAGACUC 11*45 GAAAUCACCUCUUCCG 1146 AAAUCACCUCUUCCU 114647 AAAAGACCUCUUCCUA 11 4 UC AC A U UU C U U 11650 UACGCAUUCUUACCGUUU 11651 GCUACAUUCUUCCUUU 11652 ACAUUGCCCUUG 11653 CCAAAUUCCCUUUGU 1164 CUAUUCUUCCAUUGU 11655 AUUCUUUCCUAUUGUUGG -116456 GUCUUCCUAUUGGUUCG 11641 GUAAAAUCACCAUUCC 11642 UGAAAUCACCAUUCCG 1440 11464 1441 11465 1442 11466 1443 11467 1444 1146 8 1T445 11469 1446 11470 1447 i14-71 1448 11472 1449 11473 1450 11474 1451 11475 1452 11476 1453 11l477 1454 11478 T4-55 -11479 1456 11458 1457 11459 1458 11460 1459 11461 1460 11462 1461 11659 1462 11660 1463 11661 1464 11l662 1465 11663 14-66 iTl664 1467 11665 1468 11666 1469 11667 1470 11668 1471 11669_ 1472 11670 1473 11671 1474 11672 1475 171673 1476 11674 1477 11659 1478 1T1660 UUUGAGCAGGGGCUCCCUU I3192 CUUUGAGCAGGGGCUCCCUT 3193 CCUUUGAGCAGGGGCUCCC 3194 CCCUUUGAGCAGGGGCUCC 3195 U-CCCUUUGAGCAGGGGCUC 3196 CUCCCUUUGAGCAGGGGCU 3197 GCUCCCUUUGAGCAGGGGC 3198 GGCUCCCUUUGAGCAGGGG 3199 GGGCUCCCUUUGAGCAGGG(. 3200 GGGGCUCCCUUUGAGCAGG 3201 AGGGGCUCCCUUUGAGCAG 3202 GAGGGGCUCCCUUUGAGCA 3203 GGAGGGGCUCCCUUUGAGC 3204 AGGAGGGGCUCCCUUUGAG 3205 GAGGAGGGGCUCCCUUUGA 3206 AkGAGGAGGGGCUCCCUUUG 3207 GAGGGGCUCCCUUUCAG3CU 3208 GGAGGGGCUCCCUUUCAGC 3209 AGGAGGGGCUCCCUUUCAG 3210 GAGGAGGGGCUCCCUUUCA 3211 AGAGGAGGGGCUCCCUUUC 3212 AGAAUGGUGAUUUUCUUAC 3213 AAGAAUGGUGAUUUUCUUA 3214 GAAGAAUGGUGAUUUUCUU 3215 GGAAGAAUGGUGAUUUUCU. 3216 CGGAAGAAUGGUGAUUUUC 3217 ACGGAAGAAUGGUGAUUUU 3218 UACGGAAGAAUGGUGAUUU 3219 AUACGGAAGAAUGGUGAUU 3220 AAUACGGAAGAAUGGUGAU 3221 CAAUACGGAAGAAUGGUGA 3222 CCAAUACGGAAGAAUGGUG 3223 ACCAAUACGGAAGAAUGGU 3224 AACCAAUACGGAAGAAUGG 3225 CAACCAAUACGGAAGAAUG 3226 CCAACCAAUACGGAAGAAU 3227 CCCAACCAAUACGGAAGAA 3228 -GGAAUGGUGAUUUUCUUAC 3229 CGGAAUGGUGAUUUUCUU 33 250 2005201389 01 Apr 2005 rs5855 773 11643 AAGAAAAUCACCAUUCCGU 1479 11643 AAGAAAAUCACCAUUCCGU 1479 11661 A rs58557 73 11644 AGAAAAUCACCAUUCCGUA 1480 11644 AGAAAAUCACCAUUCCGUA 1480 11662 U rs58557 73 11645 GAAAAUCACCAUUCCGUAU 1481 11645 GAAAAUCACAUCCGUAU 1481 11663 A rs58557 73 11646 AAAAUCACCAUUCCGUAUU 1482 11646 AAAAUCACCAUUCCGUAUU 1482 11664 rs5855 773 11647 AAAUCACCAUUCCGUAUUG 1483 11647 AAUCACCAUUCCGUAUJUG 1483 11665 C rs58557 73 11648 MAUCACCAUUCCGUAUUGG 1484 11648 AAUCACCAUUCCGUAUUGG 1484 11666 C rs58557 73 11649 AUCACCAUUCCGUAUUGGU 1485 169AUCACCAUUCCGUAUUGGU 1485 11667 rs58557 74 11741 AUCCAGACGUC 41 171AUUCUGICGAUUGUGU 1491 11759 rs58557 74 1 61742 GUUCAGAACGUUGCUG 149 15 1742u GUUCAUCGAUUUGCUG 1492 11609 rs58557 74 114 GGAUUGUCCC 1498 11748 AGACUUUGGUCCC 1498 116766 rs58557 7 4 11749 GACGUUUGCUCCCA 1499 11749 GACGUUU GUCCCC 1499 1167 rs5855 774 1755 AUUCUGCACCUGCC 15905 1755 UUGUCUCCACCCUGCC 15905 11773 rs5855 774 1715 AUUCUCCCACCCUGCCU 1506 11756 GC CUCACCCU 1506 11774 rs555 774 11745 GUCUCAGAACUGUUGCUG 1512. 1145CUA AACUGUUGCUG 152 11763 rs58557 74 '11746 UUUCAGAACUGUUGCUGC 153 11746 UCACUGUUGCUGC 153 11764 rs5855 774 11747 UCCAGAACUGUUGCUGCU 154 11747 UCCAGAACUGUUGCUGCU 154 1165 rs5855 774 11748 CUAGAUUGCUGCUCC 155 11748 CUAGAACGUGCUGCUC 155 11766 rs5855 74 111749 GAACGUUGCUGCUCCCC 1516 11749 GAACUGUUGCUGCUCCCC 1516 11767 rs58557 74 11750 AACUGUUGGCUCCCCCA 1517 11750 AACUGUUGGCUCCCCCA 1517 11768

CGGAAUGGUGAUUUUCUU

ACGGAAUGGUGAUUUUCU

UACGGAAUGGUGAUUUUC

AUACGGAAU)EGGUGAUUUU

j

UU

CU

UC

AAUACGGAAUGGUGAJUUUUU

CAAUA',GGAAUGGUGAUUI

CCAAUACGGAAUGGUGAU

kACCAAUACGGAii!!lill 1111], .113A ,AACCAAUACG

A

,CAACCAAUACGGAAuuuu

,CCAACCAAUACGGAAUGG

GCAACAGUUCUGAGAACUU

AGCAACAGUUCUGAGAACU

C CAACAGUU

AGAAC

GCAGCAACAGUUCUGAGAA

AGCAGCAACAGUUCUGAGA

GAGCAGCAACAGUUCUGAG

GGAGCAGCAACAGUUCUGA

GGGAGCAGCAACAGUUCUG

GGGGAGCAGCAACAGUUCU

UGGGGAGCAGCAACAGUUC

GUGGGGAGCAGCAACAGUU

GGUGGGGAGCAGCAACAGU

GGGUGGGGAGCAGCAACAG

CGGGUGGGGAGCAGCAACA

GCGGGUGGGGAGCAGCAAC

GGCGGGUGGGGAGCAGCAA

AGGCGGGUGGGGAGCAGCA

CCAACAGU

AGAACUU

GCCAACAGUUCUGAGAACU

AGCCAACA

GAAC

CAGCCAACAGUUCUGAGAA

CAGCCAACAGUUCUGAGA

AGCAGCCAACAGUUCUGAG

GAGCAGCCAACAGUUCUGA

AGCAGCCAACAGUUCUG

GGAGCAGCCAAGAGUUCU

GGGAGCAGCCAACAGUUC

GGGGAGCAGCCAACAGUU

231 3232 3233 3234 3235 3236 3237 3238::: 3239 3242 3243 3244 3245 3246 3247 3248 3249 3250 3251 3252 3253 3254 3255 3256 3257 3258 3259 3260 3261 3262 3263 3264 3265 3266 3267 L 3268 1 326L_ 2005201389 01 Apr 2005 CUCCCCAC 1518 11751 ACUGUU Icucccc 1518 11769 131U 3 I AGCAGCCAACAGU 3270 rs5855774 11751 ACUGU'J 3 izi C 3 1519 1770 GGUGGGGAGCAGCC CAG 3271 rs5855774 11752 CUGUUGGCUGCUCCCCACC 1519 117 2 CUGUUGGCUGCUCCCU GGGU AACA 3272 1520 11753 UGUUG CUGCLICCCCACCC 1520 11711 GCAGCCAAC 3273 rs5855774 11753 UGUU ji,0i3lCiJCCCCACCC I GGUGGGG rs5855774 754 GUUG AC CG 1521 11754 GUU GCUGCU( CGl3i3lUGGGGAGCAGCCAA 3274 UUGG UGCUCCCCACCCGC 16''12 11755 UUGGCUGCU(;UUUAttUk 1523 11774 GGCGGGUGGGG/kGCAGCCA 3275 rs5855774 11755 CCGCC 1523 11756 UGGCUGCUCCCCACCCGCC GAGCAGCC 3276 rs5855774 11756 UGGC CGCCU 1524 11757 GGC J U 1524 11775 AGGCGGGUGGG CAUCU 3277 rs5855774 11757 GGCU 1525 11864 CUUAC AUGU AGAU GUAAG 15 5 11846 'AAAUGUAAACAUC 3278 rs2159172 11846 AUUUGUAAGA 1526 11865 UCUUA, rs2159172 11RA7 GAU UUACAUUUGU ?A 15ZO (UiAUGUUUA 1527 1 ji-iii"'""I ACAAAUGUAAACAU 3279 IAA 848

AUGUUU

AA UU IJUGUAAGAA 3280 rs2159172 11 84tS AuoGUUACAUUUGUAA; -GlAA 152 UGUAAGAAA 1528 1 867 UUUCUUAC UGUAAACA l.'AUUUGUAAG 1528 1184 UGUUU G-UAAAC 3281 rs2159172 11849 u Co u 152 11850 GUUUACAUUUGUAAGAAAU 1529 11868 AUUUCUUACPAAU rs2159172 11850 CAUUUGUAAGAAAUA 1530 11869 UAUUUCUUACAAAUGUAAA 3282 UUUji! 11,11 i'll,"lli"LUJUGUAA AAAUA 1530 11851 UUU 1531 11870 UCUUAC UGUAA 32 rs2159172 11851 UAC JUUGUAAG UAA 1531 1185 UUACAUUUGU GAAAUAA GUUAUUU-aUUAC UGUA 3284 rs2159172 11852 UUUGUAAG AAC 1 32 11853 AUUUG AAGAAAUAAC 1532 11871 AUUUCUUACAAAUGU 3285 rs2159172 11853 UAC 533 11854 ACAUUUG AAGAAAUAACA 1533 11872 rs2159172 11854 AC UUUGUAAGAAA

AACA

UAACAC 1534 118 5 CAUUUGU G UAACAC 1534 11873 GUGUUAUUUCUUA0li'li!lJG 3286 rs2159172 1855 CAU AUU 1535 11874 AGUGUUAUUU UUA(:: IlkU 3287 1856 AUU GUAAGAAAUAACACU 1 35 11856 rs2159172 UAACACUG 1536 11875 CAGUGUUAUUUCUUAcAAA 3288 G 1536 11 UUUGUAAG/ CAA 3289 T s215917 11857 uuulljljli_ u u 4UAACACU u 1537 11858 UU UAAGAAAU CACUGU 11537 11876

AGAGUGUUAUUUCUU

?1 21 luill.13 UAACACUGUG CACAGU UUAUUUCU A 3290 rs2159172 11858 UUG G 1538 11859 UGUj 1538 11877 rs2159172 11859 UGU AACACUGUGA 1539 11860 GUAAGAAAUAACACUGUGA 1539 11878 UCACA AUUUCUUAC 3291 1 rs2159172 11860 GUAY AA 1540 11879 U CACAGUGUUAUUUCUUA 3292 11861 UAAGAAAUAACACUGUGAA 1540 11961 UAAGAAAUAACACUGU 11880 AUUCACAGUGUUAUUUCUU 3293 rs2159172 UGUGAAU 1541 11862 AA AACACUGUGAAU 1541 UAUUUCU 3294 rs2159172 11862 AAGAIAU-11-C), UG 1542 11881 CAUUCACA -U-GU rs2159172 11863 GUGAAUG 1542 11 63 A(3j!u I ACAUUCACAGUGJUAUUUC 3295 UGAAUGU 1543 11864 (3AAAUAACACUGUGAAUGU lb43 11882 UGU kACAUCU 3296 rs2159172 11864 UACAUUUGUAAA 1544 11846 A(3AUGUUUACAUUUG AAA 1544 118b4 UUUAC AAAA lb45 11865 UUUUACAAAUZ -UAAACAUC 3297 rs2159172 %-UUUACAUUUGUAAAA 1545 11847 UA UUUUUACAAALIC-1 'AAAGAI 1 3298 rs2l59172 10-41 1546 1 848 AU UUAC UUGU lb46 11866 rs2159172 1 848 AUGUUUACAUUUGUAAAAA 1547 11867 uuuUUUACAAAUUUAAATU A 24zlw UG UUACAUUUGUAAAAAA 1547 11849 UGUUUACP,,IJ UUGU 11868 AUUUUUUAC UGUAA kC 3300 rs2159172 11849 1548 11850 (3uuuAumuUUGU u 1548 UAUUJUUUACAAAUGU 3301 rs2159172 11850 GUUUACAUUUGUAAAAAAU llp 11869 rs2159172 11851 UU ACAUUUGU UA JGU UAA 1550 11870 UUUUUUACAAAUGUAA 3302 UU C UGUAAAAAAUAA liliv 1 172 11852 AUU UACA Ll c 1551 G UAUUUUUUACAikAUGUA 3303 rs2159172 11853 UACAUUUGU.Su UAAC 1551 11853 UGUUAUUUUUUAAAAUGU 3304 UAACA 1552 11854 ACAUU GUAAAAAAuAACA 155 G 3305 rs2159172 11854 CAUUUGI 'AAAAAAUAP

GUGUUAUUUUUUACAAAU

AACAC 1553 11855 AGUGUUAUuuuUUACAAAU 3306 rs2159172 11855 6R AU U UGU- tl-lIAAA &AIIAArACU 1554 11 6c; CAGUGUUAUUUU UACMA 3307 rs2159172 11856 AUUU 155,1!i 11857 1 UUU(jUAAF^MMUPv'"l=-:f AUUUUUUACAA aauo j rs2159172 1 11857 1 It JUGUAAAAAAUAAC;A(;Uu 11 cucu I i;tb ii ACAGUGUU_ rs2159172 I llk3:)ts I UUUUAAAAAAUAAL;/-XkUk--,Iu 1185fil U AACA 252 2005201389 01 Apr 2005 rs2159172 11859 UGUAAAAAAUAACACUGUG 1557 11859 UGUAACACUGGU 1557 11877 CACAGGUUAUUUUUUACAAC 330 rs2159172 11860 GUAAAAAAUAACACUGUGA 1558 11860 UA UAACACUGUGA 1558 11879 UCACAGUGUUAUUUUUUA 3310 rs2159172 11861 U AACACUGUGAA 1559 11861 AAAAAA CACUGUGAAU 1560 11880 AUUCACAGUGUUAUUUUUU 3312 rs2159172 11862 AAAAAAUAACACUGUGAAU 1560 11862 AAAAAUAACACUGAAUG 1561 11881 CAUUCACAGUGUUAUUUUU 3313 rs2159172 11863 AAAAAUAACACUGUGAAUG 1561 11863 AAAAUAACACUGUGAAUGU 1562 11882 ACAUUCACAGUGUUAUUUU 3314 rs2159172 11864 AAAAUAACACUGUGAAUGU 1562 11864 ACAU GA GC 1563 12658 CGCUGGCAGAUGAGGGU 3315 ns2237008 12640 ACCCUCAUUUCUGCCAGCG 1563 12640 ACCCUCAUUUCUGCCAGCG 1563 12659 CGCUGGCAGAAAUGAGGG 3316 rs2237008 12641 CCCUCAUUUCUGCCA 1564 12641 CUCAUUUCUGCCAGCGC 1564 12659 GCGCUGGCAGAAAUGAGG 3317 rs2237008 12642 CCUCAUUUCUGCCAGCGC 1565 12642 UUUCUGCCAGCGCA 1565 12660 UGCGCUGGCAGAAAUGAG 3317 rs2237008 12643 CUCAUUUCUGCCAGCGCA 1566 12643 UUUCUGCCAGCGCAU 1566 12661 AUGCGCUGGCAGAAAUGA 3318 rs2237008 12644 UCAUUUCUGCCAGCGCAUG 1567 12644 UAUUCUGCCAGCGCAUG 1567 12662 CAUGCGCUGGCAGA 3319 rs2237008 12645 CAUUUCUGCCAGCGCAUGU 1568 12645 UUCUAGCGCAUU 1569 1:M64 ACAUGCGCUGGCAGAAA 3320 rs2237008 12646 AUUUCUGCCAGCGCAUAUGU 1569 12646 8157 12663 CACAUGCGCUGGCAGAAA 3322 rs2237008 12645 UC UGCCAGCGCAGUU 15681971 12664 ACACAUGCGCUG GCAGAAA 3321 rs2237008 12648 UUCUGCCAGCGCAUGUG 1571 12648 AUUCUGCCAGCGAUUU 1570 12665 GACACAUGCGCUGGCAGA 3322 s2:237008 12649 UCUGCCAGCGCA-UGUGUCC 1572 12649 CUGCCAGCGAUUUU 1573 16 GGACACAUGCGCUGGCAG 3325 -s2237008 12650 CUGCCAGCGCAUGUGUCCU 1573 12650 UCCCAUGUGUCU 1574 12666 AGGACACAUGCGCUGGCA 3323 rs2237008 12651 UGCCAGCGCAUGUGUCCUU 1574 12651 UGCAGC3GCAUGUGUC 1575 12670 AAGGACACAUGCGCUGGC 3327 rs2237008 12652 GCCAGCGCAUGUGUCCU 1575 12652 CCAGCGCUGUGUCCUUU 1572 12671 GACACAUGCGCUGGC 3324 rs2237008 12653 CCAGCGCAUGUGUCCUUUC 1576 12653 CACUG UG 1573 12672 GAAAGGACACAUGCGCU G 3325 rs2237008 12654 CAGCGCAUGUGUCCUCA 1577 12654 AGCGAGUGUCCUUUCAA 1574 12673 UGAAAGGACACAUGCGCU 3326 rs2237018 12651 U GCGCAUGUGUCCUUUCAAG 1579 1267 UUGAAAGGACACAUGCGC 1 3331 s223700 12656 GCGCAUGUGUCCUUUCAAG 1579 12656 CAU UGUCCUUUCAAG 1575 12670 CUUGAAAGGACACAUGCG 3332 rs2237008 12657 CGCAUGUGUCCUUUCAAGG 1580 12671 CCUUGAAAGGACACAUGC 3333 rs2237008 12658 GCAUGUGUCCUUUCAAG 1581 12658 ACCCUCUUUCA G A 158 12672 UGCUGAAAGACACAUGCGG 3334 rs2237008 12640 ACCCUCAUUUCUGCCA 1582 12640 CC UUCUGCC 1583 1265 UGCUGGCAGAAAUGAGGG 3335 rs2237008 12641 CCCUCAUUUCUGCCACAC 1583 12641 AGCCAUGCGCAC A 1584 12673 GUGUG GGCAGAAAUGAGG 3330 rs2237008 12642 CCUCAUUUCUGCUUCACA 1584 12642 CCAUCUG CCAGCAA 158 12661 AUGCUGGCAGAAAUGG 3331 rs2237008 12643 CUCAUUUCUGCUUCACAU 1585 12643 GCA 1580 12662 AUGUGCUGGCAGAAAUG 3332 s2237008 12644 UCAUUUCUGCCAGUG 1586 12644 UAGCCA GCACAUG 1581 12663 CACUGCUGGCAGAAAUG 3339 rs2237008 12645 CAUUUCUGCUUCACAGU 1587 12645 UUCUCAGCAU 1588 12664 ACAUGUGCUGGCAGAAAU 3334 rs2237008 1 646 AUUUCUGCCAGCACAUGUG 1588 12646 UUUCUCCAGCACAUUG 1589 12665 CACAUGUGCUGGCAG 3341 2237008 12647 UUUCUGCCAGCACAUGGU 1589 12647 UUCUGCACACA UGUGUC 159 12666 ACACAUGUGCUGGCAGAA 3342 rs2237008 12648 UUCUGCCAGCACAU C 0 C 1584 12660 GACACAUGUGCUGGCAGA 3343 rs2237008 12649 UCUGCCAGCACAUGGC 1591 12649 CACAUGUGCC 1592 1266 AGGACACAUGUGCUGAG 3344 rs2237008 12650 CUGCCAGCACAUGUGUC 1592 12650 UCACAUGUGUCC U U 1593 12662 AGGACACAUGUGCUGGCA 3345 rs2237008 12651 UGCCAGCACAUG 1593 12651 GCAGCACAUGUGUCCUUU 1590 12666 AAGGACACAUGUGCUGGC 3346 rs2237008 12648 UGCCAGCACAUGUGUC UCU GCC CACAUC~A GUGUC C 1591 12667 AGGACACAUGUGCUAG 3343 rs2237008 12652 CCAGCACAUGUGUCCUUUC 1595 12653 CCAGCACAUGUGUCCUUUC 1595 12671 GAAAGGACACAUGUGCUGG 3347 253 2005201389 01 Apr 2005 rs2237 008 12654 CAGCACAUGUGUCCUUUCA 1596 12654 CAGCACAUGUGUCCUUUCA 1596 rs223700 8 12655 AGCACAUGUGUCCUUUCMA 1597 12655 AGCACAUGUGUCCUUUCMA 1597 rs2237008 12656 GCACAUGUGUCCUUUCAG 1598 12656 GCACAUGUGUCCUU UCAAG 1598 rs223700 8 12657 CACAUGUGUCCUUUCAGG 1599 12657 CACAUJGUGUCCUUUCAAGG 1599 rs2237008 12658 ACAUGUGUCCUUUCAAGGG 1600 12658 ACAUUUCCUUUCAAGGG 1600 rs36230 0 12893 CAGGUGGAACUUCCUCCCG 1601 12893 CAGGUGACUUCCUCCCG 1601 rs362300 12894 AGGUGGAACUUCCUCCCGU 1602 12894 AGGUGGAACUUCCUCCCGU 1602 rs36230 120 ACUCCCGUCGG 108190 AACUUCCUCCCGUUG 1608 rs3623 00 1901 rr' IGACUUCCUCCCGUUG 1609 128901 U CCUCCCGUUGG 1609 rs36230 0 12907 iUCCCUCGGGCGUGG 1615 128907 UCGGGUGGAGU 161 rs3623 00 128908 CCCGUUCGGGCGUAUG 1616 128908 UGA CCCGUUGCGAU 1616 rs3623 00 12895 GGUAACUUCCUCCCUU 1622 12895 GGUAACUUCUCUU 16 rs3623 00 12896 GUAACUUCCUCCCUUG 1623 12896 GAACUUCCUCCCUUG 1623 rs36230 0 12902 ACUUCCUCCCUUGCGGGU 1629 12902 ACUUCCUCCCUUGCGGG 1629 rs3623 00 12903 AUUCCUCCCUUGCGGGG 160 12903 AUUCCUCCCUUGCGGGG 160 rs362300 2904 CUCCUCCCUUGCGGGGU 161 12904 CUCCUCCCUUGCGGGGU 161 rs3623 00 12905 UCCUCCCUUGCGGGGUG 1632 12905 UCCUCCAUUGCGGGGUG 162 rs3623 00 12906 CUCCCAUUGCGGGGUGGAG 163 12906 CUCCUGCGGGGUGG AG -1633 rs36230 0 12907 UCCCUUGCGGGGUGGAGU 1634 12907 UCCCUUGCGGGGUGGAGU 1634 12672I 12673 12674 12675 12676 12911 12913 12914 12915 12916 12917 12918 12919 12920 12921 12922 1292 12924 12925 12926 12927 12928 12911 12912 12913 12914 12915 1291 12920 12922 UGAAAGGACACAUGUGCUG 34 UUGAAAGGACACAUGUGCU 3349 CUUGAAAGGACACAUGUGC 3350 CCUUGAAAGGACACAUGUG 3351 CCCUUGAAAGGACACAUGU CGGGAGGAAGUUCCACUG 3353 ACGGGAGGAAGUUCCACCU 3354 AACGGGAGGAAGUUCCACC 3355 CAACGGGAGGMAGUUCCAC 3356 GCAACGGGAGGAAGUUCCA 3357 CGCAACGGGAGGAAGUUCC 3358 CCGCAACGGGAGGAAGUUC 3359 CCCGCAACGGGAGGAAGUU 3360 CCCCGCAACGGGAGGAAGU 3361 CACACCCCGCAACGGGA 3368 UCCCACCCCGCAACAGG 3369 CACUCACCCCGCCG 3367 CCACUCACCCCGCAACG 3371 UGGAGGAUCACCUCG 3372 MIAUGGGGUCCACCU 3373 AAUGGAGGGUCCACAC 3374 CUGGGAGGAAGUUCCAC 3375 GAUGGGAGGAAGUUCCA 3376 CAAUGGGAGGAAGUUCC 3377 CCAAUGGGAGGAAGUUC 3378 CGCAAUGGGAGGAAGUUC 3379 CCGCAAUGGGAGGAAGUC 3380 ACCGCAAUGGGAGGAAG 338 CACCCGCAAUGGGAGGAA 3382 CACCCCGCAAUGGGAGGA 3383 _UCACCCG-CAAUGGGAGG 3384 _CCCCCCGCAAUGGGAG 3385 _AUCCCCCCGCAAUGGGA 3386 254 2005201389 01 Apr 2005 rs3623 00 12908 CCCAUUGCGGGGUGGAGUG 1635 129108 CCCAUUGCGGGGUGGAGUG 1635 rs36230 0 12909 CCAUUGCGGGGUGGAGUGA 1636 12909 CCAUUGCGGGGUGGAGUGA 1636 rs36230 0 12910 CAUUGCGGGUGGAGUGAG 1637 12910 CAUUGCGGGGUGGAGUGAG 1637 rs362300 12911 AUUGCGGGGUGGAGUGAGG 1638 12911 AUUGCGGGGUGGAGUGAGG 1638 rs25305 95 13022 cCCCGCUUCCUCCCUCUGC 1639 13022 CCCCGCUUCCUCCCUCUGC 1639 rs2530 595 13023 CCGCUUCCUCCCUCUGCG 1640 13023 CCCGCUUCCUCCCUCUGCG 1640 rs25305 95 13024 CCGCUUCCUCCCUCUGCGG 1641 13024 CCGCUUCCUCCCUCUGCGG 1641 rs25305 95 13030 CGCUUCCCUCUGCGGGG 1647 13030 CGCCUCCCUCUGCGGGG 1647 rs253OS 9 5 13031 GCCUCCCUCUGCGGGGA 1648 13031 GCCUCCCUCUGCGGGGA 1648 rs25305 95 13037 cCUCGGGAGGGGGA 1654 13037 Cucc CUGCGCCGGGA 164 rs25305 9 5 13038 uCUCCUGCGGGGAGGGA 1655 13038 ucc UGCGGGGAGGGC 16455 rs253059 5 13025 CUUCCUCCCUCUGGGG 1661 13025 CUUCCUCCC UCGGGG 16461 rs25305 95 1030 GUCCUCCCUCUGUGGGGG 1662 13026 GCCUCCCUCUUGGGGG 1662 rs25305 95 13032 CUCCCUCUGUGGAGGACC 1668 13032A UCCCGGGGGAGGACC 168 rs2530 9 13033 UCCCUCUGUGGGGAGGA-'C 169103CCCUCUGUGGGGAGGACCC 169 rs2530595 13034 CCUCUGGGGGAGGACCCG 1670 13034 CCUCUGGGGGAGGACCCG 1670 rs25305 95 13035 CUCUGGGGGAGGACCCGG 1671 13035 CUCUGGGGGAGGACCCGG 1671 rs25305 95 13036 UCUGGGGGAGGACCCGGG 1672 13036 1UCUGUr-rGGGGAGG(ACCCGGGU 1672~ rs2530 9 13037 CUGU1GGGAGGACCCGGGA 1673137 CUGuGGGGAGGACCCGGGA 1673 12926 12927 12928 12929 13040 13041 13044 13045 13046 13047 13048 13049 13050 13051 13052 13053 13054 13055 13056 13057 13058 13040 13041 13042 13043 13044 13045 1:3046 13047 13048 13049 13050 13051 13 052 13053

CACUCCACCCCGCAAUGGG

UCACUCCACCCCGCAAUGG

CUCACUCCACCCCGCAAUG

CCUCACUCCACCCCGCAAU

GCAGAGGGAGGAAGCGGGG

CGCAGAGGGAGGAAGCGGG

CCGCAGAGGGAGGAAGCGG

CCCGCAGAGGGAGGAAGCG

CCCCGCAGAGGGAGGMAGC

UCCCCGCAGAGGAGGMAG

CUCCCCGCAGAGGGAGGAA

CCUCCCCGCAGAGGGAGGA

UCCUCCCCGCAGAGGGAGG

GUCCUCCCCGCAGAGGGAG

GGUCCUCCCCGCAGAGGGA

GGGUCCUCCCCGCAGAGGG

CGGGUCCUCCCCGCAGAGG

CCGGGUCCUCCCCGCAGAG

CCCGGGUCCUCCCCGCAGAj U C CCG GG U C CU C C C5G CAG

GUCCCGGGUCCU--------

GGUCCCGGGUCCUCCCCGC

UGGUCCCGGGUCCUCCCCG

ACAGAGGGAGGAAGCGGGG

CACAGAGGGAGGAAGCGGG

CCACAGAGGGAGGAAGCGG

CCCACAGAGGGAGGAAGCG

CCCCACAGAGGGAGGAAGC

UCCCCACAGAGGGAGGAAG

CUCCCCACAGAGGGAGGAA

CCUCCCCACAGAGGGAGGA

UCCUCCCCACAGAGGGAGG

GUCCUCCCCAAGAGGGAG

GGUCCUCCCCACAGAGGGA

GGGUCCUCCCCACAGAGGG

CGGGUCCUCCCCACAGAGG

CGGGUCCUCCCCACAGAG

CCCGGGUCCUCCCCACAGA

UCCCGGGUCCUCCCCACAG

3387 3388 3389 3390 3391 3392 3393 3394 3395 3396 3397 3398 3399 3400 3401 3402 3403 3404 3405 3406 3409 3410 3411 3412 3413 3414 3415 3416 3417 3418 3419 3420 3421 3422 3423 342H4 325 255 2005201389 01 Apr 2005 rs25305 95 13038 UGUGGGGAGGACCCGGGAC 1674 13038 UGUGGGGAGGACCGGGAC 1674 13056 GUCCCGGGUCCUCCCCACA 3426 rs25305 95 13039 GUGGGGAGGACCCGGGACC 1675 13039 GUGGGGAGGACCCGGGACC 1675 13057 GGUCCCGGGUCCUCCCCAC 3427rs25305 9 5 13040 UGGGGAGGACCCGGGACCA 1676 13040 UGGGGAGGACCCGGGACCA 1676 13058 UGGUCCCGGGUCCUCCCCA 3428 rsl 803770 13464 CUGCUUUGC;ACCGUGGUCA 1677 13464 CUGCUUUGCACCGUGGUCA 1677 13482 UGACCACGGUGCAAAGCAG 3429 rs1803 770 13465 UGCUUUGCACCGUGGUCAG 167 13465b UUUUGCACCGUGGUCAG 1678 13483 CUGACCACGGUGCAAAGOA 3430 rsl 803770 13466 GCUUUGCACCGUGGUCAGA 16 79 136 CUUUGCACCGUGGUCAGA 1679 13484 UCUGACCACGGUGCAAAGC 3431 rsl 803770 13467 CUUUGCACCGUGGUCAGAG 160147CUUUGCACCGUGGUCAGAG 1680 13485 CUCUGACCACGGUGCAG 3432 rs1 803770 13468 UUUGCACCGUGGUCAAG 1681 1346 rUGACCGUGGUCAGAGG 1681 13486 CCUCUGACCACGGUGCMAA 3433 rs18037 70 13469 UUGCACCGUGGUCAGAGG 182136 UUGCACCGUGGUCAGAGGG 1682 13487 CCCUCUGACCACGGUGCA 3434 rsl 803770 134701 UGCACCGUGGUCAGAGGGA 1683 13470 UGCACCGUGGUCAGAGGGA 1683 13488 UCCCUCUGACCACGGUGCA 3435 rsl 803770 13471 GCACCGUGGUCAGAG3GGAC 1684 13471 GCACCGUGGUCAGAGGGAC 1684 13489 GUCCCUCUGACCACGGUGC 3436 rsl 803770 13472 CACCGUGGUCAGAGGGACU 1685 13472 CACCGUGGUCAGAGGGACU 1685 13490 AGUCCCUCUGACCACGGUG 3437 rsl 803770 13473 ACCGUGGUCAGAGGGACUG 1686 13473 ACCGUGGUCAGAGGGACUG 168 1491 CAUCCUCUGACCACGGU 3438 rs1 8037 70 13474 CCGUGGUCAGAGGGACUGU 1687 13474 CCGUGGUCAGAGGGACUGU 167 142 AAUCCUCUGACCACGG 3439 rs1 803770 13475 CGUGGUCAGAGGGACUGUC 1688 13475 CGUGGUCAGAGGGACUGUC 1688 13493 GACAGUCCCUCUGACCACG 3440 rsl 803770 13476 GUGGUCAGAGGGACUGUCA 1689 13476 GUGGUCAGAGGGACUGUCA 1689 13494 UGACAGUCCOUCUGACCAC 3441 rsl 803770 13477 UGGUCAGAGGGACUGUCAG 1690 13477 UGGUCAGAGGGACUGUCAG 1690 13495 CUGACAGUCCCUCUGACCA 3442 rs18037 70 13478 GGUCAGAGGGACUGUCAGC 1691 13478 GGUCAGAGGGACUGUCAGC 1691 139 GCU3GACAGUCCCUCUGACC 3443 rsl 803770 13479 GUCAGAGGGACUGUACU 1692 13479 GUAGGGACUGUCAGU 1692 13497 AGCUGACAGUCCCUCUGAC 3444 rsl 803770 13480 UCAGAGGGACUGUCAGCUG 1693 13480 UCAGAGGGACUGUCAGCUG 1693 13498 CAGCUGACAGUCCCUCUGA 3445 rsl 803770 13475 CAGUGUCGGGACUGUC 17071347 CGUCGAGGGACUGUCG 1707 134 93 UAGACAGUCCCUCCAG 3459 rsl 803770 13476 GUCGAGGGA CUGUCA'G, 1708 13476 GU GUCAGCUGUA 1708 13494 CGUGACAGUCCCUCCA 3460 rsl 803770 13477 UGCGCGAACU GCG 167 34775 UGGCAACUGUCAG 167 13495 CUGACCACGUCUCGCA 3461 rs1 8037 70 13478 UGUCGGACCUGGUCAGCG 17010 13478 GGUCGAGUGGGUCAGCG 17010 1346 G ~CGACCCGUCA 3462 rs1 8037 70 13479 CCGUCGGAGGGACUGGU 1711 13479 CCGUCGGAGGGACUGGU 1711 13497 AGACAGUCCCUCCGACG 3463 rs18037 70 13480 UCGGAGGGACUGUCAG 1712 13480 UUCGGAGGGAUGUCAG 1712 13498 CCUGACAGUCCCUCCGA- 3464 256 2005201389 01 Apr 2005 m1l803771 1348 CGCCCACCAGCUGU 1718 1348 CGCCCCACCAGCUG 1718 13566 AUCAGCUGGUGGGGCC 3470 rs18037 7 0 13492 CCCCACCAGCUGUG 1719 13492 GGAGACCAGCUGAUG 1719 13567 CUCAGCUGGUGGC 3471 rsl 803771 13555 CCAGACCUAAGC CUG 1725 13555 GCAGACCUAAGCCUG 1725 13573 CAGGCUUGGUGCUGG 34677 rs18037 7 l 13556 CAGACCUAAGCCUGA 41726 4135 r-A CAGAC.CUACAGCCUGA 1726 13574 UCAGGCUUGGUGCUC 3478 rs1803771 131!5517 AGACCACAGCCUGAG 1727 13557 AGk%,ACCAACCUGAA 1727 13575 CUCAGGCUUGGUGCU 3479 rsl 803771 13558 GACCUACAGCCUGIAA 172838 GCUAUCUUAA 1728 13576 AUUAAUCAG CAGUCG 3480 rsl 803771 1359 ACCUAAGCCUGAAG 19 13559 ACCUAAGCCUGAAG 1729 13577 CAUUCAGAGCUUGGUG 3481 rsl 803771 13560 CCUAAGCCUGAAGC 1 730 13560 CCUGMUCGCCUGAAGC 1730 13578 GCAUUCAGAGCUUCAUGG 3482 rms18037 7 l 13561 CACAGCCUGAAGCA 1731 13561 CUGMUAGCUUAAC 1731 13579 UGCUCUCAGAGCAUUCAG 3 G 3483 rsl 803771 13562 ACAGCCUGAAGCAA 172 13562 UGrAr-L;AUCUCUGAAGCU 1732 13580 UGCAUUCAGAGCUUCA 3484 rs1 8037 7 1 13563 AAGACCUGAAGCU 172331 363 GJIACACUCUGAAGCU 1733 13581 UUUGCAUUCAGAGCUGU 3485 rs18037 7 1 135545 GCCCCACAGACUA 1734 134 GACCCACAGCCUA 1734 13563 AGGCUCGGUGGGUC 3486 rs1807 7 1 13546 GCCACCCAGACrLLCUG 1735 13546 GGCCCCUACAGCCUM 1735 13564 UAGGUCUGGGUGGGGCUC 347 rs1837 7 1 13547 CAGCCUGACAUGCU 1736 134 AGCCCACAGCCUW 1736 13565 UAGGUCUGGGUGGGCU 34788 rs1 803 77 1 13549 CCCAGACCUAAAU G 1738 13549 CCCAGACCUAAAUCAG 1738 1357 CUAUU CUAGGUCUGG 34790 rs1 8037 7 1 13550 CCAGACCUAAAUGCGA 1739 13550 CCAGACCUAAAUGCGA 1739 135768 GCUUAGGCAUCGGUG 3491 rs1 8037 7 1 13551 ACCACGA UAUGCU 1740A 151 CAAACCUAAAUGCUAG 1740 13569 CUUAG GCUCGGGU 3492 rs1 803771 13552 CCCGACCUAAGCU 1741 135 CCAACUAAGC 1741 13570 UAAAGCAU UCGG 3493 rs1803 77 l 13553 CCGUAAUGCUUCGC 1742 135531 ACCCAGACUAAGCU 1742 13571 GAGCUUAGGAUCGG 3494 rs1803 7 l 13554 CCGA UAUGCUUCU14 35 CAGACCUA AAUGCUUCUCA 173 13572 AUGC AAGCAUU CGG 3495 rsl 803771 13555 CGAUAAUGCUUCUG 1744 13555 CC-AAC--ICUG CAAACU 1744 13573 UGUCAGAAGCAU UC 3496 rs180377l 13556 CAGACCUAAAUGAUUUA 175 356CAGACCUACUA 1745 13574 UUAGGCUUGGUGCUG 3497 rs1 803 77 1 13557 AGACCUACAACCUAG 1746 13557 AGACCUACAACCUAG 1746 13575 UAGGCUUGGUGCU 348 rs18037 7 l 135 GACCUACAACCUAA 1747 13558 GACCUACAACCUAA 1747 13576 UCUCAGAGCUUGGUGC 34899 rsl 803771 13559 CACCAAACUCUAAG 1748 13559 CACCAAACUCUAAG 1748 13577 CAAAGCAUUUAGGUUG 3500 rs18037 7 1 13560 CGCCUAAAUGCUUCUAG 1749 13560CC CCUAAAUGCUUCUC 1749 13578 GCAGAAGCAUUUAGGG 345 rs1 803771 13561 ACUAAAUGCUUUGAGAG 1750 13561 ACUAUGCUUCUGAGAGA 1750 13579 UCUCUCAGAAGCAUUUAG 3502 rs1 803 77 1 13562 CUAUGCUUCUGAGAGC 1751 13562 CUAAAUGCUUCUGAGAGC 1751 150 GCUCUCAGAAGCAUUUA 3503 257 2005201389 01 Apr 2005 rsl803771 13563 AAAUGCUUCUGAGAGCAAA 1752 13563 AAAUGCUUCUGAGAGCAAA I 1752 13581 1 UUUGCUCUCAGAAGCAUUU 3504 The 3'-ends of the Upper sequence and the Lower sequence of the siNA construct can include an overhang sequence, for example about 1, 2, 3, or 4 nucleotides in length, preferably 2 nucleotides in length, wherein the overhanging sequence of the lower sequence is optionally complementary to a portion of the target sequence. The overhang can comprise the general structure B, BNN, NN, BNsN, or NsN, where B stands for any terminal cap moiety, N stands for any nucleotide thymidine) and s stands for phosphorothioate or other internucleotide linkage as described herein internucleotide linkage having Formula The upper sequence is also referred to as the sense strand, whereas the lower sequence is also referred to as the antisense strand. The upper and lower sequences in the Table can further comprise a chemical modification having Formulae I- VII or any combination thereof (see for example chemical modifications as shown in Table V herein).

258 2005201389 01 Apr 2005 Table III: HD synthetic siNA and Target Sequences Tfarget Target SeqiD Sirna #Aliases Sequence SeqiD Pos AGAGA 586 CAAAGAAAGAACUUUCAGCUACC 3505 31993 HD:586U21 sense AGAACUUUCAGCUATT 3512 586 CAAAGAAAGAACUUUCAGCUACC 3505 31994 (586C) antisense UAGCUGAAAGUUCUUUCUUTT 3513 586 CAAAGAAAGAACUUUCAGCUACC 3505~ 199 HD:v581--2 stab04 sense B AAGAAAGAAcuuucAGcuATT B 3514 586 CAAAGAAAGAACUkUUCAGCUACC 3505 31996 HD:604L-21 (586C) stab05 antisense uAGcuGAAAGUcuuuCUuTST 3515 586 CAAAGAAGAUUUCAGCUACC 3505 31997 HD:586U21 stab07 sense B AAGAAAGAAcuuucAGCUAUT B 3516 586 CAAAG'AAAGAACUUUCAGCUACC 3505 31998 HD:604L21 (586C) stabOB antisense uAGcuGAAAGuucuuucuuTsT 3517 586 CAAAGAAAGAACUUUCAGCUACC 3505 31999c HD:586U21 inv sense AUCGACUUUCAAGAAAGAATT 3518 586 CAAAGAAAGAA0UUUCAGCUACC 3505 32000 1-116041-21 (586C) inv antisense UUCUUUCUUGAAAGUCGAUUT 3519 586 CAAAGAAAGAACUUUCAGCUACC 3505 32001 HD:586U21 inv stab04 sense B AucGAcuuucAAGAAAGAAUT B 3520 86 CAAAGAAAGAACUUUCAGCUACC 3505 32002 HD:604L-21 (5860) inv stabO5 uucuuucuuGAAAGucGAuTsT 3521 U .Da 11 nv ta07seseB AucGAcuuucAAGAAAGAAU B 3522 86 CAAAGAAAGAACUUUCAGCUACC 3505) 3204 UD0421 (586C) inv stabO8 UucuuucuuGAAAGuCGAuTsT 3523 316 CCAUGGCGACCCUGGAAAAGCUG 350 33065 HD:316U21 siRNA stab04 sense B AuGGcGAcGcuGGAAAAGCTT B 3524 591 AAAGAACUUUCAGCUACCAAGAA 3507 33066 H D:591 U21 siRNA stab04 sense B AGAAcuuucAGcuAccAAGUT B 3525 671 AAAUUCUCCAGAAUUUCAGAAAC 3508 33067 H D:671 U21 siRNA stab04 sense B AuucuccAGAAuuucAGAATT B 3526 769 AAU3CCUCAACAAAGUUAUCAAA 3509 33068 Hu: 769U21 siRNA stab04 sense B uGccucAAcAAAGuuAucAT B 3527 1 GAGGAAGAGGAGGAGGCCGAC 3510 33069 HD-ExS8:3U21 siRNA stab04 sense B GGAAGAGGAGGAGGccGAcUT B 3528 2 AAGAGGAGGAGGCCGACGCCC 3511 33070 HD-Ex58:7U21 siRNA stab04 sense B GAGGAGGAGGccGAcGcccUT B 3529 316 CAUGGCGACCCUGGAAAAGCUG 3506 33071 HD:334L-21 siRNA (3160) stabO5 GcuuuuccAGGGucGccAuTsT 3530 91 AAAGAACUUUCAGCUACCAAGA 3507 33072 HD:609L21 siRNA (5910C) stabOS5 cuuGGuAGcuGAAAGu-c-uTsT 3 5-31 antisense 6 7-1 AAAUUCUCCAGAAUUUCAGAAAC 3508 33073 HD:689L-21 siRNA (6710C) stabO5 uucuGAAAuucuGGAGAAuTsT 3532 antisense 769 AAUGCCUCAACAAGUUAUCAA 3509 33074 HD:787L-21 siRNA (769C) stabO5 uGAuAAcuuuGuuGAGG-A-TsT 3533 1 GAGGAAGANGGAG GAGGCCGAC 3510 33075 H-D-ExS8:21 L21 siRNA (Ex58-3C) GucGGccuccuccucuuccTsT 3-5-34 2 AAGAGGAGGAGGCCGACGCCC 3511 33076 HD-Ex58:25L-21 siRNA (Ex58-7C) GGGcGucGGccuccuccucTsT 316 CCAUGGCGACCCUGGAAAAGCUG 3506 33077 HD:316U21 siRNA stab07 sense B AuGGcGAcccuGGAAAAGCTT B 3536 591 AAAGAACUUUCAGCUACCAAGMA 3507 33078 HD:591 U21 siRNA stab07 sense B AGAACUUUCAGcuACCMAGTT B 3057 6-'7 1 AAAUUCUCCAGAAUUUCAGAAAC 3508 33079 HD:671 U21 siRNA stab07 sense B AuucuccAGAAuuucAGAATT B 3538 769 AAUGCCUCAACAAAGUUAUCAAA 3 50O9 3080 HD:769U21 siRNA stab07 sense B uGccucAAcAAAGUUAUcATT B 3539] 259 2005201389 01 Apr 2005 1 GAGGAAGAGGAGGAGGCCGAC 3510 33081 HD-Ex58:3U21 siRNA stab07 sense B GGAAGAGGAGGAGGccGAcUT B 3540 2 AGAGGAGGAGGCCGACGCCC 3511 33082 HD-Ex58:.7U21 siRNA stab07 sense B GAGGAGGAGGccGAcGcccTT B 3541 316 CCUGCGCCUGGAAGCG 306 33083 HD:334L21 siRNA (3160) stabO8 GcuuuuccAGGGucGCCAuTsT 3542 31 CUGGC~GAAGU 56antisense 591 AAAGAACUUUCAGCUACCAAGAA 3507 33084 HD:609L21 siRN(51)sbOcuGAcGAGUCsT 34 antisense 67 AUUCUCCAGAAUUUCAGAAAC 3508 33085 HD:689L21 siRNA (6710C) stab08 uucugAAAuucuGGAGAAuTsT 3544 antisense 769 AAUCCCAAAAGUUUCAA 50 3386HD:787L21 siRNA (769C) stab08 UGAuAAcuuuGuug.AG.~ATsT 3545 769 AAUGCCUCAAC AGUAUC 3509 33086antisense !uGcuccuuc~T34 1 AGAAGGGGCGC 31 33087 HD-Ex58:21 L21 siRNA (Ex58-3C)GuGcCUCCUCTT34 1 GAGAAGGGAGAGGCGAC 510stabOB antisense1 2 AAGAGGAGGAGGCCGACGCCC 3511 33088 HD-Ex58:25L21 siRNA (Ex58-7C) gGGcGucGGCcuccuccucTsT 3547 stab08 316 CCAUGGCGACCCUGGAAAAGCUG 3506 33089 HD:316U21 siRNA stab09 sense B AUGGCGACCCUGGAAAAGCUT B 3548 591 AAAGAACUUUCAGCUACCAAGA 3507 33090 HTD:591 U21 siRNA stab09 sense B AGAACUUUCAGCUACCAAGUT B 3549 671 AAAUUCUCCAGAAUUUCAGMAAC 3508 33091 H:671 U21 siRNA stab09 sense B AUUCUCCAGAAUUUCAGAA~rB 3550 769 AAUGCCUCAACAAAGUUAUCAAA 3509 33092 HD:769U21 siRNA stab09 sense B UGcCUCAACAAAGUUAUCAUT B 3551 1 GAGGAAGAGGAGGAGGCCGAC 3510 33093 HDE5:U1 siRNA stab09 sense B GGAAGAGGAGGAGGCCGACUT B 3552 2 AAGAGGAGGAGGCCGACGCCC 3511 339 DE5:U1 siRNA stab09 sense B GAGGAGGAGGCCGAC;GCCCUT B 3553 316 CCAUGGCGACCCUGGAAAAGCUG 3506 305HD:334L21 siRNA (316C) stablO0 GCUUUUCCAGGGUCGCCAUTsT 3554 antisense CUGACGAGUUs 591 AGAACUUUCAGCUACCAAGMA 3507 33096 HD609L21 siRNA (5910C) stablO 0UGUGUAAUCTT 671 AAAJUCUCCAGAAUUUCAGAAC 3508 33097 HD:689L21 siRNA (6710C) stablO UUAAUUGGATT 7-6-9- UGCCUCAACAAAGUUAUCWA 3509 33098 HD:787L21 siRNA (79)talUGACUGUAGATT GAGAGGGGAGCGA 31 309 Dex5:1 iN E5-C GUCGGCCUCCUCCUCUUCCTsT 3558 2 AAGAGGAGGAGGCGACC 3511 33009 HD-Ex58:25 L21 siRNA (Ex58-7C) GGCUGCUUCUTT 59 stablO antisense Uppercase ribonucleotide G 2'-O-methyl Guanosine R= u,c 2'-deoxy-2'I-fluoro U,C X= nitroindole universal base Z =sbL: symetrical bifunctional linker T thymidine Z= nitropyrole universal. base H =chol2: capped Cholesterol

TEG

B inverted deoxy abasic Y= 3',3'-inverted thymidine A =2'-O-methyl Adenosine s hosphorothioate linkage M= glycery1 Q= L-uridine A =deoxy Adenosine N= 3'-O-methyl uridine G =deoxy Guanosine P= L-thymidine 260 05-509 400/250 Table IV Non-limiting examples of Stabilization Chemistries for chemically modified siNA constructs Chemistry pyrimidmne Purine cap pSSrn "Stab 0T" Ribo Ribo TT at

S/AS

ends "Stab 1" Ribo Ribo -5 at 5'-end

S/AS

1 at 3'-end 00"Sta-"Rb Ribo -All linkages Usuall AS "tab 3"1 2'-floro Rb at 5'-end Usually S 0 4 at 3'-end In "Stab 4" 2'-fiuOro Ribo 5' and 3'--UsalS ends "Sa0" 2-loo Rb 1 at 3'-end fUsuall AS "Stab 6" 2'-O-Methyl Ribo 5' and Usually

S

"Stab 5" 2 -flu ro endsS "Stab 7" 2'-fiuoro 2'-deoxy 5' and Usually

S

ends "Stab 8"9 2'-luoro 1 at 3'-end

SA

"Sab1" '-ior M-eoh I t3-n Usually AS "Stab 12" 2'-luoo LNAo 5' and 3'ends "Stab 13" 2'-iuor RLN at 3'-end Usuallv

AS

"Stab 14"9 2'-fiuoro 2'-deoxy t5-n Usually AS 1eanden "Stab 15"V 2'-deuoxy 2'-deoxy 2 at 5'-end Usually

AS

Methy endsen "Stab 18" 2'-fuoxy '-o-y2a 5'-and 3'-ll

A

Meth 1t ends "Stab 19" 2'-luoo 5' and YUs/ayS Methy end "Stab 20" 2'-fluoro 2'-eo 5' and Y-Usually

S

"Stab 25" 2'-fluoro* Yt3-end

S/AS

Meth 1* I'St 20" 2'-luor 2'deox 3' sua261A 05-509 400/250 0"Stab 26"1 21-fluoro* 2-0

SA

0 Meth 1*SA (Ni "Sab27 29-fluoro* 3'-end "Stab 2Meth 1* "Stab 28"1 2)-fluorO* 3'-end

SA

Meth 1* 1a 'edSA "Stab 29"1 2'-fluoro* 1a 'edSA Meth 1*

SA

cl "Stab 30"1 21-fluoro* 00 Meth 1*

SA

"Stab 31"1 2'-fluoro* 2- 3'-end

SA

MVethyl*

SA

In"Stab 32"1 2'-fluoro Mt "Stab 33"1 2'-fluoro 2'-deoxy* 5' and 3'--UsalS endsUsalS "Stab 34"1 2'-fluoro 25-0- 5' and 3'- "Stab 4F"I 2'-OCF3 Ribo 5' and Usually

S

"Stab 5F"I 2'-OCF3 Ribo I at 3'-end Usually

AS

"Stab 7F" 2'-OCF3 2'-deoxy 5an3'- Usually

S

"Stab 8FP 2'-OCF3 2-U- I at 3'-end /i Meth I Ia -n salA "Stab hIF" 2'-OCF3 2'-deox -1a3-ed Usually AS "Stab 12P" 2,-OCF3 LNA 5' and 3'-Uual% "Stab 13F" 2)-OCF3 LINA I at 3'-end Usuall.

AS

"Stab 14F" 2'-OCF3 2'-deoxy 2 at 5'-end Usually

AS

1 at 3'-end "Stab 15F" 2'-OCF3 2'-deoxy 2 at 5'-end Usually

AS

I at 3'-end "Stab 18P" 2'-OCF3 5' and Usually

S

Meth I ends "Stab 19P" 2'-OCF3 3'-end

S/AS

Meth

I

"Stab 20F" 2'-OCF3 2'-deox 3'-end Usuallv

AS

"Stab 21P" 2'-OCF3 Ribo 3'-end Usually

AS

"Stab 23F"I 2'-OCF3* 2'-deoxy*, 5' and Usually

S

ends "Stab 24F" 2'-OCF3* 1 at 3'en

S/AS

"Stab 25F" 2'-OCF3 2'O 1 t 3-en

S/AS

"Stab 26F"I T20 OCF3* 262 05-509 400/250 o IVI L S/AS Methy l

S/AS

S I "Stab 32F" 2'-OCF3 Meth IUsually "Stab 33F" 2'-OCF3 2'-deoxy* 5' and 3ends Usually S "Stab 34F" 2'-OCF3 5' and 3- Methyl* ends CAP any terminal cap, see for example Figure All Stab 00-34 chemistries can comprise 3'-terminal thymidine residues All Stab 00-34 chemistries typically comprise about 21 nucleotides, but can vary as described herein.

S sense strand AS antisense strand *Stab 23 has a single ribonucleotide adjacent to 3'-CAP *Stab 24 and Stab 28 have a single ribonucleotide at *Stab 25, Stab 26, and Stab 27 have three ribonucleotides at *Stab 29, Stab 30, Stab 31, Stab 33, and Stab 34 any purine at first three nucleotide positions from 5'-terminus are ribonucleotides p phosphorothioate linkage 05-509 400/250 Table V A. 2.5 pmol Synthesis Cycle ABI 394 Instrument Reagent Equivalents Amount Wait Time* DNA tWait Time* 2-O-methyt Wait Time*RNA Phosphoramidites 6.5 163 L 4 se; 2. mi 7. mi S-Ethyl Tetrazole 23.8 238 pL 45 sec 2.5 min. i Imidazole 21sc1se 00 TCA 176 2.3 mL 21 sec;2 e Iodine 11.2 1.7 mL 45 sec 45 sec 45 sec Beaucage 12.9 645 pL 100 sec 30sec 300 sec Acetonitrile NA 67mL NA NA

NA

l~nB. 0.2 pmol Syntesis Cycle ABI 394 Instrument Reagent Equivalents Amount Wait Time* DNA WatTie -O-methyl Wait Time*RNA (N1 Phosphoaidts 15 31 IJL 45 sec 233 sec; 465 sec S-EthylI Tetrazole 38.7 31 pL 45 sec; 233 min 45 sec Acetic Anhydride 655 124 IJL 5 sec 5Ssec 5 sec N-Methyl 1245 124 pL 5 sec 5 sec; 5 sec Imidazole TCA 700 732 pL 10 sec 10 sec; 10 sec; Iodine 20.6 244 iL 15 sec 15 sec; 15 sec Beaucage 7.7 232 pL 10sc30ec300 sec Acetonitrile NA 2.604 miL

NAAN

C. 0.2 pmol Synthesis Cycle 96 well Instrument Reagent Equivalents: DNA/ Amount: DNAIT'-O- Wait Time* DNA Wait Time* Wait Time* Ribo 2'-O-methyIRibo methyl/Ribo methyll Phosphoramidites 22/33/66 40/60/120 iJL 60 sec 180 sec 36Osec S-Ethyll Tetrazole 70/105/210 40/60/1 20 pL 60 sec; 180 min 360 sec Acetic Anhydride 265/265/265 50/50/50 pL 10 sec 10, se'0 sec N-Methyll 502/502/502 50/50/50 pL 10 sec; 10sc0sec Imidazole TCA 238/475/475 250/500/500 ilL 15 sec 15 sec 15 e Iodine 6.8/6.8/6. 8 80/80/80 pL 30 sec;3 e 3 e Beaucage 34/51/51 80/1 20/1 20 100 sec; 20 ec20 sec IAcetonitrile INA 1150/1150/1150 liL N4A NA

NA

Wait time does not include contact time during delivery.

Tandem synthesis utilizes double coupling of linker molecule

Claims

05-509 400/250 n The claims defining the invention are as follows: O S 1. A chemically synthesized double stranded short interfering nucleic acid (siNA) Smolecule that directs cleavage of a huntingtin (HD) RNA via RNA interference, wherein: 00 M 0 a. each strand of said RNA molecule is about 19 to about 23 nucleotides in length; b. one strand of said RNA molecule comprises nucleotide sequence having sufficient complementarity to said HD RNA for the RNA molecule to direct cleavage of the HD RNA via RNA interference; and c. at least one strand of said RNA molecule comprises one or more chemically modified nucleotides. 2. The siNA molecule of claim 1, wherein said siNA molecule comprises no ribonucleotides. 3. The siNA molecule of claim 1, wherein said siNA molecule comprises ribonucleotides. 4. The siNA molecule of claim 1, wherein one of the strands of said double-stranded siNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence of a huntingtin (HD) gene or a portion thereof, and wherein the second strand of said double-stranded siNA molecule comprises a nucleotide sequence substantially similar to the nucleotide sequence or a portion thereof of said huntingtin (HD) gene. The siNA molecule of claim 4, wherein each strand of the siNA molecule comprises about 19 to about 23 nucleotides, and wherein each strand comprises at least about 19 nucleotides that are complementary to the nucleotides of the other strand.
6. The siNA molecule of claim 1, wherein said siNA molecule comprises an antisense region comprising a nucleotide sequence that is complementary to a nucleotide sequence of a huntingtin (HD) gene or a portion thereof, and wherein said siNA further comprises a sense region, wherein said sense region comprises a nucleotide sequence substantially similar to the nucleotide sequence of said huntingtin (HD) gene or a portion thereof. 05-509 400/250
7. The siNA molecule of claim 6, wherein said antisense region and said sense region S, each comprise about 19 to about 23 nucleotides, and wherein said antisense region comprises at least about 19 nucleotides that are complementary to nucleotides of the sense region.
8. The siNA molecule of claim 1, wherein said siNA molecule comprises a sense region and an antisense region, and wherein said antisense region comprises a nucleotide 00 sequence that is complementary to a nucleotide sequence of RNA encoded by a Shuntingtin (HD) gene, or a portion thereof, and said sense region comprises a nucleotide sequence that is complementary to said antisense region.
9. The siNA molecule of claim 6, wherein said siNA molecule is assembled from two separate oligonucleotide fragments wherein one fragment comprises the sense region and the second fragment comprises the antisense region of said siNA molecule. The siNA molecule of claim claim 6, wherein said sense region is connected to the antisense region via a linker molecule.
11. The siNA molecule of claim 10, wherein said linker molecule is a polynucleotide linker.
12. The siNA molecule of claim 10, wherein said linker molecule is a non-nucleotide linker.
13. The siNA molecule of claim 6, wherein pyrimidine nucleotides in the sense region are 2'-O-methyl pyrimidine nucleotides.
14. The siNA molecule of claim 6, wherein purine nucleotides in the sense region are 2'- deoxy purine nucleotides. The siNA molecule of claim 6, wherein the pyrimidine nucleotides present in the sense region are 2'-deoxy-2'-fluoro pyrimidine nucleotides.
16. The siNA molecule of claim 9, wherein the fragment comprising said sense region includes a terminal cap moiety at the 5'-end, the 3'-end, or both of the 5' and 3' ends of the fragment comprising said sense region.
17. The siNA molecule of claim 16, wherein said terminal cap moiety is an inverted deoxy abasic moiety.
18. The siNA molecule of claim 6, wherein the pyrimidine nucleotides of said antisense region are 2'-deoxy-2'-fluoro pyrimidine nucleotides 05-509 400/250
19. The siNA molecule of claim 6, wherein the purine nucleotides of said antisense Sregion are 2'-O-methyl purine nucleotides. S 20. 20. The siNA molecule of claim 6, wherein the purine nucleotides present in said antisense region comprise 2'-deoxy- purine nucleotides.
21. The siNA molecule of claim 18, wherein said antisense region comprises a 0 phosphorothioate intemucleotide linkage at the 3' end of said antisense region.
22. The siNA molecule of claim 6, wherein said antisense region comprises a glyceryl modification at the 3' end of said antisense region. S23. The siNA molecule of claim 9, wherein each of the two fragments of said siNA molecule comprise 21 nucleotides.
24. The siNA molecule of claim 23, wherein about 19 nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule and wherein at least two 3' terminal nucleotides of each fragment of the siNA molecule are not base-paired to the nucleotides of the other fragment of the siNA molecule. The siNA molecule of claim 24, wherein each of the two 3' terminal nucleotides of each fragment of the siNA molecule are 2'-deoxy-pyrimidines.
26. The siNA molecule of claim 25, wherein said 2'-deoxy-pyrimidine is 2'-deoxy- thymidine.
27. The siNA molecule of claim 23, wherein all 21 nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule.
28. The siNA molecule of claim 23, wherein about 19 nucleotides of the antisense region are base-paired to the nucleotide sequence of the RNA encoded by a huntingtin (HD) gene or a portion thereof.
29. The siNA molecule of claim 23, wherein 21 nucleotides of the antisense region are base-paired to the nucleotide sequence of the RNA encoded by a huntingtin (HD) gene or a portion thereof. The siNA molecule of claim 9, wherein the 5'-end of the fragment comprising said antisense region optionally includes a phosphate group. 05-509 400/250 0 31. A pharmaceutical composition comprising the siNA molecule of claim 1 in an acceptable carrier or diluent. S 32. A chemically synthesized double stranded short interfering nucleic acid (siNA) molecule that directs cleavage of a huntingtin (HD) RNA via RNA interference Ssubstantially as hereinbefore described with reference to any one of the examples. Dated 1 April, 2005 oo 00 Sirna Therapeutics, Inc. f Patent Attorneys for the Applicant/Nominated Person SSPRUSON FERGUSON