CA2771228A1 - Treatment of 'c terminus of hsp70-interacting protein' (chip) related diseases by inhibition of natural antisense transcript to chip - Google Patents

Abstract

The present invention relates to antisense oligonucleotides that modulate the expression of and/or function of 'C terminus of HSP70-Interacting Protein' (CHIP), in particular, by targeting natural antisense polynucleotides of 'C terminus of HSP70-Interacting Protein' (CHIP). The invention also relates to the identification of these antisense oligonucleotides and their use in treating diseases and disorders associated with the expression of CHIP.

Description

TREAT:MEN` ' OF `C TERMINUS OF H:SP70-I=NTO RA.C'FIN+G PROTEIN' (CHIP) RELATED
DISEASES
BY INHIBITION OF NATURAL 'l;.NTISENSE TRANSCRIPT TO CHIP
FIELD OF THE INVENTION
[00011 The present application claims the priority of U.S. provisional patent application No. 6I f2235 85 filed August

2 1,:2009 Which. is incop)or tcd.1:erein by reference in its en.à reà ,.
100021 nthoditnents of the Invention comprise oti'oanueleoÃides modulaÃi. g expression and/or function of CHIP and assodtated molecules, BACKGROUND
[00031 DNA-RNA and RNA-RNA hybridization are important to many aspects of .nucleic acid ftnction including DNA replication, transcription, and translation. 1-lybrid.ization is also central to a variety of technologies that either detect a particular nucleic acid or alter its expression. Antisense nucleotides, for example, disrupt gene e:spaession by hybridizing to target RN A, thereby nterkring with RNA splicing , transcription, translation, and rep] ication, Antisense DNA has, the added feature that DNA-RNA hybrids serve as a substrate .foa digestion by riboll uclease .H. an activity that is present in most cell types. Ant sense molecules can be delivered into cells, as is the case for olitigodeo yn.acleotdes (O. N's), or they can be expressed fom endogenous genes as RNA molecules. The FDA
recently approved an antisease drug, VITRAVENE"' (far treatrnent of cvtomega.lovirus reti:titis), reflecting that antisense has therapeutic ufflity.
SUMMARY
100041 This Summary= is provided to present a swinutr) of the invention to briefly indicate the nature and substance of the inivention. It is submitted e pith the uridersÃand1mr that it will not be used to interpret or limit the scope or meaning of the claims-100051 In one embodiment, the invention provides methods for inhibiting the action of a natural antisensc transcript by using aartisense oligonucleotide(s) t irgeted to any region of the natural antisense transcript resulting an up-regulation of the corresponding sense gene. .It is also contemplate. herein that Inhibition of the natural antisense transcript can be achieved by siRNA, ribo v nines and small molecules, which are considered to be within the scope of the present invention.
100061 One embodiment: provides a method of modulating: linetion and'or expression of a CHIP polynnucleoÃide in patient cells or tissues in vivo or in vitro comprising contacting s<a: d cells or tissues with. an arttisense. hl ro:attac.leot ale.
to 30 nucleotides i.n length wherein said o.ligonueleotide has at least 50 %, sequence identity to a reverse complement of a polyiiucleotide comprising to 30 consecutive nucleotides within nucleotides I to 2074 of SEQ ID NO: 2 and I to 1237 of SEQ ID NO: _a thceby modulating finction and-or expression of the 0-11-P polynuclcotide in patient cells or tissues in vivo or in 34i1'a, I

[00071 In another preferred embodiment, an oligonuc eotide targets a natural antisense sequence of CHIP
polvnucleotides, fà r exampl _. nucleotides set forth n SEC1 ID NOs: 2 and 3, and any variants, alleles, ho ologs, mutants, derivatives, fragments and. complementary sequences thereto- Examples of autisense oligonu.clcottdcs are set forth as SEQ ID aNO`'S: 4 to .11, I.0008l Another embodiment provides a method of modulating function and/or-expression of a C}-11P polyrtucleotide in patient cells or tissues in Vivo or in vitro comprising contacting said cells or tissues with an antisense olil onucleotide to 30 nucleotides in length w.hercir=:a said. ol:igonuc.leotide has at least 50% sequence identity to a reverse complement of the an antisense of the CHIP poly-nucleotide~ thereby modulating function and/or expression of the CHIP
poly<nueleotide in patient cells or tissues in vivo or in vitro.
[00091 Another embodiment provides a method of modulating function, and/or expression of a CHIP polyrrucleotide in patient cells or tissues in vivo or in vitro comprising contacting said cells or tissues with an antisense oligorucleotide to 3f1 rntreleotides in. length ,--herein said. oltgonucleotide has at least 50$';, sequence identity, to an as ttisense oligoatncleotide to a. CHIP antisense polynucleotide; thereby modulating function and or expression of the CHIP
polynucleotide in patien cells or tissues in vivo or in vitro.
[00101 In a preferred embodiment, a composition comprises one or more arnisense oli xorauclootides which bind to sense and/or- antisensc CHIP porhnucleotides.
[00111 In another preferred embodiment, the oligonucleotides comprise one or more modihe i or substituted nucleotides.
10012 Ira arrrother l refer ed e.rnltodirtrent, tlac oli or:aareleotides coamapris e~tte or raaore. artrr.,lif ed boucle.
10031 In yet another embodiment, the modified nucleotides comprise modified bases comprising phosphorothioate, methylphosplaonate. peptide nucleic acids, 2'-O-r ethyl, fluorox or carbon, t tethylene or other locked nucleic acid (LNA) ntolectrles, Prefer-ably, the modified nucleotides are locked nucleic acid molecules, including [0014) In another preferred ernbodimea t, the oligonucleotides are administered) to a patient subeutarteously, irrtrartztasctrl1r14 Intravenously or Intrapentoneally, [001.51 In another preferred embodiment, the oligonucleotides are administered in a phat aaaceutical composition. A.
treatment rÃ.g.imen comprises administering the antisense compounds at least once to patient; hop ever., this treat raermt can be modified to include multiple doses over a period of time, Th .
treatment can be combined with one or more other types of ther'apic;s.
['001.61 In another preferred ertatrodimer t, the oligonucleoti:des are encapsulated in a. iiposo:n:re or attached to a carne molecule (e.g. cholesterol, TAT peptide).
100171 Other aspects are described infra.
BRIEF DESCRIPTION OF THE DRAWINGS

[00181 Figure f is a graph of real tin:me PCR results showing the fold chzwge.
standard deviation M CHIP n RN.A.
after treatnacnt of ZR75-1 cells with phosphorothioate ofigonucleotdes introduced using Lipp-atcctamine 2t.s[3Ã), as compared to control. Real time P C..R results show that the levels of CHWI m RNA in ZR75-1 cells am significantly increased 48 h after treatment with two of the siRN.As designed to l Xt?lll N9.i (CÃ. R-0314 and CUR-Ã1:316). Three phosphorothioarte .aaatisense ohios designed to Us.::3V l (C:L1R-Ã} r+z - CUT-08833) also significantly lip regulated C::11IF1 mRNA le els. Bars denoted aas C X_iR-t1 +1 , C R.-0316, CUR -0879, C.1.i:R-0 i Ã3 and C'1 R-#11 3 correspond to samples treated with SEQ lU NOS: 4 to and 9 respectively.
10091 Figure 2 . is a graph. of real time PCR results showing the fold change standard deviation in ClIfl? mRNA
after treatment of ZR75-1 cells with phosphoroth oate oligonucleotides introduced using Lipofectami:ne 2000, as compared to control. Real time PCR results show that the levels of C'i-HPI
na:RN'1 in Z:R75-1 cells are significantly increased 481i after Ãreatmenat with one of the phosphorothioate antisense olivos designed to Hs.5337 i 15 (CUR-0$80).
Bars denoted as CUR-0' 14, CUR-03 16. CUR-0879. CUR-0880, CUR-0883, CUR-08$2 and CUR-0884 correspond to sample, treated with S.EQ ID .NOS: 6 to l 1 respectively.
Sequence Listing Descript on:
100201 SEQ ID NO: 1: Homo sapiens STIPI homology and U-box containing protein I (STUB!), mRNA. (NCBI
Accession No.: NM 00586.1) SEQ ID (0 2: Natural CHIP antisense sequence (Hs.53377i ); SEQ ID NO,, -3: Natural CHIP aantisense sequence (BXÃIS8969); SEQ ID NO : 4 to 11: Antisense oligo.racucleotides '- indicates phos hoÃh oate bond and 'r ' indicates RNA.
DETAILED DESCRIP'T'ION
(00211 Several aspect`s of the invention are described below with reference to example applicatÃons for allustation, It should be understood that numerous specific details, relationships. and n--methods are set forth to provide a full, understanding of the in . ention. One having ordinar-y> skill in the relevant art. however, will readily recognize that the invention can be practiced Without one or more of the specific details or with other methods. The present invention is not limited. by the ordering of acts or events. as so:me acts may occur in di fer .nà orders and or concurrently With other aets or events. f'urthemaor s, not all illustrated acts or events aa:re required to implement a methodology in accordance with the present invention.
100221 All genes, gene. names, and. genc products disclosed herein are intended to correspond to ha n'aologs from any species for which the compositions and methods disclosed herein are applicable. Thus, the terms include, but am. not limited to genes and gene products .fom humans and mice. It is understood that when a gene or gene Product from a particular species is disclosed, this disclosure is intended to be exemplary only, and is not to be interpreted as a limitation unless the context in which it appears clearly indicates. Thus, for example, for the genes disclosed herein, which in some embodiments relate to mammalian nucleic acid and amino acid sequences arc intended to encompass homologous and:/or orthologous genes and 4gene products from other animals including, but not limited to other

3 mammals, fish, amphibians, reptiles, and birds. In preferred cnilaardimen s, the genes or nucleic acid sequences are humaaaan.
i)c in{r{orr 100231 l'he terminology used .herein is for the purpose of describing particular embodiments only and is not i:tntcndcd to he limiting of the invention. A.s used herein, the singular franns rats tw ee and "tlrea, are intended to include the plural forms as well, unless the context clearly indicates othemise. I= rthermore, to the extent that the tens s "i ncluding a rcludes", "havintra "'lraas", "Z4`rtla". or variants thereof aar'e used ara either the detailed description aarad aa' tlr( e laatarrs, such terms are intended to be inclusive in a manner similar to the term "coaarpa isin~~.
100241 The term "about" or "approximately" means Within an acceptable error range for the particular value as, determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system, For example, "about" can mean within I or more than 1 standard deviation, per the practice in, the an. Alternatively, "about" can mean as range of up to 20'.''o, prof raably tap to Y)%, more preferably tap to 5%-,Q. and more preferably still. tip to i % of a given value. Alternatively, particularly With respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a Value, Where Particular values are described in the application and claims, unless otherwise stated the term "about" aaaeaanang within an acceptable error r aange for the particular value should be assumed., [00251 As used herein7 the tour "mRaNA` means the presently l no'-n rRNA
transcript(s) of a Targeted gene. and any farther- transcripts ,,hich may be elucidated.
100261 By "aantisense. oligoraaacleotide..s" or "aantiserase compound" is meant an RN--A or DNA molecule that binds to another RNA or DNA (ta'arg~et RNA, DNA). For example, if it is an RNA
ohgonacleotide it binds to another RNA target by means of NA-RNA interactions and alters the activity of the target RNA. An aantasense of-Wo:nucleotide can ripregulate or downregulatc expression and/or function of a particular polyraueleotide_ The definition is meant to include any foreign RNA or DNA molecule which is useful t:roirt a tlieraapeutic, diagnostic, or other. -viewpoint. Such molecules include, for example. antisense RNA. or L)N A molecules, interference RNA
(RNAi), micro RNA, decoy RN:
molecules, si N'A, en matie RNA, therapeutic. editing R\ and aagoni.st and aantaagonist RNA, anti ease of romcri:e Compounds, '1116 'sense oligonueleotides, external guide sequence (EGS) of:igonuclcotides, alternate splicem primers, probes, and other oligome:ric compounds that hybridize to at least a portion of the targget nucleic aacid..rA.s such, these:
compounds may be. introduced in the firm of single-stranded, double-stranded, partially single-stranded, or circular oligorareric corarpounds.
100271 In the context of this invention, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid #RNA) or deoxyribonucleic acid (DNA) or mimetics hereof The term.
"oligonucleotidc", also includes linear or circular oligonmers of natural and/`or modified monomers or link ages, including deoxyribonueleosides, r'ibonueleosides, substituted and a alpha-aanomeric forms thereof, peptide nucleic acids ÃPNA), locked nucleic acids (LNA),

4 phosphorothioaatc, nlethylpho phonate; and the like. Oligon:ncleotides are capable of specifically binding to a target l?olyneacleotide by way of a regular pattern of onom -t -aa7a~a ran e~
interactions, such as Watson-Crick type of base pairing, Hotygsteen or reverse Ho6gsteen types ofbase pairing, or the like.
[00281 i'he olirrontrckotide may be. "chimeric". that is, composcd of different regions. in the context of this invention "chimeric" compounds are oligonticleotides, which contain isvo or more chemical regions, for example, DNIA
region s)t RNA region(s), PNA region (s) etc. Each chemical region is made Lip of at least one monomer unit, r c., a nucleotide in the case of an oligonucleotides compound. These oligonucleotide.s typically comprise at least: one region wherein the olicgonucleotide is modified in order to exhibit one or more clesi:a .d properties. T e desired properties of the oli{yonucleotide include, but are not limited, for example, to increased resistance to :nuclease degradation. increased cellular uptake, and/or increased binding affinity for the target nucleic acid. Different regions of the oli onucleot de may therefore have different properties. The chimeric ohgonucleotides of the present invention can be formed as mixed structures of two or more oligonueleotides, modified. oligonucleeottides, oligoa:aaacleosides and,-or oligonuc:i tide. analogs as described above.
1.00291 The ohgonucleotide can be composed of regions that can be linked in "register", that is, when the monomers are lurked consecutively, as in native DNA, or linked via spacers. The spacers are intended to constitute a covalent 'bri.dge" between the regions and have in preferred Cases a length not exceeding about 100 carbon atoms. Fhe spacers may carry. dift nr=ent functionalit:ies, for example, having positive or neg ati}e charge, carry special nucleic acid binding properties (intercalators, { aoove binders. toxins, f'luorophors etc.), being lipaphilic, inducing special, secondary structures like, for exaample, almine containing peptides that induce alpha-helices.
100301 As used herein ''CHIP" aand `C terr:ninaas of H"SP7O-interacting Protein- are. inclusive of all family=- me tubers, mutants, allelesõ fragments, species, coding and. noncoding sequences, sense and. antisense polynucleoti:de strands, etc.
[00311 As used herein, the words T terminus of l-fSP70-lrateracti:nf~
Protein", Antigen NY-CO-7, Carboxy terminus of Hsp10-int m cuing protein, CHIP, C"LL-associated antigen KW'-5, E3 ubicluitin-protein ligase CHIP, HSP I.BP7, N'V-CO-7, 11'1131.. SDC'CAGc7, STIP 1 homology and C lax-ca_saztainin~ protein 1, Ut.BOXI, are considered snare in the literature and are used interchangeably in the present application.
[00321 As used herein, the terra. "olig nucleotide specific for" or "oligonucleoti:d which targets" refers to an oligonucleotide having a sequence (1) capable of fbrr rinõ a stable complex with a portion of the. targeted gene, or (ii) capable of forming a stable duplex with a portion of a mRNA transcript of the targeted gene. Stability of the complexes and duplexes can be determined by theoretical calculations and.1or in vitro assays. Exemplary assays for determining stability Of hyb.n J7 atio.n complexes and duplexes, are described in the Examples below-.n As used herein, the term "target nucleic acid" encompasses DNA, RNA
(comprising p` nrRNA and ml NA) transcribed from such DNA, and also cD)\ 1 derived from such R.NA, coding, noncoding sequences, sense or aantisen`e polynucieotides. I'lre specific h y bridiz ation. of an oligomeric compound with its target nucleic acid interferes with the S

normal f=unction of the nucleic acid. This modulation of function. of a tar t nucleic acid by ccatnpounds, which specifically hybridize to it, is generally referred to as "antisensc". The f nctiÃtns of DNA Ão be interfered include, for example, replication and transcription. `l"he fit ctions of R\A to be interfered,. include all vital functions such as, for exaumple, translocatioa of the RNA to the site ofpr atein traarislation, translation of protein from the RNA, splicing of the R\. A. to yield one or more, mR\ A ,peen s, and catnlsrtic activity- which niay, b engaged in of Facilitated b the RNA.
The overall effect of such interference with target nucleic acid function is modulation of the expression of an encoded product or oligfonucleotides, 341 RNA interference "RNAi" is mediated by double stranded RNA (dsRNA) i ole.cules that. have sequence-specific homology to their "target" nucleic, acid sequences. In certain embodiments of the present invention., the mediators are 5-25 nucleotide "small interfering" RNA duplexes tsIRNAs. The siRNAs are derived from the processing of dsRN-A by an RNase enzyme known as Dicer. siR\A duplex products are recruited into a multi protein siR\'A complex tcrmcd..RISC (RNA Induced. Silencing Complex), Without wishing to be bound by any particular theory-, a RISC is then believed to be guided to a target nucleic acid (su.ittbly mRN:,), where the siR\A duplex interacts in a sequence-specific wi ayr to mediate cleavage in a catalytic fashion. Small interfering RNA s that can be used in accordance with the present invention can be sy"ntlhesized and used according to procedures that are well known in the art and that will be familiar to the ordinarily skilled artisan. Small interfering R\ As for use i,n the methods of the present invention suitably comprise between about l to about 50 nucleotides (tit). In examples of non limiting en:ibodim rents, siRNAs can comprise about 5 to about 40 at, about 5 to about 30 nt, about 10 to about :)t? ut, about t5 to about 25 at. or about 2(-25 nucleotides.
(00351 Selection of appropriate oligonucleotides is ffacilitated by using computer programs that automatically align nucleic acid sequences and indicate regions of identity or homology. Such programs tyre used to compare nucleic acid sequences obtained, for e.xanpple., by searching databases such as ClenBanlc or by sequencing PCR products.
Comparison of nucleic acid sequences from a range of species allows the selection of nucleic acid sequences that display an appropriate degrx of identity between species. In the ease of genes that have not bear sequenced, Southern blots are performed. to allow a determination of the degree of identity bets ween genes in target species and other species.
By performing Southe:trt blots at v try=in? degrees of stringency, as is we:l.l know~.in in the art, it is possible to obtain an approximate measure of identity. These procedures allow the selection of oligonucleotides that exhibit a high degree of complenicntarity to target nucleic acid sequences in a subject to be controlled and a lower degree of compleme.ntarity to corresponding nucleic acid s q,: aces in other species. One skilled in the art will realize that there is considerable latitude in selecting appt opri etc regions of g lie's for use in the Present invention.
100361 By "enzymatic RNA" is meant an RNA molecule with enzymatic activity (Czech, (1988) "1.. American, .1 4s#.
Assoc-260, 3030-31335 ). Enz yruatic nucleic acids (ribozyrnes) act by first bir:idin x to a target RNA. Such binding occur through the target bindin4g portion of an enzymatic nucleic acid which is held in close proximity to an enzymatic.

portion of the i- olecule that acts to cleave the target RNA. Thus, the enzyrmatic nucleic acid first recopuzes and then binds a target RNA through. base pairing, and once bound to the correct site, acts euzynuttically to cut the target RNA.
100371 By. "decoy RNA" is meant an RNA molecule that mimes the natural binding domain for a. ligancl. The decoy RNA therefore competes with natural binding target for the binding of a specific 1.igaa d- For example, it has been sfho n that o e~ expression of FHV trans activation response (TAR) RNA can act as a "decoy" and efficiently binds T-t:1V tat protein, thereby preventing it from binding to TAR sequences encoded in the H IN' RNA. This is meant to be a specific example, Those in the art will recognize that this is but one example, and other emboditlicuts can be readily generated usineg techniques generall known in the art.
100381 As used herein, the tern "monomers" typically indicates mono-mien, linked by phospbodiester bonds or analogs thereof to form oligonucleotides ranging in size from a :few .monomeric units, e.g., from about 3-4, to about several hundreds of monomeric units. Analogs of phosph?diestcr linkages include:
phosphorothioate, phosphorodithioaate, methylphosphornates, phosphoroselenoate, phosphoramidate, and. the like, as more fully described below.
[00391 The term "nucleotide" covers naturally occurring nu leotides as Well as rYr~.rana#a:tr~illy occurring nucleotides. It shouuld be clear to the person skilled in the art: that various nucleotides which previously have been considered "non-naturally occur ing" have subsequently been found in nature. Thus, "nucleotides" includes not only the known purine and pyrimidine heterocycles-containing molecules, but als_s heterocycl e analogues and tautomers thereof illwttrative examples of other types of nucleotide s are molecules containing ardenine7 guanine, thy mine, cy tosirae uracil, pa:rritte xanthine, diaminopur-ine, 8-oxo- t`+ 6--methyladeninc ;. 7 dearaaxanthinc, 7-de rza<guaninie, N4.,N4-etthano vtos.in. N6,N6-cthano-2,6- diaanainoprtrir:ae, 5-r:nethylcyrtosine, 5-(C3-C6)-atlkynvlev't()sirre,, 5-fluorouracil, 5-bromouracil, psetrdoi oe:yts sira<., ?-1t c~toxy'_ -r aetl yl- -iii<tzolop 'Ã-clisa, isocytosine, isoguarrin, inosine and the "noà -naturall .y occurring" nucleotides described in Benner et al., U.S. Pat No. 5,412272-1.
The ter-um "nucleotide" is intended to cover every and all of these examples as well as analogues and tautome.rs thereof Eslecially> interesting nucleotides are these containing adenine, gguanine, thy<n-ainc, cytosine, and uracil, which are considered as the: naturally occurring nucleotides in relation to therapeutic and diagnostic application in hun-aans, Nucleotides include the natural 2'-clcoxy and. 2'-hydroxyl sugars..:.4g., as described in Koornber=g and .Baker, DNA
Replication, 2nd Ed. (Freeman, San Francisc(:i. 1992) as well as their analosg~s.
100401 "Analogs" in reference to nucleotides includes synthetic nucleotides having modified base moieties and/or modified sugar moieties (see e.g., described generally by Scheit, Nucleotide Analogs, John Wiley, Now York, 19,W-, Freier & Altmann, (1997) Acid Resc ., -25(22).4429.444 3,'Toulmc, i-:1 , (2001) attire iotechnolm-, 19:17-14;
Ianotaaraar 1sT.. (199 9) iocJr +3 a1 "er et {afai hrri a Ada .1493:117-1. 9;
Freier S. NI., (1997) Nucleic 25: 29r444 , t_?hlmaan, F., (20t)0) .Doug 1 iscmvr e~ I r'vc/> r errt, 1203-213, llerde~~ira P., (2000) Amliseusc &
Nucleic Add Drus> Dc'av., 10:297-310)1 2'-O, 3'-C-linked 132,01 bieyeloarraabinonuelcosrcles. Such analogs include sy rthetie :nucleotides designed. to enhance binding properties. eg., duplex or triplex stability , spec. f city, or the tike.

[00411 As used herein; "hybridization" naeaans the pairing of substantially conaplenaentary strands of oiigonieric con-tpoaands. One mechanism of pairing ins olvcs hydrogen bonding, which may be Watson-Crick, HoogsÃcen or re. eas l Hasoosteeaa hydrogen lxiraelaaa , between. eoanplean ntaay a7aacleosicle or nucleotide bases (nucleotides) of the strands of oli oneric compounds. :For example, adenine and thymi:inc are complementary nueleoti:des which pair through the formation ofhytdrogen bonds. Hybridization can occur under vary-irm circumstances.
421 An antisense compound is "specifically hy(ridizable" when binding of the compound to the tar et nucleic acid interferers r ith the normal function of the target nucleic acid to cause a modulation of function and/or acti4 ity, and there is a sut c:icaat degree of complenaentarity to avoid non-speecifIc binding of the antisense compound to non-target nucleic.
aid sequences under conditions .in which specific binding is desired, i.e., under physiological conditions in the. case of in vivo assays or therapeutic treaatriaent, and under conditions in which assays are performed in the case of in vitro assay s.
[00431 As used herein, the phrase " stringent hybridization conditions" or "stringent conditions" refers to conditions under which a compound of the invention will hybridize to its target sequence, but to a :minimal number of other sequences. Stringent conditions are scclaacrace-t e pt.raclt.rat and will be different in different circumstances and in the context of this iris ention, ' stti.aa ;cant conditions" under ~.laie it oligonicnc compounds hybridize to a target sequence are determined by, the nature and composition of the oligoar Brie compounds and the assays in a. hich they ate. being;
invesÃigated. In genes-;al, stringent hytbridizaition conditions comprise low concentrations (<0.15M) of salts with inorganic cations such as Na-++ or K i.c., low ionic strength), tea: per ature higher than 20'C - 25" C. below the Tux of the oligomerie eo:-npound:targct sequence complex, and the presence of denaturants such as format-side,.
dimethylfonnarnide, dimct vl sulfoxide, or the detergent sodium etodecyl sulfate (SDS). For example, the hybridization rate decreases .l.1% for each I.% forrnamide..An example of a high stringency hybridization condition is 0.I.X sodium chloride-sodium citrate buffer 4 SSQ/0.1`} t= SDS at 60' C. for 30 mim sites.
100441 "Coniplementaary," as used herein, refers to the capacity fbr precise pairing bets coil. two nuclei tides on one or two oligo-aierie strands. For example, if a nucleobase at a certain position of all antiscnse compound is capable of hydrogen bonding a vifh a nucleob.a_se at as certnin position of as target nue.lei.c acid, said Ãarget nucleic acid being as DNA, RNA, or oligonucleotide molecule, then the position of hydrogen bonding between the oligoa:aucleoÃ:id - and the target nucleic acid is considered to be as compleanentary> position. The oli{gome:ric compound and the further DINA, RNA, or oligonuclcotide molecule are complementary to c ach other when a sufficient number of complementary positions in each molecule are. occupied by nucleotides which can hydrogen bond with each other. `thus, "specifically h;ybridizable"
and t"ceanaplenaeaataay" are terms which are used to indicate a sufficient degree of precise pairing or conmaplenmtentarity over a sufcient number of nucleotides such that stable and specific binding occurs between the oligonmae:ric con pound and a target nucleic acid.

F 4 1 It is understood in the art that the sequence of an oligoaneric compound need not be 100'%, complementary to that of its target nucleic acid to be specifically hybridizaable:, kloreo er, an oligonuc1eotid<. may, hybridize over one or more segments such that interR,craing or adjacent segments are not involved in the hybridization ea-ent ie. ., a loop structure, mismatch or hairpin structure). '.bile oligom.cric compounds of the present i:nvention comprise at least about 70%, or at l :ast ahc ut 75%, or at least about 80%, or at least about 85%. or at least about 9Ã0t-0it, or at least about 195 %, or at least about 99'= sequence compkmcntarity- to a target region. within the target nucleic acid sequence to which they are targeted, For example, an aaatisense compound in w. vlaich 18 of 20 nucleotides of the antisense compound are complementary to a target te(gioon, and % ould therefore specifically hybridize, woaald represent 90 percent complemenmrity. In this example, the remaining noncomple:mentary- nucleotides may be clustered or interspersed with coat .pletnentaary nucleotides and need not be contiguous to each other or to complementary> nucleotides, As such, an antisense compound. Which is 18 nucleotides in length having 4 (four) noncornplernentaty, nuclcotidcs ezhici} are flanked by two. regions of complete compltmentarity with the target nucleic acid would have i 7.8% overall complenmentarity with the tamer nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an an isense compound with a region of a target :nucleic acid can be determined routinely using BLAST programs (basic local. alignment sea ch tools) and PowerBLAST programs known in the art, Percent homology-,, sequence ide.attity, or contplenaentaaraty, can. be determined.
by., for exanmple, the (Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, l University% Research Park:, Madison Wis.), using, default settings, which uses the algorithm of Smith and Water-man (4t1a. ,4pp!. 11olk, (1981 -2,482-489).
100461 As used herein, the term "Thermal Melting Point (fm) refers to the temperature, under defined ionic strength, pl 1. and. nucleic acid. concentration, at which 50% of the oligonucle tides complementary- to the target sequence hybridize to the target sequence at equilibrium. '-1'vpically, , stringent conditions will. he those in Which the salt concentration is at least about 0.0 1 to 1.Ã1 M Na, ion concentration (or other salts) at p1-1 7.0 to 83 and the temperature is at least about 30"C for short oligonucleotides (e: g., 10 to 50 nucleotide).
Stringent conditions may also be achieved with the addition of destabilizing agents such as form a nidc.
[00471 As used .herein, "modulation" means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene.
100481 The term "vaariatat," when used in the context of a polynucleottde sequence, may encompass a polynticlcotade sequence related to a wild type gene. `1'h:i definition may, also include, for example, "allelic.", "Splice," "species.'" or polymorphic" variants. A splice variant may have significant identity to a reference :molecule, but will. generally have a greater or lesser number of po.lvilucleotiles due to alternate splicing of ,axons during niRNA. processing. The corresponding polypeptide may possess additional functional domains or an absence of don ains. Species v a:riaants are poly-nucleotide sequences that vary from one species to anoth r. Of particular utility in the invention are Variants of wild type gene products. Variants may result from at least one mutation in the nucleie acid sequence and may- result in altered as.RNAs or in polyp ptides whose structure or function may or may not be altered, Any given riatunil or recoanbinant gene may have none, one, or mans, allelic forms. Conraaaon mutational chan'g.es that give rise to variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes Imlay occur alone,. or ira coa~abina:tion ~~i.tl:a the others, one or more.
times in a g:i ven sequence.
100491 The resulting po ypeptides generally will have significant: anxino acid identity relative to each other. A
polymorphic variant is a variation in the polynucleotide sequence of a particular gene beÃween. individuals: of a given species. Poly>rnorphic variants also may encompass "single, nucleotide palymorphisms" (SNPs) or single base nout,itions in which the polynucleotide sequence varies by one base. The presence of SNPs may be indicative of., for example, a certain population With a propensity for a disease state, that is susceptibility versus resistance-100501 Derivative polynucleotides include nucleic acids subjected to chemical modal cation.. for exan:mple, replacement of hydrogen by an alkyl, acyl, or amino group, De:rivaatives, e.g,, derivative oligoraucleotides, may comprise non-naturally-occurring portions, such as altered sugar moieties or inter-su rar linkages. ExcmplaÃy among these are phosphoroth.ioate and other sul.fiar containing species which are know =n in the art. Derivative nucleic acids may also contain labels, including radionucleotides, eaazvrnes, fluorescent agents, L
.hemilun unesa ern agents, chrornogenie agents, scibsta ates, cofactors, inhibitors, ma gneti:c particles, and the like.
10051.1 A "derivative" polypeptide or peptide. is one that is modified, for e ample, by gl cosylation l egylation, phosphorylaationa. saulf"tioaa, reduction/"al.k rl.aatioa net l atiÃaaa, chemical coupling, or mild #ornialin Ãreaatme_n. t. A derivative may also be modified to contain a detectable label, either directly or indi.aectly, including, but not limited to, a radioisotope, fluorescent, and enzyme label, [00521 As used herein, the term "aa:nimal or "Patient is meant to include, for example, humans, sheep, elks, deer, anule deer, minks, nurnniaal , monaL ys, horse's cattle, pigs, goats, dogs, cats., rats, mice, birds, chicken, reptiles, fish, insects and arachnids.
101531 "Maaaanaal" covers warm blooded mammals that are typically under medical care (e fg., humans and domesticated animals). Examples include feline, canine, equinne, bovine, and human, as well a.s,just huunan.
100541 "Treating" or "treatment" covers the treatment of a disease-state in a mama?a al, and includes: (a) preventing the disease-state from occurring in a mammaal, in particular, lit a . saich maninial is predisposed to the diseaa<se-state but has not yet been diagnosed as having it: (b) inhibiting the dismse-state, e.g..
arresting it development and/or (c) relieving the disease -state, e.g.. causing regression of the disease state. until a desired endpoint is reached. Tr e.atinng also includes the amelioration of a symptom of a disease (0.-R-7 .lessen the pain or discomfort), wherein such aamelioratiOil may or may not be directly affecting the disease (e.g. cause, tra smission, expression, etc. i.
100551 As used herei a; cancer" refers to all types of cancer or neoplasm or malignant tumors found in maraana:als, including. but not limited to: leukemias, lymphomas, melanomas, carcinomas and sarcomas.. The cancer manifests itself at. a "tumor" or tissue compnisin maalignaant cells of the cancer.
Examples of tumor include sarcomas and carcinomas such as, but not liniited to: fil_??osarc.orna, c? ~<arc c? a ~, lipt?s.arcoi?ma., chondros< x? ? a: osÃeo en c sa:rc:oiiaa, clrordonaa ara"Yiosar orraa., endotheliosarcotr?aa, lympl?an Yiosarcorna, lym han Yio c- dotl?clios arco ? a, synoviorrmaa, mesogtheliornaa, E4vrngs tumor, lciorrryosarcorna, rhabdox?nyosarcorxaa, colloan carcirlotna, pancreatic Cancer, breast Cancer, Ovarian caa?.cer, Prostate cancer, sC uaa?:aous coil carcinoarra, basal cell carcinoma, ade'r? caa'crt?o?a?a, sweat `Aland carenaornaa, sebaceous hind carcinorraa, paipil.la.rt Carcinoma, api.llirr tacla nocarcinom;as, c y' idenoctrrc-i onia, medullary ca_rciraomaa, broarchogenic carcinoma, renal cell carcinorr:aa.:
lrc:patoma.: bile duct carcinoma, clrc?rac?e atcinoma, serrrirroraaa, embryonal carcinoma, Wilins' tumor, cervical cancer, testicular tumor, lung c:aarcinonraa, small cell lun=a caarcino:ar:aa, bladder carcinoma, epithelial carcinoma, ghoarra, astr-ocytonia, meduliohiastoma, cr'anioph'a i:a4gacnna=
ependyrrmo aaa, pinealorn a, he ?mangioblastoma, acoustic :neuror?a, ol:igodendrogliorr aa, me:ningio;ma, melanoma, nerraoblastonraa, and retiaoblastonia. Additional cancers which can be treated by the disclosed corn position according to the invention include but not limited, to, for example, Hodgkin's Disease, Non-Hodgkin's Ly-mphorna, multiple mycloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdor?ayos arcomaa, primary tluaonlbocytosis, prUnaary raracrogltil?ul nemia, small-cell lung tur:arors, pri:naar train turrmors, stomach car, cer, colon cancer, malign ant pancreatic insulanoma, malign ant carcinoid, uri.nary bladder cancer, prey aliõtaant skin lesions, testicular caaucer, lyraaplrorraaas, tl? roicl dancer, r caaroblaa tear a, esophageal cancer, genitourinary tract cancer, malignant hyperc-;alccraria, cervical cancer, endometrriaal cancer, adrenal cortical cancer, and prostate cancer.
õl ecrrc loalc<al fist asc c?r disc?rtler"' ac fers Eo < nw disease c?r disorder cif tl e pert ocap system and i?r i i u>al sr tcara.

"Neurolomeal disease or disorder" include disease or disorders that involve the central. nervous system (brain, bra:insten:r and cerebellnara), the peripheral nervous system (including cranial nerves), and the autonomic nervous system (parts of vvlrich are located i.n l c?ti-a central and peripheral nen'ous system). Examples of neurologic al disorders include but are not limited to, headache, stupor and c.o:maa, doinentiaa, seizure,, sleep disorders, trauma, infections=
neoplasrar , neuroopthaaln?.o.log `, r?:rovement disorders, dcnxyelinratang diseases: spiuat cord disonlers, arid disorders, of peripheral nerves, n :uscle and neuromuscular _junctions. Addiction and mental illness, include, but are not limited to, bipolar disorder and schizophrenia, a arc. also included in the definition of neurological disorder. The l"orilowing is a list of several neurological disorder's, symptoms, signs and sy daz-rrrtnes that can be treated using compositions and methods according to the present invention: acquired epilcptiforan aphasia; acute dissemi. ated. eneephaalorl-.t clÃtis;
adrenoleukod ,strolls; age-.re.larted a ?au ulaii degenerationageneso of (he corpus c.dllosura ; agnc?s_i<?., ..icaudi syn-dronr c Alexander disease; Alpers' disease; alternating henriplegiaa; Vascular dementia; arnyotrophic lateral sclerosis;
anencephal-y; Engelman syndrome; angiomaatosis; anoxia; aphasia; atpraxia;
aaraachnoid ctists; aaraaclrnoiclrtis; r` rrroa?l-Chiari maalfor ration; =a:rtenovenous nxaalfbra ra :i.on; Asl erger syndron?.e, ataxia telegiecrosia; attention deficit hyperactivitiy, disorder autisn?.; autonoinic dy:sftu.ictiorr; back pain Batten disease .iBel?cot's disease; Bell's palsy; benign essential l?lc plaaarcrslaaasra ; benign [c~c.aal; aura carol?lay; benign i.ratracr'<rrr.i.aal hypertension; l tasc>4aara. er"s disease;
blcphaarospasm; Bloch Sulzber4Yer syndrome, brachial plexus njur-y; brain abscess brain, injury; brain tumors (irae.lr.adin glioblastoma maaitiforme); spinal tumor; Brown--' equard syndaonae; Canavan di aase, carpal tunnel, syndrome;
causalgia: central pain syndrome; central poaltinc nnyelinolysis cephalic disorder; cerebral a teurysm; cerebral arteriosclerosis; cerebral atrophy; cerebral gigantism; cerebral palsy; Char e>t- 'larie-Iootlt disease. el}eattotherapy-induced neuropathy and nea.rropathic pain; Chan maiforatnaation; chorea;
cht3;?atic inllatanrxmatory demyclinaatiatg po.lyneaaropathy; chronic pain; chronic regional pain syndro.av; Coffin LoNNw svudrome, coma, including persistent vegetative state; congenital facial dip1egia corticobasal degeneration;
cranial arteratis; craniosya:nostosis, Creutzfeidt-Ja kob disease; cu m ul.;:ative trauma disorders, Ctishing's syndrome.-c:yiomegaiic inclusion body disease cytomegalo ims infection; dancing, eyes-dancing feet syndra_siuc; DaandyWalk :r syndrome; (yas son disease; )c 's,forsier s syndre me::T e jerine-Itha Tike palsy; dementia; dens atortlyositis; diabetic aaeuropathy; diffuse scleroslS, d ytsaa.atoa on na; dysg ;aphia, dyslexia; dystoa ias; early= inf urti.ic epileptic: u.icephalopathz; w:mpty sclia s' ndrorne;
encephalitis; e.nceplaailcacc les encephalottigertainal a rngioraaatosis;
epilepsy; Eaft`s palsy. essential treaxmor; l"abry's disease-, Fakirs syn.dromaae; fainting; familial spastic paralysis, febrile seizures; Fisher syndrome, Friedacich's ataxia;
fionto-tcmporai dementia and other "taaaopathies"; Ciaaucher's disease;
Gerstaaaaaan's syndrome; giant cell artet tis: giant cell inclusion disease; globoid cell leu odystrophv; Cinillain-Barre sw.idrome 1ITL.V-I-associated raayelopathy;
Ha11er' ordcn-Spatz disease; head i.n uty: head ache; l:tenaifacial spasm.;
hereditary> spastic paraplegia.; heredopathiaa atactic a polynaeuritiformis: herpes zoster oticus; herpes zoster; H rayama syndrome; .HlVassociated dementia and neuropathy (also neurological manifestations of AIDS); holoprosencephaly;
Huntingtcin s disease and other poly<glutamine repeat diseases, ltydra_neaaccpltaly: hydrocephalus;
hypcrcortisolisaaa by ao~s:ia.; imam une-mediated encephalon ayelitist inclusion body nryositis, incontinentia pigrta.enti;
infantile phytanic acid storage disease; infantile refsuaam disease; infantile spassmaas-, inilan aaaatory trtyopatlay;
inatracran.ial cyst; intracranial hypertension; Joubert syndrome; K. arns San e , -ndrunie: Kennccty> disease. Kin boa ne s' ndaoa iea Kl ppei F it synadroinc; rabhe disease,, Kugelhe..rg-\ Blander disease; kuru; Lafora disease; Lambert-1 aeon :na >astheatic syndrome; I...anciatat T icf axcr syndrome;
lateral medullary (NVailenberg) sy<nclac~-me.; learning disaabilitaes;
I:,ugh's disease-, Len-Gusta:ut syndrome; T csch-? yl-tan syndrome: leukodystrophy; Levy body dementia; Lissencephaly; locked-in syndrome; Lou Gelvicw`s disease (i.c., motor neuron disease or ainvotrophic lateral sclerosis); lun3:bar disc disease; Lyme di ease--neural gical seciuelae Machado-Joseph di eaa e; ta:macrencephaly'ineg alencephaly, lLlcllcca so:aa-Rosenthatl syndrome: M:eniercs disease;
n cningxns; l :tenkcs disease-, tnetuchron-aauc leukodysnrophs na.icrocephaly n-u rains- Miller Fisher sva drone; mini-strokes; rnitochondrial nayopa:thics; Mobius syndronme; ra:mono mmelic aarnyotrophy; motor neuron disease, Moyarnoya disease; a wcopolysaacel:hatidoses; milti-in_Cahrct den)enntia; naultiiocal motor nearr'opatla:y: mulÃiplc sclerosis and other demyelm hinny; disorders; multi le stun atrophy with postural hypaterlsion: p .niauscula dystrophy- nr tsthc:aaiaa em is;
niveliuociastic dil"firse sclerosis; nmyoclomaic criceplt.alopathy~ of itg:t nt;a taryocloraus, t3:m opatlhhy rrx<<otonia. congctnttii narc.olcpsy; taeti#rolil~rcaata~ttca ais; ncaaroleptic malignant syndlronae;
neurological iaaanifstations c f AIDS; neurological sec raclaac ofltaptas; neuron y'oWatia; neuronal ccroid lipofusei:nc?sis; new-anal migration disorders." ienmamata-Pick disease, 'Sulliramn=McL od sz.ndrornc; occipital neuralgia; occult spinal dysrcÃph-isrn sequence, Ohtah< i s ytxdroa ;
oli c?p ~~tt cerehellatr atrophy; opsoclon us my.x.' onus; optic ncuritis;
orthosiuaÃie hy=poÃcnsiott overuse sy=ndrotat ;
paresÃhesia. ' t.Etrc~ ege.ne 4ttiie disease or disorder (Parkinson's disease, HuntingÃon's disease, AIzhcirner's disease, antyotrophic lateral sclerosis (ALS), dementia, nmitipls sclerosis and other diseases and disorders associated with neuronal cell death); patranryotoni<t congen.itd; parttieoplasà c diseases;
parox snial attacks; Pan= Roniberi syndrome;
Pelizacus-:i er7bachcr disease; periodic paralyses; peripheral tetra?patl y;
painful neuropaathv and n :urt pathic p tin persistent ti egctative state; pervasive dcvclopmentnl disorders; phoÃic si eezee reflex; phy Lath c acid storage disease flick's disc ase: pinched ncr =c; Pituitary Ãtirnors; polvt3-aj=osit: s; pot nccphai : ; post-polio syridr'on-le, pos ltct'pcti:c neuralgia; postinpcctious encephalon clitis postural hy1,otensi.on; Pradcr-Willi s ndromo, pri.inary Lateral sclerosis:
pr-ion diseases; pro ressitc: hcrzril:acial attt?ph ; progte sip i multi focal lettl z?4trccl?l ztlc?l~aaÃhy>; pro, rt.ssive sc.lctc?3i.n poliody-straphy; progressive sttpratatucleatr palsy, pseudoturnor cerebri;
Ratnsay-Hunt syndrome (types I and fl)-, Rasrnussen's encephalitis; reflex sympathetic dystrophy syndr rne; Rofsum di ease; repetitive motion disorders;
repetitive stress injuries; restless leis syndrome; ra trees irus-associated niyelopathy Rett syndrome, Rey 's syndrome;
Saint Vitus dance; Sauc hoff disease; Schildcr's disease, schizenceplhaaly, , septo-optic dysplasia shaken baby. swidrome;
shingles; Slry 1?raager scr dram ; ` jo~~r r s syndrome; ales p a l nea; Sotos sa=nclrc?tttc; ala~tsÃicity; sl inabi-lida; spinal cord injury: spinal. cord tumors; spinal muscular atrophy: Stuff-Person. syndrome;
stroke- Sturge-Weber syndrome; subacute sclerosing pane rccphalitis; subcortical arteriosclerotic eneephalopathy, Sydenham chorea; syncope; sy~ringomyeiia;
tardive dyskincsi t lay-Sachs disease; temporal ar-teritis; tethered spinal cord syndrome; Thorsen disease; thoracic outlet syndrome; Tic Douloureux; Todd's paralysis. Touu'e to sy ndrmne transient isehe ie attacl ; transmissible slit?rr gi:form encephaalop ethics, transverse my~clitis traumatic brain itttttr tic rrac?E'; ÃÃ tgcrrtirral neural iaa; tropical SI? spastic paraparesis; tuberous sclerosis; vascular dementia (multi-infarct dementia);
vaasca:lms including temporal atteritis; Vol).
I lippe.l-l.:indau cliseawc; \ ' llenherg's syndrome, \ erdttig-Tloi ran disease; West mndronne'. whiplash', Williams syndrome; Wildon's disease, and Zell %veger syndrome.
100571 An "Inflammation" refers to sy=stemic in:fanimaatoayy conditions and.
conditions associated lrcall,- with mitgration and attraction of monocy"ies, leukocytes and/or ne ttroph:i.ls.
Examples of inflammation include, but are not limited to, Tnfl animation resulting f-on infection with pathogenic organisms (:including gram-positive bacteria, gra:n-t-ncgativ=e bacteria: viruses, tngi, and parasites such as protozoa and hcl:minths), transplant rejection (including rejection of solid organs such as kidrnc\', ii er, heart, lung or cornea, as well as rejection of bone marrow transplants including graft-versus-lhost disease (GVHD)), or fiom localized chronic or acute autoiaxamune or allergic reactions. Autoimmune diseases include acute g-lomerul.onephuitis: rheumatoid or reactive arthritis;
chronic. ~~le?r re rarlonelnhrrtis; inflammatory, bowel diseases such as Crohn:-r's dise,aase, ulcerative colitis and necrotiring cnterocolitis; hepatitis; sepsis; alcoholic liver disease; non-alcoholic steatosis; grauuulocyte transfusion associated syndromes; inflammatory derntttoses such as contact dermatitis, aatopic dermatitis, psoriasis; systemic lupus er'y,tlh:cmatosus (SLE), aautoimmtnune thyroiditis, n ult:iple sclerosis, and some forms of diabetes, or any other atrtoirnnrunc state where attack by tltw subject's o : as itnnmune system resa:tlts in pathologic tissue destruction. Allergic reactions include allergic asthma, chronic bronchitis, acute and delayed hypersensitivity. Systemic irrfammaÃory disease states include intlarntiration associated with traxttrra, burn}s, r ;perfusion fdlloicving rscherrric et ents # e.g. thrombotic events in heart, brain, intestines or peril5heral t asculaWre, including r avocardial infarction and stroke), sepsis, ARDS or multiple organ dysfunction , ndrotne.. Inflanxnlatotyy cell.
recruitment also occurs in atherosclerotic plaques. inflammation includes, but is not limited to, Noa=I={odgkin"s l ymplrornaa, Nra'cgcncr's granulonratosis, Ilashirraoto s thymidids, hcpatoce lulatr carcinoma, Ãhyrrius atrophy, clrionic pancr'catitis., rheumatoid aanfiriti,. tvactivc l 'mphcud hypcrplasia, c stcoartlt itis, at.le.~rati~:c colitis. Ã lla cat'cirs~_o-t sa=
Crohn's dismse, ulce:raatir e colitis, acute choleevstitis, chronic choleeystitis, cirrhosis, chromic sialaadenitis, peritonitis, acute pancr eatitis, chronic pancreatitis, chronic Ãiastritis, adenornyosis, endonaetriosis.. acute cervicitis, chronic clay icitis, lymphoid lryperplasiar, multiple. sele.rosis, a pcrtrophy secondary to idiopathic tlr; c oil sc?co tc+pi.tiic ptat tcta, prinrary IgA nephropathx, systemic ful us cr ytherraatosus, psuuriasis, pulmonary emphysema, chronic pyelonephritis, and chronic cy.Sm s.
100581 .A cardiovascular disease or disorder includes those disorders that can either cause ischernia or are caused by reperfusion of the heart. Examples include, but are not limited to, atherosclerosis, coronary artery disease, 4graanulonaatous rn oeat~litis, chronic n voc.arditis (raora-?Ãanrtloaaaatorrsl printar hypet'tr:ophic cardtornvopathv, peripheral artery disease. (PAD), Ivripher.l atscular disease, venous thromboemholiam, pulmonary embolism stroke;
angina pectoris; m'yo'cardial infarction, cardiovascular tissue dim ge caused h cardiac. arrest, cardiovascular tissue damage caused by cardiac bypass, cardiogenic shock, and related conditions that would be known by those of ordinary skill i.ta the art or which involve dysfunction of or tissue damage to the heart or vaseulaturc especially, but not limited to, tissue claraaauc related to CHIP activation. CVS diseases include, but are. not limited to. atherosclerosis, granulomaatous navocarclitis..mycocartiiaal infarction, myocardial. fibrosis secondary to valvular heart disease, niyoeardial fibrosis without itn.harctiotn., primary hypertrophie cardiomvol athv, and chronic r:nyoearditis, (non-granulomatous).
[Oft ,591 Examples of diseases or disorder, associated with oxidative stress include but not limited to a atherosclerosis, Parkinson's disease, heart failure, iu ocardi l 1ntiarctiota .l lucita3et'y d s;.ats4., chromic fatigue sviidri me Ati.i onophic Lateral Sclerosis, (ALS), chronic obstructive pulmonary disease (C'OPD), mul'tiplee sclerosis, hepatic diseases or disorders, gastrointestinal diseases or disorders, diabetes, cancer, autoimmunity, immune related diseases or disorders, neurological diseases or disorders, raccarode enerartive diseases or disorders, nerve repair and paralysis, rneurocndocrirne dif :i;.rentiation, inflammatory d: seases, am uscular diseases or disorders, diseases or disorders associated with infectious organ:isms, and the like.
f'o/z m0kolide one/ Oiigomwle lode' ComImsi ion>: anti/I..fokvuuks.

[00601 t1 gets: In one e nbodirnent, the taargct;s comprise nucleic acid.
sequences of `C to minus of I-ISP7O-hitc aactin<
Protein' (CHIP), including without limitation sense andior antiacansc noancodinL, and/or coding sequences, associated with CHIP, E'00611 Carboxy terminus of Hsp 7 ()-.interacting protein WHIP) promotes the pmteaso:mal degradation of other proteins implicated in neurodegenerative diseases such as tau. AA u,,~nuclein oliõoruers and proteins with expanded polyp=Iaatanrine repeats. CHIP regulates leuci.nerich repeat kinaase 2 gene (LRRK2) ubiquitination, degradation and toxicity, CHIP hinds to both. wild-type and mutant ILRRl 2 and promotes the zrhiquiriination and proteasomal degradation of LRRK2. There are two indeperndent means of CHIP binding to LR.RK'?; an rndirect interaction between the \-terminal tetratr:icopeptide, repeat MR) of CHIP and the armadillo (A.RNI) domain of LRRK2, likely via Hsp9O, and an interaction between the char;. ed domain of CHIP and the Ras of complex f.R(X') domain of LRRK2, which is either direct or indirect via a common adaptor protein, [00621 `I:lne carboxyl. ter minnus of heat-shock cognate (H.sc)70-interacting protein (CHIP) is a. ubiquitin E3 ligase that can coll<aboa ate With molecular chaperones to facilitate protein folding and.
pre ern. protein aggregation. Prey ions studies showed that, together s >ith heat-shock protein (ffsp)70, CITIP can regulate tau uhiquitination and degradation in a cell culture sy>;stem. I_Jbiquiti.n ated tau is one component in a neur'ol"ibrillary tangles (NFTs), which arc a major histopatholowcal. feature of Alnccimner`s disease (AD). Increases in CHIP may protect against NF.1' formation in the earl y stages of AD.
100631 In preferred embodiments, anti sense oli can art leotides are used to prevent or treat diseases or disorders associated with CHIP family ramembeÃs. Exemplary' C. terminus of I-HSP 7O-Interacting ('rotein' (C:f lll'1 mediated diseases and disorders which. can be treated with cell tissues regenerated from stein cells obtained using the aantiserase compounds comprise: a disease or disorder associated with aahnomiaal. function and/Or expression of CIll_P, a neurological disease or disorder- a naeuroinflaanrrinator'' disease or disorder, an aatoirnnmuie disease or disorder, obesity, diabetes, atherosclerosis; Cancer, AIDS, atherosclerotic plaque, gliolalastonna, a disease or disorder associated with arar 'lord deposition, clnoriocageinoanna, astroe. tornna9 aann Ioidos s, hy'periipidenria. nca.arcncle.pencr'atisfra, aaer>plasti:e transformiation, AI.DS..metastasis, myocardial infarction, pulmonary fibrosis, iantl<aninaation, gglion a, a vascular disea e or disorder, cell damage:, Nonsnn: ali cell lung carcinoma ( SCCs , by crcholesteroleaamaiaa, liposarcoma, a cardiovascular disease or disorder, irnmunodeficiency, glonerulonephritis, venous thrombosis, a pathological process, cellular stress, as diseaase or disorder associated with oxidative stress, a polygiutamine disease, a disease or disorder associated with impaired functioning of agsgresonme pathway, a disease, disorder or condition associated with aging, a disease or disorder associated. with aggregation of n isfaldr d of proteins, a disease or disorder associated with misfolding and/or aggregation of alpha sy.nuclein; and a disc ase or disorder associated with be t .a-aamy'loid (Abeta) manetaabolism.

.5 [00641 Inn an embodiment, anodulation of Cl-HP by one or more antisense olie,-ooucleotides is administered to a patient in need thereof, to prevent or ti-cat any disease or disorder related to CHIP
abnomutl expression, function, activity as compared to a a omial cc=.ntrol.
1'00011 In a preferred embodiment, the 01WOntacleotides are specific for polyaaaacleotides of CHIP, which includes, without limitation noncoding regions, The CHIP targets comprise variants of C:1-HIP; mutants of CHIP, including SNPs;
no acodin 7 sequences of MR :alleles, fragments and the like. Preferably the oligonucleotide is an antisense I N
molecule..
[00021 In accordance with embodiments of the invention, the target nucleic acid :molecule is not limited to CHIP
polynucleotides alone but extends to any of the isoforan.s, receptors, homology, non-coding regions and the like of CHIP.

In another pa (erred embe~claaatent, an oli~t ttuc.lcc tide tar ?ets a natural ~ttitisensc sequence ( aÃtaral a t sense to the coding and n:ora-coding, regions) of CHIP targets, including, ti ithout limitation, varimits, alleles, homologs, aautants, derivatives, fragments and complementary sequences thereto. Preferably the oligonucleotide is in antisense RNA or DNA molecule, [00041 In araothe prof.brred embodiment, the oligo aeric compounds of the present invention also include variants in Which a ditfereait base is present at one or more of the nucleotide positions in the compound., or exa:mpie, if the first nucleotide is an adenine, variants may he produced w ,h ch contain tl yrnidine, guanosi;ne, cytidine or other natural or unnatural nucleotides at this position. This may be done at any of the positions of the antasense compound. These compounds are then tested using the methods described herein to detern-fine their ability to inhibit expression of a target nucleic acid.
1'00051 In some embodiments, homology, sequence identity or com leave a:t<arity, between the antisea:ase compound and tar4uet is from about 5t:}`4, to about 6W-10. In some emboditncn{s, htarnolog\\, sequence .identity` or cot aplea aentazit >, is from about 60'...N to about rtl` ,, In some embodiments, homolozfL sequence-identity or eomplen:m:.ntarÃty, is from about 70`'ea to about W%',. In some embodiments, homology, sequence identity or complementaiity,, is from about 80% to about 90%, In some err-dxodiments, .homolog , sequence identity or comple:menÃanty. is about 90%, about 92%, about 9411'0. about c ti`e' r, about 96%, about 97%, about 98y'%u, about 99% or about 100061 An antisense. compound is specifically hybridiz a.bl'e when binding of the compound to the target nucleic acid interferes with the. normal Ãunction of the target nucleic acid to cause a loss of activity, and there is a sufficient degree of complcmentarity to avoid non-specific binding of the a:ntiscnse compound to von-tar get nucleic acid sequences under conditions in which specific binding is desired. Such conditions include, i.e., physiological conditions in the case - It, ofiaa, vi' o assays or therapeutic treatnient, and conditions in which assays are performed in the case of in vitro assays, 100071 An antisense compound, whether -I)NA, RNA, chimeric, substituted. etc, is specifically hybridirable When binding of the compound to the target DNA or RNA molecule interferes with the normal t anction of the target DNA or RNA to cause a loss of utility, and there is a suf=ficient degree of complementarily to avoid non-specific bitiditu, of the antiseiise compowid to non-targct sequences under conditions in which specific binding, is desired; Le., under physiological conditions in the case of in vivo assays or therapeutic trQatrrie1rt, and in the case of in vitro assays, wider conditions in which the assays are perftarriicd.
100081 In another preferred eiiabodinierit, targeting of CI-LIP i icleadinfg without arrtisense sequences which are identified and expanded, lasing for ex:iiaiple, I'[;R, hybridization etc., one or more of the sequences set forth as SEQ
ID NOS: 2 and 3, and the. litrf,% modulate the expression or function of CHIP, In one ernbodinierit, expression or function is up-regulated as, compared to a control. In. another preterned enlibodiment, expression or function is down-renulated as compared to a control.
100091 In another preferred embodiment, oligoni.rcleot.ides comprise nucleic acid sequences set forth as SEQ ID NOS:
4 to I I including aaritisense sequences which are identified and. expanded, using for example. Pt :R, hybridization etc.
These oligoiucleetides can, comprise one or more modified nucleotides, shorter or longer fragments, modified bonds and the like. Examples of modified bonds or iilternlacleotde linkages comprise l hosf?liorotli:ioate. plloslihorodi.tliioate or the like, In another preferred embodiment, the nucleotides comprise a phosphorus derivative. The phosphorus deri><ative (or modified phosphate group) which may be attached to the sugar or sugar analog moiety iii the modified oligoneucleotides of the present invention may be a nio ophosphate, diphospha.te,, t iphosphate, atl.ky. lphosphate, alkaanephiispha:te, p liosphorcitliioate and the like, The preparation of the abov .-noted phosphate analogs, and their incorporation into nucleotides, modified ii:ucIcotidcs and ohgonucicotidcs, , er se, is also known and need not be described here.
100101 "f'lie specificity and sensitivity of aaiÃisense is also harnessed by those of skill in the art for therapeutic uses.
Antrseri_se oligonueleotide:s have been employed as therapeutic moieties in the treatment of disease: tae in animals and man. Antisense oli :onueleotides have been sal-ely and effectively administered to humans and numerous clinical trials are presently urnderway. It is thus established that oligonueleotides can be useful therapeutic modalities that can. be configured to be useful In treatment regiriie.s for treatment of cells, tissues and animals, especially licaiiians.
100111 In embodiments of the present invention olivomeric antisense compounds, particularly oligonucleotides, bind to target nucleic acid molecules and modulate the expression and/or function of molecules encoded by a target gene.
The fiaiict:ions of DNA to be interfirred comprise, for exaaniple, replication and transcription, The functions of RNA to be interfered comprise all vital functions such as, for example, tr`arlslocation. of the RNA, to the site of proteini.
translation, translation of protein fi ini. the RNA, splicing of the RNA to y=ield one or more niRNA species, and catalytic activity which may be engaged in or facilitated by the RNA.. The functions nu ay he up-regulated or inhibited depending on the functions desired.
10Ã1121 The antisen e compounds, include, antasense oligomeric compounds.
antise nse oligoslucleoudes, external guide sequence (.EGS) oli<gonucleotides, alternate splicers, primers, prbes, and. other oli~einerie compounds that hybridize- to at least a portion of the target nucleic acid. As such, these compounds may introduced in the fb:rn-i of sirs tlc-str4aridetl, douhic-stranded, partially single stranded, or circular oligoineric compounds, [00131 `Targeting an antis .nse eoaiifiorriacl to a paitieiilrti iaracieic :acrid aai~ilec le, in tfae: ctiaitext of this in etititiai, Cati a multistep process. The process usually begins with the identification of a target nucleic acid. evhosc function is to be modulated. This target nucleic acid may tie-:l-or example, a cellulargeinne (or niRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state:; or a nucleic acid molecule from. an i'{afecÃious agent..
In the present invention, the target nucleic acid encodes 'C terminus of I-ISP7O-Interacting Protein' (CI-UP).
[004j The targeting process usually also includes deterriiination of at least one target region- ser inent. or site: with-11-1 the target nucleic acid for the arttasetise interaction to occur such that the desired effigyct, 0-.&7 :modulation of expression, Will result. Within the Context of the present invention, the term "region" is defined as a portion of the target nucleic acid having at least one idenÃifiable. structure, function, or characteristic.
Within regions of target nucleic acids are segriients. "Segments" are defined as smaller or stale-portions of regions within a target nucleic acid. "Sites," as used it) the present invention, are defined as positions within a target nucleic acid, (0015[ In a preferred en-mbodiment, the antisense oliyonucleotides bind to the natural antisense sequences of 'C.
terminus of IISP70-Interacting Protein' (CHIP) and modulate the expression and or finiction of 'C terminus of I-ISP 7O-interacting Protein' ((.'.HIP) (SEQ ID NO: 1). Examples of autisense sequences include SEQ ID NOS; 2 to l 1, [0016 In another preferred en-bodiment, the antisense oligonucleotides bind to one or moaee segments of `C terminus of i-lSP70-InteracÃiny Protein' (Cl-UP) poly>nucleotides and modulate the expression and:/or function. of "C terminus of HSP70-Interacting Protein' (CHIP). The segments comprise at least five consecutive nucleotides of the 'C..' terminus of HSP70-Irnteraeting Protein' (CHIP) sense or aritisense polyi:tucic otides.
[00171 In another preferred embodiment, the antisense oligonucleotides are specific for natural antisense sequences Of 'C; terminus of 11SP70-interacting Protein' (CHIP) wherein binding of the olio, onucieotides to the natural antisenise sequences of CHIP modulate expression and or function of `C terminus of t-ISP7O-Interacting Protein' (0-11p).
[00181 In another preferred eniliodiment, oliponueleotide compounds comprise sequences set forth as SEQ ID _NOS: 4 to 11, antisense sequences which are identified. and espatnded, using for example, PCR, hybridization. etc Thee oliyonucleotides can comprise one or more modified nucleotides, shorter or longer fragments, modified bonds and the like. Examples of modified bonds or internuc.leotide. linkages comprise phosphorothioate, phosphorodithioatc or the l:ike...In another preferred embodianent, the nucleotides co.-nip rise a phosphorus dea:ivative. The phosphorus derivative (or modified phosphate group) which may be attached to the sugur or sugar analog iiioietc iii the modified ohyonuc:.leotides of the present i vei.ition n:iay be a raicinophtispliate, diph isphate, triphosphaite, allcylphosphait alkanephosphate, phosphoroÃ:hioaÃ:e and the like. The preparation. of the above-noted phosphate analogs, and their incorporation into nuclcotidcs, modified nucleotides and oligonucleatides, per se, is also known and need riot be described. here, is [00.1.91 Since, as is knc?tvn in the art, the translation initiation codon is typically 5'-,AUG (in m--inscribed -n--iRNA
molecules-, 5"-ATG in the cor.rc.sponding DNA molecule), the to anslattion initiation colon is also rcf~r-red to as the "AUG colon.," the "start eoodon" or the "A.UCi start codon". A minority of genes, has a translation initlation codon 1?a ing the RNA sequence 5-tiUG, 5'-U G or 5 -E: Uhf; and 5'-:AUA, 5' .A.f:;G
and 5'-CTJC have been shown to flanction in vivo. Thus, the terms "translation initiation colon" and "k tart cmlon" can encon1pass many codoll sequences, even though the initiator amino acid in each instance is typically rn? thioni.n - (in cuk rvoics) Or font?y lalae hiorairie. (in prokaryotes). Eukaar-yotic and prokaryotic: genes 11-lay have two or more adtemaativc Start codons, arid, one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In tae context of the invention, "tart codon"
and traanslation initiation codon" refer to tile codon or codons that are used in vivo to initiat : translation of ml.ty..\A
transcribed from a gernc encoding `C terminus of HSP 0-lntcractina Protein' (CHIP), regardless of the segiicrace(s,) of such codons. A translation termination codorr (or. ,'stop codon") of a gone n?ay= have one of three sequences s, Y'-UAA, 5' UAG and 5'-UGA (tile cormsponding D.\i.A sequences are 5'-J 1... 5"_ TALC and 5-FGA, respectively).

I.00201 The terms "start codon region" and "translation initiation codon region" refer to a portion of such an .n.iRNA. or gene that encompasses .from about 25 to about 5Ã3 contiguous nucleotdcs in either direction (i.e., 5' or ') from a translation initiation colon. Similarly, the terms "Stop codon region" and "translation termination codon re4g"ion" refer to of such an mRNA or c;ene that encompasses from about 25 to about 50 contiguous nucleotides in either a portion direction (1,e., 5' or 3') from a translation termination codon_.
Consequently, the "start colon region" for "translation initiation codoa region") and the. "stop codon region" (or "translation termination colon region") are all regions that may be targeted efecti vely with the antiseilse compounds of the present invention.
[0021 The open reading frame (ORE) or "coding region," which is known in the art to refer to the region bet veen the.
translation initiation codon and tlae translation termination co flora, is also a region which .may be targeted effectively.
Within the context of the present invention, a targeted rs Mion is the i:ntragenic region encompassing the translation initiation or termination codon of the open reading frame ((I .F) of a gene.
[00221 Another target :region includes the 5' unfit?nslated region '1>,TR ), known it), the art to refer to the portion. of mi mRNA in the 5' direction from the translation initiation cocoa, and thus ii c ltacline? nuclei?tines between the. 5' cap site and the translation initiation codon of an mRNA (or corresponding nucleotides on the gent). Still another target region includes the 3 untranslaated region (3'i_'.l'R),, known in the art to refer to the portion of an mRNA in the 1' direction from the translation termination codon, and thus including nue.leotides bets een the translatim ter ?rination codon and 3 " end.
of an :niT N.A (or cc?rresf?c?a?.cfii? nucleotides on the gene)_ The 5' crap site of in mRNA comprises an N7-n?.cthyl:aated guarralasinc residue joined to the 5most residue r?i'tl?e n~'` . pia a :?`-5`
fait?hc~sl?laartc linkage. The 5' cap region of an mRNA is considered to include the 5' tap structure itself as well as the first 50 nucleotides adjacent to the caap site.
Another target region for this invention. is the 5 cap region.

[00231 Although some eukaa votic niRNA transcripts are directly translated, rnan y contain one or more regions, knoss-n as "introaas " which are excised from a transcript before it is translated. The renaairting (and therefore tr"anslaated) , regions are known as "cxous" and are spliced together to form a contia3ttc~aas ]RNA sequence. In one embodiment targctirae splice sites, i.e.., i:ntran-eXon ,junctions or exon-intron junctions, is particularly usefi:tl in situations where.
:aberrant. splicing is implicated. in disease, or where < Overproduction of a Particular splice product is . i plicated in disease. An aberrant fusion junction due to rearrangement or deletion is another embodiment of a target site. mR,, A
transcripts produced via the process of splicing oft o (or aiaort 3 niR As from different gene sources are known as "filsion transcripts" herons can be effectively targeted using arttiserise compounds targeted to, for example, DNA or pre-mRN:A.
100241 In another preferred embodiment, the watiseuse oligonucleotides bind to coding and/or non-coding regions of as target polyTiucle.otide and. modulaate the. expression and/or function of the target molecule.
[0025j In another preferred. .mbodiment, the intisense oliponurcleotides bind to natural '111tiscris-0 poly-nucleotides Mid modulate the expression and/or ft action of the target molecule.

1.00261 In another preferred eaa:mbc inaent, the antisense oligonucicotides bind to sense polynucleondcs and modulate the expression arid/ or :function of the target molecule.
10027 Alternative RNA transcripts can be produced from the same 4ge.nontic region of DIVA. These alternative transcripts are generally known as "variants". le lore specifically. "pre-niRNA variants" aare transcripts produced froni the same genornic DNA that differ from other transcripts produced from the same genor:nic DNA in either their start or stop position and contain both intronic and c xorric sequence.
100281 Upon excision of one or more exon or r:atron regions, or portions thereof during splicing, pre-mRNA variants produce smaller "mRNA sari ants". Consequently, nmR`N A variants are processed p:re-.niR' A variants and each unique pre-mRNA variant must always produce a unique rRNA variant as a result of splicing. These mRNA variants are also known as "alternative splice variants". If no splicing, of the pre-mRNA
variant occurs then the pre-mRNA variant is :identical to the mRNA var iarnt.
[00291 Variants can be produced. th-rough the use of a alternative signals to start or stop transcription, Pt mP-N.-As and mRNAs can possess more than one start cation or stop codon. Variants that or-iginaate from a pre-mRNA or mRNA that use alternative start codons are known as "alteniativ>e start v.atr-iants" of that pr -mRNA or ÃtmRNA, Those transcripts that use all. alternative stop codon are knos at as "alternative stop variants" of that pre-n RNA or rt3. _ti'A. One specific type of alternative stop variant is the "poly. variant" in which the multiple transcripts produced :result :from the altermitive sole Ãitira of bare of ÃIre. "poly' stop signals" by the uaraacrilttion zaaacliin4r , thereby sroditcirx trarastri its that ici nmate.
at unique Ittil -A sites, Within the context of the invention- the types of variants described herein arc also embodiments of target nucleic acids.

[00301 The locations on the tar, et nucleic acid to which the antisense compounds hybrkiizc are defined as atleast a ..
nuelc.ot.idc low), portion of a tare et region to rOuch: an active jai-Itis.iaae; compound is to geted, 1003-1.1 While the. specific sequences of certain exemplary target segnlerats arc set forth hen in, one of skili in the art.
will recognize that those serve to illustrate and describe particular embodiments within the scope of the present invention. Additional target. Segments are reality Identifiable b one has i.m, ordinary skill in the an in v ieA- of this disclosure.
100321 Target segments 5-100 aiueleotides in length cot;mprising a stretch of at least. five (5) consecutive nucleotides selected from within the illustrative preferred target segments are considered to be suitable for targeting as well.
10033 Target se meats can include DNA or RNA sequences that comprise at least the 5 consecutive nucleotides from the 5-terminus of one of the illustrative preferred target segments Ãthe remaining nucleotides being a consecutive stretch of the same DNA or RNA beginning immediately upstream of the 5'-terminus of the target segment and continuing until the DNA or RNA cotataaiiis about S to about 100 nucleotides).
Similarly preferred target segments are represented by :FICA or Rlr.A sequences that comprise at least the 5 consecutive nucicotides from the 3'-terminus of one of the illustrative preferred target segments (the r maini:nig nucleotides being a consecutive stretch of the sane DNA or RNA beginning irumedi ately downstream of the 33'-tertaainus of the tarxet segment and continuing until the .l) i.A or RNA contains about. S to about 100:nt.ue.leotides). One having skill in the an armed with the target segn c is illustrated herein will be able, without undue experimentation, to identify further pre&..rred target segments.
[0034 Once one or more target regions, segments or sites have been identified, antisense compounds are chosen which are sufficiently complementary to the target, i,e,, hybridize, sufficiently well and with sufficient specificity, to give the desired effect.
00351 In embodiments of the invention the oligonucleotides 'bind to an aantisense strand of a particular target. The oligonucleot.ides arc at least 5 nucleotides in length and can be synthesized so each oli onucleotide targets overlapping sequences such that oligotnicleotides are synthesized to cover the entire length of the target polynucleoude. The targets also include coding as well as non coding regions.
[00361 In one embodiment, it is preferred to target specific nucleic acids by aan ,isenre ollgonucleotides.. Targeting an antisense compound to a particular nucleic acid, is a multistep process. 'Mc process usually begins with the identification of a nucleic acid sequence whose function is to be modulated.
This may be, for example, a cellular gene (or mRNA transcribed from the gene) Whose expression is associated With, a particular disorder or disease state, or a non coding polvnucleotide such as for example, non coding .RNA (neR.NA).
[00371 RNAs can be classified into (1 ) mess anger RN As (mRNAs), which are translated into proteins, and (2)non-protein-coding RNAs (ncRNAs). neRNAs comprise ztaicroRN'l.s. antisen:se-transcripts and other Transcriptional Units (l[;) containing a high density of stop codons and lacking any exterrsne.
"O;pen Reading Frame", \'l.aamly tncRNA.s appear to start from Initiation sites in 3' trntxanslatt cl regions (11TRs) of protein-coding loci. ncR N. As am often rare.

and. at least half of the .ncRN. s that have been sequenced by the FANTOM
consortium seem .not to he polyad :ny tatted.
Most researchers have. for obvious reasons focused on poNaadenylated mf\:1s that arc. processed and exported to the cytoplasm, Recently, it was shown that the set of non-polvaderaylated nuclear RNAs may be very large, mid that mans, such transcripts arise from So-called intergenic regions. The nrechaanism. by which neRNAs may regulate gene expression is by base pairing with target Ãr anscripts.. The RNAs that function by base pairing can 'be grouped into (1. ) cis encoded RN s that are encoded at the same genetic location, but on the opposite strand to the RNAs they act upon and therefore display perfect complementarity to their t,`ar4get, and :' trans-encoded I NAs that are encoded at a chromosomal location distinct from the RNAs they act upon and generally do not exhibit perfect base-pairing potential with their targets.
IOO38I Without wishing to be bound by theory, perturbation of an wntisc nse firerÃynr.aelw.ot.ide by the antiseuse oligontaeleotides described herein can alter the expression of the corresponding sense messenger RN < s. However, this regulation can either be discordant (atnaseaase knoekdozsii results in messenger RNA elevation) or concordant (antisense knockdown. results in conconaitaaant messenger RNA reduction). In these cases. annsense oligonarcleotides can be targeted to overlapping or non overlapping Parts of the aantise.nse transcript resulting in its knockdown or sequestration. Coding as well as non-coding antrsense can be targeted in an identical naanmer and that either- category is capable of regulating the corresponding sense transcripts - either in a concordant or disconeor'daant trtanner. The strategies that are employed in identil ing new oligonr.ucleotides for us against a target can he based on the knockdown of antisense RNA transcripts by antisense oligonueleotides or any other means of modulating the desired target 100391 Strategy I: In the case of discordant regulation, knocking down the aantisense transcript elevates the expression of the conventional (sense) gene. Should that latter gene encode for a known or putative drug target, then knockdown of its amisense counterpart could conceivably mimic the action of a receptor agonist or an enzyme stimulant.

100401 Stmvcgv 2: in the case of concordant rcuulaation, one could concomitantly- knock down both antisense and sense transcripts and thereby achieve synergistic reduction of the cony enrtionaal (sense) gene expression. If, ferr example, an antisense oligonueleotid:e is used to achieve knockdozvta, then this strategy can be used to apply one anti ense caligonatclewttide targeted to the sense transcript and another aant:i,eanse ofiionucleotide to the ccrr'esi?caaxcl:in . anÃr ease transcript, or as single energetically- sy%mnnrettic antisense oligonueleotide that simultaneously targets overlapping sense and antrsense transcripts.
1004.1.1 According to the present invention, antisense compounds include atrtisense oliCgonucleofides, ribozynies, external guide sequence (f G) olagonueleotides, siR"--.\"A conrpoar& single-or double-straanded RNA interference (RNA.i ) compounds such as siRNA compounds, and other oligome.rie compounds which hybridize to at least a portion of the target nucleic acid and modulate its 1-unction. As such, they may be DNA, RNA, DNA-kilo, RNA-dike, or mixtures thereof,, or nraav be nr.imetics of one or more of these. These compounds may be single-stranded, doublestranded, circular or hairpin oli~gonterie compounds and may contain structural elements such as internal or terminal bulges, mismatches or loops. Antisense compounds are routinely prepared linearly but can be ,;joined or other ise prepared to be circular and/or branched. "Vitisense eoanpoa.ands can include constructs such as, for cmimple, to strands hybridized to farm as wholly or partially double-str-madcd compound or a single strand with sufficient self conaplernentarity to aallo v for hybridization and fornaaaÃic_aaa of a fully or partially double-stranded co Drpound. The two strands can be linked internally leaving free 3 or 5' terraaini or can be linked to fonn a continuous 'lhaairpin. strncttare: or loop, The hairpin structure r nay contain an overhang on either the 5' or 3' terminus producing an extension of single traanded claaaracter. The double stranded compounds optionally can include overhangs on the ends. Frail=
modifications can include conjugate groups attached to one of the. ter ini.
selected nucleotide positions, sugar positions or to one of the internuc.leoside linkages. Alternativ-e.1Y7 strands can be linked. via a non-a ucleic acid moiety or linker group. When for maed from only one strand, dsRNA can Like the f:otni of a self complemeumi-v haa.irpira-type molecule that doubles back on itself to form a. duplex, Thus, the dsRNAs can be fully or partially double stranded.
Specific modulation of gene expression can be achieved by stable expression of dsRNA hairpins in taunsgenic cell lines, however, in some embodiments, the c ;ne expression or function is up regulated. When formed, from two strands, or a single strand that takes the form of a s W-complement ary hairpin-type molecule doubled back on itself to fbrm a duplex, the two strands or duplex-fbrnaing regions of a a single strand) are complementary RNA strands that base pair in Watson-Crick fashion.
100421 Once introduced to a system, the compounds of the invention may elicit the action of one or more enzymes or structural protein" to effect cleavage or other modification of the target nucleic acid or may 1Nvoa via occupaancy-based mechaiaisms. In general, nucleic acids (includiaa calm=c~nucleotidcs, may be described as "DNA-like," (Le., generally having, one or more 2'-deer .y sugars and generally, T rather than Ii: bases) or "RNA-like" (Le_ generally having one or more 2'- hydroxy-l or "'-modified. sugars and, generally pi rather than 'i bases)- Nucleic acid helices can adopt more than one type of structure, most commonly the A- and B-forms. It is believed that, in general, oligonucleoti=des which have B-form-like structure are " ly\.A-like" and those which have: :A-faunlike structure are "RNA-like." In some (chimeric) embodiments, an antisense-- compound. may contain bode A- crud 9-form regions.
100431 In another preferred embodianent, the desired oligonuc eot:i.des or antisense compounds, comprise at least one of: antisense RNA, antisense DNA, chimeric antisense oligonucleotides, antisense oligonucicotides comprising modified linkanes, interference RNA (RNAi}, short interfering RNA (siRNA); a micro, interfering RNA (miRN.A): a small, temporal RNA (stRNA)or a short, hairpin RNA (hRNA ; small RNA-induced gene acti-wition (RNAa); small activating RNAs (saRNM), or combinations thereof.
100441 dsRNA can also activate gene expression, a mechanism that has been termed "small RNA-induced gene activation" or RN.Aa. dsRNAs targeting gene promoters induce potent t anscr.-iptional acts ation of aassociaÃe ge=nes.
R.NAaa was demonstrated in hu.m aaa cells using synthetic dsRNAs, termed "si-mall activaatin;. RNAs" (sa.RNAs). It is currently not known whether RNAaa is conserved in other or';gaaa3isms.

[00451 Snxrall doable- Ãranded RNA (dsRN.A), such as small interfering RNA
(siRNA) arid n?_icroR.NA. (miR]A).
have been found to be the trigger of art evolu.tiona:ry conserved rrreehanisnr known as RNA irr,tcrfa rencc (R ' Ai). RNAi if -U iabiy leads to gene silencing pia remodeling cl? ~?a to i z Ãcs therch r sfl.tppres transcription, degrading complementary nrRNA, or blocking protein tranaslat on. However, in instances described, in detail i the examples section which l plc?Ws, oligonucleotides are shown to increase the expression and ter fimction of CHIP po!ynuckeo des and encoded products thereof. dsRNAs may also act as small activating RNAs Ã
aRN:A.#. Without sisl?.iota to be hound by theory, by targeting sequences in gene promoters, saRNAs would induce target gene expression in a phenomenon referred to as dsRNA-induced transcriptional activation (RN A.a).
100461 In a f arà er cr?ahc?tiia?acnt, the "preferred target segments"
identified herein r may be employed in a screen for additional compounds that modulate the expression of ` : terminus of HHSP7O-Interacting Protein' (CHIP) polynuclcoticles. "Modulators" are those compounds that decrease or increase the expression of a nucleic acid molecule encoding 'C terminus of HSP 7O-lnteraeting Protein' (CHIP) and which comprise at least a 5-nucleotide portion that is complementary to a preferred target segment. '1110, screening method comprises the steps of contacting a preferred target segment of a. nucleic acid molecule encoding sense or natural antisense polynucleotides of `C terminus of HSP7` -interacting.. Protein' (CHIP) with one or more candidate r aodulators, and selecting for one.. or more candidate modulators ..:icii decrease or increase the expression of a nucleic acid molecule encoding `C terminus of HSI' O-Interact-i:ng P'ro -in' (CHIP) pol:ynue.leotides, e.g. SEQ TD :! OS: 4 to l .1.Once it is shown. that the candidate modulator or modulators are capable of modulating (e.g. either decr asirng or increasing) alas: expression c?fa nucleic acid molecule.
encoding `C tern inus of H:S1'7O-lntcracting Protein' (CHIP) polyxnuclcotides the modulator may then be employed in further investigative studies of the function of 'C terminus of HS170-hrteractiag Protein' (CHIP) polynucleotides, or fur use as a research, diagnostic, or therapeutic a<gent in accordance with the present im.-ention, 100471 Targeting the ratan- al antisense sequence preferably modulates the functi rra of the target gerne., For example, the CHIP ;=c rte (e. g. arc cession number NM ('05861), In a pr .fer cd cmbodiÃtrent, the target is an anti::ernse polynuc-leoticlL
of the CHIP ggenne. In a preferred embodiment, an antisense oligoan:ucleoÃide targets sense annd/or natural anti. ense sequences of 'C Ã.eraniims offHS.P70-Interacting Protein' (Cl- P) px?lynrucleotides (e.g. accession nun-lber NMQO586I), variants, alleles, isoformms, homologs, mutants, derivatives, fragments and complementary sequences thereto. Preferably the oligonuclcodde is an autisense m rolecule and the, targets include coding and nonce?cling regions of <antisens - and/or sense CHIT' poly>=nucleotides.
0048 The preferred t arg;et segments of the present invention may be also be combined with their respective complementary anntisense compounds of the Present invention to fomi stabilized doul le-stnn ded (duplexed) oliuonrucleot.ides.
100491 Such double stranded of ig onneleotide. moieties have been shown in the art to modulate tar get expression and regulate translation as well as.RNA processing Via an anntisense mechar ism.
Moreover, the double-stranded moieties may he subject to chemical i-aodifacations. For example, such dotable -stranded moieties ha-:>e been shorn to inhibit the tang et by thc classical hybridization of aaratiseanse str4arad of the duplex to the target, (hereby tail e,ring enzymatic degradation of the target.
[0050 In a preferred embodiment, an aantisense ol.igontrcleotide targets `C
terminus of HSP7O-Interacting Protein' (CHIP) polynzucleotides (c._g. accession number NM 005861), variants, alleles, isofornns. homolots, nn.artants, derivatives, fragments and cornplemernÃ< sequences 'thereto. Preferably the oli<gonucleotide is an. antisense molecule.
100511 In accordance With. embodiments of the invention, the target nucleic acid molecule is not limited to 'C
terrnrirrars of HSP7t) la teraactrrn4Y Protein' (CHIP) aslone but extends to any of the isoforrrrs, receptors, homology and the like of 'C terminus of HSP7O-Interacting Protein' (CHIP) molecules .
[00521 In another preferred embodlianment, an oligorlucleot#de targets a natural anttisense sequence of CHIP
polynucleotides, for examample, pol yfnueleotides set forth as SEQ ID NOS: 2 and 3, and any variants, alleles, homolotgs, mutants, derivatives, fragnients and. complementary sequences thereto.
Examples of intisensc oligonucleotides are set forth as SEQ ID NOS: 4 to .I I .

I.0053I In one embodiment, the of gonucleotides are, coaxalalementaa to or bind to nucleic acid sequences of"(' terminus of HSP O-ltxteraactim, P.rotei.n' (C.l-1:IP) antisense, including without limitation noncoding sense a and/ar-anti.sense sequences associated with 'C; terminus of HSP70-T:anteractin4g Protein' (CHIP) polynnr:acleotidcs any modulate expression and/or- function of `C terminus of t-TS.P7O-Interacting Protein' (C'.l-I.IP) molecules.
[00541 In another preferred embodiment, the oligon acleotides are complementary to or hind to nucleic acid sequences of CHIP natural aantisense, set forth as SEQ ID NOS: 2 and 3 and modulate expression and/or function of CHIP
molecules.
00551 In a preferred embodirmnent, oligonuel.eotides comprise sequences of aat least 5 consecutive nucleotides of SEQ
ID NC)S: 4 to 11 and modulate expression and/or firnctior t M., T terminus of l-iSP 7fr-Interacting rotein' (CHIP:) molecules.
10056 The poivnucleotide targets, comprise CHIP, including family menabeis thereof, variants of CHIP; mutants of CHIP, including SNPs; noncoding sequences of CHIP; alleles of C l-II.P;
species variants_ fragments and the like. Preferaabl > the o _igonauclcotide is an antisean_se molecule.

0057.1 In another preferred. ernnbodir:nent, the oligor:aueleotlde targeting `C' terminus of 11SP7O4nteraetintg Protein' (C.HIP) poly :anwleoÃides,_ comprise., antisense RNA, interferenc RNA
(R.N`A:i), hort: interfering RNA (s RNA) :micro interfering RNA. (miRCN:A); a small, temporal RNA (stRNA); or a short., Inaairpin RNA (shRNA); smaall.R;NA-i:ndueed gene activation (RNAa); or, small activating RNA (sa.RNA).
100581 In another preferred errnbodunent, targeting of 'C terrain s of 1-ISP7O-interacting Protein' (CHIP) Ixn.lyniucleotides, e.g. SEQ 117 NOS: 2 and. 3 modulate the expression or function. of these targets. In one embodiment, expression or function is up-.reg 1laÃed as compared to a control. I.n anoÃ:her. preferred embodiment, expression or function is down-regulated as compared to a. coinrol, [Oft,591 In aarcaftacr preferr'cd crtifac~diarae.art, aartisense ctararptaaarads ee arapr ise se u~aices set for as S.E:Q IL) .NOS: 4 to 1 t .
These oli oaraaclctvtitle, can comprise one or more modified nucleotides.
shorter or longer fro ments, modified bonds and the like.
100601 in another pref rred en-ibodiraaent, SEQ ID NOS: 4 to 1 I comprise one or more LNA nr1ClcOtides.
10061.1 'f lie modulation of a desired taQ,,ct nucleic acid can be carried out in several ways known in the art. For example, antisc:nse oligonucleotides, siRN.A. etc. Enzy-m at c nucleic acid molecules e.g., (ribozyme) are nucleic acid molecules capable of catalyzing one or more of a variety of aeact_ions, including the ability to repeatedly cleave other separate nucleic acid molecules in a nucleotide base sequence sp ci.fie :raaannea. Such enzymatic nucleic acid molecules can be used, for exaaraple, to Ãargeà virtually any RNA transcript.
100621 Because of their sequence-specificity. trans-cleaving e azyniatic nucleic acid mot ules show promise as therapeutic a agents for human disease (t_ sman & McSwiggen, (1995) Ann. Rep.
., A,6 Chem, 30, 285-294:
Christof ersen and Man-, 0 995) J Med. ('hem. 38, 2023-2037,,). Enzymatic nucleic acid molecules can be designed to cleave specific RNA targets within the background of cellular RNA. Such a cleavage event renders the mRNA raon-functional. and abrogates protein expression from that RNA. In this manner, synthesis of a protein associated with a disease state can be selectiv el > inhibited.
[006131 In ;general, enzymatic nucleic acids with RNA cleaving activity act by first binding to a target RNA. Such binding occurs through the target binding portion of an enzymatic nucleic acid which is held in close proximity to an enz matic portion of the molecule that aact a to cleave the target RNA, I'hus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base pairing, and. once bound to the correct site, acts ea-a:z maaÃia:all ` to cut the taarget RNA, Strategic cleavage of such a target.R :\ will destroy its ability. to direct synthesis of an encoded protein. After as enzymatic nucleic acid 1-t as bound and cleayrdt its RNA t::rrget, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.
100641 Several approaches such as in Vitro selection (evolution) strategies (Orgel, (1979) . 'roc, R. Soc. London, B
205435) have been used to evolve new nucleic acid catalysts capable of catalyzing a variety of reactions, such as cleavage and ligation of phost hodiester linkages and amide linkages, [006 51 The development of ribozy:mes that are optimal. for catalytic activity would. contribute significantly to any strafe y that , raaplo\:, RNA-cleaving ribcszynacs for the l ar rose of rc aalaatia3.? gene expressiort.- The hammerhead rihozyme, for example, functions with a catalytic rate: (kcat) of about 1 min-1 in the presence of saturatin it) axa 'l}
concentrations of'Mg2 co~lacÃor. An artificial "RNA iigasc.,' rilre+ arac has been shown to catalyze the corresponding self=modificatio a reaction with a rate of about I(A) wiir-l. In addition, it is known that certain modified hammerhead.
riboz;r:ares that have substrate binding arms made of D'..-\A catalyze RNA
cleavage with multiple tun-ove:r rates that a app roacli. 100 rgrin-1. Finally, .replacement ofa specific residue within the catalytic core of the hara:amc head -\ ith certarin nucleotide analogues gives modified. ribozymaes that show as much as a lil-fold improvement in catalytic ate. Tese findings demonstrate: that ribozymcs can promote: chemical transfix-nations with catalytic rates that are sitgnificantl ,, greater than Those. displayed. in, vitro by most natural self-cleaving nbozymes. It. is then possible that the structures of certain seifeleaying rihoxyanes may be optimized to give ai xinmaal catalytic.
activity, or that entirely new RNA motifs can be made that display si n ficautly faster rates for RNA hosphc. is: ster cle age.
100661 bate rmolecuular- cleavage of an RNA substrate by an RNA catalyst that fits the "hammerhead" model wk'-as First shown in 1987 (Uhlenbecl , 0, C. (198,') ahf e, 328: 596-600). The RNA
catalyst was Ãe overed and reacted w th multiple RNA raaolecules, demonstrating that it was truly catalytic.
100671 Cataly=tic RNAs designed based on the "hammerhead" armotif have been used to clea ~e specific target sequences by making appropriate base changes in the catalytic RNA to maintain necessary base. pairing with the target sequences.
This has allowed use of the catalytic RNA to cleave specific target sequences and indicates that catalytic RNA
designed according to the "haamnacrheaad" model may possibly cleave specific substrate R.NA.s i:n vivo-100681 RNA interference (RNA.i) has become a Powerful tool for modulating gene expression in mammals and mammalian cells. This approach requires the delivery of small interfering RNA
(siRNA) either as RNA itself or as DNA, Lasing an expression plasmid or virus and the coding sequence for small hairpin .K'' As that are processed to siRNAs. This system enables efficient transport. of the pre-siRNAs to the cytoplasm, where they are active and permit gene expre asi.can.
the use of regulated aand tissue specific promoters for 100691 In a. preferred embodiment, an oligonucleotide or antise ase compound comprises an oligomer or polymer of ribonucleic acid (RNA) mici/'oà deoxy=ribonucleic acid. ( N:A), or a mimetic, chimera, analog or homology thereof This term includes oligonucleotides composed of naturally occurring na.acl.eotides, sugars and covalent internaacleoside (backbone) linkages as well as oligonucleotides having non-:naturally occurring portions which function similarly- Such modified or substituted oligonucleotides are often desired over native forms because of desirable properties such as, for example, enhanced cellular- uptake, enhanced affinityy, ffor a tam-et nucleic acid and increased stability in the presence of nucleases.
[00701 According to the present invention, the oligonncleotides or "antisense coanpounds" include. auntÃiseuse oligonracleotidcs (e.g. RNA, DNA, mimetic, chimera, analog or homolo y thereof), ribozy acs, external guide secparence (fGS) oligonucleotides, siRN.A comtpounds, single- or double-stranded RNA
interference (RNAi:) compounds such as siRNA compotandss, bail'., aiRN:A, aand other oligomeric compounds which hyibridiz to at least as Portion of the target nucleic. acid and modulate its function. As such, they may be DNA, RN A, -D-NA-like. RNA-like., or mixtures thereof, or may be mi.metics of one or more of these. These compounds may be single-strrarlded,, double-stranded., circular or hairpin oli.gonaeric compounds and may contain structural elements such as internal or terminal bulges, mismatches or loops. Antisense compounds are routinely prepared linearly but can be joined or otherwise. prepared to be circular and/or branched. Antisense compounds can include constructs such as, for exarnp'e, two strands hybridized to form a wholly or partially double-stranded compound or a single srr uid with sufficient self-com lccx entarity to allow for by ridization and formation of a fully or partially double-stranded compound.
The two strands can be linked internally leaviiag, fee. 3' or e` tctniiri or can be linked to form a continuous hairpin structure or loop. The hairpin structure may contain an overlhang on either the 5" or 3' terminus producing an extension of single stranded charat er. The double stranded conipounds optionally can include overli.aa s on the ends. Further artodificatioanis can include conjugate groups attached to one of the termini, selected nucleotide positions, sugar positions or to one of the iiiternueleoside linkages, ,Alternatively-, the two strands can be linked via a taon-nucleic acid moiety or linker group. When fbrti ed from only one strand, dsR.NA can take the form of a self-complemeanvir h :ai:ppin-type molecule that doubles back on itself to form a duplex, Thus, the dsRNAs can be fully or partially double stranded. Specific modulation of gene expression can be achieved by stable expression of dsRNA hairpins in transgente cell lines, When formed from two strands, or a single strand that takes the form of a scl.f eotatple.ntentsaay hairpin-type molecule doubled back on itself to form a duplex, the tai o strands (or duplex-forming regions of a singe strand) are complementary RN,A strands that base pair its Watson-Crick fashion, 100711 Once introduced to a system, the compounds of the invention tiaaav elicit the action of one or more enzymes or structural proteins to e f :ct cleavage or other modification of the target nucleic acid or may work via occupancy-based mechanisms. In _enertl, nucleic acids (including oligonucleotidis) may he described as "MA-like` (r e_, generally having one or more 2'-dcoxy sugars and geiierally>, T aat:her than U bases) or "RNAnt.ike" (i e., gene rally having one or more 2'n hydroxyl or 2'-modificd sugars and, generally U rather than `1"
bases), Nucleic acid helices can adopt more than one type of structure, most commonly the A- and B-forms, It is believed that, n general, oligotiucleoticfes which ha e -fian-n-like structure are "DNA-like" and those which have A-fbrmlike structure are "RNA-like:.." in some. (chimeric embodiments, an antisense compound ma-,,, con both A- and fi-forixi regions.
721 The antisense compounds in accordance with this invention can comprise art aritise:nse portion .from about 5 to about 80 nucicotidcs (i.e. from about S to about W linked nucleosides) in length. This refers to the le.:tngth of tllc anti ense strand or portion of the antisense compound. In other words, a sing le-stranded .anÃi ease compound of the .invention comprises f':rom 5 to about 80 nucleotides, and a double-strnded antisense compound of the invention. (such as a dsRNA, for example) comprises a sense and an antisense strand or portion of 5 to about 80 nucleotides in length. .
One of ordinary skill in the art will appreciate that this comprehends antisense portions of 5, 6, 7,8, 9, 10, 11, 12, ]3, 14, l5, 16, 17 18, 19, 20, 21, 22 , `?3, 24 25, 26,27,28, ` 7, 28, 20 3Ã , >1, 32, 3-1, 34, 35, 36, 38, 39, 40, 41, 42, 43 , 44 451 46, 4748, tc , 50,51 . 52, , 55, 56, 57,58, w , 60 ,61.. #i?, eti: , F? ih.
66, 676,S (its, 70, 71. 2, 'x, 74- 7 i if 78, 79, or 80 nucleotides in length, or any range thecewithin.
100731 In one embodi.nienat, (fie, aiatiscnse compounds of the invention have antisense portions of 10 to 5tl nucleotides in length. One having ordinary, skill an the. art will appreciate that this embodies oligonucleotides having antasense portions of 160 11 . 12, 13, 14, 1 ?. 16, 17. 18, l 9,'-?0,211,22,23,24,"-)'-;,26,27,28,-?9,30,31., 32, 3 _ 34, 35 t f?, ;+, 8, 39, 40, 41, 42, 44, 45, 46, 47. 48.49, or 50 nucleotides in length, or any range then within. 1 n sonic Caribod.iancnts, the oligoaaucleotid:es are 15 nucleotides in length.
[00741 in one emhodinaeiit, the antisense. or oligoaaucleodde compounds of the invention have antisense portions of 12 or 13 to 30 nucleotides in l: ngtla One, having ordinary shill in the art will appreciate that. this ciaahcadic antisense compounds having a ntiseir portions of 12, U. 14. 15, 16, 17, 1,8, 19, 20. 21, 212-3, 24, 25, 26, 27, 28, 29 or 30 n icleotides in length, or any range t]-terewithin.
[00751 In another pr&rred embodiiaaent, the oligonaeric compounds of the present invention ailso include variants in which a different base is laaesent at one or more of the :nucleotide position in the coaaaiaoti:tad. l or exarn il4 it the fia t nucleotide is an. adenosine, variants may b : produced which contain thytriidine, guanosine or cytidine at this position.
This may be clone at any of the positions of the antisense or dsR` -A
compounds, These compounds are then tested uslnL, the methods described herein to determine their ability to inhibit expression of a target nucleic acid.
[00761 in some enabodiaaaLnts, homolog sequence identity, or complenaentanty, beta Len the a:ntisense compound and target is from about 40% to about 60"J,. In some eaaabodiane.nts, hotaaology, sequence identity or conaplenatntarity, is f oiaa about 6W-% to about 7W/,D. In some embodiments, homology, sequence rdenÃity> or coanplementarity, is from about 70% to about 80%. In some embodiments, homology, sequence identity or complcnierataaity, is from about 80% to about 90%. In some er bodianents, hoinolo y. sequence identity or comple lentari y, is a bow 90%, about 92%, about 94%, about 95'`o. about 96';, about 97%, about 981%%, about W%:%, orabout (0077[ In another preferred) embodiment, the antisense. ol#gonuc=leotides, such as for example, nucleic acid molecules set forth in SEQ ID NOS: 2 to 1 comprise one or more substitutions or modifications. In one embodiialent, the nucleotides are substituted with locked nucleic acids {l. ` ).
[(00781 In another preferred embodiment, the oligomicleotides taqet: one or more regions of the nucleic acid molecules sense and"'or- antisense of coding arid/or non-codhig sequences associated with CHIP and the sequences set forth as SEQ l.tM INNS:.I to 3. The oligeanueleotides are also targeted to overlapping regions of SEQ ID NOS: 1 to 3.
[00791 Certain preferred oligonuclcotides of this invention are chimeric oligonucleotides. "Chimeric oligoiaiacleoticles"
or 'Viimeras," in the context of this invention, are oligonucleotides which contain two or more chemically distinct regions, each made up of at least one nu.c eotidc. These oligon icleotides typically contain at least one region of modified nucleotides that confers one or more bene fic:ial. properties (such.
as, for example, increased nuclease resistance. incaeased uptake into cells, increased binding affinity for the target) and a region that is a substrate for enzymes capable of cleaving RN:DNA or RNA:RNA .hybrids. By -way of example, RNase ff is a cellular endonuclease Much cleaves the RNA strand of an RNA.DNA duplex, Activation of RNase -f-l, thereibre, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of aantisense modulation of gene expression.
Consequently, comparable results can often be obtained with shorter oligonueleotides when chimeric oligonuelcotides ,ire itsed compared to h s l?ht?r thii? tt rice? c?li t~~atai l Licit s h briclizing to the same targ et re1g ion. CIeav>a4 e of the R ,-\ taigct can be routinely detected by gel ciectrophoresis and, if necessary, associated nucleic acid hybridization tecl-tniclucs known err the ass. In one preferred embodiment, a chimeric oligonucteotide comprises at least one region modified to increase target binding aaf=fin.ity>, acrd, usually, a region that acts as a substrate for RNAse H. Affinity of air oligonucleotide for its target,in this case, a nucleic acid encoding, rasa is :t ?utinely determined by measuring the Tm. of an c?li; onucleotide'taq-,et pair, which is the temperature at ti hick the oligonuc:leodde and target dissociate; dissociation is detected sp+ectrophotoramett eallcf. The high r the Tilt, the greater is the affinity of the oligornueleotide for the target.
801 Chinicrie aritisense compounds of the invention Inky be foraned as composite struettires of two (-,I]- more oli{gonuclcotides, modified oligonucl.eotides, oligonuc cosides and/or-oligonucleotides mimetics as described above.
Such; cotupounds have also been referred to in the art as hybTids o:rgapmxters. Representative United States patents that teach the preparation of such hybrid structures comprise, but are not limited to, US patent nos. 5,013,830; 5,149,797; 5, 220.007; 5,256,775, 5 3f?6,87 5,403,711; 5,491,131, 5,565,350: 565 1 0;

5,623,06 5,652.55; 5,657.,356 and 5,700,92-2.
each of which is herein incorporated by reference.
1OO811 In another preferred embodi.t?:rent, the region of the. oligoni teotide which is modified comprises at least one nucleotide mr odified. at the T position of the sugar, iraost lyre e.:r-ably a 2'-Oalkyl. -O-alkyl-O-alkyl or -fluoro-modified nucleotide. In Other Preferred embod:imebnts, I N.A. modifications include 2'-fuoro, 2.'-amino and 2.' O-methyl modifications on the ribose of pyrninridines, abasic residues or an inverted base at the :3` end of the .RNA. Such modifications are routinely incorporated into oligonucleotides and these olifonucleotides have been shown to ha e a higher Trn (i.e., higher target binding affinity) tharn; ?'~clecrxyoli c?rrtielcc?tielcs against a given target, The of f ct of such increased affinity is to greatly enhance. RN-Ai oligco mete otide Inhibition of gerne expressiorn. RNAse H is a cellular e ndonuclease that Cleaves the RNA strand of RNA:DN,A. duplexes; activation of this cnz me therefore results in cleavage of the RNA target, and thus can greatly= enhance the efficiency of RNAi inhibition, Cleavage of the RICA
tartget can be routinely demonstrated by gel electtophor~esis. In another preferred embodiment. the chimeric oligontreleoticle is also mortified to enhance ruck:se r'csistance. Cells contain a variety of exo- and e.ndo-nucleases can degrade nucleic acids.: number of nucleotide and nucleoside:.
tnodifications have been sho ;e n to make the oligonueleotide into which they are incorporated more resistant to nuclease digestion. than tire.- native alit eodeoxy<nucleotrde:.. Nuclease resistance is routinely measured bye incubating oligonucle otides with cellular extracts or isolated nuclease solutions and measuring the extent of intact oligonucleoÃide remaining over time, usually by gel electrophoresis. Oligorrucleotides which have been modified to enhance their nuclease resistance survive intact for a longer time than irun?c?c ificcl t?lis,oriucl.e ?tit s s. A variety of tali c n:trel ec?ticlc modifications have been demonstrated to enhance or confer nuclease resistance. Olige :nucleotides which contain at least one phosphorothioate modification are presently more preferred. In some cases, oligonucleotide modifications which enhance target binding affinity are also, independently, able to enhance nuclease resistance.

6 PCT/US2010/046102 [00821 Specific examples of some preferred of g onueltoÃides envisioned for this invention include those comprising modified backbones, fir example., phosphorc-thioatcs, phosphotriesters, methyl plaosphr~raaÃes, short chitin alkyl or cr-cloaalkyrl intersugar- iinka~.Fes or short chain heteroatomi : or heteroevelic iritersugar linkaLges. Most preferred are oligouucieotides With phosphorothioate backbones and. Those with .heteroatom bac:kbor es, particularly CH2 --NH--O--C112, C:1-12, C1-1,- N(CH3)--C --0112 Ikaao w as a methy.lene(methviir ino) or kl. li backbone], C112 --O--N (013)_-012, 0142 -' (('1-13).,_x; (CI-13)--01-12 and 13..-N (CH3)-õ('112 0--112 backbones, wherein the native phosphodiester backbone is represented as C.1--P--C--CH). The amide backbones disclosed by Dc Mesr7aaeker c t al, (1995) Ac . (w m.
Ras. 28:366-374 are also preferred. Also preferred are olio?;_,r trelcoÃitles having morpholirro backbone str=uctcrr:es (Su_mmerton and Weller, U-S. Pat.. No. 5,034,506). In other preferred embodiments, such as the peptide nucleic acid.
(PNA) backbone, the phosphodiester backbone; of the of gonucleotide is replaced with a polyamide backbone., the nucleotides being Found directly or indirectly to the aza nitrogen atoms of the polyan-ride backbone. Oligonucleotides may also comprise one or More substituted sugar moieties. Preferred oligo:nueleoÃides comprise one of the following at the 2` position: OH7 51.1, SCH.3, F, (K N, 0(..H.3 00.13, 0[;'1-13 O(CH2)n C'H3, 0tCH23rn NH2 or O(CH2)n 043 where ra is from I to about 10; C.l to ('10 lower alky=l, tll:o <:rll`rraz s bstituted lower alkyl, a karyl or ar aiky.i; Cl: Br;
CN; CF3 : OCF3: 0--, S--, or N'-alkyl; 0--. S--, or N-alkenyl; SOCHI: S02 CH
3; ON02: N02, N3; N H2;
heterocycloalkyl. heterocycloalkaryl; amino-alkylar-raino; poly-a1kylan3mo;
substituted stl:-l; an RNA cleaving 4groly- a reporter group; an irnter alator:, a group for improving the phaarmac-okinetic properties of an oliponucicotide; or a group for improving the phtrmacodynamie prop rties of an oligomiclcotide and other substituer:rts having similar properties.
A preferred modification includes T-methoxyethoxy> [2'-O-012 0112 OC:1-13, also known as ? _Ã) (' rrtatlaf?xyetlat )_ =
Other preferred modifications include 2'-r7aetl;to .y (2'-0--(_'H_ I), 2'-propoicy (2+-0[112 CH2CH3) and 2`-IluoÃo (2'-F).
`similar :ramr fificati,>:i s nay also be made at other positions on the olig-onuc.leotide., particularly the 3' position ofth:e sugar oil, the 3'' terminal .nucleotide and the 5' position of S' teaminal nucleotide. Oligonucleoti:des may also have sugar mimetics such as eyelobutyrls in place of the peutofumnosy>l group.
1008 31 Oligonuclcotides may also irnclude., additionally or a.lterrrativ-ely, nuclcobase: (often referred to in the art simply as ""Lase") modifications or substitutions. As used herein. "unmodif ed" or "natural" nucleotides include adenine (A), gutaniuc (0), thymine (1'), cytosine (C) and uracil (U). Modified nucleotides include nucleotides found only infrequently or transiently in natural nucleic acids, e.g., hy1manthine. 6-:methyladeaine, 5-Me pyrimidines, particularly 5- methylcytosine also referred to as 5-mctiayrl-2' dcoxycytosine and often referred to in the art as Me-C':), 5-ir= dr o yraretlryleytosi:rac (HNIC) Ldycosyl H' IC and gentobiosyl HM C as well as synthetic nucleotides, e,g 2-aniinoadenine ?-(naethyl.trnira.c~}aelcrairie, 2-(iii caazc)lylal'ky.iladeniuc, 2- (<arr iriosrl_141ysrrr3:iaac?}atderxirre or other heterosubstituted alkyladenines, 2-thiouraaeil, 2-thiothyminc. 5-brornouracil. 5-hydroxyrriethylarrÃrc il, kaza;~rr< nine, 7-de.ammian ic, N6 (6-ar:tritnohexyl)adei;tinne .aracl 2,t rli rtaaiuol uritae.
A "universal" base known ià the art, e.g., inosule., zz.

may, be included, 5-Me-C: suhstitutior s, have been shown to increase nucleic acid duplex stability by O.6-1.2`'C . and are prescntly piefen-cd base substitutions.
[04:84j ;another modification of the oligor ucl.eotides of the invention involves chern,ically linking to the oligonueleotide One or snore moieties or Conjugates w vhieh enhance the activity or eelhrlar uptake of the i)ligonucleOtide. Such moieties include but are not hrn.ited to lipid moieties such as a cholesterol moiety, a cholesteryl moiety, an aliphatic chain, t. u., dodecandiol or undecy>l residues, as prolyanaine or a poi }'cth ie-cg of chain, or Ndar rarrtaare acetic acid. Oligonucleotides comprising lipophilic moieties, and methods for preparing such oli.t onucl.eotides ar-e known in the art, for exanmple, I i.'. Pat. Nos. O4 51"218,1 O'S and5,459,255, [00851 It is not necessary for all positions in a given of gonucleotide to be:.aanfifornil y modified, and in fact: more than one of the aforementioned modifications may he incorporated in a single olifgonucleotide or even at within a single nuclcoside within an c~li c a aaeleoticfc. The present invention also includes oligonudcotides which are chimeric oligonucleotid: s as hcreinbefore defined.
[0086j in another embodifrment., the nucleic acid m lecule of the present invention. is con.#a.a4?ratetl with arac t er rtaaaietS
including but not limited to ahaasic nucleotides: polyetlrer, polyarnine, polyamides, peptides, catrbolaydfaatcs, lipid, or polyhydrocarbon cor porands. Those skilled in the art will recognize that these: molecules can be linked to one or more of any nuleoÃides comprising the nucleic ac d molecule it several Positions on the. sugar,, base or phosphate 4grocaip.
[00871 The tale*onucleotides used in accordance with this invention may be conveniently and routinely made through the wellrcknm\-n technique of solid phase synthesis. Equipment .for such synthesis is sold by several. vendors including Applied Biosystcnrs. Any other means for such synthesis may also be employed;
the actual synthesis of tlrc oligonucleotides is well within the talents of one Of ordinary skill in the art. It is also well known to use similar techniques to prepare Other olio oar.ucleotides such as the phosphor:othioates and alky hated derivatives. It is also well.
known to use similar techniques and commercially available modified arnidites and controlled-pore glass (CPG) products such as biotin, fluorescein, acridi.ne or psoralen-modified amidites and or C: PG (available from Glen Research, Sterling VA) to synthesize f uorescently labeled, b:iotiny .ated or other modified oligonu.c.eoti d.es such as cholesterol-modified o.lig onucleotides.
[00881 In accordance with the invention, use of modifications such as the use of LNA. monomers to enhance the potency, specificity and duration of action and broaden the routes of {ailrfr _aaistfation Of oligonueleotides comprised of current chemistries such as .MICE, ANA, FAA, I'S etc. This can be achieved by substituting some of the aafononiers in the current oligonucleotides by I.NA mono mrers. The LNG modified oligonucleotide may ha-_ a size similar to the parent compound or may be larger or preferably smaller. It is preferred that such LNA-nmaodified oligonu:cleoiides contain less than about 70%, more preferably less than about W/0, most preferably less than about St3!'% LNA
, mono ers and that their sizes are between about 5 and 25 nucle +tides,. more preferably between about 1?. and 20 nucleotides.

[00891 Preferred modified oligonucicotide backbones comprise, but not fin i.tcd to, phosphorot1i oates, chiral phosphorothioates, phosphorodithioatc:s, phosplaotriesters, a~~ainoalk >lphosp tà i~ si: rs, in :.thyl and other alkyl phosphonates comprising 3alkylenc phosphonates and chiral phosphonates, phosphinates, phosphorarnid ates conprising) Y -amino phos hot amidaatc and ami.tioalkylphosplroiana:idates, Cltion pto+aslal car tta~i~ at s.
It c a n e tll4 l h c a phcanat s, tla:icataza all: >lpliawpla triesters, m d 1?or<anc l lacas faates ia:t in normal Y-5' 13311 a s es. 2'-5' lined analogs of these, and those having 1-averted polarity wherein the aad,iacent pairs of nucleoside units are linked 3rr5, to 5`-3' or 2 -5' to 5'-2'. Various salts, mixed salts and tree acid forms are also included.
[00901 Representative United States patents that teach the preparation of the above phosphorat conÃaining linkages comprise, but are not .limited to, US patent nos 3,687,(R08; 4,469,86-3r, 4,476, 331; 5023,243; 5 1777196; 5,188,897;
5,264,423.1 5,2276,019- 52718,302.. 5-286,717- 5,321,131-1 1. 5,399,676: 5,405 939. 5,453,496; 5,455, 233. 5,466,6771 5,476925' 5,519,126; 5,536,821- 5,541_34)6, 06, 5,550,111, 5563. 2153- 5,57 1,7799- 5,587,361 ; and 5,625,050, each of which is herein incorporaÃed by reference, [00911 Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are ibemed bye short chain alkyl or cycloalkyl intemuel aside linkages, mixed heteroaiom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heturoatomic or heterocyclic intemucleoside linkages. These comprise those having raoipholino linkages (formed in past from the sugar portion of a nueleoside); silox tae.
backbones; sulfide, sulfoxide and sarlfone backbones formacetyl and thioforaraacett l backbones; na.ethyl ne formaacei vl and thiofonuaacetyl backbones: aalkene containing backbones-, srilf amaate backbones; methyleneimianao and rriethylenehydr azino backbones; si_tlforiatc and stilfbananridc backbones, amide backbones- and others having mixed N, 0, S and. CH2 component parts.
1'00921 l .cprc gent iaa , e L iait~d States p rte asÃ.s Ãlrat teat It tlac prepaara i.on of tlrs abcat c cali~~carrtaclecasidcs comprise, brat are not limited to, US patent Taos. 5,034,506, 5.1667315- 5,18-5, 44-, 5,214,134, 5,216,141, Ã 6,14.1; 5,215,033-, 5,264, 562; 5, 264,564; 5,40,938-, i,434,257, 5,46077- 5,470,967-, 5,489,677; 5,54007-5,561,22-5, 5,596, 086; 5,602,240;
5,610 289 5,602,240; 5,608,046, 5,6113.289, 5,618,704-1 5,621, 070; 5,663,3 l 2; 5,633,360, 5,677,437; and 5.671,439.
each. of which is .herein incorporated by reference.
[00931 In other preferred oligonucleotide mimetic:, both the sugar and the .iarteantaelccaside linkage, i.e..: the backbone, of the nuclcotidc. units a are replaced with novel groups. The base units arc.
maintained fbr hybridization With an al tars-priate nucleic acid target compound. One such olitgoraaerie. compot nd, an ohiLgona:aclecftide taaiaate.tic. that has been shozwaa to have excellent hybridization properties, is referred to as a peptide nucleic acid. (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced c.:itlr an amide containing backbone, in particular an a m noethy>lal} cine backbone. The .nucleobases aaa e retained and are bound directly or indirectly to aza nitrogen atoms of they aatnide portion of the backbone, Representative United States patents that teach the preparation of PNA compounds comprise, but are not limited. to, US patent :nos. 5,539,082; 5,714, 331; and 5,719.,262, each of a vhich is herein incorporated by reference . Further teaching, of RNA compounds can. be .found in Nielsen, art al. (19 1) Science 254, 1497-1500.

[00 941 In another prefe rcki e tbodim.e t ofÃt the invention. the o1igouuc1coÃidcs with phosph ii~othhlsoaxe baackbsonnes and oligoaaucleosides with hcteroatom backbones, and in particular- C.H2 NIA.-C) C.H2__-C.H2-' (CH3) {)-Ã'H2-knows as a methylene (araethyliaaairao) or I' INII backbo e. 0112-O- C1122-and-C3-N(013.) C142.0-12- -,vherein the native phosphodiester backbone is represented as- C3-.pr()-':1-12- of the above referenced US
patent no. 5.489,677, and the amide backbones of the above ref rented US
patent no, 5,6Ã12;240, Also pref rred are oli.tgotauc.i.cotides haavin< morpholino backbone structures oftlhe above-referenced US patent no. 5,04,506..
100951 l :lodified tsl.i oaaraclct?ticks may also contain one or more substituted sugar moi ties. Preferred olimmucleoticles comprise one of the fhllovv-ing at the 2' position: 01-1;1?, 0-, S or N-alkyl;
0-, S-, or N-alkenyl; 0-, S-or N-aalkynvl, or 0 alkyl-0-alkyl, wherein the alkyl, alkenyl and aalkynyl may be substituted or unsubstituted C to CO alkyl or C2 to CO
alkcn l and vlkyaayl. Particularly preferred are. 0 (C:H')ta C3a aC:i13, 0(C:H
)aa C;H3 [3(C:111)n .Hy', O(( H2)nC;H3 O((:H2)nONH27 and O(CH2aa:C?,N(CH?)nCH3 -' where a and m can, be from 1 to about 111. other- preferred of gsonucleotides comprise one of the :following at the. 2' posiÃion: C to CO, (lower alkyl, substituted lower alky ], aalkarv 1. aaralkyl, 0-aalkaarvl or C3-aral:kyl, SR, SC113, (KN, Cl, fare CN, CF3, CXT3. SOCH3. S020-13, ON 2, N02., X13, N H2, heteroc cloaalkyl. heterocycloalkaz- 1, aminoalkylami o, polyaalkvl.aamiuo, substituted si,lyl, an RNA cfcaavi:ng ;,group, a reporter group, an intercalator, a group for improving the harm acoka.neti.c properties of an oligonucleotide, or a group for improving the phaarmacodvuan:aic properties of an ol.igonucl otide, and other substituc-nis having similar properties. A preferred modification comprises 2'yraac tla.ta syetlaoxy (2`-O-C 1 2Cf f2C)C'H_ , also known as T-C3-(2-methmyethyl) or 2'-R1C3I .) i.e., an aIkox4 alko_ y group, A further pref .rred modification comprises 2'-diaaaetlaylaataiaaoo ycila y. i.e. , a O((H2)2O\(CH3)2 gn up, also known as 2'-1.)MA.0E, as described in examples herein below, and 2"- dimethvl.aaaaaiaaoethoxyethoxy (also .known in the art as 2'-t3-ci ara~Ãhy`laa3anraetlxca vetlxyl or 2'-D:MAEOE)i.e., T-0-0-12-0-0-12 -N ((,l12)2.
100961 Other preferred modifications comprise 2.+-methoxy (T-O C H3), 2'-aria opropoxv (`'-0 C.H2CH2CH2N'H2) and 2'-fluoro (2'-k). Similar modifications may also be made at other positions on the oligonucleolide., particularly the 3` position of the sugar on the 3' teramninaal nucleotide or in 2'-5' linked oligonucleotides anad the 5' position of 5' terminal, nueleot.ide. C3ligonucleotides r ivy also have sugar n iaaaerics such as cyclobutyl moieties in place of the pea'sÃofhraanosyl sugar, Representative United. States patents that teach the preparation of such modified sugaar structures comprise,but are not limited. to, US patent nos. 4,981,957; 5,118,800; 5,319,080. 5,359,044-5,393,1178- 5,4.46,137- 5,4W786,5,514, X6,786: 5,51 , 785 `.51k3 l i4; 5. 67,51.1 w, 76 1?7; `,591.77"? a t t etta'3, 610,300:
5,627,053:5,639,873', 5M6, 265; 5,658,873:
5,670,613: and 5,700,920, each of which is herein incorporated by reference.
100971 C3i<gonucleotides may also comprise arucleobasG (often referred to in the art simply as "base") modifications or sta.bstitaatioaas.: As used heroin, "zaaaagaoelified" or "natural" nucleotides c and a est. the l a:arine bases Mcnine (A) and gaaan:iale {C_ ),and the py<ri.araidinc bases thynrine (T}, cytosine C) and nrdcil (.T:'). Modified nucleotides comprise other synthetic arid natural naac;leotidcs such as ~!-meÃlhylcytusinc (5-ra c-C), " - I r t h o x y r n e t h =1 evÃws nc, xandiinc., h poxamzinc., 2-air inoadenine, 6-methyl and other alkyl derivatives of aaclcnine and guanne, 'prop 'I and caber all i de vat s of adetrin.e. and guaaanine, 2-thiouracd. 2-thiothyarrinc and 2-thiocytosine, 5-halo,raacii and cytosinr.e, 5-propytryl uracil and CSto'ir1e, t -a.?i) arraruil, cytosine and thvararne, 6.t.aracil (pseudo-uaac:il), 4-thiotarac:il, 8-b o, 8-amino, S-thiol., 8-thioalkyI, 8-hydroxyl and other 8-sabat.ituted adenines and mian Ines. 5-halo particariarly 5.-bronmo, -triflaroromethy.1 and other 5-saiLrstitaatec$ air arils and ytosines, ' raretlt5'ltlaaanine and 7 metlaylasla.rairac, S-;a z :a xtaaa nw and ' -{azra<adeniare. -deazaguaninc and 7-dear aaaaden.:are raarci. ;T-clca a aararria e raa d era :aaadcni ac.
1'OO98 Further, nucleotides comprise those disclosed in United States Patent No. 3,687,80S., those disclosed in The Concise Encyclopedia of Polymer Science `nd 1 ar .ina c a iar x', pafges 858-859, Kroschwitz, J.i., cd. John Wiley & y ons, 1990, those disclosed by Engdisch c:i. ar.L, 'Angp.vandle Chenrie, International .Edition', 1991, 30, page 613, and those disclosed by Sanglivi, ,a> S., Chapter 15, Antisensc Research and Applications', pages 289-302, Crooke, S. T. and I..ehleu. T . ea., CRC Press, 1993. Certain of these nucleotides are particularly aaseftil for increasing the bindilig, affinity of the oligorr-reric compounds of the invention. These comprise 5-substituted pyri:n:aidines, 6- a azapvriniaidines and N-2, N-6 and 0-6 suubstituted puri.rncs, comprising -rat aiatf~T~raal?slaacla rairac, 5- propynylur aril and 5-propyrnylcytosirne. 5 methylcy-tosine substitutions have been slim m to increase nucleic acid duplex stability by 0.6-I.2'C Sanghv Y.S., Crooke., S. T. mad l ehleu, B., eds., ': ritisense: Research and _'\pplic ations', CRC Press Boca Raton. 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly i hs a combined with 2'-Omer oxyethyl. sugar modi:fica.tions.
(00991 Representative United States patents that teach the preparation of the above noted modified narcIcotidc , as Well as other modified nucleotides comprise, but are not limited to, US patent nos.
3,687,808, is well as 4,845,205.-, 5.130 02 5,134,066; 5J75, 273: 367,0665,43,272; 5.457,187. 5,4-59,255.-5,484,908 5,502,177. 5,52?, 711, 5,552 540 5,5877,409; 5,596,091; 5,614,61--, 5.750.692. and 501,941, each of which is herein incorporated by reference.
[00.1.00] Another rxrodihcaation of the oligonucleotides of the invention involves chemically linking to the oli4aonucleot.ide one or more moieties or conjuga .tes, Which enhance the actil ity, cellular disÃribut_ion,, or cellular uptake of the oligonucleotide.
10Ã310.1.1 Such moieties comprise but are not lairtitA to, lipid moieties such as, a cholesterol moiety, ch(-Aic acid, a thioetl:hcr, e.g.. hesyl-S-tritylthiol, a thiochole.tcrcii, an aliphatic drain, e.g., dodecaandiol or undee -l resid arts. a phospholipid. e.g., di-he.xadccvi-rac-glyrcero.l or triethylanrmon um I ?-cli-C?kl s ~aci. cy>l-ram-~rly ceacr >-11-phc sl hc?n<rtc, a poly carmine or a poly: ethylene glycol chain, or Ada n antane acetic acid., to paalmiÃ:yl moiety, or an octadc.evlaa:rarinc or hexylam .no-carbonnyl-t o.xycholesterol moiety.

~35 [00.1.02] Representative United States patents that teach the preparation of such oiiegonuc1eotid coi1jugaÃ:cs con-1prise, but. acs not limited to, US patent iii:+s. 4,$28,979 4 .,948,8821 5,2 18,10.
5,52-5,465- 5$1,3133; 5,545>73O 5,551, >38;
x,578,7.17, 5,580,731'. {.580,731 5591-584" 5,1.091,1 24; 5,11,8,90-2; 5.13$
045; 5,414,0777', 5,486, 603 5'512,439:
'578,71 S;
,t 0 ,0 6; 87,11 1,60 77351 4,(267,025; 47762, 779- 47789,737-, 4,8247941;
4,835,2637 4,876,335,-584- 4744, 5 l 90582; 4,958,013; 138' ls::i}, 5,112.963, 5,214.136, 5,082,15:}0R 1.1 963:
5,214,1 {ti, 5, ') 3, -45,022-. ?,?
54,4 69:
5,.258,506-.258 506'. 5,26-2.536-1 5,272,250, . 5,29,873: 5,317,098-, 53'7 1,241, 5-391, 723, 5.416,203. 5.451,463; 5.510475:
5.:? "1',667 5,514,785-, 5 >52: 5,567,810- 5,571142' =~~i3,481, 5,587,371 t 5 }7,6t}_6- 5,54t c } ?`i3 ~ .~;?_ 5,595,726, 5.599, 928 and 57688,941, each of which is herein incoapo:ra:ted by. reference.

1001031 .l.:ir og ci over y: The compounds of the present invention can also be applied in. the areas of drug discove. y and tar et validation. The present invention comprehends the use of the comp funds and preferred target se gents identified herein in dma discovery effiirts to elucidate relationships that exist between 'C terminus of .1-151'7O-l:nteracting Protein' (CHIP) polytaucleoti.des and a disease ,gate, phenotype, or condition, These methods include detecting or modulating, 'C terminus of HSP70-Interacting Protein' (CHIP) poly-nucleotides comprising co.ntactn g a sample, tissue, cell, or organism with the compounds of the present invention, measuring the, nucleic acid or protein level of 'C termini.ts of 1-ISP7O=hateracti.ng Protein' (CHIP) l olynuc:leotides and/or a relate cl pheno pie or chemical endpoint at some time after treatment, and optionally comparing the measured value to a non-treated sang ple or sample treated itlz a further compound of the invention. These methods can also be performed in. parallel or in combination with other experiments to determine: the function of unknown. genes for the process of target validation or to deternmiine the validity of a particular gene product as a. target for tieatt-rment or prevention of a particular disease, condition, or phenotype.
ss: ssbi 1 r ~ru/ tion ?r' Irahthllion o; G ine~ , rpivssio 1001041 Transfer of an exogenous nucleic acid into a host cell or organism can be assessed by directly detecting the presence of the nucleic acid in the cell or org"ataism. Such detection can be achieved by several methods well known in the art. For ex mple, the presence of the exogenous nucleic acid can be detected by Southern blot or by, a. polymerise chain reaction (PCR)' technique using primers that specifically anmtplify nucleotide sequences associated with the nucleic acid. Expression of the exogenous nucleic acids can also be mea cured.
using? conventional methods including gene expression analysis. For instance, mRNA produced from an exo=genous nucleic acid can be detected and quantified. using a Northern blot and reverse transcription PC'R (RT-PC'R).
1'OO1O51 Expression of K NA from the exogenous nucleic acid. can also be detected by measuring an enzymatic activity or a reporter protein activity. For example. antisense modulatory; activity can be measured indirectly as a decrease or increase in target nucleic acid expression as an indication that the e xoegenous nucleic acid is producing the effector RNA. Based on sequence conservation, primers can be designed and used to amplify ceiling regions of the target genes, initially, the most hi4ghlR, expressed coding region from each gene can be used to build a model control gene, although any coding or non coding rcgiota can be used. Each control geoc is assembled by inserting each coding region b .twecn a reporter coding region and its poly(A) signal. These plasmids would produce an RNA with a reporter gene in the upstream portion of the gene and a potential RNAi target in the 3` non-coding region. The cffecÃiveriess of individual aatatisetase oligonuel.eotides would be assayed by modulation of the reporter gene, Reporter genes useful in the methods of the present invention include, neeÃt hydroxyacid synthase (A1-1AS), alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase (GUS),. ehloraamphenieol accty>ltransfcrase (CATS, green flue rescent protein (GFPt, red fluorescent protcm (UP), yellow fluorescent protein ('`FP), can Iuorese:cnt protein (CFP), horseradish peroxidase MR.P), lueiferase (Luc), nopaltt e synthase (NOS), octopine synthasc (OC: S), and derivatives thereof.
Multiple selectable markers are available that confer resistance to aarnpicillin, btennivcin, eh.loramphenicol, gentamycin, hytfgronmiyein, ka:namycin, linconlycin, methottexate, phosphinothricin, puroaxa yc:in, and tetracyclirae. Methods to determine modulation of a reporter gene arc well known in the art, and include, but are not limited to, f uorometri0 methods (e~g~ 11uoreseence speetroscopy, Fluorescence :activated Cell Sorting (F ACS), f1:uoresccncc micro s yaps ), antibiotic resistance deterarri:naation.
1001061 CHIP protein and inRNA expression can be assayed using methods kn viia to those of skill in the art and described elsewhere herein. For exata ple, inurninoassays such as the FLISA
can be used to measure protein levvels.
CH111 E'LISA. ass-,ay kits are available comme.a ially, e.g., from Il&I) Systems (`clinneapolis MN).
1001071 Tn embodiments, CHIP expression (e.g., mRNA or protein) in a sample Cells or tissues in vivo or in vitro) treated using aaaa tunas nse oligonucleotide of the invention is evaluated by comparison. with C1711:P expression in a control sample, For example, expression of the protein or nucleic acid can be compared using methods known to those of skill in the art with that in, a mock-treated or untreated saaample, Alternatively, comparison with a sample treated with a control antisense o.ligoaa.ueleotide (e.., one having an altered or different sequence) can be made depending on the information desired, In another embodiment, a d fT-erence in the expression of the Cl-HP protein or .nucleiic acid in a treated vs, an untreated sample can be compared with the difference in expression of a dif) rent nucleic acid (including am, standard deemed appropiiaÃe by the researcher, e.g., a housekeeping gene) in a treated sample vs. an untreated saample.
1OOiO81 Observed differences can be expressed as desired, e.g., in the foram of a ratio or fraction, for use in a comparison with control. In embodiments, the level of CHIP mRNA or protein, in a sample treated with an antisense oligonucleoticle of the present invention, is increased or decreased by about I.25-fold to about 10--fold or more relative to an untreated. sample or a sample treated with a control nucleic acid. In embodiments, the level of CHIP mRNA or protein is increased or decreased by at least about 1..26-fold, at least about I.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about I.6-fold, at least about I.74=old. at least a about 1 .l-iiold, at least about 24old,, at least about 2.5Y
bld, at least about ,-ibid, at least about 3.5-fold, at least about 4-fold, at least about 4.5-find, at least about 5-ibid. at icaast about 5.5-fold, at least about 6-fold, at least about 6>5 fold, at least about 7-fold., at least about 7.5 .foist. at kasÃ:
about 8-fold, at least about 8.5--fold, at (east both 9-fold, at lesa.sà about 9.54ali< or at least about 10-fold or niorc.

Kits Research Reqgews, and [001091 Tlic compounds of the present invention can be utilized for ding iostics_ therapeutics, and prophylaxis, and as research reagents and components of kits. Furthermore, antisense oligon ucleotides, which are able to inhibit gene expression with exquisite specificity. are often used by those of ordinary skill to elucidate the .fiaamctitn of particular genes or to distinguish bet Y-cen functions of various members of a biological pathway, 1001101 For use in kits and diagnostics and in various biological systems. the compounds of the present invention, either alone or in combination. with other compounds or therapeutics, are useful as tools in differential ,an l or combinatorial analyses to elucidate expression patterns of a portion or (lie L
nt:irc complement of genes expressed wih n cells and tissuu:es.
[001111 As used herein the term "biological system" or "system" Is defined is any organism, cell, cell culture or tissue that expresses, or is made competent to express products of the `C; terminus of H.SP70-interacting Protein' (CHIP) . These include, but are not limited to, humans, transgenic artinaaÃs, cells, cell cultures, tissues, .xeno rafts, genes transplants and combinations thereof 1001121 As one non limiting example, expression patterns with-in cells or tissues treated with one or more antisen e compounds a are compared to control cells or tissues not treated widi antisense compounds and the patterns produced are analyzed for differential levels of gene expression as they pertain, for example, to dis aas aassoci aÃion, si tzaili-a?
pathway, cellular localization,, expression level, size, structure or function of the genes examined, These analyses can be performed on stimulated or auastimulated. cells and in the presence or absence of other compounds that affect expression patterns.
1001131 Examples of methods of gene expression atudysis known in the art include DNA arrays or anci'oarravs, SAGE (serial analysis of ens expression). READS (restriction enzyme amplification of digested eDNAs). TOGA
00M Lenc: express. EST. sec uenc:ing, subtractive aoia aataraysts), protein arrays and 1?r-atcon~aacs, c.xl~at-cssc.~l sequence tax-- 1 7 RN,A. fingerprinting (SuR1), subtractive cloning, dittcreaatial display (Dl)), comparative genonuc hybridization, FIS11 (fluorescent .in situ hybridizationa.) techniques and mass spectroneà y methods.
001141 The compounds of the invention are useful for research and diagnostics, because these. compounds hybridize to nucleic acids encoding V terminus of HSl. 7tl-lntcractinL- l rotein' (CHIP). For example, oligonaucleotides that hybridize with such of Iciencyand under such conditions as disclosed herein as to be effective `C to-minus of HSP7O-Interacting Protein (Cl-1IP) modulators are effective primers or probes under cc?raclitioits .ttt~f?ring gene amplification or detection, res aectiv ely.. These primers and probes are useful in methods requiring the specific detection of nucleic acid molecules encoding `C.: terminus of I-HP7t}=1n terac.ting Protein' (0-UP) and in the amplification of said nucleic acid molecules for detection or for use in ether studies of `C terminus of HSP70-I
ateracting .l rotei:n' (CHIP).

i-lyybridization of the antiser:tse oligor:rudeotides, particularly the primers and prof es, of the invention. with a nucleic acid encoding ii.. ternainÃrs of 11SP7O-interacting Protein' Wf-ilP) can be detected by means known in the an. Such means rrraay include co ju ration of mi enzyme to the oiigor t:tcleotide, radiolabding of tile, Ã li goriueleot de, or any other Suitably detection means. Kits using such detection means for detecting the level of 'C
terminus of HSP7Ã)-1ntc'racting Proteinn' (C'1-1IP) in a sample: may also be prepared.
1001151 The specificity and sensitivity of antisense are also harnessed by those of skill in the art for therapeutic uses.
ntisense compotuads have been employed as therapeutic moieties in the treatment of disease states in animals, includin humans. Antisense ohgonuclcotide drugs have been safely and el ietively administered to humans and numerous clinical trials are presently ruaderway. It is thus established that antiseuse compounds can be useful therapeutic n .odalities that can be configured to be useful in treeatrtment regimes for t1 le treatment of cells, tissues and animals, especially humans, 100161 For therapeutics, an animal, preferably a human, suspected of having, a disease or disorder which can be treated by modulating the expression of iC tenninus of HS.P70 ateraetin4g Protein' (CHIP) polyrtnucleotides is treated by administering antisense con-rpaounds in accordance with this in vention.
For example, in one non-limiting enmbÃxlirmre:ut, the methods comprise the step of ad ministering to the animal in need of treatment, a therapeutically effective amount of `C terminus of HSP7O-Interacting Pr`otein' (CHIP
modulator. The 'C terminus of .HSP70-Interacting Protein' (C.11.IP) modulators of the present invention, effectively modulate the activity of the 'C term minus of i t P7ff-lnteractina Protein' (011P) or modulate the expression of the 'C ter minus of 1-I SP 0-interacting Protein' (CHIP) protein, In one embodiment., the activity or expression of iC terminus of HSPP70-interacting Protein' (CHIP) in an animal. is inhibited by about 10 %i, as compared to a control. Preferably, the activity or expression of 'C terminus of .-1SP70-Interacting Protein' (CHIP) in am animal is inhibited by about >f}`' Q. More preferably, the activity or expression.
of 'C terminus Ãrf it'SP"[y-lrrtc.rarctin<mP .Protein' (C1-.HIP) in an animal is inhibited by 5tt`.l or more. Th:tas, d e oligon eric compounds modulate expression of 'C; terminus of HSP"0r1.11teracting Protein' (CHIP.) naR .A by at least 10%, by at least 5f)zr , by at least 25t:
, ~. by at least >f}i C)> by at least 40:fi, by at Ic.ast. hW'.i:, by at.
feast 60f/'0, by at least 70%, by at least 7by at least 80%, by at least 85%, by at least à 0'1',, by at least 95%, by at least 9811-', by at least 99%, or by t.t]Ã)'.%% as compared to a control.
1001171 In one erraboclirraent the are.ti-, ity or expression of `C- terminus of llala"tf inter n:Ãi r Protein' (CHIP) and, or n an animal is increased. by about 10 %'%% as compared to a control, Preferably, the activity or expression of 'C'. terminus of HSP 7[i-lnteraeting Protein' (CHIP) ira arr. animal is increased by about >W,'('9, More preferably, the acts vity- or expression of 'C terminus of 11SP70nteracting Protein` (C.1-1IP) in an animal is increased by 50% or n-im.. Thus, the ol.igo:tueric compounds modulate expression of 'C terminus of IiSP70-Interactinng Protein' (cl-l:1P) tnRNA by at least by at least 50%, by at least 25,` 1, by at least by at least 40 /u, by at least 50%, by at least à dt{'-i,,, by at least ; 770u` f), by at least 71%, by at least Sttca,~, by at least b .y at least 90y %D., by at ].cast 951:f4,, by at least 98%,,, by at least 99%, or by 100% as compared to a control.
[00118) For exaample7 the reduction of the expression of 'C tea-minus of F .SP

7 0-Interacting Protein' (CHIP) may tv pleasured in serum, blood- adipose tissue, liver or any other body :fluid, tissa:ac or organ of the anima]. Preferabil", tile cells contained within said fluids, tissues or organs being analyzed contain a nucleic acid molecule encoding 'C
terminus of l-lS: 7O4nÃeracting Protein' (WHIP) peptides and/or the `C
terminus of l-lSP70-Interartiaayg :Protein' (0-11P) protein itself.
[00119) The compounds of the invention can be utilized in pharmaceutical, compositions by adding an effective amount of a compound to a suitable phala taaaceutical.ly aaccept<able diluent or carrier. Use of the compounds and methods of the invention may. also be useful prophylactically, (. opt ua?atew [00120] Another modification of the oligotaueleotides of the invention involves chemically= linking to the olioaaucleotide one or more moieties or conjugates that enhance the activity,, cellular dist-ibution. or cellular uptake of the oligonucleotide. These moieties or conjugates can include conjugate roups cca ale tl r hound to functional coups such, as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalaators, reporter molecules, poly am3-aines, polyarnides, polyethylene glycols, poly-ethers, groups that enhance the phaartnacodynataaic properties of oligomers, and groups that enhance the phara:aaaaeolkinetic properties ofoli c a at.:rs, Typi.ca.lconttr ~atc groups include cholesterols, lipids, lahospholil id a biz?t n hcnaa ine, folate, phenaanthridine, a aaathracluitnc>ne, acridine, fluor esceins, rhodan ines, coumaria s, and dyes. Groups that enhance the harrnacodynaniic properties, in the conle?et of this invention, include groups that iniproove uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization -%%6th the target nucleic acid.. Groups that enhance the phaarm acoà inetic properties, in the context of this invention, include groups that improve uptake, distribution, .metabolism or excretion of the compounds of the present invention. Representative conjugate groups are disclosed in lntemaÃional Patent: Application No.
PC T US92.09196, filed Oct. 23, 1992, and U.S. Nit, N. 6,28 ,860, v hich are incorporated herein by, reference.
Conjugate moieties include, but are not limited to, lipid moieties such as a cholesterol mo ety, cholic acid, a thioether, e.g.. hexyl-5- tritylthiol, a thioc.holesterol, an aliphatic chain, cg., dodecandiol or undecyl residues. a phospholipid, e.g., di-hexadec.y l-rac-glycerol. or triethylammoniunx I ,2-di-O-hc a atlccyl a as--õl ccrc~-fall slxc?sphssaxate, a polyamine or a polyethylene glycol chain, or Adaraaantaaac acetic acid, a palmityl moiety, or an octadecylaarnine or hexylam no-carhoaayl-oxycholeste.rol trio ety. Oligonucleotides of the invention may also be conjugated to active drug substances, for example, aspirin t <rrfirriu.. phenylbut.aaz ane, ihaaprofen, suprofn, feaabufen, ketopmfen, (S) (+)-pr anoprofen, carprofen, dansyyisarcosinc 2 3,5-triiodolaenzoic acid, fufeaaamic acid, folinic acid, a bc- othiaad.iaa: idc, chlorotl uaaride, a diazepine, i.aadorraethicin, a barbiturate, a cephalosporin, a sul-fa drug, an a autidiaalaetic:, an antibacterial or an asÃntibiotic-[001.2.1] Representative United States patcnts that teach the preparation of such oligonueleoti:des conjugates include, but are not limited to, U.S. Pal. Nos. 4 82$. X79, 4,918,882' 5,218,105-5,525465; 5,541,31-1- 5545,730- 5,552538-51,578,71-1, 5.58Ã,731; 5,580,731. 5591,584, 5,.109,124; 5,1 1 8,802, 5.1.39;015; 5,414,077, 5,486,6Ã33, 5,512439, 608.1146; ,587 04 1, 05 73 5; 4667,025- 4,7627779; 47789,737- 4,824,941 4,135,2 4 3;15:
578,71 S;
5, 4,67 63; ,876,335-4,904,5822; 4,958,9113; 5,082,830 5,11'.96 3; 5, 214,136, 5,Ã82,83Ã3;
5.112,96" 5,214, 1 36; 5,245,022 5'2'54'469;
5,258,506- 5262,536 - 5,292,873; 5-3171,098, 5,371,241, 5,_' }1; 223' 1.6,2 03 - , 5,45 1-463: 5,510,475, 5,512,667; .514.7X5; 5.565,552 5,567,810; 5,574,142, 5,585,481, 5,587.371;
5,59-5,726; 5,597,696 5,599 923' 5.5993 928 and 5,688,941.
I"ormuhilioins 1001221 The compounds of the invention a a:aay also be admixed, encapsulated., conjugated or ?then-Vise associated with other 11-molecules, molecule structures or n-fixtures of compounds, as forexample, liposomes, receptor4tar'gctcd mole ules, 3oral, rectal, topical or other fon. nulat ons, for assisting its uptake., distribution and/or absorption.
Representative l.. nited States patent" that teach the preparation of such uptake, distribution. and/or ahsorption-assisting 1ortnulauons include: but are not limited to, US, Pat. Nos.
5,108,9215,354,",,,443- ,416,01(; 5,A59,127-'. 552L2911-1 5.543,165; 5,547,932-1 5,583020 5,591,721; 4,426,330; 4,534.899; 5.013,556;
5,108.321 5,21-1,804-1 5,227,170.;
5,264,221; 5,356,633, 5,395,619: 5,416,016; 5,417,978; 5,462,854; 5,,469,854;
5,512,295, ?,295, 5,5217,528-, 55.4 259:
5,543,152; 5,556.948, 5,580.5-5; and 5,595,756, each of which is herein incorporated lby reference.
[001.23] Although, the a-ntisense oligonucleoticies do not need to be adnai:nistered in the context of a vector in order to modulate a target expression and/or function, embodiments of the invention relates to expression vector constructs for the expression of aantiscnse oligonucleotid.es, con: prising promoters, hybrid promoter gene sequences and possess a strong constitutive promoter activity, or a promoter activity which can be induced in the desired case.
1.00124] in an embodiment, invention practice involves administering at least one of the formoin. antisense ol.ia;==Ãaraa.tcleotides with a suitable nucleic acid delivery system. In one embodiment,, that system includes a non-viral vector operably linked to the polyrt 1cl.cotide. lsxa:mples of such nonZ%iral vectors include the oligonueleotide a.lone (e.g.
,ant % one or more of SEQ 11) NOS: to I -1) or in combination with a sui.t<
ble. protein, fatale s fieclaabt iclt or lipid tbnnulaation.
10Ã11251 Additionally suitable nucleic acid delivery s -stems include viral vector, typically sequence from at least One of an adenovi_rus, adetatasints-associated virus (AAV), helper-dependent adenovirus, re.trovirus. or hemaggiutinatin irus of .latpan-Iiposome (H4.1) complex Prcferab.lY, the. viral vector comprises as strong eukat= =otic Promoter- operably linked to the polynucleotide e,g,, a cytomegalovira.as tC;MV:l promoter.
1001.26] Additionally preferred vectors include viral vectors, fusion pm ems and chemical conjugates. RetrovIral vectors include Moloney murine leukemia viruses and HTV-based viruses. One preferred. H-1'V -bated r iral vector comprises it least W "o vectors wherein the gag and pot genes are from an H.1\' genome and the my,= gene is from another virus. DNA >i. na1 vectors are preferred, These vectors, include oi vectors such as o t1? ? s . or ? i f?a Vectors' herpc svirus vectors such as a herpes simplex l virus (HSV) cctor, Ade ro` t.r-u-s Vectors and Ad no-associated Virus Vectors.
[{101271 =i1ic autisense compounds of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters. or any other compound ?-1iach_ upon administration to an anima!, including a I nm? an, is capable of providing (directly or indirectly) the l iologica:lly active metabolite or residue thercof [0028) The term "pharmaceutically acceptable salt" refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention. i_e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. For oligonucleotides, preferred examples of pharmaceutically acceptable salts and their uses are litrther described in US, Pat. No.
6,287,860, which is incorporated herein by reference, [0019] The present invention also includes pharmaceutical compositions and foaanaulations that include the antisense compounds of the invention. 111c,. Pharmaceutical compositions of the present invention may be, administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated.
Administration m ty be topical. (including ophthalmic and to mucous mend raries including vaginal and rectal deli erv)), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by anebt:ilizer; intrr:traehea.l, intianasal, epidermal and trap dennaaall, oral or parenteral. Parcnnteral administration includes intra .erious, intnaarterial, subcutaneous, iritra1writc?real or intramuscular injection Or Infusion-, or iutraact.tuial, e.g., itatriiÃ:hecaal or irttr, at entrictilar, admiriistra:tioan.
(001301 For treating tissues in the central, nervous system, administration can be made by, c.g., injection or anlusion into the cerebrospinal fluid. Administration of aantise.nse RNA into cerebrospinal fluid is describe , e.g , its t_ .S. Pat.
App. Put?, No. 2(K)7/01 1 77.72, "`c-lethods for slowing familial lL.S disease progression," incorporated herein by r krence is its c ntire.ty.
100-1.3.1.1 When it is intended that the antisense olisgoaaucleotide of the present intention be administered to cells in the Central nervous system, administration can be with one or more agents capable of promoting penetration of the subject a3iatisense oligonuclootidi across the blood-brain barrier. Injection can be made, e.g., in the entorhinal cortex. or hippocannpus. Delivery ofrieurotrophic factors by administration of all aadeenovirus vector to motor neurons in is?uscle tissue is described in, e.g.. U.S. Pat. No. 6,6 2427, < ~denovi tl-~ectoi-tatctl aitect gene transfer into medullary motor neurons," incorporated herein by reference. Delivery of vectors directly to the brain, e.g.. the striatum, the thalamus, the hippomaampus, or the substaantia nigra, is known in the art and described, e.g., in U.S, pat. No. 6, 756,52D, ".Adenov.irus vectors for the transfer of foreign genes into cells, of the central nervous system particularly in brain,-incorporated lhereirn by reference.. =- idrininistraation can be rapid as by ii jeetion or made over a period oftime as by slow irif ision or administration of slow release formulations.

[00.1.321 The suhjcct ana.tisense oligonudlcotides can also be linked or conjugated titla agents that prkwide desirable phaniraeentical or pharmacod:yna: iic properties. For cxa nple, the stnÃisense oligonw !eoiide. can be, coupled to any suhstancc, kno i In the art to, p: -rnaote penetration 0a transport across the blood--brain barrier., such as an antibody to the tr-a.ns:Ãerrin rt eptor, aand administered by intravenous injection. ".Uhe artt scosc conwo and can be linked with ,a viral vector, for Crr araaple, that makes the antisense compound more efTective and"'or increases the transport of the antrsense compound across the blood-brain barrier. Osmotic blood brain barrier disruption can also be accomplished by, c.g., Infusion of sears includa p. but not limited to, meso crythrito+l, xylitol, D(-;-) galactose, D(i lactose, D1-Ã-' xslosc, dulcitol., myo-iravsitol, 1.(-) fructose, i)(-) mannitol, D(+) glucrrse D(-) a:rabinosc, T-)Ã-_t a.rahitruse:, cellob:ose, L)(+) rnaaltose, W-) rahrrose., U+) r.han-rnosc, T)() mebibiose, i3(-) ribose, adonitol, D(+) arabitol7 1,(1--.) aarabitol, D(+) fucose, L(-) fucose, D(-) lyxose U-+-) lyxose, and U-) lyx se, or amino acids including, but not limited to, glutarn.ine, lysine., argininc, asparaaginc. aspartic acid., cysteir:rc:, glutamic acid, 4lycine, histidine, lcucine, rnetlaionine, plrearylalarlirrc, praline, serine, thr.onine, tyrosine., valine, and. taurine.. Methods and materials for enhancing blood brain barrier penetration are described, e.g., in L:. S..Patent No. 4;866,042, a Method. for the dei:rrcry of genetic material across the blood brain barrier," 6,294,52M 520, material for passage through the blood-brain barrier," and 6,936,589, "Parenterail delivery systems,"all. incorporated herein by reference in their entirety.
[001331 The subject antise.aase compounds may be admixed, encaapsulated, conjugated or alter ; ise associated with otlrcr molecules, molecule structures or mixtures of compounds, fir es ample, l iposo:naes, rr ce pto:rktarge to at molecules, oral, rectal, topical or other fomiulationr, for- assisting in uptake, distribution and/or absorption. For example, cationic lipids may be included in the formulation to facilitate oligonucleotide uptake, One such composition shown to facilitate uptake is LIPOFECTIN (available from. (II CO BRL, Bethesda. MD).
[001341 Olivgonueleot.ides with at ].east one '2'-O-rmnethoxyethy1 modification are believed to be particularly useful for oral administration, Pharmaceutical compositions and formulations for topical administration may, include transderrraal patches, onaÃrtaents,, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pbarmaceaatic:al carriers, aqueous. p,~-rwder or oily bases, thickeners and the like r-my be necessary or desirable. Coated condoms, gloves and the like may also be u-seful.
1001351 The pharmaceutical formulations of the present invention, 'u/hich.
r:naay conveniently be presented in unit dosage fbnn, may be prepared according to con ventionaal techniques well known in à ie pharmaceutical industry, Such techniques include the. step of bringing into association the active ingredients with the Pharmaceutical carrier(s) or exeipierr.t(s). In g-ene ral, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid. earners or finely divided solid carriers or both, and then, :if necessary, shaping the product.
1001361 The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syraps, soft gals, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueoars, non-aqueous or mixed. media.

Aqueous susperlsicans x nay further contain substances that increase the viscosity of the suspension including, for e aatatple. sodicttta earhox ntethylccilulose, so bitol and ,"or clextran. The suspension may also coaataiat stahilizcrs.
[001371 Pharmaceutical coaaiposition, of the present rove ttion inehadc., but are not limited to, soi utions, cmulsiou,, foams and l posome-containitu formulations. The pharmaceutical conapositio:n:s and formulations of the present invention may comprise one or more 7 netr ation Cna}:haiacers, ea Tiers, exc pie:ats or otl`aer active or inactive irigredicrim [00138] Emulsions are typically heterogeneous systems of one liquid dispersed in another in the .fbran of droplets usually exceed new t).1 gm in diaaaneter, Emulsions may contain additional components in addition to the dispersed phases, and the active drug that may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. '~ficroentaalsions are included aas aan embodiment of the present invention. Emulsions and their uses are well known in the art and an farther described in US, Pat. No. 6,2$7.$f0.
1001391 Fora- ulations of the present invention include lipcasoanal for ulations. As used. in. the present inventi:on, the terra "hposome" means a v esicle. composed of amphiplhi.lie lipids aarrangged in a spherical bilayer or bilaayers. Liposomes are a.unailaattellar or :mialtilaagaellar vesicle which have a ntcatibrarae foamed from a lilaolala:ilie material and an aqueous interior that contains the composition to be delivered, Cationic liposotnes are positively charged liposonies that are belie ~ved to interact with negatively charged DNA molecules to form astable-complex. L.iposomes that are pli-:sensitize or negativel char4ed are believed to entrap DNA rather that complex With It, -Both eationic and noaacationic liposolnes have been used to deliver DNA to cells.
[{1{1140] Litxasomes also include "sterically stabilize 1" lipasomes. a term which, as used herein, refers to liposota.es comprising or e or more specialized lipids. When incorporates into liposornces., these specialized lipids resale in liposomtes with enhanced Circulation lifeti.n:mes relative to liposoÃneslackiu such specials ed lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle fttrtaaita lipid portion of the liposome comprises one or more gxlycolipids or is derivatized with one or moan hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. Liposomes and their uses are f .trtlter described in U. . Pat, No 6,287,860, 10Ã14.1.1 The pharmaceutical. formulations and compositions of the Present invention. may also include suafiactants. The use of surfactants in drug products, formulations and in emulsions is ".-ell known in the art- Saar ict ants and their uses are further described in kJ. i. Mat. No, 6,287,860. which is incorporated herein by reference.
001421 lit one embodiment, the present: invention employs -various penetration enhancers to effect thee, efficient de .iveryy of nucleic acids, particularly oli4gonuc. eoti des. In addition to aiding the. if .tasion of non- ilto; lilac drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs. Pe titration enhancers :may he classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, c.helatinf agents, and non-chelating norisurfactants. Penetration enhancers and their uses are further described in L.4. Pat. No. 6,287,860. which is incorporated herein by reference.

[00.1.43] One ofskill in the art ,. rill n::cogn c that fb inlations are routinely designed according to their intended use, .c, mute of admi nistra do al, 1011441 Pi-eferred fonaraalations for topical administration include those in which the oligonucleotides of the invention are in adraaiztrare With a, topical delivery arc rat such as lipids, liposor acs, fat: aacids, tatty acid esters, steroids. cl c-l.aatiaa4 agents and su rf acÃatits. Preferred lipids and liposonies inelaade neutral (e.g. diol oyl-phosplx atidyl DOPE ctliallolasnine, clirray ristcrvlplac splaaatidvl choliaac 0 ^iPC, distearolvphosphatidyl choliare) negative (e.g. dirnyristoylphosphaatidvl glyrcera l DMPG and cationic w. g. cli:vleoyfte.taaaaarctlaylaa3aiaac+l a p l 1 1: P and clitala,ca l l lroa hatiti idyl ethanolam'Me DOTMA).
[001.45] For topical or other a administration, oligon ucleotides of the invention. may he encapsulated within l po acmes or may forma complexes thereto, in particular to cationic liposornes.
Alternatively, oligonucleoti:des .nmv be complexed to lipids, in paaticular to cationic lipids. Preferred fatty acids and esters, pharmaceutically acceptable salts thereof; and their use are farther described in U.S. Pat. No, 6,257,860 [001.46] Compositions and fbramulatioars for oral administration include powders or granules, is ic.ropaarnculates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, ~. e.l capsules, cachets, tablets or nairtitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Preferred oral foraanalations no those in '" hich oligonucleotides of the ni ention are administered in con'juaunction with one or more penetration enhancers surfactants and chclators. Preferred surfactants include fatà acids and/or esters or salts thereof, bile acids and/or salts thereof. Preferred bile acids.'salts and fatty acids and their asses are .further described in. U.S. Pat.
No, 6,28 7x,860, which is incorporated, herein by refi rence. Also preferred are combinations of penetration enhancers, for example., fatty acids/salts in combination with bile texas salts. A
particularly preferred combination is the sodizanr salt of laurie acid, capric acid and l:_ .DCA. Further penetration enhancers, include l cal os c tla le_tar - -lxaata yl ether, polyoxy>cthvlene-20-ceÃyl ether. Oligonaacleotides of the invention may be delivered orally, in gran alar fbrria including sprayed dried particles, or corriple.xed to fbrnr m icro or nanoparticles, Olig onucleotidc conaplexi:ng agents and their uses are further described in U,S. flat. No, 6.287,860, which is incorporated herein by refbnmce.
[001.47] Compositions and fomuarlations for parcnteral, intr'athecal or inaraven ricarlar administation may include sterile aqueous solutions that mayr also contain buffers, diluents and other suitable additives such aas., but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or cxcipie.nts.
[ 0II481 Certain embodiments of the invention provide pharmaceutical compositions containing one or more oligonaerie Cora prsaaards and one or more other chemotherapeutic agents that function. by a non-antisense r.a3echanism.
Examples of such chemotherapeutic agents include but are not limited to cancer chemotherapeutic drugs such as daurnorubicin, daunontvcin, dactinonrycin, doaortibicin, epiruhicin, idarubicin, csorrabicin, bleoraaycirn, niaafos~fWnide:, ifbsl~aar itlc, cytosiare as abinoside, bischloroethyl- rri:trosntcar, busu.lfart, raaitonrvcin C, actinoatrycin D, ralitirraaaayc:iar,.
prednisone 1a ,drerxypro steroaae testosterone, taarmoxifin, daciffivine, proc<irbazine. lar,_xanae.ttrsrlra claa3ai:t}c=

aataaaattiayiancianaine naitoxazatrone, amsacrine., clalurtatnl ncii ntcth l . icl c is itac~sttr ~t, nitre ~t z ta~ttrcl , anciphalan, cyclophusphanu , 6-rnercaptoput-ia1c, 6-drio;;Fuamnc, cytar4abinc, 5- azacytidinc, h d oxyurc;a, eax rcolbra iycin, f hydrnx vps;:mxycyc.lea-pliosl-alaoa`aaniclc, 5-flu rouracil (5 F ), 5 f1uorod.oxyuaidinc (5 Ffr.d.R), a1)Cthot rexatC (\1T ) colchicine, taxol., t incr tine., vinbl tiitc, eÃoposklc (VP-11), triretrexate, ia'inotecan, topotecan, genicitabine, teniposide, cisplatin and diethylstilbestrol (DES). 'Viieaa used with the compounds of the invention, such chemotherapeutic zasnents may be used individually (e.g., 5 -FU and oligonucleoÃtdet, sequentially (c.&' 5-RJ and oligonucleotide fib` a period of time fiallowed lay. INITX and oligonuckotide3, or in combination with one or more other such the otherapeutic agents (e.g., :5-F1, , MTX and oligonucleotide, or 5-Ri, radiotherapy. and oligonuclesot:idc). Anti-inflammatory drugs, including, but not limited to nonsteroidal anti-:inflammatory drugs and, corti.costeroids, and antiviral dugs, including but not limited to ribivirin: v.idarabine, acyciov:ir and ganciÃ..lovir, may also be combined in compositions of the invention, Cora-rl inactions caf ttaatisensc: compounds and a tlrca non~araÃiscuse druggs are, also within the scope of this invention. Two or more combined compounds may be used together or sequentially.
[001491 In another related embodiment, compositions of the invention may contain. one or more antisense compounds, particularly olig:oaaucieotides, targeted to a first nucleic acid and one or more additional antisense co ripounds targeted to a second nucleic acid target. For exaanple, the first target may be a particular anÃrsense sequence of `C terminus of HSP704nterarti:ng Protein' (CHIT'), and the second t artget may be a region from another' nucicotide sequence..
Alternatively, compositions of the invention may contain two or mow antiscnse compounds targeted to different regions of the same 'C` terminus of I-SP7O--Interacting Protein' (Cl-i l?) nucleic acid target. Numerous examples of antisense compounds are illustrated herein and others may be selected from among suitable compounds known in the art. Two or more combined compounds may be used together or sequentially, 1'001.501 The formulation of therapeutic compositions and their subsequent administration (dosing) is believed to be within the skill of those in the art. 'Dosing is dependent on severity and responsiveness of (lie disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal. dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleoddes, and can generally be estimated based on ..C50s found to be effective in vilro and in v vo animal models. In general, dosage is from 0.0 .1 lt`e to 100 g per kg of body weight, and may he given once or more daily, Zv eekl y, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the an can easily estimate repetition mates for dosing based on measured residence tinges and concentrations of the drug in bodily fluids or tissues..] ollo i.a3;~ successful treatment, it may be desirable to have the psttiertt undergo maintenance therapy to prevent the recurrence of the disease state.

wherein the t? FS f3.F7Eif 1LE?t9C i is administered in maintenance doses, :FaF?pIFIg from 0,01 ig to 100 g pe q? of body weight, once or more daily, to otice_ e ve_ 20 years.
[001511 In embodiments, a patient is treated,e-ith a dosage of drag that is, at bast about 1, at least about 2, at least about 3, at least about 4. at bast about 5 at .least about 6, at least about 7, at least about S at least about 9, at least about 1.0, at least about 15, at least about ?0, at least about 2?. at least about 30, at least about 35, at least about 40, it least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100 nib;%k body x4e:i liÃ. Certain itije ted'. dosages of aritisctise c~ligon clet~tides are described, e, g., in US, Pat, 'dc.
7.563,884, "Antisense modulation of .PTT. I B expression," incorporated herein by reference in its entirety..
1001521 While various embodiments of the present invention have been described above, it should be understood that they have been presented by Ray of example only, and not limitation. Nuincrous changes to the disclosed embodiments can be made in accordance with the disclosure herein without departing from the spirit or scope of the invention. Thus, the breadth and scope of the present invention should not be limited by any of the above described einbodiÃiienits.

1001531 All documents :mentioned herein are incorporated hereiai, by reference. All publications and patent documents cited in this application are incorporated by reference for all purposes to the same extent as if each individual publicatimi or patent document were so individually denote. By t leir citation of various references in this docuinc.m.
Applicants do not admit any particular reference is " prior art" to their invention. Embodiments of i:tii entive compositions and methods are illustrated in the following examples.
EXAMPLES
1001541 The following non=lirniÃing Examples serve to ill.F:tsÃratc selected embodirients of the in eritisfn. It will be appreciated that variations in proportions and ailternatives in elements of the components shown will be apparent to those skilled in the art and are within the scope of embodiments of the present invention.

k n.-un le I: L)eecign ofanfir nse T1fl`3FT?IIC.'leollder spec fT /br 1:d nucleic acid molecule anti nee to a fC l nmmnus of /I.SP O-.I ictat't ag Pi otein ` (CH/'i't erta r7 e bc:,ist Strand of f , fo?=rni mrn n/ /S1'10-irnkiac'f/rag Protein' (C."711P) poly arc~ieoticfe [00 .551 As indicated above the term "ol.lgonuckotide specific for" oor "oiigonticleotide targets' refers to an oligonucpeotide having a sequence Ã.i1 capable of forming a stable complex with a portion of the targeted e.ne, or ill capable of forming a stable duplex with a portion of an mRN A transcript of the targeted g ne.
[00-1.561 Selection of appropriate cilaatoaiaiele.oticles is facilitated by F:rsin computer pro, rams that automatically align nucleic acid sequences and indicate regions of identity or homology-. Such programs are used to compare nucleic acid sequences obtained, for cxaiiap?la., by searching databases such as Cietil3arik or by sà quaiuing PC'R products.
Comparison of nucleic acid s quences from a range of species allows the selection of nucleic acid sequences that display an appropriate degree of identity between species, In the case of genes that have not been sequenced. Southern blots are performed. to allow a determination of the degree of identity between genes in target species and other species, By perfbrmiantg Southern blots at varying degii es of stringency, as is well known in the art, it is possible to obtain <
approxiniaate aalaeaasUre of ideaatity. These procedures allow the. selection ofoligonuclcotidcs that exhibit a high degree of compleancata:rity to tar ,.wt nucleic acid sequences in a subject to be controlled and a lower degrec of conaplenacaataarit , to corresponding nucleic. acid S q,;imccs in other species.. One skilled in the art will realize that there is considerable latitude in selecting appa opriaate re ;icons of 4 lie's for use in the present invention. 1001571 L ,n aaritisense compound is specifically laybridizaable" when binding of the compound to the target nucleic acid interferes ww1it.h the normal function of the target nucleic acid to cause a modulation of -uncti on and.,or aact vity, and there is a sufficient degree of c o~aapleaaaeaata:rit to avoid :non-opts f c binding of the antisensc compound to norn-Target nucleic acid sequences under conditions in which specific binding is desiaed, a.e., under physiological conditions in the case of in w iw. o assays or therapeutic treaatment, and under conditions in which assays are perfbn`ned in the case of in vitro assays 1001581 The hybridization properties of the olagonucleotides described herein can be deteraamined by one or more it) vitro assays as known in the an. For example, the properties of the ol:igonucleotides described herein can be obtained by determination of binding strenfall between the tauget:.naatural autisense and a potential drug, .nmmlccules using melting curve assay.
1001.591 The binding strength bet. a een the target natural aantisense and a potential drug noolecule lolec ul ) can be estimated using any of the established methods of measuring the strength of intermolecular interactions., for example, a melting curve ZassaaV.
1001601 Melting curve assay determines the temperature at which a rapid transition from double-stranded to single-stranded. conÃbrmaati:on occurs fbr the natural a3aat sense/ lolecule complex, 'flits temperature is widely accepted as a reliable .measur of the interaction strength between the ÃA o molecules.
1001611 A melting cu ve assay can be performed using a c 3 \ A. copy of the actual n atural antisen ae .N.A molecule or a synthetic DNA or RNA nucleotide co responding to the binding site of the Molecule. Multiple kits containing all necessary reagents to perform this assay are aw, ailaable (e.g.Applied.
Baosystems inc. MeltD)octor kit). These kits include a suitable buffer solution containing one of the double strand DNA (dsDNA) binding dyes (such as AM HR-`,\1 dyes, SYBR Green, SYTO, etc,). The properties of the dsDNA dyes are such that they emit almost no fluorescence in free form, but are highly fluorescent when bound to dsDl A, 10Ã1621 To perform the assay the ei)NA or a corresponding oli~tonlae;.leoticlc. ire nailed with Molecule in concentrations defined by the Particular nmanufaacturer's protocols. The mixture is heated to 95 t:_': to dissociate all p:re-foaxaaed ds DNA complexes, then slowly cooled to room temperature or other lower temperature defined by. the kit manufacturer to allow the DNA molecules to anneal. The newly= formed complexes are theca slowly heated to 94; 'C
with simultaneous continuous collection of data on the amount of fluorescence that is produced by the reaction, The fluorescence intensity is inversely proportional to the amounts of d,-,DNA
present in. the reiction. The data can be collcctc d using a real time PCR instrua:nent con pa.tible with the kit (e.g.A131.'s StepOne Plus Real Time PCR. System or L.ightTyper instrtuncrrt, Roche 'Diargrrostics, Lewes, UK), [001631 Melting peaks are constructed by Plotting the negative dca'ivative of fluorescence with respect to temperature #;-ai(l laaoa .suerre );d'T') on the V-axis) against temperature tx-axis}
ursine appropriate soil .vare (for example Li4ghff' q)er (Roche) or SDS Dissociation Curve, A131). The data is analyzed to identify the temperature of the rapid transition from dsDNA complex to single strand nmolecules. This temperature is called Tm and is directly proportional to the strength of interaction between the two molecules, Typically, Tm will exceed 40 C.
l ~rrrr r!c 2 l Frzc elation f Cill=' pot;'t; trc'ieoudt r f t?[7ti3'l 'ti o /'!L' t? / cc/L' S#'If i anfisense oii1 tknn e e, 100164 La-75 cells were grown in RPMI (ATC. C.: 430-2001 ) 1-109 t, VI3 frcrricrllrrr steel torrryc rr at 37C and 5%, CO. One day before the experiment the cells were rcplaatcd at the density of 15 ,.,. It., nil into 6 well plates and incubated at 37 C and 5% CO-- On, the day of the experiment the receding. ill the 6 well plates was changed to fresh <nrowtla media. All aantiseaase oligonucleotides were diluted to the concentration of 2Ã3 pkl. Two 1-t1 of this solution was incubated with 400 gl of Opti-MENI media (Gibco cat: 31985-070) and 4 ld of Lipofcctarreioe 2[101) (hivitrogen cat#
11668019) at rooare temperature for 20 eerier and pplied to each well of the 6 well plates with Z R75-1 cells. A Sir? ilar mixture including 2 td of water instead of the oligonucleotide solution was used. for the asrock-transfected controls, After 3-18 h of incubation at 37'C and 5 % CO the media was changed to fresh growth. media. 48 l). after addition of xurtiscrrse otigonucleotides the media was removed and RNA was extracted from the cells usi-no SV Total RNA
Isolation System from Pronrcgõa (cat Z31.05) or RNeasy Total RNA Isolation kit from Qiagen (cat.' 74181) following the rrraanufacturers' instructions. 600 ag of RNA was added to the reverse transcription reaction pertbrnred using Verso eD.NA kit :from Thermo Scientific (caaViAB14531B) or High Capacity eDNA
Reverse 1'r inscription limit (cat# 4368$13) as described in the manufacturer's protocol. The eDNA from this reverse transcription reaction was used to Ivor itor gene expression by real time PCR using ABI TacIrnaan Gene Expression Mix (cat 36951f0) and primers,/probes designed by ABl (Applied. Biosysterers 'I'zrela:rmatn Gene .h xpression Assay:
HsO0195 300 real by AApplie d Biosti,stcrr-is 1rrc., Foster City CA.). The follo~~ at .l'CR cycle was used: 50'C for 2 niin, 95 C_:
for 10 mm . 40 cycles of (95 C for 15 seconds, 60'C' for I min) using Mx4000 thermal cycler (SStratagene)..Fold chg.
ange in gene, expression after treatment with antisense oligonucleotides was calculated based on the difference in 185-normalized dC't values between treated and nmock-trans 'eeted samples.
Result, 1001651 Real time PC R results show that the levels ofCT-1IP1 mRNA. in ZR 75-1 cells me significantly increased 4811 after treatment with two of the siRN.A designed to :X088969,1 (C1: Rõ03114 and CUR-0316). Three phospho.rotliioate antiserrse oligos designed to I-ls,5337715 (CUR-0879 - CUR-0883) also significantly up regulated CHIP1 nmR A
fe els (Fig U.

[00.1661 Real time PCR results show that the levels of CHIP1 rRNA in ZR.75-1 cells are signit=i:cantly increased 4S lx after treatment With orae of the hosphorotlhio ite antiscrisc oligos des fined to Hs.S337715 (CUR-08(8.0) (Fig. 2).
0Ã31671 Although the invention has been. Illustrated and described with respect. to one or more implementations.
s quiv.alent altercations and modifications Will. occur to others skilled in the art upon the reading and understanding of this specification and the annexed drawings. In addition, while a particular feature of the invention may have been disclosed with respect to only one of several a mplenicrn.tation:s. such feature may be combined with one or .more other features of the. other r plementtr.tions as may be desired rrrd advantageous for any given or particular application.
[001.681 The Abstract of the disclosure will allow the reader to quickly ascertain the nature of the technical. disclosure.
It is submitted with the a nderstan.ding that it will not be used to interpret or limit the scope. or meaning of the following Claims,

Claims (37)

1. A method of modulating a function of and/or the expression of a 'C terminus of HSP70-Interacting Protein' (CHIP) polynucleotide in patient cells or tissues in vivo or in vitro comprising:
contacting said cells or tissues with at least one antisense oligonucleotide 5 to 30 nucleotides in length wherein said at least one oligonucleotide has at least 50% sequence identity to a reverse complement of a polynucleotide comprising 5 to 30 consecutive nucleotides within nucleotides 1 to 2074 of SEQ ID NO: 2 and 1 to 1237 of SEQ ID NO: 3; thereby modulating a function of and/or the expression of the 'C terminus of HSP70-Interacting Protein' (CHIP) polynucleotide in patient cells or tissues in vivo or in vitro.

2. A method of modulating a function of and/or the expression of a 'C terminus of HSP70-Interacting Protein' (CHIP) polynucleotide in patient cells or tissues in vivo or in vitro comprising:
contacting said cells or tissues with at least one antisense oligonucleotide 5 to 30 nucleotides in length wherein said at least one oligonucleotide has at least 50% sequence identity to a reverse complement of a natural antisense of a 'C terminus of HSP70-Interacting Protein' (CHIP) polynucleotide; thereby modulating a function of and/or the expression of the 'C terminus of HSP70-Interacting Protein' (CHIP) polynucleotide in patient cells or tissues in vivo or in vitro.

3. A method of modulating a function of and/or the expression of a 'C terminus of HSP70-Interacting Protein' (CHIP) polynucleotide in patient cells or tissues in vivo or in vitro comprising:
contacting said cells or tissues with at least one antisense oligonucleotide 5 to 30 nucleotides in length wherein said oligonucleotide has at least 50% sequence identity to an antisense oligonucleotide to the 'C terminus of HSP70-Interacting Protein' (CHIP) polynucleotide; thereby modulating a function of and/or the expression of the 'C terminus of HSP70-Interacting Protein' (CHIP) polynucleotide in patient cells or tissues in vivo or in vitro.

4. A method of modulating a function of and/or the expression of a 'C terminus of HSP70-Interacting Protein' (CHIP) polynucleotide in patient cells or tissues in vivo or in vitro comprising:
contacting said cells or tissues with at least one antisense oligonucleotide that targets a region of a natural antisense oligonucleotide of the 'C terminus of HSP70-Interacting Protein' (CHIP) polynucleotide; thereby modulating a function of and/or the expression of the 'C terminus of HSP70-Interacting Protein' (CHIP) polynucleotide in patient cells or tissues in vivo or in vitro.

5. The method of claim 4, wherein a function of and/or the expression of the 'C terminus of HSP70-Interacting Protein' (CHIP) is increased in vivo or in vitro with respect to a control.

6. The method of claim 4, wherein the at least one antisense oligonucleotide targets a natural antisense sequence of a 'C terminus of HSP70-Interacting Protein' (CHIP) polynucleotide.

7. The method of claim 4, wherein the at least one antisense oligonucleotide targets a nucleic acid sequence comprising coding and/or non-coding nucleic acid sequences of a 'C terminus of HSP70-Interacting Protein' (CHIP) polynucleotide.

8. The method of claim 4, wherein the at least one antisense oligonucleotide targets overlapping and/or non-overlapping sequences of a 'C terminus of HSP70-Interacting Protein' (CHIP) polynucleotide.

9. The method of claim 4, wherein the at least one antisense oligonucleotide comprises one or more modifications selected from: at least one modified sugar moiety, at least one modified internucleoside linkage, at least one modified nucleotide, and combinations thereof.

10. The method of claim 9, wherein the one or more modifications comprise at least one modified sugar moiety selected from: a 2'-O-methoxyethyl modified sugar moiety, a 2'-methoxy modified sugar moiety, a 2'-O-alkyl modified sugar moiety, a bicyclic sugar moiety, and combinations thereof.

11. The method of claim 9, wherein the one or more modifications comprise at least one modified internucleoside linkage selected from: a phosphorothioate, 2'- Omethoxyethyl (MOE), 2'-fluoro, alkylphosphonate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate, carboxymethyl ester, and combinations thereof.

12. The method of claim 9, wherein the one or more modifications comprise at least one modified nucleotide selected from: a peptide nucleic acid (PNA), a locked nucleic acid (LNA), an arabino-nucleic acid (FANA), an analogue, a derivative, and combinations thereof.

13. The method of claim 1, wherein the at least one oligonucleotide comprises at least one oligonucleotide sequences set forth as SEQ ID NOS: 4 to 11.

14. A method of modulating a function of and/or the expression of a 'C
terminus of HSP70-Interacting Protein' (CHIP) gene in mammalian cells or tissues in vivo or in vitro comprising:
contacting said cells or tissues with at least one short interfering RNA
(siRNA) oligonucleotide 5 to 30 nucleotides in length, said at least one siRNA oligonucleotide being specific for an antisense polynucleotide of a 'C terminus of HSP70-Interacting Protein' (CHIP) polynucleotide, wherein said at least one siRNA
oligonucleotide has at least 50% sequence identity to a complementary sequence of at least about five consecutive nucleic acids of the antisense and/or sense nucleic acid molecule of the 'C terminus of HSP70-Interacting Protein' (CHIP) polynucleotide; and, modulating a function of and/or the expression of 'C terminus of HSP70-Interacting Protein' (CHIP) in mammalian cells or tissues in vivo or in vitro.

15. The method of claim 14, wherein said oligonucleotide has at least 80%
sequence identity to a sequence of at least about five consecutive nucleic acids that is complementary to the antisense and/or sense nucleic acid molecule of the 'C terminus of HSP70-Interacting Protein' (CHIP) polynucleotide.

16. A method of modulating a function of and/or the expression of 'C terminus of HSP70-Interacting Protein' (CHIP) in mammalian cells or tissues in vivo or in vitro comprising:

contacting said cells or tissues with at least one antisense oligonucleotide of about 5 to 30 nucleotides in length specific for noncoding and/or coding sequences of a sense and/or natural antisense strand of a 'C
terminus of HSP70-Interacting Protein' (CHIP) polynucleotide wherein said at least one antisense oligonucleotide has at least 50% sequence identity to at least one nucleic acid sequence set forth as SEQ ID
NOS: 1 to 3; and, modulating the function and/or expression of the 'C terminus of HSP70-Interacting Protein' (CHIP) in mammalian cells or tissues in vivo or in vitro.

17. A synthetic, modified oligonucleotide comprising at least one modification wherein the at least one modification is selected from: at least one modified sugar moiety; at least one modified internucleotide linkage; at least one modified nucleotide, and combinations thereof; wherein said oligonucleotide is an antisense compound which hybridizes to and modulates the function and/or expression of a "C terminus of HSP70-Interacting Protein' (CHIP) gene in vivo or in vitro as compared to a normal control.

18. The oligonucleotide of claim 17, wherein the at least one modification comprises an internucleotide linkage selected from the group consisting of: phosphorothioate, alkylphosphonate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate, carboxymethyl ester, and combinations thereof.

19. The oligonucleotide of claim 17, wherein said oligonucleotide comprises at least one phosphorothioate internucleotide linkage.

20. The oligonucleotide of claim 17, wherein said oligonucleotide comprises a backbone of phosphorothioate internucleotide linkages.

21. The oligonucleotide of claim 17, wherein the oligonucleotide comprises at least one modified nucleotide, said modified nucleotide selected from: a peptide nucleic acid, a locked nucleic acid (LNA), analogue, derivative, and a combination thereof.

22. The oligonucleotide of claim 17, wherein the oligonucleotide comprises a plurality of modifications, wherein said modifications comprise modified nucleotides selected from:
phosphorothioate, alkylphosphonate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate, carboxymethyl ester, and a combination thereof.

23. The oligonucleotide of claim 17, wherein the oligonucleotide comprises a plurality of modifications, wherein said modifications comprise modified nucleotides selected from: peptide nucleic acids, locked nucleic acids (LNA), analogues, derivatives, and a combination thereof.

24. The oligonucleotide of claim 17, wherein the oligonucleotide comprises at least one modified sugar moiety selected from: a 2'-O-methoxyethyl modified sugar moiety, a 2'-methoxy modified sugar moiety, a 2'-O-alkyl modified sugar moiety, a bicyclic sugar moiety, and a combination thereof.

25. The oligonucleotide of claim 17, wherein the oligonucleotide comprises a plurality of modifications, wherein said modifications comprise modified sugar moieties selected from: a 2-O-methoxyethyl modified sugar moiety, a 2-methoxy modified sugar moiety, a 2'-O-alkyl modified sugar moiety, a bicyclic sugar moiety, and a combination thereof.

26. The oligonucleotide of claim 17, wherein the oligonucleotide is of at least about 5 to 30 nucleotides in length and hybridizes to an antisense and/or sense strand of a 'C terminus of HSP70-Interacting Protein' (CHIP) polynucleotide wherein said oligonucleotide has at least about 20% sequence identity to a complementary sequence of at least about five consecutive nucleic acids of the antisense and/or sense coding and/or noncoding nucleic acid sequences of the 'C terminus of HSP70-Interacting Protein' (CHIP) polynucleotide.

27. The oligonucleotide of claim 17, wherein the oligonucleotide has at least about 80% sequence identity to a complementary sequence of at least about five consecutive nucleic acids of the antisense and/or sense coding and/or noncoding nucleic acid sequence of the 'C terminus of HSP70-Interacting Protein* (CHIP) polynucleotide.

28. The oligonucleotide of claim 17, wherein said oligonucleotide hybridizes to and modulates expression and/or function of at least one 'C terminus of HSP70-Interacting Protein' (CHIP) polynucleotide in vivo or in vitro, as compared to a normal control.

29. The oligonucleotide of claim 17, wherein the oligonucleotide comprises the sequences set forth as SEQ ID
NOS: 4 to 11.

30. A composition comprising one or more oligonucleotides specific for one or more 'C terminus of HSP70-Interacting Protein' (CHIP) polynucleotides, said polynucleotides comprising antisense sequences, complementary sequences, alleles, homologs, isoforms, variants, derivatives, mutants, fragments, or combinations thereof.

31. The composition of claim 30, wherein the oligonucleotides have at least about 40% sequence identity as compared to any one of the nucleotide sequences set forth as SEQ ID NOS: 4 to 11.

32. The composition of claim 30, wherein the oligonucleotides comprise nucleotide sequences set forth as SEQ ID
NOS: 4 to 11.

33. The composition of claim 32, wherein the oligonucleotides set forth as SEQ
ID NOS: 4 to 11 comprise one or more modifications or substitutions.

34. The composition of claim 33, wherein the one or more modifications are selected from: phosphorothioate, methylphosphonate, peptide nucleic acid, locked nucleic acid (LNA) molecules, and combinations thereof.

35. A method of preventing or treating a disease associated with at least one 'C terminus of HSP70-Interacting Protein' (CHIP) polynucleotide and/or at least one encoded product thereof, comprising:

administering to a patient a therapeutically effective dose of at least one antisense oligonucleotide that binds to a natural antisense sequence of said at least one 'C terminus of HSP70-Interacting Protein' (CHIP) polynucleotide and modulates expression of said at least one 'C terminus of HSP70-Interacting Protein' (CHIP) polynucleotide; thereby preventing or treating the disease associated with the at least one 'C terminus of HSP70-Interacting Protein' (CHIP) polynucleotide and/or at least one encoded product thereof.

36. The method of claim 35, wherein a disease associated with the at least one 'C terminus of HSP70-Interacting Protein' (CHIP) polynucleotide is selected from: a disease or disorder associated with abnormal function and/or expression of CHIP, a neurological disease or disorder, a neuroinflammatory disease or disorder, an autoimmune disease or disorder, obesity, diabetes, atherosclerosis, cancer, AIDS, atherosclerotic plaque, glioblastoma, a disease or disorder associated with amyloid deposition, choriocarcinoma, astrocytoma, amyloidosis, hyperlipidemia, neurodegeneration, neoplastic transformation, AIDS, metastasis, myocardial infarction, pulmonary fibrosis, inflammation, glioma, a vascular disease or disorder, cell damage, Nonsmall cell lung carcinoma (NSCLCs), hypercholesterolemia, liposarcoma, a cardiovascular disease or disorder, immunodeficiency, glomerulonephritis, venous thrombosis, a pathological process, cellular stress, a disease or disorder associated with oxidative stress, a polyglutamine disease, a disease or disorder associated with impaired functioning of aggresome pathway, a disease, disorder or condition associated with aging, a disease or disorder associated with aggregation of misfolded of proteins, a disease or disorder associated with misfolding and/or aggregation of alpha-synuclein; and a disease or disorder associated with beta-amyloid (Abeta) metabolism.

37. A method of identifying and selecting at least one oligonucleotide for in vivo administration comprising:
selecting a target polynucleotide associated with a disease state; identifying at least one oligonucleotide comprising at least five consecutive nucleotides which are complementary to the selected target polynucleotide or to a polynucleotide that is antisense to the selected target polynucleotide; measuring the thermal melting point of a hybrid of an antisense oligonucleotide and the target polynucleotide or the polynucleotide that is antisense to the selected target polynucleotide under stringent hybridization conditions; and selecting at least one oligonucleotide for in vivo administration based on the information obtained.