USOO66641 05B1 (12) United States Patent (10) Patent No.: US 6,664,105 B1 Pecker et al. (45) Date of Patent: *Dec. 16, 2003

(54) POLYNUCLEOTIDE ENCODING A (58) Field of Search ...... 536/23.1, 24.1, POLYPEPTIDE HAVING HEPARANASE 536/24.5; 514/44; 435/320.1, 455; 530/350 ACTIVITY AND EXPRESSION OF SAME IN GENETICALLY MODIFIED CELLS (56) References Cited (75) Inventors: Iris Pecker, Rishon le Zion (IL); Israel U.S. PATENT DOCUMENTS Vlodavsky, Mevaseret Zion (IL); Elena 5,968,822 A * 10/1999 Pecker et al...... 435/325 Feinstein, Rehovot (IL) 6,177.545 B1 * 1/2001 Pecker et al...... 530/387.3 (73) Assignees: Insight Strategy & Marketing Ltd., OTHER PUBLICATIONS Rehovot (IL); Hadasit Medical Sudhir Agrawal, AntiSense oligonucleotides: towards clini Research Services and Development cal trials, TIBTECH, vol. 14, Oct. 1996, pp. 376-387.* Ltd., Jerusalem (IL) Alan M. Gewirtz et al., Facilitating oligonucleotide delivery: Helping antisense deliver on its promise. PROC. NATL. (*) Notice: Subject to any disclaimer, the term of this ACAD SCI. USA, vol. 93, pp. 3161-3163 1996.* patent is extended or adjusted under 35 Douglas W. Green et al., AntiSense Oligonucleotides: An U.S.C. 154(b) by 0 days. Evolving Technology for the Modulation of Gene Expres sion in Human Disease, J. AM. COLL. SURG., pp. 93-105 This patent is Subject to a terminal dis 2000.* claimer. * cited by examiner (21) Appl. No.: 09/435,739 Primary Examiner Ram R. Shukla (22) Filed: Nov. 8, 1999 ASSistant Examiner Joe Zara (74) Attorney, Agent, or Firm-G. E. Ehrlich (1995) Ltd. Related U.S. Application Data (57) ABSTRACT (63) Continuation of application No. 09/258,892, filed on Mar. 1, 1999, now abandoned, which is a continuation-in-part of A polynucleotide (hpa) encoding a polypeptide having application No. PCT/US98/17954, filed on Aug. 31, 1998, heparanase activity, Vectors including Same, genetically which is a continuation of application No. 08/922,170, filed modified cells expressing heparanase, a recombinant protein on Sep. 2, 1997, now Pat. No. 5,968,822. having heparanase activity and antisense oligonucleotides (51) Int. Cl." ...... C12Q 1/68; C12P 19/34; and constructs for modulating heparanase expression are C07H 21/02; CO7H 21/04; C12N 15/00 provided. (52) U.S. Cl...... 435/320.1; 435/6; 435/91.1; 536/23.1; 536/24.1; 536/24.5; 536/23.5 5 Claims, 34 Drawing Sheets

U.S. Patent Dec. 16, 2003 Sheet 2 of 34 US 6,664,105 B1

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Fig. 13

Inouse CTGGCAAGAAGGTCTGGTTGGGAGAGACGAGCTCAGCTTACGGTGGCGGT 50 | | | | | | | | human CTGGCAAGAAGGTCTGGTTAGGAGAAACAAGCTCTGCATATGGAGGCGGA lll:5 Inouse GCACCCTTGCGTCCAACACCTTTGCAGCTGGCTTTATGTGGCTGGATAA OO | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | human GCGCCCTTGCTATCCGACACCTTTGCAGCTGGCTTTATGTGGCTGGATAA 1165 CS ATTGGGCCTGTCAGCCCAGATGGGCATAGAAGTCGTGATGAGGCAGGTGT 50 | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | human ATTGGGCCTGTCAGCCCGAATGGGAATAGAAGTGGTGATGAGGCAAGTAT 125

no use TCTCGGAGCAGGCA ACACCACTAGTGGATGAAAACTTTGAGCCTTA. 2 OO | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | human TCTTTGGAGCAGGAAACTACCATTTAGTGGATGAAAACTTCGATCCTTTA. 265

Iose CCTGATTACTGGCTCTCTCTTCGTCAAGAAACTGGTAGGTCCCAGGGT 25 O | | | | | | | | | | | | | | | | | | | | | | | | human CCTGATTATTGGCTATCTCTTCTGTTCAAGAAATTGGTGGGCACCAAGGT 315 Icouse GTTACTGTCAAGAGTGAAAGGCCCAGACAGGAGCAAACTCCGAGTGTATC 300 1 human GTTAATGGCAAGCGTGCAAGGTTCAAAGAGAAGGAAGCTTCGAGTATACC, 365 IIouse TCCACTGCACTAACGTCTATCACCCACGATATCAGGAAGGAGATCTAACT 350 | | | | | | | | | | | | | | | | | | | | hunan TTCATTGCACAAACACTGACAATCCAAGGTATAAAGAAGGAGATTTAACT 4.5 Inouse CTGTATGTCCTGAACCTCCATAATGT CACCAAGCACTTGAAGGTACCGCC 400 | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | human CTGTATGCCATAAACCTCCATAACGTCACCAAGTACTTGCGGTTACCCTA 1465 mouse TCCGTTGTTCAGGAAACCAGTGGATACGTACCTTCTGAAGCCTTCGGGGC 450 | | | | | | | | | | | | | | | | | | | | | | human TCCTTTTTCTAACAAGCAAGTGGATAAATACCTTCTAAGACCTTTGGGAC 1515 Inouse CGGATGGATTACTTTCCAAATCTGTCCAACTGAACGGTCAAATTCTGAAG 500 | | | | | | | | | | | | | | | | | | | | | | | | | | human CTCATGGATTACTTTCCAAATCTGTCCAACT CAATGGTCTAACTCTAAAG 1565 Thouse ATGGTGGATGAGCAGACCCTGCCAGCTTTGACAGAAAAACCTCTCCCCGC 550 | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | human ATGGTGGATGATCAAACCTTGCCACCTTTAATGGAAAAACCTCTCCGGCC le5 OS e AGGAAGTGCACTAAGCCTGCCTGCCTTTTCCTATGGTTTTTTTGTCATA 600 | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | human AGGAAGTTCACTGGGCTTGCCAGCTTTCTCATATAGTTTTTTTGTGATAA 1665 Inouse GAAATGCCAAAATCGCTGCTTGTATATGAAAATAAAA 637 | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | human GAAATGCCAAAGTTGCTGCTTGCATCTGAAAATAAAA l702 U.S. Patent Dec. 16, 2003 Sheet 14 of 34 US 6,664,105 B1

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U.S. Patent Dec. 16, 2003 Sheet 31 of 34 US 6,664,105 B1

fit t t g t c : q caata at at g (33 g a gqia ?ca gatt (; tra gat at gateag at A 34 f) () aaaaat (qtta at, ga ("a at t t eig agg Coga ciga cyatt C1 (t a caa ... t. t. a ca 435 (0 at tact 3 tai at gadat. tiga t t t gt. C&l a gagqat a caat t t t (a gada a Ca C 4 3bb () cca at accittata actot, ct attaat (Cttgct t t t t it ?tacct t t ct, t 43 600 (Ctt (ft. t. t. Cagttggga a gCttittgg (t.gcaagta a Caga in a Ct c (ta at 4 3650 t caa at gg Ctta a gcaata agga a catgitat at t. CCC a Cat-3 act a gacqt. A 3700 tcaaa Cagg Coaggct Coag Cact toagtacgt. caccagg gatctgggitt 43750 ct toccagct citctgctctgc.cat Ctttagcgctgg Ctt cattct cagac 4.3800 t ct go tag cat. gatggct g tag ct q t t t catgg gcc Cottcaaacct cat 43850 agcaaccagaggaagaaaatgagccatttitttgag to t. CCtt Cataga Ct. 43900 togaataact ctitt t t cagagctt ct cacagcaaacct ct cot catgtctic 43950 ct catgtct tattott Cagaaatggg taatgtggc.cattt Caccagt cac: 44000 togccaacaacaacgaggitt. CCtata attgttct.ctgagta a CCCtttggaa 44.050 tgga gagggit gttggt. Cagt. Ct. a caaact gaa cact.g. Cagttctg.cgctit 44 l OO tit.taccagtgaaaaaatgt.aattatt.t. tcc.cct cittaaggattaatatt c. 44150 titcaaatgitat gcct gt. Latggata tagta t. Ct. t.taaaatttitt tattitt 44200 aa tagct.t. tagggg taca Cactt.tttgct tacaggggtgaattgttgtagt 44250 ggtgaag act cqg Cttittaatgtacttgt. Cacctgagtigatgta cattgt 44300 accoaatagg taatttitt Catccatta Ccct cott cog ccct ct t coctt 44350 ct gagt. Ct. Cica a catcCCttata CCaCtgtg tatgttcttgttgtacctac 44400 agctaagct tccacttataagt gaga a Catgcag tatttggttitt.ccatt 44450 cct gag titact tocct taggataa.ca.gc.ccc.cagttc.cg to Caagttgct. 44500 gCaaaata Cattatt Ctt Ctt tatgg Ctgagtaatagt CCatggta cata 44.550 tat acca cattitt Cttta t. Coca Ct-tat cagttgat gga cact taggittaa. 44 600 titccattcaat.tt catt.ca atttaagtatatttgta aggagctaaagctg. 44.650 aaaattaa attittagat Ct.t. t. Caatact Cltaaattittatatgta act guy 44700 t. t t t a tatttit. Ca Cat - tigaaataaagta att. ... t. tata a Cttgata t t 447 's O cy at Cactatt. Ct. t t tag taatgitaa a gr:cta caq act Cota Catttgga 44 800 acca C. tag togt. gttgttit Ca CCCct tottata citat caggat.cct cqa 4 4898

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U.S. Patent Dec. 16, 2003 Sheet 33 of 34 US 6,664,105 B1

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Fig. 18 U.S. Patent Dec. 16, 2003 Sheet 34 of 34 US 6,664,105 B1

MIRSKIA, I.M.I.L.E.GPLGPS PAI.PRPAAQOWW) LDFFTQEPLHLWSPSFLSWT , ) PHD EEEEE HE EEEE EEE

IDANLATDPRFLILEGSPKLRTLARGLSPAYLRFGGTKTDFLl FDPKKESTFEERSYWOS 120 PEIL) EEE EEEEE HHHHHH HHIE EEEEE HHHHHH

QVNQDICKYGS PPDWEEKLRLEWPYOEQI.L.I.REHYQKKFKNSTYSRSSVOVLYTFANCS BO PID HHHHHHHH HHHHHH HHHHHHHHHHHEEH EEEEEEEEEEEE

GLDE, FG.NA, RADI.QWNS SNAOLLLDycsSKGYNISWELGNPNSFLKKADI FINGS 240 PHD HHHHHHHHHHHHHHHHHHHHHHHHHHHHHH EEEEE HHHHHHH EEEE

QLGEDYIQLHKLLRKSTFKNAKLYGPDWGQPRRKTAKMLKSFLKAGGEVIDSWIWHHYYL 300 PHD HHHHHHHHHHHHHHHHH HHHHHHHHHHHHH EEEEEEEEEEE

|NGRTATREDFINPDV LiDJ FISSVQKVFQVVESTRPGKKWWLGETSSAYGGGAPLLSDTFA 360 (i) ; HEEEEEEEEEEEE EEEEEE HHHHHHH

AGFMWLDKLGLSARMGIEVVMROVFFGAGNYHVIDENFDPLPDYWLSLLFKKLVGTKVLM 420 PHI) HHHHHHHH HHHH HHHHHHHHH EEEEE HHHHHHHHHHHH EEEEE

ASVQGSKRRKLRVYLHCTNTDNPRY KEGDI.T.I.YAINLHNW'KYIRI.JPYPFSNKQVDKYI, 480 PHD EEE E. EEEEEEEE EEEEEE, EEEEE Hili HHHHH

RPLGPHGLLSKSWQLNGLTLKMVDDQI'll PLMEKPLRPGSSLGLPAFSYSFFV RNAKVA 540 PHE) HH EEEEEEE EEEEF, EEEEEEEE EE

ACT 43 PHD

Fig. 19 US 6,664,105 B1 1 2 POLYNUCLEOTDE ENCODING A endothelial cell borders and migration through the breach in POLYPEPTIDE HAVING HEPARANASE the endothelium toward the exposed underlying BM (9). ACTIVITY AND EXPRESSION OF SAME IN Once located between endothelial cells and the BM, the GENETICALLY MODIFIED CELLS invading cells must degrade the Subendothelial glycopro teins and of the BM in order to migrate out of the vascular compartment. Several cellular (e.g., This is a continuation of U.S. patent application Ser. No. collagenase IV, plasminogen activator, cathepsin B, elastase, 09/258,892, filed Mar. 1, 1999 now abandoned, which is a etc.) are thought to be involved in degradation of BM (10). continuation-in-part of PCT/US98/17954, filed Aug. 31, Among these enzymes is an endo-B-D-glucuronidase 1998, which is a continuation of Ser No. 08/922,170, filed (heparanase) that cleaves HS at Specific intrachain sites (6, Sep. 2, 1997, U.S. Pat. No. 5,968,822. 8, 11). Expression of a HS degrading heparanase was found to correlate with the metastatic potential of mouse lym FIELD AND BACKGROUND OF THE phoma (11), fibrosarcoma and melanoma (8) cells. INVENTION Moreover, elevated levels of heparanase were detected in The present invention relates to a polynucleotide, referred Sera from metastatic tumor bearing animals and melanoma to hereinbelow as hpa, encoding a polypeptide having 15 patients (8) and in tumor biopsies of cancer patients (12). heparanase activity, vectors (nucleic acid constructs) includ The control of cell proliferation and tumor progression by ing Same and genetically modified cells expressing hepara the local microenvironment, focusing on the interaction of cells with the (ECM) produced by nase. The invention further relates to a recombinant protein cultured corneal and vascular endothelial cells, was inves having he paranase activity and to antiSense tigated previously by the present inventors. This cultured oligonucleotides, constructs and ribozymes for down regu ECM closely resembles the Subendothelium in vivo in its lating heparanase activity. In addition, the invention relates morphological appearance and molecular composition. It to heparanase promoter Sequences and their uses. contains collagens (mostly type III and IV, with Smaller proteoglycans: Heparan Sulfate pro amounts of types I and V), proteoglycans (mostly heparan teoglycans (HSPG) are ubiquitous macromolecules associ 25 Sulfate- and dermatan Sulfate-proteoglycans, with Smaller ated with the cell surface and extra cellular matrix (ECM) of amounts of chondroitin Sulfate proteoglycans), laminin, a wide range of cells of Vertebrate and invertebrate tissues fibronectin, entactin and elastin (13, 14). The ability of cells (1-4). The basic HSPG structure includes a protein core to to degrade HS in the cultured ECM was studied by allowing which Several linear heparan Sulfate chains are covalently cells to interact with a metabolically sulfate labeled ECM, attached. These polysaccharide chains are typically com followed by gel filtration (Sepharose 6B) analysis of deg posed of repeating hexuronic and D-glucosamine disaccha radation products released into the culture medium (11). ride units that are Substituted to a varying extent with N- and While intact HSPG are eluted next to the void volume of the O-linked Sulfate moieties and N-linked acetyl groups (1–4). column (Kav-0.2, Mr-0.5x10'), labeled degradation frag Studies on the involvement of ECM molecules in cell ments of HS side chains are eluted more toward the V, of the attachment, growth and differentiation revealed a central 35 column (0.5

Expression of the 592 Amino Acids HPA 1O Example 9 Polypeptide in a Human 293 Cell Line Chromosomal Localization of the hpa Gene The 592 amino acids open reading frame (SEQ ID NOS:13 and 15) was constructed by ligation of the 110 bp Chromosomal mapping of the hpa gene was performed corresponding to the 5' end of the SK-hep1 hpa cDNA with utilizing a panel of monochromosomal human/CHO and the placenta cDNA. More specifically the Marathon RACE 15 human/mouse somatic cell hybrids, obtained from the UK PCR amplification product of the placenta hpa DNA was HGMP Resource Center (Cambridge, England). digested with SacI and an approximately 1 kb fragment was 40 ng of each of the Somatic cell hybrid DNA samples ligated into a SacI-digested pCHP6905 plasmid. The result were Subjected to PCR amplification using the hpa primers: ing plasmid was digested with Earl and Aati. The Earl hpu5655'-AGCTCTGTAGATGTGCTATACAC-3', SEQ ID Sticky ends were blunted and an approximately 280 bp NO:22, corresponding to nucleotides 564-586 of SEQ ID Earl/blunt-Aat I fragment was isolated. This fragment was NO:9 and an antisense primer hp 1171 ligated with pFasthpa digested with EcoRI which was blunt 5'-GCATCTTAGCCGTCTTTCTTCG-3', SEQ ID NO:23, ended using Klenow fragment and further digested with corresponding to nucleotides 897-876 of SEQ ID NO:9. Aati. The resulting plasmid contained a 1827 bp insert The PCR program was as follows: a hot start of 94 C-3 which includes an open reading frame of 1776 bp, 31 bp of 25 minutes, followed by 7 cycles of 94 C-45 seconds, 66 3' UTR and 21 bp of 5' UTR. This plasmid was designated C-1 minute, 68 C-5 minutes, followed by 30 cycles of pFastLhpa. 94 C-45 seconds, 62 C-1 minute, 68 C-5 minutes, A mammalian expression vector was constructed to drive and a 10 minutes final extension at 72 C. the expression of the 592 amino acids heparanase polypep The reactions were performed with Expand long PCR tide in human cells. The hpa cDNA was excised prom (Boehringer Mannheim). The resulting amplification prod pFastLhpa with BSSHII and Not. The resulting 1850 bp ucts were analyzed using agarose gel electrophoresis. AS BSSHII-Not fragment was ligated to a mammalian expres demonstrated in FIG. 14, a single band of approximately 2.8 sion vector pSI (Promega) digested with Mlul and NotI. The Kb was obtained from chromosome 4, as well as from the resulting recombinant plasmid, pSIhpaMet2 was transfected 35 control human genomic DNA. A 2.8 kb amplification prod into a human 293 embryonic kidney cell line. uct is expected based on amplification of the genomic hpa Transient expression of the 592 amino-acids heparanase clone (data not shown ). No amplification products were was examined by Western blot analysis and the enzymatic obtained neither in the control DNA samples of hamster and activity was tested using the gel shift assay. Both these mouse nor in Somatic hybrids of other human chromosome. procedures are described in length in U.S. patent application 40 Ser. No. 09/071,739, filed May 1, 1998, which is incorpo Example 10 rated by reference as if fully set forth herein. Cells were harvested 3 days following transfection. Harvested cells Human Genomic Clone Encoding Heparanase were re-suspended in lysis buffer containing 150 mM NaCl, Five plaques were isolated following Screening of a 50 mM Tris pH 7.5, 1% Triton X-100, 1 mM PMSF and 45 human genomic library and were designated L3-1, L5-1, protease inhibitor cocktail (Boehringer Mannheim). 40 ug L8-1, L10-1 and L6-1. The phage DNAS were analyzed by protein extract Samples were used for Separation on a Southern hybridization and by PCR with hpa specific and SDS-PAGE. Proteins were transferred onto a PVDF vector Specific primers. Southern analysis was performed Hybond-P membrane (Amersham). The membrane was with three fragments of hpa cDNA: a PvulI-BamHI frag incubated with an affinity purified polyclonal anti hepara 50 ment (nucleotides 32–450, SEQ ID NO:9), a BamHI-NdeI nase antibody, as described in U.S. patent application Ser. fragment (nucleotides 451-1102, SEQ ID NO:9) and an No. 09/071,739. A major band of approximately 50 kDa was NdeI-XhoI fragment (nucleotides 1103–1721, SEQ ID observed in the transfected cells as well as a minor band of NO:9). approximately 65 kDa. A similar pattern was observed in Following Southern analysis, phages L3, L6, L8 were extracts of cells transfected with the pShpa as demonstrated 55 Selected for further analysis. A Scheme of the genomic in U.S. patent application Ser. No. 09/071,739. These two region and the relative position of the three phage clones is bands probably represent two forms of the recombinant depicted in FIG. 15. A 2 kb DNA fragment containing the heparanase protein produced by the transfected cells. The 65 gap between phages L6 and L3 was PCR amplified from kDa protein probably represents a heparanase precursor, human genomic DNA with two gene Specific primers while the 50 kDa protein is suggested herein to be the 60 GH pull 3 and GHplL6. The PCR product was cloned into the processed or mature form. plasmid vector pGEM-T-easy (Promega). The catalytic activity of the recombinant protein Large Scale DNA sequencing of the three Lambda clones expressed in the pShpaMet2 transfected cells was tested by and the amplified fragment was performed with Lambda gel Shift assay. Cell extracts of transfected and of mock purified DNA by primer walking. A nucleotide Sequence of transfected cells were incubated overnight with heparin (6 65 44,898 bp was analyzed (FIG. 16, SEQ ID NO:42). Com ug in each reaction) at 37 C., in the presence of 20 mM parison of the genomic Sequence with that of hpa cDNA phosphate citrate buffer pH 5.4, 1 mM CaCl, 1 mM DTT revealed 12 exons separated by 11 introns (FIGS. 15 and US 6,664,105 B1 41 42 16). The genomic organization of the hpa gene is depicted in designed and a Marathon RACE was performed using a FIG. 15 (top). The sequence include the coding region from Marathon cDNA library from 15 days mouse embryo the first ATG to the stop codon which spans 39,113 (Clontech) and from BL6 mouse melanoma cell line. The nucleotides, 2742 nucleotides upstream of the first ATG and mouse hpa homologous cDNA was isolated following Sev 3043 nucleotides downstream of the stop codon. Splice site eral amplification Steps. A 1.1 kb fragment was amplified consensus Sequences were identified at exon/intron junc from mouse embryo Marathon cDNA library. The first cycle tions. of amplification was performed with primers mhpl773 and Ap1 and the second cycle with primers mhpl736 and AP2. Example 11 A 1.1 kb fragment was then amplified from BL6 Marathon cDNA library. The first cycle of amplification was per Alternative Splicing formed with the primers mhpl152 and Ap1, and the second Several minor RT-PCR products were obtained from with mhpl83 and AP2. The combined sequence was homolo various cell types, following amplification with hpa Specific gous to nucleotides 157-1702 of the human hpa cDNA, primers. Each one found to contain a deletion of one or two which encode amino acids 33-543. The 5' end of the mouse exons. Some of these PCR products contain ORFs, which 15 hpa gene was isolated from a mouse genomic DNA library encode potential shorter proteins. using the Genome Walker kit (Clontech). An 0.9 kb frag ment was amplified from a Dral digested Genome walker Table 1 below Summarizes the alternative spliced prod DNA library. The first cycle of amplification was performed ucts isolated from various cell lines. with primerS mhpl114 and Ap1 and the Second with primers Fragments of Similar sizes were obtained following mhpl103 and AP2. The assembled sequence (SEQ ID amplification with two cell lines, placenta and platelets. NOs:43, 45) is 2396 nucleotides long. It contains an open reading frame of 1605 nucleotides, which encode a polypep tide of 535 amino acids (SEQ ID NOs:44, 45), 196 nucle Cell type Nucleotides deleted Exons deleted ORF otides of 3' untranslated region (UTR), and an upstream 25 Sequence which includes the promoter region and the Platelets 1047-1267 8, 9 -- 5'-UTR of the mouse hpa cDNA. According to two promoter Platelets 1154-1267 9 predicting programs TSSW and TSSG, the transcription start Platelets 289-435, 2, 4 562-735 site is localized to nucleotide 431 of SEQ ID NOS:43, 45, Sk-hep1, platelets, Zr75 562-735 4 -- 163 nucleotides upstream of the first ATG codon. The 431 Sk-hep1 (hepatoma) 561-904 4, 5 upstream genomic Sequence contains the promoter region. A Zr75 (breast carcinoma) 96-2O3 1 (partial) TATA box is predicted at position 394 of SEQ ID NOs:43, 45. The mouse and the human hpa genes share an average homology of 78% between the nucleotide Sequences and Example 12 81% similarity between the deduced amino acid Sequences. 35 Search for hpa homologous Sequences, using the Blast 2.0 Mouse and Rat hpa server revealed two EST's from rat: AIO60284 (385 EST databases were Screened for Sequences homologous nucleotides, SEQ ID NO:46) which is homologous to the to the hpa gene. Three mouse EST's were identified amino terminus (68% similarity to amino acids 12-136) of (accession No. Aa177901, from mouse spleen, Aao67997 human heparanase and AI237828 (541 nucleotides, SEQID from mouse skin, Aa47943 from mouse embryo), assembled 40 NO:47) which is homologous to the carboxyl terminus (81% into a 824 bp cDNA fragment which contains a partial open similarity to amino acids 500–543) of human heparanase, reading frame (lacking a 5' end) of 629 bp and a 3' untrans and contains a 3'-UTR. A comparison between the human lated region of 195bp (SEQ ID NO:12). As shown in FIG. heparanase and the mouse and rat homologous Sequences is 13, the coding region is 80% similar to the 3' end of the hpa demonstrated in FIG. 17. cDNA sequence. These EST's are probably cDNA frag 45 ments of the mouse hpa homolog that encodes for the mouse Example 13 heparanase. Prediction of Heparanase Active Site Searching for consensus protein domains revealed an amino terminal S homology between the heparanase and 50 Homology Search of heparanase amino acid Sequence Several precursor proteins Such as Procollagen Alpha 1 against the DNA and the protein databaseS revealed no precursor, Tyrosine-protein kinase-RYK, Fibulin-1, Insulin Significant homologies. The protein Secondary Structure as like growth factor binding protein and Several others. The predicted by the PHD program consists of alternating alpha amino terminus is highly hydrophobic and contains a poten helices and beta sheets. The fold recognition server of tial trans-membrane domain. The homology to known signal 55 UCLA predicted alpha/beta barrel structure, with under peptide Sequences Suggests that it could function as a signal threshold confidence. peptide for protein localization. Five of 15 proteins, which were predicted to have most The amino acid Sequence of human heparanase was used Similar folds, were glycosyl hydrolases from various organ to Search for homologous Sequences in the DNA and protein isms: IXyZa- from Clostridium Thermocellum, databases. Several human ESTs were identified, as well as 60 lpbga-6-phospho-beta-Ö-galactosidase from LactococcuS mouse Sequences highly homologous to human heparanase. Lactis, 1amy-alpha- from Barley, 1ecea The following mouse EST's were identified AA177901, endocellulase from Acidothermus Cellulolyticus and 1 qbc AA674378, AA67997, AAO47943, AA690179, A122034, alpha chain, glycosyl . all sharing an identical Sequence and correspond to amino Protein homology Search using the bioaccelerator pulled acids 336–543 of the human heparanase sequence. The 65 out Several proteins, including glycosyl hydrolyses Such as entire mouse heparanase cDNA was cloned, based on the beta-fructofuranosidase from Vicia faba (broad bean) and nucleotide sequence of the mouse EST's. PCR primers were from potato, phlorizin hydrolase from human, Xyla US 6,664,105 B1 43 44 nases from CloStridium thermocellum and from Streptomy in all mammals, while faint bands were detected in chicken. ces halstedi and from CloStridium thermocellum. This correlates with the phylogenetic relation between Blocks 9.3 database pulled out the active site of glycosyl human and the tested animals. The intense bands indicate hydrolases family five, which includes from vari that hpa is conserved among mammals as well as in more ous bacteria and fungi. Similar active site motif is shared by genetically distant organisms. The multiple bands patterns Several lysosomal acid hydrolases (63) and other glycosyl Suggest that in all animals, like in human, the hpa locus hydrolases. The common mechanism shared by these occupy large genomic region. Alternatively, the various enzymes involves two glutamic acid residues, a proton bands could represent homologous Sequences and Suggest donor and a nucleophile. the existence of a gene family, which can be isolated based Despite the lack of an overall homology between the on their homology to the human hpa reported herein. This heparanase and other glycosyl hydolases, the amino acid conservation was actually found, between the isolated couple Asp-Glu (NE), which is characteristic of the proton human hpa cDNA and the mouse homologue. donor of glycosyl hydrolyses of the GH-A clan, was found at positions 224-225 of the human heparanase protein Example 16 Sequence. AS in other clan members, this NE couple is 15 Characterization of the hpa Promoter located at the end of a B sheet. The DNA sequence upstream of the hpa first ATG was Considering the relative location of the proton donor and Subjected to computational analysis in order to localize the the predicted Secondary Structure, the glutamic acid that predicted transcription start site and to identify potential functions as nucleophile is most likely located at position transcription factors binding Sites. Recognition of human 343, or at position 396. Identification of the active site and PolII promoter region and Start of transcription were pre the amino acids directly involved in hydrolysis opens the dicted using the TSSW and TSSG programs. Both programs way for expression of the defined catalytic domain. In identified a promoter region upstream of the coding region. addition, it will provide the tools for rational design of TSSW pointed at nucleotide 2644 and TSSG at 2635 of SEQ enzyme activity either by modification of the microenviro ID NO:42. These two predicted transcription start sites are ment or catalytic Site itself. 25 located 4 and 13 nucleotides upstream of the longest hpa Example 14 cDNA isolated by RACE. A hpa promoter-GFP reporter vector was constructed in Expression of hpa AntiSense in Mammalian Cell order to investigate the regulation of hpa transcription. Two Lines constructs were made, containing 1.8 kb and 1.1 kb of the hpa promoter region. The reporter vector was transfected A mammalian expression vector Hpa2Kepcdna3 was con into T50-mouse bladder carcinoma cells. Cells transfected Structed in order to express hpa antisense in mammalian with both constructs exhibited green fluorescence, which cells. hpa cDNA (1.7 kb EcoRI fragment) was cloned into indicated the promoter activity of the genomic Sequence the plasmid pcDNA3 in 3">5" (antisense) orientation. The 35 upstream of the hpa-coding region. This reporter vector, construct was used to transfect MBT2-T50 and T24P cell enables the monitoring of hpa promoter activity, at various lines. 2x10 cells in 35 mm plates were transfected using the conditions and in different cell types and to characterize the Fugene protocol (Boehringer Mannheim). 48 hours after factors involved regulation of hpa expression. transfection cells were trypsinized and Seeded in Six well plates. 24 hours later G418 was added to initiate Selection. Although the invention has been described in conjunction 40 with Specific embodiments thereof, it is evident that many The number of colonies per 35 mm plate following 3 weeks: alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all Such alternatives, modifications and variations Antisense No insert that fall within the Spirit and broad Scope of the appended 45 claims. T24P 15 60 MBTTSO 1. 6 LIST OF REFERENCES 1. Wight, T. N., Kinsella, M. G., and Qwarnstromn, E. E. The lower number of colonies obtained after transfection (1992). The role of proteoglycans in cell adhesion, migra with hpa antisense, as compared with the control plasmid 50 tion and proliferation. Curr. Opin. Cell Biol., 4,793-801. Suggests that the introduction of hpa antisense interfere with 2. Jackson, R. L., Busch, S.J., and Cardin, A. L. (1991). cell growth. This experiment demonstrates the use of Glycosaminoglycans: Molecular properties, protein inter complementary antisense hpa DNA sequence to control actions and role in physiological processes. Physiol. Rev., heparanase expression in cells. This approach may be used 71, 481–539. to inhibit expression of heparanase in Vivo, in, for example, 55 3. Wight, T. N. (1989). Cell biology of arterial proteogly cancer cells and in other pathological processes in which cans. Arteriosclerosis, 9, 1-20. heparanase is involved. 4. Kjellen, L., and Lindahl, U. (1991). Proteoglycans: struc tures and interactions. Annu. Rev. Biochem., 60, 443-475. Example 15 5. Ruoslahti, E., and Yamaguchi, Y. (1991). Proteoglycans as 60 modulators of growth factor activities. Cell, 64, 867–869. Zoo Blot 6. Vlodavsky, I., Eldor, A., Haimovitz-Friedman, A., Hpa cDNA was used as a probe to detect homologous Matzner, Y., Ishai-Michaeli, R., Levi, E., Bashkin, P., sequences in human DNA and in DNA of various animals. Lider, O., Naparstek, Y., Cohen, I.R., and Fuks, Z. (1992). The autoradiogram of the Southern analysis is presented in Expression of heparanase by platelets and circulating cells FIG. 18. Several bands were detected in human DNA, which 65 of the immune system: Possible involvement in diaped correlated with the accepted pattern according to the esis and extravasation. Invasion & MetaStasis, 12, genomic hpa Sequence. Several intense bands were detected 112-127. US 6,664,105 B1 45 46 7. Vlodavsky, I., Mohsen, M., Lider, O., Ishai-Michaeli, R., 22. Ishai-Michaeli, R., Svahn, C.-M., Chajek-Shaul, T., Ekre, H.-P., Svahn, C. M., Vigoda, M., and Peretz, T. Komer, G., Ekre, H.-P., and Vlodavsky, I. (1992). Impor (1995). Inhibition of tumor metastasis by heparanase tance of size and Sulfation of heparin in release of basic inhibiting Species of heparin. Invasion & MetaStasis, 14, fibroblast factor from the vascular endothelium and extra 290-302. cellular matrix. Biochemistry, 31, 2080-2088. 8. 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SEQUENCE LISTING

<160> NUMBER OF SEQ ID NOS: 47 <21 Oc SEQ ID NO 1 <211 LENGTH 27 <212> TYPE DNA <213> ORGANISM: Artificial Sequence <22O > FEATURE OTHER INFORMATION: synthetic oligonucleotide <400 SEQUENCE: 1 ccatcctaat acgactic act at agggc 27

SEQ ID NO 2 LENGTH 24 TYPE DNA ORGANISM: Artificial Sequence FEATURE OTHER INFORMATION: synthetic oligonucleotide <400 SEQUENCE: 2

gtagt gatgc catgitaact g aatc 24

SEQ ID NO 3 LENGTH 23 TYPE DNA ORGANISM: Artificial Sequence FEATURE OTHER INFORMATION: synthetic oligonucleotide <400 SEQUENCE: 3

act cactata gggctcqagc ggc 23

SEQ ID NO 4 LENGTH 22 TYPE DNA ORGANISM: Artificial Sequence FEATURE OTHER INFORMATION: synthetic oligonucleotide <400 SEQUENCE: 4

gcatcttagc cqtctittctt cq 22

US 6,664,105 B1 S3

-contin ued gggalactagg caatgaac ct aac agitttcc ttaagaaggc tgatatttitc atcaatgggit cgcagttagg agaagattat attcaattgc ataalactitct aagaaagttcc accittcaaaa. 840 atgcaaaact citatggtoct gatgttggto agc citcgaag aaagacggct aagatgctda 9 OO agagct tcct gaaggctggit ggagaagtga ttgattcagt tacatgg cat cactactatt 96.O tgaatggacg gactgctacc agg galagatt ttctaaa.ccc tgatgtattg gacatttitta O20 tittcatctgt gcaaaaagtt titcCaggtgg ttgagag cac caggcctggc aagaaggtot ggittaggaga aacaagctict gcatatggag gcggagc gCC cittgctatoc gacaccitttg 14 O cagotggctt tatgtc.gctg gataaattgg gcc totcago CC gaatggga atagaagtgg 200 tgatgaggca agtattottt ggagcaggaa actaccattt agtggatgaa aactitcg atc 260 citttacctga ttattggcta totcttctgt tdaagaaatt ggtgggCacc aaggtgttaa 320 tggcaa.gc.gt gcaaggttca alagagaagga agctt.cgagt attacctitcait tgcacaaaca citgacaatcc aaggtataaa gaaggagatt taactctgta tgc cataaac citccataacg 4 40 to accalagta cittgcggitta cc citaticcitt tittctaacaa. gCaagtggat aaatacctitc 5 OO taag acctitt ggg accitcat ggattactitt ccaaatctgt ccaacticaat ggtotaactic 560 taaagatggit ggatgatcaa accittgccac citttaatgga aaaac citctic CggcCaggaa gttcactggg cittgccagot ttcticatata gttttitttgt gataagaaat gccaaagttg 680 citgcttgcat citgaaaataa aatatactag toctdacact g 721

<210> SEQ ID NO 10 &2 11s LENGTH 543 212s. TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 10

Met Lieu. Lieu Arg Ser Lys Pro Ala Leu Pro Pro Pro Leu Met Telu Telu 1 5 10 15

Leu Lieu Gly Pro Leu Gly Pro Leu Ser Pro Gly Ala Leu Pro Arg Pro 25 30

Ala Glin Ala Glin Asp Val Val Asp Lieu. Asp Phe Phe Thr Glin Glu Pro 35 40 45

Lieu. His Lieu Wal Ser Pro Ser Phe Leu Ser Wall Thir Ile Asp Ala Asn 50 55 60

Leu Ala Thr Asp Pro Arg Phe Lieu Ile Leu Lieu Gly Ser Pro Lys Lieu 65 70 75 8O Arg Thr Lieu Ala Arg Gly Lieu Ser Pro Ala Tyr Leu Arg Phe Gly Gly 85 90 95

Thr Lys Thr Asp Phe Leu Ile Phe Asp Pro Lys Lys Glu Ser Thr Phe 100 105 110

Glu Glu Arg Ser Tyr Trp Gln Ser Glin Wall Asn Glin Asp Ile 115 120 125

Tyr Gly Ser Ile Pro Pro Asp Val Glu Glu Lys Leu Arg Lieu Glu Trp 130 135 1 4 0

Pro Tyr Glin Glu Glin Lieu Lleu Lieu Arg Glu His Tyr Glin Lys 145 15 O 155 160

Lys Asn. Ser Thr Tyr Ser Arg Ser Ser Val Asp Val Leu Tyr Thr Phe 1.65 170 175

Ala Asn. Cys Ser Gly Lieu. Asp Lieu Ile Phe Gly Lieu. Asn Ala Telu Telu 18O 185 190

Arg Thr Ala Asp Leu Gln Trp Asn Ser Ser Asn Ala Glin Leu Telu Telu US 6,664,105 B1 SS

-continued

195 200 2O5 Asp Tyr Cys Ser Ser Lys Gly Tyr Asn. Ile Ser Trp Glu Lieu Gly Asn 210 215 220 Glu Pro Asn. Ser Phe Lieu Lys Lys Ala Asp Ile Phe Ile Asn Gly Ser 225 230 235 240 Glin Leu Gly Glu Asp Tyr Ile Glin Lieu. His Lys Lieu Lleu Arg Lys Ser 245 250 255 Thr Phe Lys Asn Ala Lys Lieu. Tyr Gly Pro Asp Val Gly Glin Pro Arg 260 265 27 O Arg Lys Thr Ala Lys Met Leu Lys Ser Phe Lieu Lys Ala Gly Gly Glu 275 280 285 Val Ile Asp Ser Val Thr Trp His His Tyr Tyr Leu Asn Gly Arg Thr 29 O 295 3OO Ala Thr Arg Glu Asp Phe Lieu. Asn Pro Asp Wall Leu Asp Ile Phe Ile 305 310 315 320 Ser Ser Val Glin Lys Val Phe Glin Val Val Glu Ser Thr Arg Pro Gly 325 330 335 Lys Llys Val Trp Leu Gly Glu Thir Ser Ser Ala Tyr Gly Gly Gly Ala 340 345 350 Pro Leu Lleu Ser Asp Thr Phe Ala Ala Gly Phe Met Trp Lieu. Asp Lys 355 360 365 Leu Gly Leu Ser Ala Arg Met Gly Ile Glu Val Val Met Arg Glin Val 370 375 38O Phe Phe Gly Ala Gly Asn Tyr His Lieu Val Asp Glu Asn. Phe Asp Pro 385 390 395 400 Leu Pro Asp Tyr Trp Leu Ser Lieu Lleu Phe Lys Lys Lieu Val Gly Thr 405 410 415 Lys Val Lieu Met Ala Ser Val Glin Gly Ser Lys Arg Arg Lys Lieu Arg 420 425 430 Val Tyr Lieu. His Cys Thr Asn. Thir Asp Asin Pro Arg Tyr Lys Glu Gly 435 4 40 4 45 Asp Lieu. Thir Lieu. Tyr Ala Ile Asn Lieu. His Asn Val Thr Lys Tyr Lieu 450 455 460 Arg Lieu Pro Tyr Pro Phe Ser Asn Lys Glin Val Asp Lys Tyr Lieu Lieu 465 470 475 480 Arg Pro Leu Gly Pro His Gly Lieu Lleu Ser Lys Ser Val Glin Lieu. Asn 485 490 495 Gly Leu Thir Leu Lys Met Val Asp Asp Glin Thr Leu Pro Pro Leu Met 5 OO 505 510 Glu Lys Pro Leu Arg Pro Gly Ser Ser Lieu Gly Lieu Pro Ala Phe Ser 515 52O 525 Tyr Ser Phe Phe Val Ile Arg Asn Ala Lys Val Ala Ala Cys Ile 530 535 540

<210> SEQ ID NO 11 &2 11s LENGTH 1721 &212> TYPE DNA <213> ORGANISM: Homo sapiens &220s FEATURE <221 NAME/KEY: CDS <222> LOCATION: (63) . . (1691) <400 SEQUENCE: 11 citagagctitt cqactcitc.cg ctg.cgcggca gctgg.cgggg ggagcagoca ggtgagcc.ca ag atg citg citg cqc tog aag cot gcg citg cc.g. cc g cc g citg atg citg US 6,664,105 B1 57 58

-continued Met Leu Lleu Arg Ser Lys Pro Ala Lieu Pro Pro Pro Leu Met Lieu 1 5 10 15

citg citc. citg ggg cc.g citg ggit cc c citc. to c cct ggC gcc citg cc c cqa 155 Teu Telu Telu Gly Pro Teu Gly Pro Telu Ser Pro Gly Ala Teu Pro Arg 20 25 30 cct gCg Cala gca cag gac gto gtg gac citg gac titc. titc. aCC Cag gag Pro Ala Glin Ala Glin Asp Wall Wall Asp Telu Asp Phe Phe Thr Glin Glu 35 40 45 cc.g citg cac citg gtg agc ccc. tog titc. citg to c gto acc att gac goc 251 Pro Telu His Telu Wall Ser Pro Ser Phe Telu Ser Wall Thr Ile Asp Ala 50 55 60

aac citg gcc acg gac cc.g Cgg titc. citc. atc citc. citg ggit tot cca aag 299 Asn Telu Ala Thr Asp Pro Arg Phe Telu Ile Teu Teu Gly Ser Pro Lys 65 70 75 citt cgt. acc ttg gcc aga ggC ttg tot cct gcg tac citg agg titt ggit 347 Teu Arg Thr Telu Ala Arg Gly Telu Ser Pro Ala Teu Arg Phe Gly 8O 85 9 O 95 ggC acc aag aCa gac titc. cita att titc. gat ccc. aag aag gaa toa acc 395 Gly Thr Lys Thr Asp Phe Teu Ile Phe Asp Pro Lys Lys Glu Ser Thr 1 OO 105 110 titt gaa gag aga agt tac tgg Cala tot Cala gto aac cag gat att togc 4 43 Phe Glu Glu Arg Ser Trp Glin Ser Glin Wall Asn Glin Asp Ile Cys 115 120 125

a.a.a. tat gga to c atc cct cct gat gtg gag gag aag tta cgg ttg gala 491 Gly Ser Ile Pro Pro Asp Wall Glu Glu Lys Teu Arg Leu Glu 130 135 1 4 0 tgg cc c tac cag gag Cala ttg cita citc. cga gaa cac tac cag aaa aag 539 Trp Pro Tyr Glin Glu Glin Teu Telu Telu Arg Glu His Glin Lys Lys 145 150 155

titc. aag a.a. C. agc acc tac toa aga agc tot gta gat gtg cita tac act 587 Phe Lys Asn Ser Thr Tyr Ser Arg Ser Ser Wall Asp Wall Teu Tyr Thr 160 1.65 170 175 titt gca a.a. C. tgc toa gga citg gac ttg atc titt ggC cita aat gc g tta 635 Phe Ala Asn Cys Ser Gly Teu Asp Telu Ile Phe Gly Teu Asn Ala Leu 18O 185 190 tta aga aCa gca gat ttg cag tgg a.a. C. agt tot aat gct cag ttg citc 683 Teu Arg Thr Ala Asp Teu Glin Trp Asn Ser Ser Asn Ala Glin Telu Telu 195 200 2O5

citg gac tac tgc tct to c aag ggg tat a.a. C. att tct tgg gaa cita ggc 731 Teu Asp Tyr Cys Ser Ser Lys Gly Tyr Asn Ile Ser Trp Glu Leu Gly 210 215 220 aat gaa cct a.a. C. agt titc. citt aag aag gct gat att titc. atc aat ggg 779 Asn Glu Pro Asn Ser Phe Teu Lys Lys Ala Asp Ile Phe Ile Asn Gly 225 230 235 tog cag tta gga gaa gat att Cala ttg cat a.a.a. citt cita aga aag 827 Ser Glin Telu Gly Glu Asp Ile Glin Telu His Teu Teu Arg Lys 240 245 25 O 255

to c acc titc. a.a.a. aat gca citc. tat ggit cct gat gtt ggit cag cot 875 Ser Thr Phe Asn Ala Telu Gly Pro Asp Wall Gly Glin Pro 260 265 27 O cga aga aag acg gct aag atg citg aag agc titc. citg gct ggt gga 923 Arg Arg Lys Thr Ala Met Telu Lys Ser Phe Teu Ala Gly Gly 275 280 285 gaa gtg att gat toa gtt a Ca tgg cat cac tac tat aat gga cqg 971. Glu Wall Ile Asp Ser Wall Thr Trp His His Asn Gly Arg 29 O 295 act gct acc agg gaa gat titt cita a.a. C. cct gat gta gac att titt 101.9 Thr Ala Thr Arg Glu Asp Phe Telu Asn Pro Asp Wall Teu Asp Ile Phe 305 310 315

US 6,664,105 B1 63 64

-continued agacctittgg gacctdatgg attacttitcc aaatctgtcc aactcaatgg totaacticta 1740 aagatggtgg atgatcaaac cittgccacct ttaatggaaa aacct citcc g gcc aggaagt 1800 toactgggct tcc cagottt citcatatagt tttitttgttga taagaaatgc caaagttgct 1860 gcttgcatct gaaaataaaa tatac tagtc. citgacactg 1899

<210> SEQ ID NO 14 &2 11s LENGTH 592 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 14 Met Glu Gly Ala Val Gly Gly Val Arg Arg Arg Asn Gly Ala Glu Glu 1 5 10 15 Arg Arg Lys Gly Arg Trp Gly Ser Ala Gly Gly Ser Ala Arg Ala Lieu 2O 25 30 Asp Ser Pro Leu Arg Gly Ser Trp Arg Gly Glu Gln Pro Gly Glu Pro 35 40 45 Lys Met Lieu Lieu Arg Ser Lys Pro Ala Lieu Pro Pro Pro Leu Met Lieu 50 55 60 Leu Lleu Lieu Gly Pro Leu Gly Pro Leu Ser Pro Gly Ala Lieu Pro Arg 65 70 75 8O Pro Ala Glin Ala Glin Asp Val Val Asp Lieu. Asp Phe Phe Thr Glin Glu 85 90 95 Pro Leu. His Leu Val Ser Pro Ser Phe Leu Ser Val Thr Ile Asp Ala 100 105 110 Asn Leu Ala Thr Asp Pro Arg Phe Leu Ile Leu Leu Gly Ser Pro Lys 115 120 125 Leu Arg Thr Lieu Ala Arg Gly Lieu Ser Pro Ala Tyr Lieu Arg Phe Gly 130 135 1 4 0 Gly Thr Lys Thr Asp Phe Lieu. Ile Phe Asp Pro Lys Lys Glu Ser Thr 145 15 O 155 160 Phe Glu Glu Arg Ser Tyr Trp Glin Ser Glin Val Asn Glin Asp Ile Cys 1.65 170 175 Lys Tyr Gly Ser Ile Pro Pro Asp Val Glu Glu Lys Lieu Arg Lieu Glu 18O 185 190 Trp Pro Tyr Glin Glu Gln Leu Lleu Lieu Arg Glu His Tyr Glin Lys Lys 195 200 2O5 Phe Lys Asn Ser Thr Tyr Ser Arg Ser Ser Val Asp Val Leu Tyr Thr 210 215 220 Phe Ala Asn. Cys Ser Gly Lieu. Asp Lieu. Ile Phe Gly Lieu. Asn Ala Lieu 225 230 235 240 Leu Arg Thr Ala Asp Leu Gln Trp Asn. Ser Ser Asn Ala Glin Leu Lieu 245 250 255 Leu Asp Tyr Cys Ser Ser Lys Gly Tyr Asn. Ile Ser Trp Glu Lieu Gly 260 265 27 O Asn Glu Pro Asn. Ser Phe Leu Lys Lys Ala Asp Ile Phe Ile Asn Gly 275 280 285 Ser Glin Leu Gly Glu Asp Tyr Ile Glin Lieu. His Lys Lieu Lieu Arg Lys 29 O 295 3OO Ser Thr Phe Lys Asn Ala Lys Lieu. Tyr Gly Pro Asp Val Gly Glin Pro 305 310 315 320 Arg Arg Lys Thr Ala Lys Met Leu Lys Ser Phe Lieu Lys Ala Gly Gly 325 330 335 US 6,664,105 B1 65

-continued Glu Val Ile Asp Ser Val Thir Trp His His Tyr Tyr Leu Asn Gly Arg 340 345 350 Thr Ala Thr Arg Glu Asp Phe Lieu. Asn Pro Asp Val Lieu. Asp Ile Phe 355 360 365 Ile Ser Ser Val Glin Lys Val Phe Glin Val Val Glu Ser Thr Arg Pro 370 375 38O Gly Lys Llys Val Trp Lieu Gly Glu Thir Ser Ser Ala Tyr Gly Gly Gly 385 390 395 400 Ala Pro Leu Lleu Ser Asp Thr Phe Ala Ala Gly Phe Met Trp Lieu. Asp 405 410 415 Lys Lieu Gly Lieu Ser Ala Arg Met Gly Ile Glu Val Val Met Arg Glin 420 425 430 Val Phe Phe Gly Ala Gly Asn Tyr His Lieu Val Asp Glu Asn. Phe Asp 435 4 40 4 45 Pro Leu Pro Asp Tyr Trp Leu Ser Lieu Lleu Phe Lys Lys Lieu Val Gly 450 455 460 Thr Lys Val Lieu Met Ala Ser Val Glin Gly Ser Lys Arg Arg Lys Lieu 465 470 475 480 Arg Val Tyr Lieu. His Cys Thr Asn. Thir Asp Asn Pro Arg Tyr Lys Glu 485 490 495 Gly Asp Lieu. Thir Lieu. Tyr Ala Ile Asn Lieu. His Asn Val Thr Lys Tyr 5 OO 505 510 Leu Arg Lieu Pro Tyr Pro Phe Ser Asn Lys Glin Val Asp Llys Tyr Lieu 515 52O 525 Leu Arg Pro Leu Gly Pro His Gly Lieu Lleu Ser Lys Ser Val Glin Lieu 530 535 540 Asn Gly Lieu. Thir Lieu Lys Met Val Asp Asp Glin Thr Lieu Pro Pro Leu 545 550 555 560 Met Glu Lys Pro Leu Arg Pro Gly Ser Ser Lieu Gly Lieu Pro Ala Phe 565 570 575 Ser Tyr Ser Phe Phe Val Ile Arg Asn Ala Lys Val Ala Ala Cys Ile 58O 585 59 O

<210 SEQ ID NO 15 &2 11s LENGTH 1899 &212> TYPE DNA <213> ORGANISM: Homo sapiens &220s FEATURE <221 NAME/KEY: CDS <222> LOCATION: (94) . . (1869) <400 SEQUENCE: 15 gggaaag.cga gcaaggaagt aggagaga gC C gggCaggcg ggg.cggggitt ggattgggag 60 Cagtgg gagg gatgcagaag aggagtggga ggg atg gag ggc gCa gtg gga ggg 114 Met Glu Gly Ala Val Gly Gly 1 5 gtg agg agg cqt aac ggg gcg gag gala agg aga aaa gag cqc togg ggc 162 Val Arg Arg Arg Asn Gly Ala Glu Glu Arg Arg Lys Gly Arg Trp Gly 10 15 2O tog gC g g g a gga agt gct aga gCt citc gac tot cog citg cqc ggc agc 210 Ser Ala Gly Gly Ser Ala Arg Ala Lieu. Asp Ser Pro Leu Arg Gly Ser 25 30 35 tgg C9g ggg gag Cag cca ggit gag CCC aag atg Citg Ctg cqc tog aag 258 Trp Arg Gly Glu Glin Pro Gly Glu Pro Lys Met Leu Lleu Arg Ser Lys 40 45 5 O 55 cct gcig citg cc g c cq ccg citg at g citg citg citc citg g g g cc g citg ggt 306 Pro Ala Lieu Pro Pro Pro Leu Met Leu Lleu Lleu Lleu Gly Pro Leu Gly

US 6,664,105 B1 71 72

-continued cc.ggg.cgctt goatcc.cggc catctocqca cccittcaagt gggtgtgggt gattitcgtaa 480 gtgaacgtga Ccgc.ca.ccgg ggggaaag.cg agcaaggaag taggaga gag ccggg Cagg C 540 gggg.cggggt toggattggga gcagtgggag ggatgcagala gaggagtggg aggg 594

<210 SEQ ID NO 17 <211& LENGTH 21 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: synthetic oligonucleotide <400 SEQUENCE: 17 cccCaggagc agcago atca g 21

<210> SEQ ID NO 18 <211& LENGTH 21 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: synthetic oligonucleotide <400 SEQUENCE: 18 aggctt.cgag cqcago agca t 21

<210 SEQ ID NO 19 <211& LENGTH 22 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: synthetic oligonucleotide <400 SEQUENCE: 19 gtaatacgac toactatagg gc 22

<210> SEQ ID NO 20 &2 11s LENGTH 19 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: synthetic oligonucleotide <400 SEQUENCE: 20 actatagggc acgc.gtggit 19

<210> SEQ ID NO 21 <211& LENGTH 21 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: synthetic oligonucleotide <400 SEQUENCE: 21 cittgggctica cct ggctgct c 21

<210> SEQ ID NO 22 &2 11s LENGTH 23 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: synthetic oligonucleotide <400 SEQUENCE: 22 agctctgtag atgtgctata cac 23 US 6,664,105 B1 73 74

-continued SEQ ID NO 23 LENGTH 22 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: synthetic oligonucleotide

<400 SEQUENCE: 23 gcatcttagc cqtctttctt cq 22

SEQ ID NO 24 LENGTH 23 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: synthetic oligonucleotide

<400 SEQUENCE: 24 gag cagccag gtgagcc.caa gat 23

SEQ ID NO 25 LENGTH 23 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: synthetic oligonucleotide

<400 SEQUENCE: 25 titcgatcc.ca agaaggaatc aac 23

SEQ ID NO 26 LENGTH 23 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: synthetic oligonucleotide <400 SEQUENCE: 26 agctctgtag atgtgctata cac 23

SEQ ID NO 27 LENGTH 24 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: synthetic oligonucleotide <400 SEQUENCE: 27 toagatgcaa goagcaactt toggc 24

SEQ ID NO 28 LENGTH 22 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: synthetic oligonucleotide <400 SEQUENCE: 28 gcatcttagc cqtctttctt cq 22

SEQ ID NO 29 LENGTH 24 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: synthetic oligonucleotide US 6,664,105 B1 75 76

-continued SEQUENCE: 29 gtag to atgc catgtaactg aatc 24

SEQ ID NO 30 LENGTH 22 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: synthetic oligonucleotide

<400 SEQUENCE: 30 aggcacccita gagatgttcc ag 22

SEQ ID NO 31 LENGTH 24 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: synthetic oligonucleotide <400 SEQUENCE: 31 gaagatttct gtttccatga cqtg 24

SEQ ID NO 32 LENGTH 25 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: synthetic oligonucleotide <400 SEQUENCE: 32 ccacactgaa totaatactg aagtg 25

SEQ ID NO 33 LENGTH 22 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: synthetic oligonucleotide <400 SEQUENCE: 33 cgaagctotg gaactcggca ag 22

SEQ ID NO 34 LENGTH 22 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: synthetic oligonucleotide <400 SEQUENCE: 34 gccagotgca aaggtgttgg ac 22

SEQ ID NO 35 LENGTH 23 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: synthetic oligonucleotide

<400 SEQUENCE: 35 aacaccitgcc tdatcacgac titc 23

SEQ ID NO 36 LENGTH 22 US 6,664,105 B1 77 78

-continued

TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: synthetic oligonucleotide <400 SEQUENCE: 36 gcCaggctgg cqtcgatggit ga. 22

SEQ ID NO 37 LENGTH 22 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: synthetic oligonucleotide <400 SEQUENCE: 37 gtcgatggtg atggaCagga ac 22

SEQ ID NO 38 LENGTH 22 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: synthetic oligonucleotide <400 SEQUENCE: 38 gtaatacgac toactatagg gc 22

SEQ ID NO 39 LENGTH 19 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: synthetic oligonucleotide

<400 SEQUENCE: 39 actatagggc acgc.gtggit 19

SEQ ID NO 40 LENGTH 27 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: synthetic oligonucleotide

<400 SEQUENCE: 40 ccatcc taat acg act cact atagggc 27

SEQ ID NO 41 LENGTH 23 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: synthetic oligonucleotide

<400 SEQUENCE: 41 actcactata gggcto gagc ggc 23

SEQ ID NO 42 LENGTH 44848 TYPE DNA ORGANISM: Homo sapiens <400 SEQUENCE: 42 ggatcttggc ticactgcaat citctgcctcc catgcaattic titatgcatca gcc to citgag 60

US 6,664,105 B1 83 84

-continued gtoaa.gcticc cc.gcaattga citalacacccc cctaacacgt agaaattic.ca atctgcaatt 4860 tagt gaggat gatacctitta ttcttcttaa. attacatctot toattitcc.ca gag cacccitt 4920 tittitcc ccto citctgcacct ttttgttaaa gactggagta taatgaaata ccaagaga.gc 4.980 ataa.catgtg attacataaaa. citttittittct ggitttacaaa acagttcatt cittgtccata 5040 cgtgcttcto to caaggctg gctgctgtct gttccagccc gctitcgcttg gag aggC Cat citgccatacc tgctocccag acgcatcgac aag cacaccc agagtgttat citgctaagac 5 160 citaaaagagg gaggaaccoc citctic citcait citaag accita gcttctaaat tagagtgtga 5220 gggtocatct CCC Caggagg ggCacagggC ccaaacagoc cagocatcto agaagacaac 528 O actaagctitt gtaggggtcC acagtagagg agagtaagac gcctgttgtt taatttatta 5340 cagttcctca aaagtgaaga tgttgttggg.cg ggatggcaag agctgag Cag acgaaagctg 5 400 aaggaataag gaaaga gagg aggacacaaa cagotgacac titcctcagtt cittgtcattt 546 O gcctggcc ct gttctaagca ccttctaggit attaatccat ttagt cittgg citacaiacact 552O gtgagtaact agttttgtca cc.cccattitt aaaaatgaag aaagtgaggc to agg gaggit 558 O taagtaactt ggccacagtt tgaaactaga citctgatcac atgagataat agtgcc.cata 5640 aaaagggaaa gcagattata ttttittaaag gaaagagagt aggatatggit agaaaaagat 5700 tgtttggaaa ggaattgaga gattgatata atgaaaagaa gcattcacat gag agtaa.ca 576 O. gtaticagggc ccaaacct tc atctaaggta cittcaaagag gcc talagcaa. acttagtcac 582O tggcgtggitt citagtotcca tgatggcaaa tacattgttgt acagoccaac to cacacaaa. 588 O actitaaaitac caatgataga gcaatctaaa atttgaaaga aaaaatctitt caatttgtcg 594 O tottcccaga ggg acttaat caagaalacca atcaaaaitac titcctaag.cc taactgttgtg 6 OOO cagaactc.ca aagaga.gc.cc agcc.ctaaat caacactgtc caatggaaat attaatataat 6060 gtgggcctica tatgcaaggit catatgtaat tittaaattitt citagtag coa tattaaaaag 61.20 gtaaaaagaa acaagtgaaa ttaattittaa. taattittatt tagttcaata gatccaaaat 618O gttittctoag catgtaatca attataaaaat attaatgagg tatttattat to cittittctic 624 O aalaccalagto tattoctataa. totgg.cgtgt attatttaca gcacttctoa gactatattt 6300 cittitc.tttct tittitttitt to cgagacaatt ttgctcittgt cacccaagct agagtacaat 6360 ggcgttacct cggcto acto caaccitcc.gc citc.ccgg gtt caagttatto toct9.ccitca 642O gtotcc caag tagctdggac tagaggcatg caccaccacg cctggctaat tgttgtattitt 64.80 tagtagagac agg gtttcac catgttggcc aggctaatct caaactoctd agcto aggtg 654. O atatgcccac citcgg cctoc caaagtgttg ggattacagg cgtgagccac tgcaccc.ggc 6600 citcagattaa citatattit.ca agc gttcagt agccacatgt agctagtgct atgg tagtgg 6660 acagtacaga totgcatttc aattalaga.ca cgtatacaag catagttcac taatgcacgg taaaaaaaag tatagtgctg agtcggtggit agaaatccta aatactgcag agcaaaagtg 678 O. gtacgaacag caatctoagt gataatgcaa. ccatgcttgc ttittcattgc aatttgctta 6840 tittitcc titca gcaaagttca to catttittg ccalattcaat aaatatttac tgataaaaac 69 OO tittcaatatt agattottgc atcttcatag acagagttgc ttittcacatt tagaaaatta 696 O cittatcaatg ttaalacacac gttittgataa ccagtgttgg aaagaggtgc agacitcc cca tgttgccitatt gatggCagaa atattoacag ccaaagggaa acaaagg got ggggacaatc 708O acacaccitca tgtctoctaa citcctgggaa gtgctgtc.cc totgatt gag citctitatitat 714. O tgcctitcc cc actaaccotg to cactgtgc cctggagccc tittgcagggit tacctgctot US 6,664,105 B1 85 86

-continued gtoctoctoa cagaatatot ccitctacctic cittgtccaag citacaacttg gctattotot 726 O gatgacactg tottcc ctgt ag.cccttittg agtaatggct gcatattotc ccatagtc.ca 732O gttcttitt.cc tgttctocag totggcttct ggatgacago ccactagttt gaacticcata citgctatagt tdaagttcc ct tittgacttgt taccttgggc aaattacctic cittttgttca 440 ggttccittgt ttgtaaaatg acgataataa tgccatttgc ttcagtgggit tattittgaaa 7500 ttgagtgaaa galagg.cgggt agctitcccta cacgctcagt gtag act agc citgatgtgca 756 O ttacgggtga tgc catgact cagtgtgttt to citcatc.tc. cacatctggc totcatccag tgctcctgct tacggcactc tgtc.ccccitc titact tactic cc cct tatta actgaag act 768O ggcact gatc toacagtttc citctic cactit cctag totca ccatcatcct agatgactitc 774. O aagttcaccita gataaactgt citcagtttct toacticacat titttittataa. cagataatgt 7800 tacacticaag ttgtaacaga accag ctitat ccagotcatg aaatgtatgc attitcatcto 786 O aactctgtat toagtgacat cctgtgggta totggaaatc agc catggtg agaatattta 7920 ccatggaaat tggcaaatac taaaaag.cag agcaccittitt tittctgagag ccagaccata 798O gctottctac to catagdac ccatcataac aatttittaala taccticcact galacagottc 804. O titccitcticitc. tactitctitcc. atatotgatt tgagcttctt aattitat cat gtgaaccact 8100 cittgtaataa taa.ccc.caaa. tocctgttcc attgttctitc citgctaaaat actaalacctg 81 60 gtttagtc.ca accatattitt citctotttgg aatctacagg gtggcc.caaa aacctggaaa 8220 tggaaaaata ttacittatta attittaatgt a tattaataa. gcc attittaa tgcttcattt ccagtctoag tggccaccct gtatagotgg gctattgagc tottgcggga ggagggagtg 8340 gacagt citcc cagocacaca gactgatgtt gcaccaaaca ttttittagot to cag acttic 84 OO cctgg.cccitt agtgttaccc ttalacticitcc atttctotgc cittitcacatt citctacttitt 84 60 taaaaatcto tgacticcacc titcaccittat cattcttagc acatgac cat acttctgctt 852O cc caaagaaa atgagcaatt act tcc.ttitt cCtttitcc.tc. citgtcatcaa atctgcagac atgtcatgcc taagtc.ca.gc titt.ccitcCtt totctgatct cagtctgctt cittccatttic 864. O tgcc citgaat ccc.gtocc ct cocca acco caagg acttic gctictatoag to accitc.ttic 87 OO cctcitcctgt atcttcaact ccitcc cattt tactggctitc titcctcaagic cittitcc.ccala 876O gcctittccca totcaattac citcctc.gcac atgccitctg.c agaalaccacc cc.gtttctitc 882O cctocc citcg gcagoctott cittcctgttc tg.cccitcatg atggcaccat cattgttgtca 888O citaaaatcaa. totctocq ac atcatcaatg gcctitcc titt gttgggaaac citaataaa.ca 894 O ctittatctta tittggtottt gttatgggitt gaatgaggitt accoc galaat ccatattaga 9 OOO agtcctaacc cccagtacct cagaatgtga citttatttgg gaatagggto attgcagacg ttattagtta ggatgaggto: atactggaat gtgatgggct gcttatctaa tatgact gat 912 O gtoctitataa caaggagaaa tittggaga.ca gacacgcaca tagg gagaat accatgtgat 918O gacaggagtt atggagttgg agtcaaaaag citatgggaac ttaggagaaa gacctggaac 924 O aaatcc tittc citgc.gc.ctag agagggagta tggcc cit gcc actaccttga attcaacgtt toggctttitc aaaact gtaa gacaatacat ttctgttgtt caaaccalatt agtttgcagt 936 O actctg.cgiac tgcagoccita acaaactaat acagtctott ggagg cattt ggcaaggttg 9420 acaatggaag cactitt citta cccctittagg totgtc.gc.ct ttcttgttgg ggggtgttitt 94.80 citaacaattic citc.tcCatct citc.tc.tc.tct agtttgttctt aaacattggit gttctitcaga 954. O

US 6,664,105 B1 89 90

-continued tgcc togcag agt cagcctg caacaaatat ttgttgaata aattalacaga tggctittatc 2OOO toctitaagta aatcttgctt ttittcaccita ttaaaacaga cgcacaggcc aggttggtg 2060 gcc catgcct gtaatcc.cag cactittggca ggctgaggtg ggcggat CaC citgaggtoag 2120 gagttcaaga ccagoctogc caa.catggtg aaa.ccc.catc totaataaaa. attacaaaaat 218O tagctdggca tggtggtggg tgcgtatagt cccag ctact agg gaggctg aggcaa.gaga 2240 atc.gcttgaa CCC aggaggc agaggtggca gtgagcc gag atcatgccac tgtactic cag 2300 cctggatgac agaga.ccctg totcaaaa.ca cacacacaca CaCaCaCaCa cacacacaca 2360 cacacacaca cacacacacic aagttgtata atttaaaata taacgtgctt gttatggaac 2420 acttgtaaaa tacaggaaag taatgaaaaa gtotaccatc tagctcacca cataatgacc 24.80 attgctatoa to citgg cata attcticitc.cit gtatataaat attatattott ttattgttaa 2540 aattacacta tgagtact at titattitatitt tactgtggca aaatgc.gcaa. aa.cataaaat 2600 cittgccattt taagg tatgc agtttggtgc attcaccaca citcacattgt tgtgcaaata 2660 toaccactat citatctoaga acttctitcgt citt.cccaaac tgaaactctg tacccattaa. 2720 acaatagtgc atcctotgtt titc.ccc.tc.cc. tacaattitat ttittatttgg gtttgtacca 2780 aactgaaaat agctgcttct toctitacitta gttcagatta gcatttccat ttatttagoc 284 O gtggttittga ggatgc catg acagatgc.ca toctitcc tag agctotttgg ggctgtcagg 29 OO tatttcagtc agggtgaatt cgg gttgata acattittaala atctoactitt attctgaggit 2960 toctagtgtc agagcc cacc gtatttittag ggacitcc caa gttacaaaca aaaatatggit 3020 gaggaggaat cactgaagtt ttaacacaag agacittacat tttgttcaat ttctatotitt tagtttattt cctaag cata aagaaatact ttgaaaattit tacatag cat tatacatatt 314 O taattalagca tgagcacatc ttaaaactitt aaattittaga toagatctitt aatticcitagg 3200 atattalagag gtactggcaa. tittggccagg tgtggtggitt cacgcctata atc.ccalacac 326 O tittgggaggg tgaagtgggC gaattgctag agccCaggag gtggaggctg caatggcctg 3320 agat cacgcc atcgtacticc agcctggatg atgagaatga aatcctgtct Caaaaaaaaa. 3380 aaaaaaaaaa. aaaagaagaa gaagaagitat tggcaatcag tgcto cagga attaattitcct 34 40 gacittgaaat aalaccitacat gtag acaaac taattaggcc attccaagag ttgctag cat 3500 tggitttaata tgtttitcaga gcatt.ccagg aag cagtgtg gcc agcattg catgtttgat 356 O actitcagaaa tgitatgacag gtgtttctot tacccaggto ttctgtttitc ttagttittgc 362O tdatgtaaat atttatgaac atcct catct ttittgaggga agggattata gatcattcta 3 680 attic cattitt ctagoatttg gtaccattct aag cacatga taggcacco a tittggag cat 3740 ttittggcttg acagaatatg catttagaat tgttcaaatt agaggtgtca gtgatgggaa 38 OO ttagaatact attataattct aagtcatttg act taalaitac aaaagaatga tttitccttgg 3860 tggggaatgg tgaagggagg caggagittaa gala gaggaga agagatccta agtcatttat 392 O aaacttctict ggaaagacag gtgtgtgaag actittittaala aagttcattca ccaaattgtg 398O tgtgtgttgtg tgtgtgttgtt ttaaatagac tittattittitt agagcagttt taggttcaca 4040 gcaaaattga atgcaaggac agagattitcc catalaa.cccc ctg.cccacac acatgcatag 41 OO cctic cctocat tat caa.catc cccaccagag aggtgtttgt totagttgat galaccitacac 416 O tgacacatca titatica.ccca aagtccatag ttcacgg cag ggttcactot cggtgtacat 4220 totatoggtt tgagcaaatg tataatgaca tgitatccacc attatagitaa catacagagt US 6,664,105 B1 91 92

-continued attittcagtg ccctgcaaat ccc.ctgttct ccaccitatto atc.ccticcot citctgcattt 434 O ccaccoccag cc cctogtaa cc.gctgatct ttttactgtc ccatagitttc ggacgatcta 4 400 tttittcagac agacacagag citgtc.tttcc cittagtttct attctatocat ttctittcticc 4 460 ccatccatca taaaaggcta tgagttttitt ttaagtgttg alacac catcc tacttgttcaa 4520 gttaaaac at aagctoctogg citgggtacag tggct catgc citgitaatcto agcattttgg 4580 gaggctgtgg cagaag catc actitgaagcc agaagtttga gaccagoct ggcaa.catag 4 640 caag accoca toccitccaca CaCasaCaCa cacacacaca CaCaCaCaCa cacacacaca 47 OO cacacacaca CaCaaaaa Ca agctottgcc agaattagag citacaaattg cccitcaggitt 476 O ccitagaagat cagtccttca attagattca gattgagatg cittccitctitt taaacaatga 482O titcc ctittct atcatgcc.ca ataagaaaac aaataaaaat taaacaatac tgcctgtaat 488 O citcagotacc Caggaggcag aag cagaact gcttcaacco ggcaa.gcaga agttgcagtg 494. O aagtgagatc gcqccactgc acticcagoct gggaalacaga gcaagattot gtotcaaaaa 5 OOO caaaacaatg tgattitccitc citctaagttcc tgcacaggga aatgttalaga aataggtoca 5060 ccaggaaaga aggaagtaag aatgtttgac tag attgtct tggaaaaaat agittatactt 5 120 tottgcttgt citt.ccitaa.ca gttctocaaa gctitcgtacc ttggccagag gcttgtctoc 5 18O tgcgtacctg aggtttggtg gcaccalagac agactitccta atttitcg atc ccaagaagga 5240 atcaacctitt galaga.gagaa gttactggca atctoaagttc alaccagggtg aaaatttitta aag attcact citatattitta attaacgtca gtocgtoatg agaatgctitt gagaaaactg 536 O ttatttctica caccitaacaa. ttaatgagat taact tcc tic tocccitcatc tgacctgtgg 542O aggaatctga acalagaggag gaggcagtgg gcaggtttcc ttatcatgat gtttgtcatg 54.80 ttcagtgttga ggc citcacaa aaaaaaaaaa. aaaaaaaaaa. ggcgtcCtgg atata actoga 554. O gagcto attg tacagtaaat attaataaaa. cagtgattgt agctgaagga tagaactgct 5 6.OO tggagggagc alagtggg tag aatc.gc.gtca aactaaagag catttctago caaag acaca 566 O atgatagatt gaaggatatt tattotalaat atagaatatg ggtgaac gag atctgtggac 572O ttctgggcto caacgttaga ttctgattitt agcaa.gcttg to aggggatt citgatattga 578O aaggctgtgg ccttcaccitg agaaacct gc CCtagggggc catgaaaatt tgtc.ctgtct 584 O ttcagaagtg citatcagaca toaaatggaa gttaaatcgt. atcttaacaa. ttacitagg at 59 OO ggg.cgcagtg acticacacct gtaatcc.caa cactittggga ggctgaggca ggaggat CaC 596 O ttgagcc.cag gagttcggga C CagcCtggg caa.catagag agacgttgtc totattittitt 6O20 aataatttala agagaaaaaa atactgaaaa tattgtatac accactgaat tataataatg 608 O tgtatataat gtatatatto attat gagga atatttgatt attitcatata titatatotitt 614 O toctitctgtt tattittatcc agittatgaag tatttagaac aattcatcag taattggggc taaattgaca gaatagtaat cagagaaaat agaaaaagac agatgggitta totttgaata 6260 ccaggttgga gttgtttatg ggtttgttitt ttgttittggg gg.cgttttitt tag acagagt 632O cccactctgt tgcc.caggct ggagtgcagt ggcacaa.gca tggcc cactg catccittgac 6,380 citcttgggct caa.gcaatct toccaccitta gcc to citgag tagctgggac cacaggtgca 6 440 tgtcaccaca cccagotaat titttittattit tttgtagaga cagtctttct atgttatcca 6500 ggct gatcto aaactoctg.c acticaagtga tocccctg.cc ttggcgt.ccc aaagtattgg 656 O gattataggc atagocacca cacccalacct agtttctatt tag acttggc cctitt.cccac cagtcatttg tgtccaaaag atctoataaa. tgtag acagg aaactgtc.cit ttgctcatca 6680

US 6,664,105 B1 95 96

-continued titttgtattt tittagtagag acagg gtttc accatgttgg ccaggct coa ggctogtotc gaactcct ga cct caggtga to cacccacc tdagcctccc aaagttctgg gattacaggc 914 O gtgagccacc acticcitggcc acaatcc titt tittaactatg aaatatattit ttatctgaag 920 O tittgatgttt attacccaact gagggatgat gttcc catat citcagittaaa gaaataacct 9260 gctcagatac ttcaagctct tottttgact tittgaaaata aatgatcttg aagttacitat actttgtttg ggittagttaa cattatttala agtatattat tittaattaat tatctttgta 9380 agattittact gtatactacc tggagttcaa tgitat cagat ggatttcaaa tittatgtaca 944. O titttittatgt atatggtaca gaaaaaaatg tgatccataa gaaatcagaa aatagog cat 95 OO atgctaatag ctaatgttgt cctictaaaaa. act tatttitt gcatttittaa gagggggata 956 O tactctgaca citttaataag tgtaattaat tattgacitgg aatttgg cat gaggcagggc 962O catttcag at cc cattaaag gaatgacaca taccagagaa ccacagaagt aaggccacat 968O ttgtaataaa toattatago totgctagga galaga.cccag ttgtattagg taattaatgg 974. O atttgctott aaaacacatg toccggaaga tataggtgag tottgggggg cc.gcattaaa 98OO cattatacca atgitatctta catttctaag aaagttttac tactttacag gatctittctg 986 O ttaccaaaat ggalaggtttc caacticcagg acttggctitt catagttcct acacCagggg 992 O aaatgcctitc citttgctaac tatgcaacca ggittagttag tgtaagttcca gcc accotgt 998O tggcaatgct aaaaggtaca acaaacacag aattittattt gcatttgtaa acatttgatt 2004 O totggctoga aattittcagt titt catgggc acgtoatgga aacagaaatc ttctgttgttt 2010 O agtttgggca cc tact catt gtagtgacaa atatttcaga agccaatagg ggatt.ccaca 2016 O aattgttctg aacct gtggc tgagacitggit aatggct gag tgacatgggg acataccaca 2O220 aaagaagagg tag caaaagg citgct gagat aagga catgt toattgctta gctagtggcc 2028O tgcaccctta aaacacatgt CCC aggctgg gtgctgtggC toacgcctgt aatcc cago a 2O340

Ctttgg gagg Ctgaggcggg tggattacct gaggtoagga gttcgag acc aacctggcca 2O4 OO acatagtgaa accitcatttic tactaaaaat acaaaaatta gcc aggCatg gtggcggg.cg 2O460 cctgtagtoc cagotactica ggagg CaggC aggagaatta cittgaatctg ggaggcagag 2O520 gttgttggtga gcc gagattg cgccaccgca cgctagocto ggC gaCaaag tgagacitctg totcaaaaaa. a Caaaaaaa. aaaaaaa Ca a Caaaaaac a Cala Caa.ca aaaaaacggg 2O640 tatc.ccagaa gatacaggta agttittctaa cacaggtoct cittgtatggit gcgttccact taagtagaag atgacaaaaa catttgtcat gagaatatag acticacattt taaacctgtt tgag Caggaa aaggaa.gcaa. tgttacagat gtaattctgg gtgtgacitoc agaaaggatg actic cctitat taaagtag to atcctgagtg agctaactct ttgtactitcc tottctic citc. citgttcc.cct catcaccc.ca ttctt.ccgtt gcctacacco aggcc cacat tggatgctga 2O 940 catagacitta catggtacag to caagggaa agatctgcca tittittittcaa. tgttgtcatct 21 OOO tggittatctt cattccaagg atctotccac totttataca gtaagagatg agagtctgga 21 O60 aaggattggg aataagataa tgaattgtaa gttittaaatt gttctitcgta ttittggggaa 21120 ggagtaggct aggtggtoct totgttttitt titttgtttitt ttttittaaag tagatgtggc 2118O cagacgtggit ggctoacgcc tgtaatccca gcactittgag aggct gaggc aggtggatca 2124 O cittgatgtca ggagttcaag accagoctogg cca acacagt gaaacco cqt citt tactaaa. 21300 aatacaaaaa. Ctagc.cgggC ttggtggcgt. ccaccitgtag toccagotac tgcagaggtg 21360 gagg Caggag aatcacttga accCgggagg tggaggttgc agtgagccala gatcatgcca 21420 US 6,664,105 B1 97 98

-continued ttgtactcca gcc toggg.cga. cagaacaata citctgtctoa aaaaaaaaga gaaaagaaaa 21480 gaaaaaaaga atggatttga acticagtcgt. caatagocto tatto cagga gatgttacag 21540 ttgattatgt tatagggggt gtataataga attitcgagct atgtaaattic caagtgcatt 21600 tggaagaatg aagaaatgga ggalagggtaa agtatgagtg caag cattcc aggittttittg 21660 aaaatgctat aatctttgtt cagggctagt acaaagtgct atttagotgt aagggitttitt 21720 tgttgatttac agacagttitt cacatgtgtc atttcaacct tggittittatg gCgalagg Cat 21780 gtgatggtgc ttgtcc cagg actittagatc catat citgag gttcc togtog ggcaaagata 21840 ttacccctga toatattata gtotataagt gggagagttg tgcctggagc tdaagttctta 21900 tgatttctga to cagggcac titcc tacaac atgattittgc aatataaaag cctataatgt 21960 gtgactaaag caggtoactic accocittgta acagacticta gtaatggtac tgccaccaaa 22 O20

Cggctg.cgtg atattgggca aag acttacc ttatttgaat citcagtttcc toctagaaaa 22080 atgagggtgg aggittaa.gca taggctgatg atcctaaag.c citccatactg ccctaaactg 221 4 0 tggctotalag atccagtaga atgctgggto acaggacitct agg gagctitt toaaa.cccala 22200 atgtctgtca titccttgatg gtagg cagca gtttatggaa gtggg.cgaca cagdaaatat 22260 caaaatacct aaag.cagott gcaa.gagttg tittctg.ccta gtggtottta tagittaatat 22320 taaatagitta attittitttitt tttittgagac agagt cittgc totgttaccc aggctgcagt 22.380 gCagtggCaC aatctoggct cactgcaacc to caccitccc gggtttgagc aattctgtct 224 40 cagoctocca agtagctggg actacaggtg catgccactg caccoagcta attitttgtat 225 OO ttittagtaga gacggggttt caccatattg ggCaggctog totcgaactic ttgacctcag 22560 gtgatccacc tgc citcagcc toccaaagtg citgggattac agg catgagc cactgcaccc 22 620 agcttaaata gctaatattt aat attatto tatagittatt caagtaattic aggccaaaga 2268O cittagaaa.ca aaacaaaaag ccacttittaa. ggagaaaggg tgtaagtttg ccagatagat 2240 agagatctitt cittittittaac tacaagagtt caggaatgaa titactictitta acaaacg act 22800 atagatatac atgaaaattg gaaggacitta titatgcatat gataatcaat ttaaagacaa 22.860 cact taaaat tatattgttg ccact citcaia aaagtggtaa tagaacagot aatggtttaa 22920 aaag cagagt acagaagttc ccaaactitat ggcaccittaa tat cqcagaa aactittittaa. 22.980 agcatgccita ggccacaaaa aatacctgta ttittgattat taaattgtaa ggtotacaca 23040 acctaatagt aataggtoca atagtaatgc tgtccaatag atgttgatgt tittitt to citt 23100 gcaaacttaa aagatccitac agtgc citctg taaatagdac tgcctggitta gagttgaatt 2316 O toagataaat aattitttittc atgttaatta tittitt cittitt citt tacttitt tttitttgttt 23220 titttgtttitt ttgtttittitt ttittgaga.ca gggtotcatt citgttgccoa ggctgctgtg 2328O caatgg catg atcatggctc actgcagoct tgaccitccct gggct Caggit gatccitccca 2334 O cctdag cotc ccaagtagct agctgggact acaggtgctt accatcatgc cc.ggctaatt 234 OO tttgttgttitt ttgtagagat gtggttittgc catgttgccc aggctggtot tgaactcctg 23460 ggctcaagtg atcc.gc.ccgc citcggcctcc caaagtgcta ggatgacagg catgagccac 23520 tgcaccitggc CCCt99g.cga. agtatttctt aatggittaca tagga catac actaalacatt 2358O atttattgtc tatatgaagt tdaagtttaa citaggtgccc tgcacttitta gttgctaaat 2364 O cctgtagctg tacccatgca ttcactggtg citccc.cagot tgccttgcac agagtttgga 237 OO alaccatagtc citatalactict aggccaatitt tittaatgitaa aatttgattc attittaaatt 23760 US 6,664,105 B1 99 100

-continued aataaataat aac aggaatt tittittaaaaa. ttgttittaaa tataattaaa. attatcaaaa. 23820 tattittittaa. citgaacttgt gacitagagat atttagatta tgaagagtgg ggitttatgct 2388O aactaatgac agtctggcta tgcatgtgga gcactgagct ataaattgttg gctitccccaa 2394 O ttctoctoat gtoactitgaa caaalaccitaa gtgtcag acc agagcttctg gtatctitcca 24 OOO tgggatttca ttcaacagot ggagcaaatg aagtcagatt gatttitttitt aatttgtc.ca 24O60 attttgttgt citcaaaaa.ca taattataat catttattag aactagaatt tottcagttt 24 120 aacaacagaa atagittatto attatgaaaa gcqaatctgg aggccitt cat tgtggtgcca 24, 18O atctalaccat taaattgttga cgtttittctt ttaggaagct citgtagatgt gctatacact 2424 O tittgcaaact gcticaggact ggacittgatc tittgg cctaa atgcgittatt aagaacago a 24.300 gatttgcagt ggaacagttc taatgctoag ttgcticcitgg actactgctc titcca agggg 24360 tatalacattt cittgggaact aggcaatggit gagtaccc.ca gggaacaatt cattaataag 24 420 gagatticc cc actago atta titt cittittct titt cittittitc. ttittctittitt tittitttittitt 24 480 gagacagagt citc.gcactgc tgcc.caggct ggagtgcagt ggcgccacct cggctcactt 24.54. O gaag citctgc citcc.caaaac gcc attctoc tgccticagoc toccgagtag citgggacitac 24 600 aggcaccc.gc caccgc.gc.cc ggctaattitt tittitttittitt tittitttittitt tttittittgca 24 660 tttittagtag agacggggtt to accgtgtt agcCaggatg gtottgatct cct gaccitcg 24.720 tgatctg.ccc toctoggcct cc caaagtgc tgggattaca ggcgtgagcC accaggc.ccg 24780 gctago atta tittctitatga cacttitttitt tttitttittga gacggagtot cgctotgtc.g 24840

CCC aggctgg agtgcagtgg cgc.catctog gct cactgca agctocacct cc caggttca 249 OO cgc.cattcto citgccticago citc.ccgagta gctgggacta cacgcac cog ccaccacgc.c 24960 cggctaattit titttgtattt ttagtagaga cggggtttca cc.gtgttagc caggatggto 25 O20 totatatoct gaccc.catga totg.ccc.gc.c toggcctccc aaagtggtgg gattacaggc gtgagccact gcgc.ccggCC aac actictitt ttatt attag caaatatact totgcctggg 25 140 cacattcttg caagtgctica acaatgcaac ttittggaagt gcatgtggca gaaactcctg 25200 citgitattitat to cagaacct attattgcta atcccagttt atgttacatt tgaagtgaga 25260 accagttgga gcc agcaacg titcccagotc caaagttccc ttgagattitt cagaatcact 2532O talaccctatt atgcttggca acctggactic agcaaaactg ggalagtoagic agtttgttitt 25380 attcatcc ct toctittctica gtttctoaaa tgtgtcagtt aatctoagta accocattgc 25440 alacctitcaitt acctg.cccala gcqgtctaga actitgcc agt atagaatcct acgtggg to a 255 OO agct cotgac tgttctoctitc titcactic titt ttittgcaaag aacttgtaaa ttittalactat 255 6.O aagtattoat gattc.gc.cac atttattoaia aacatagagt gctttittcca catatoagcc 25 620 aatggaaata aggattaaat gggaaatgaa atgtagtaat aggataagca caagttcttct 25 680 toctgctdaa actitttittitt tittitttittitt cagacaagat cittgctotgt tacccaggct 2574 O ggagtgcagt ggcgtgttca tagct caatg talaccitccala citcctgggct catgcaatct 258OO citcacaccitc agcc.ccct ga ttagotagga citacactatg cctag coaat tittittitt citt 2586 O ttgttctggitt gtgttgcc.ca ggctgtc.tc.g atctoctdgc citcaagtaat cctcctgcct 2592 O cggccttcta alagtgctggg attataggca tgagccactg tgc.ccggtot caaac cittitt 2598O tittccaaagt aaatgaagtt attagatatg gaatatagtc tagttcc cag altatic catat 2604 O ccattggittt attaccctica ttattalactit caaattgttt aatagaccot catatotcag 261 OO ttatacagtt aaaatttittg titttgtttitt citggagtatc ttatttataa. citatgagttt 26160 US 6,664,105 B1 101 102

-continued tactitt actt atttattitta tttitttgaga cagacgcttg citctgtcact Caggctggag 26220 tgcggttgcg tgatcatggc toactatogc citcg acctitc tgggctdaag tgatcctcitc 26,280 cctdag cotc ccaagctgag actacaggca tgcaccacca catctagota attittitttitt 2634. O titcc.ccatgg aacaaggctt tactatgtta CCC agagtgg totcaaactic citggcct cag 264 OO gggatcctico tgtctoagico taccaaaatg citgggattac agg catgagc catago.gc.ca 264.60 gacctggttt tacttittctit gactittgaat tacaagttitt tgtaatttgg aaaatgttitt 2652O gttgcttitta aatact gctg tatgtttgct tittaaataca acatttctog attatatattit 26.580 tgagaattgc tgtctittcag aacctaacag tittccittaag aaggctgata ttittcatcaa. 2664 O tgggtogcag ttaggagaag attittattoca attgcataaa cittctaagaa agtccaccitt 267 OO caaaaatgca aaactotatg gtoctogatgt tgg to agcct cgaagaaaga cggctaagat 2676 O gctgaagagg taggaactag aggatgcaga atcactittac ttittctitc.tt titt.ccttittg 26820 agacagagtc toactctgtc agccagacitg gagtgcagtg gtacaatcat ggcto acto c 2688O aactitcg acc toccaggcto aa.gcaatcct cccatctoag toccacalaat agctggg act 26940 acaggtgcac atcaccacac citggctactt taaaaaaatt ttitttgtag a gatggggtot 27 OOO ccctgtgttg CCC aggctgg totcttgaat toct9tgcto aagccatcct tocaccitcag ccitc.ccagag tgc.caggatt acagg catga gccaccacac ccagocacca cittitt cittaa. 27 120 aaaaaaaaaa. agattototc tggtag acaa toctoaatag tocacatgtt attaaacaat 27 18O citgctgcctg aatacatgat ttaccaaaaa. aaggaaattit tgacgggttc agaatat caa 2724 O gggatctgag gcaaatgtca cctatgataa aatttgctat caaaattagg aagtttgttgt 273OO ttacct gatc citaaag cagt alaccagocca tittctaggga ataaaactict catgc gtata 2736 O ttgttgcatat atatgtatta tatgactgag tgataataaa attittitttitc. tagctitcctg 2742O aaggctggtg gagaagtgat tgattcagtt acatggcatc agtaagtatg totcc tatto 27480 ttaatactag gaaagtaagg ctagotttat ttattaccta gtattoaaaa agittagttca 2754 O tittaactgcc aattgactgc agttcaaata agaaacaaat agtgtc.tcaa gtag cactdt 276 OO actic caatitt taatattaat aaaaaaaatt ttaagttatt ttaaataatg tagtggtttc 27660 tataaagatc actittata.ca gaagaacagt gccaattaac ccatggaa.ca tataagtagc 2772O taaaac caat tgcttgccala agaaccagta accoaggagt acatgtccitt gcc actotgt 2778O tttittcaaga cagagtaact gatttctagt tacttgcata gaatggactic citcct catala 2784 O citcc ct tcca tottggtott toccitagtag aacttctacc tttittittagt aac aggtgag 279 OO tgggagaggit aagaaggaga atalaggtoag caattalacct aaaag cagaa agtaaaattit 2796 O gttattittitt ttctgaatat tittctgttgta atttagotac tatttgaatg gacggactgc 28 O20 taccagggaa gattittctaa accotgatgt attggacatt tittattt cat citgtgcaaaa 28 080 agttittccag gtaatagt ct ttittaalactit tittaatgitaa alaccagaatc cittatttitat 2814. O agtctagota gttctaaatt citataggitat gtatatttac atgtttittct aattittagag 282OO aacaag cact atgactitatc cactgttagt titt.ccc.citta gcattgggto ttaccccatg 28260 tacgtgatta gaaatttgaa a tattitccala tagcctittag tagaattaac tdacatagat 2832O gataagaatg ggttggttca cittcatgttc cittccacago citact attitc aataaaagaa 28380 agttitcccaa gacctaaatg actatogaa.ca tattittataa. citatatagga ggggtgggtc. 28440 taggaataca aagttittgaa tgctgttaat cittcaa.cacc acagttgaaa ccacagg to a 285 OO

US 6,664,105 B1 105 106

-continued aattitcctica gttataattit ttgcaaaggc ggitttcagtc ccagotactt gggaggctga 3O 960 gacaggagga ttaatggagc ccaggagttt gaggttgcag agagctatoga toacgc.cact 31 O20 gcactc.ca.gc citgggtgaca gagtgagaCC citgtctotaa ataaataaat aagtaaataa 31 O80 ataaatacat aaataaaatc aagatggtgt gcaattagaa ttgagcgatt ttgtttccaa 311 40 acct caagaa agcttggtot tgctotgtcc Caggtggctg gataaattgg gcc totcago 31.200

CC gaatggga atagaagtgg tgatgaggca agtattottt ggagcaggaa actaccattt 31260 agtggatgaa aactitcgatc citttacctgt aagtgac cat tatttitccita attctagtgg 31320 agtagattaa agt caactica ggaccitctgg tgtta accto citatgaacag toagtccitct 31380 cagtaactag ccaaatcatg agatgatgaa ttagaaggag ccittagatag catccaatct 3.1440 aa.catttittt tgtgtgtttg aag agaagaa atcaagagct aggaataact ttittaaaggit 315 OO aa.gc.catttg cagtatagtg tggattttgt ttaaaagggg ataatttgaa attittatgac 31560 toattata.ca agacaaaata agttggattt toaaatgttt tacaaagtaa atcaaagtta 31620 taattgccta cagtacgcaa. agcttcaaaa catttittitat gttatgaaat tgtaattitat 31 680 ttalaccittaa. aatgagccag taccatgttgt ttgcttaaaa atctoatgct aagaatttac 31740 tatgttgtta attaatctt.ca agatattitat gaataaagtc ttatttctaa toctitccitcc. 31.800 aactgtatct ggtgctaaat caggaaatgt ttctt.cccala aaag.cctic git ggaagatctg 31860 tatgtctaaa tatatgtcag ggataataca gatgtagccc tgc galag cat gaccttgatt 31920 tittatagtct aaaatgtcat ttgcagatat citattittcta agaataattic citaaaagaat 31.980 tatttgaatg ttgtaggaaa gctaagaaat tittgcaaaga gcgtacgtga aaatataagc 32040 taggcttittg tggtttgttgg atag actitcc caacaaaatt gctttittatc tatagtgatc 32100 caagcttgtg galacat atta gtdatcttitt tittagaaaat tottagaaaa gtgat cittgc 3216 O aaaaatggaa tittatctittc cc caagtata ttctgtcatg tatagagitta aactaag cat 32220 agtaattitca ccagacaaac attcaaaatc tactcctgac citttittatct catccalaatt 3228O titcc cagggc ccagacataa acctittgcct tacgaactot ttgtatatgc actaaatatg 32340 cittctic ctitc. aaggttctica gtoagctaga aaaatgtgca agagtaaatg gtacccttct 324 OO cacttgtaga to caa.gagaa ttagacittaa acticacticta catgtctgttg actittattitt 324 60 atttgcatca cagtcc totg aggtgcaag gcaggitatct tggatccatt ttittagataa 32.520 ggaagttcaa attgagaaga ggttgcatca tttacaggaa gccatactgt agtccitatgt 32580 tact cittaaa. aatcc cattc aaatcctgct totgaggcct gcatactittc tacccitacca 32 640 gtoattgacc catgctitatg totcctittga aaacattgat to cactcittg totccagtga 327 OO aaaagtggaa tittaag caga gaaacaaaag ccatttgttct tgttaagttct actitt.ccctic 3276 O. tactittcaag aaggaaagtt gggg tatgtg ttgaatggtg atttattitat ttatt tatta 3282O ttittaaaaat tgatacaagg tottactgta ttgttgcaggc tggtotcaaa citcctgggct 32880 caagtgatca toccaccitca gcc toccagt gttgggatta cagoatgaac cattgttgccc 329 40 accacc gatc cgcagtttitt taagaaaaac titt tactata gaaaattitta atcatataca 33 OOO aaatacagag gaaagtatat gaaccoactt taggagacita gaatatgc.ca cc.ccaaaata 33060 tgcc actittg gcataaggat tatttcgagc taaaggcaac tgggaagaala cacatagaag 33 120 aaaagttcto tgtc.cttcto catttgccta aaag.caggac atgaatctta aaagttcc.ccc 33 18O toctitc.ccitt totaccagga aaaacaagag ttaatcactg aagataactt cag accotta 3324 O US 6,664,105 B1 107 108

-continued toagtgtaga gatggcacta gaagaatcta tattacatac to attitatitt toctitcc cac 333OO aacttgccac cccagagact aaaaatcc tit titcctttgtc atgtctottg to caaaaatt 33.360 tgctctataa gctggagttc taagccacct citttgagaat tacttgttcc citgg tattitt 33 420 citgttaacat acatgitatta atatacatgt taacaag citt citgtttgttt ttctoctgtt 33480 ttctgtcttg ttacagaggit ccatcc.caac taagaactaa agagtaggag gaaaatataa 33540 tittccitcctg catactittga tottgtttaa tocgtaacco titc.ccactitt to accticcita 33600 cctattagat tactittgaag caaattitcag attatattact titatictataa. atatttcagt 33660 atgtgctagg tgtggtggct cacacctgta atc.ccalacac tittgggaagc tgagg Cagga 33.720 ggat cacttg agccCaggag ttcaagacca gctacggcaa. caaaaaatca aaaactitatic 3378O tggg catggit ggcacatgcc tgtggtocca gctacatgag aggct gaggc aggaggatcg 33840 citttagcc.ca ggaggttgag gctgcagtaa gctgcattca caccactg.ca citc.ca.gc.ctg 339 OO ggtgacagag taagaccatg totcaaaaaa. attacatattt tag tatgtat ccttitttgta 3396 O aaalacacaat acttittatca tactittaaat aataacaata attccittagt atcaccalaat 34 O20 attttgtcag tgtctdacat titt.ccittatt gtotaaaata ttgttgatag ttattocaaat 34 080 cagaatccala acaaggtoca tat attacat ttggttgaca agtctottaa gtttgttcat 34140 citttaagttc titcc.tc.cctic totttcatct cittgtaattit attaatgtoga aaaaa.caggt 34,200 aatttgttct atagtattitc citacattata gagtttgcta catttattoc citatgatato 34,260 atttagcatg titccitctgtc ccctgttgttt cctgtaaact ggtagittata ccitagaagct 34320 tgagtttatt caggtttitta attgtattitt ttittgcaaga attctittatt atctgcttct 34380 ggalagcacag aatgtctggit tgtgtctggit tittgatcttg acago tactg atgaccattg 34 440 ccitaatcc at tactittattg gggtgggggg aataaggttt taaaataaat tittittittaaa. 34500 gattitttitta actgttattt tgaga cagtg totcatttcg tittcc caggc tggagtgcag 345 60 tggcacaatc acggct cact gcago cittga cct cotggga to aggtgatc ttcticaccitc. 34 620 agccitcctgg gtacctggaa citacaggtgc acaccaccac acctggctaa tttitttgtat 34 680 tttgttgtaca gaaggggttt catcatgttt cccagacitgg tottgaactic citgggttcaa 3474. O gtgatctacc cactitcagot toccaaaatc citgggattac actittggc.ca cc.gtgcc togg 34.800 cctaaatgaa attatttgtc totaalacaga cagaagttitt actittaaaaa. tttgttctittg 34.860 tgtgtacatg tgtttgttgta tgtgtgttgtg totaaaagtt tggctittgag citttgctittg 34920 aattcttgga tgaacaataa ccaagaatac ttaaactctg atcattcttg acagatato c 34.980 cctacaggct atggccttitt gaattgttgtc citc.ca.gtgat aaaaag.ca.gc aag cacgata 35040 citgctotcag att catggtg gtocacatgtg aggtgaaaaa aaaaaaaaag atgaatccta 35 100 tittaaatgcc cc caggataa cagtgatact citttgtagga taactatttg cittgccactg 3516 O gtttcattaa ataaggacat aagtaaagat citattitttgt citc.tttctoc ccalaccacca 35220 caac taggat tattggctat citcttctgtt caagaaattg gtggg Cacca aggtgttaat 3528O ggcaa.gcgtg caaggttcaa agaga aggaa gctitcgagta tacctitcaitt gcacaaacac 3534. O tgacaagtaa gtatgaaa.ca caccctttac caatcatcaa. gttittagtgg gtaagcctdt 35 400 aactitt actic aaacaccctg ttgcatgttgt citatacattg cataagtata ggcagttgca 3546 O atttagtaaa gttittataca acg atttitat tittatttitat ttittagalaga aaaatgctac 35.520 titttgttgtt gttgttttitt gaga.cggggc citc.gctogto acco aggctg gagtgcagtg 3558O gtgcaatcto agctoactgc aacctcc.gc.c toccgggttc aagtgattot tgaagaggag 35 640 US 6,664,105 B1 109 110

-continued aacaataata acaacaatat tattittcaaa. agttgttgacc gcagtttctg gagttgagaa 357 OO gacatc gaga ttitttgtagc citcaitactict tgctittaggit agcaaaaaat gttcctaaat 3576 O. citcaggaata ttctotag at aggtttcaat citatic attcc tgataagatg atgctgaaat 3582O actaatticta gccaaaaaag accagotacic attitcc.gatt gttggggact gggaactctg 3588 O gatagtgagg acco cagtag galagtagcga. ggggaatggit ttgaatggat aaatt catala 3594 O aaaatgtcag tag atttaat titt cittatac atttcagtct ttittataagg citaggaaaag 36 OOO cccctgttitt tatggitttat aatttgaatt cacat galacc cacaaaattit gccttittacc 36060 titccitatgtc tgaaaatgga tag totggct ggcct cittaa caa.cccagot ggcagagctg 361.20 tgaggatcto agtgtgctict agcc.ca.gaca ttggtag cat gaacggcaac atttittaatt 3618O gtgttttcaa aataggagca cactagoggit citaaaacgat cataaaagaa ggatactaag 3624 O agggcc cact gtoattatgg atcc taatac ttaggatgca titatggattg to attatgga 36,300 tactaatact taggat caca tttgtaattg agtttittaat tgcttaaatt agatacatat 36360 ttctattaag ttalaccticitt tgcttittagt ccalaggtata aagaaggaga tittaactctg 3642 O tatgccataa accitccatala tgtcaccalag tacttgcggit tacccitatcc tittittctaac 36480 aa.gcaagtgg ataaatacct totaag acct ttggg accitc atggattact titccaagtaa 3654. O gtaattittcc ttgttcattc caaactitt.ca atalaattitat tggtgttitat cagaatagag 36600 agtttggaca gggagcaaaa gacaaagttca actatatocaa. gttctaataa ttcttaatat 36660 to aggaaatt tatgtatgaa tact tactaa tatgagtata acticatccita agagtictaaa 36 720 gcaaaaggat gtgaacacaa actagoagtt atcttagaga ataagtttgc atttcaaaat 3678 O aacttgacat atcaagat.cc acticaacgca tittaaattat titactictaaa. aag acataat 36840 tottggitaac acattcacta aa.gcaaaata tacctittata taattgctat caaagg tatg 369 OO tgggttggta taaaat atca taccatgttga gatcagtgtg attcc tittac agcattaatt 36960 tittattggitt agagtaagaa aaagaatago tagagtatat ttcttaagta gattctoata 37020 cactittggitt toaaaalacca attattgact acatcttata aaag.cct gta ttcaatggag 3708 O tgccaaaaaa tgactatoag tottaaagag ttagg catat aaatattitta aggtttctgt 37140 tdaatgtatg ttggaaggag titcc tittctic atgactatto toatattgga gcataaaaag 372OO agtttacagg cittggcgcag tggct catgc citgitaatccc aatactittgg gaagctgaag 3726 O cagg cagatc actitcagocc aggagtttga gaccagocto ggcaatatgg caaaact citc 3732O totacaaaat attaccaaaat tagcc aggcg tggtggtgca tgcct gtagt cccag ctact 3738O tgggaagctg aggtggagg attgcttgag CCC agggggg tdatggctgc agtgagctdt 37.440 gatggtgcct citgtcaccca gcc togggtga Cagagtgaga ccctgtctoa aaaaaataaa. 375 OO taaataaaaa. ttaag agttt acaaaattct caccatctoc toccatctitt gcaaatgcca 375 6.O catalagtgat gtgttcCagg act attagcc toggalacctg agg cagtaca gtaag cacgc 3762O tittctic caaa. gtoctatocc ccacagacaa acattattta cactgggtac tgctotttta 3768 O titttitt.cccd. totatgctitt attittacitat alactataatc atataacatg taataggaaa 3774. O alagg Cagggit cgggggaga.g atccagaagt cittcc caaga gccttitccaa catag cotct 378OO gtag acattt tittctittctit cittittitttitt tittitttittitt ttctgaga.ca gag to tcact 37860 citgttgtc.ca ggctagagtg Cagtggcgtg atctaggctc actgcaacct cc.gc.citcctg 3792 O ggttcaagca attctic ccac citcag cotcc citagtagctg ggattagagg catgcatcac 3798O US 6,664,105 B1 111 112

-continued cacgc.ctggc taattitttgt atttittagta gagatgaggt ttcaccatgit gggcCaggct 3804. O ggtottgaac toctdaccitc aagtgatcca cctg.ccttag cct cocaaag tgctaggatt 381OO acac gagtga gcc accgtgc cctg.ccccta ttacattctg atcacacatt tdatgttitta 381.60 taattggaaa actggtgaaa ttatagacaa tgttttgttc ccctaaattic totttgatga 38220 gtatatatta cittacactict totgtctitta aaattittgca aaatagitatc citagataagt 3828 O titatgagtgc acagtctgta cgcttactica tattaatgac citcggagagt taaacaa.ca.g 38340 to acctittaa. aaattattac tat cattatc attatttittg aggcgggggit citcattctgt 384 OO citcc caggct ggagagtagt ggtgcggtca cagotcactg cagocaccoc tacctgggct 384 60 caagtgatcc titcc.tc.citca gcc ttctgag tagct gagac cacaggctta tgctaccaca 3852O cctggctaat tittitta actt tttgtagaga cgatgtc.tca titatgttgcc caggctggto 3858O toaaactic ct aagctdaagt gatctitccitc agcct cocaa agtgctggga ttacagg cat 3864 O gaaaaactgc accoagcc ct aaaaattatt agggtoctoc atagtaagac tittaataaat 387 OO atttaaatga acatctggitt tittittaaaaa. aaaaatagag acaaggtotc actatattgc 3876O ccaagctggit citcgaacticc tgg actoacg caatcctgct gccittagcc.g cc caaagtgc 3882O tgggattaca ggcatgaccc accitcatctg ggctgagtga acatatttitt aacataaagg 3888 O cc.gtattitta tatttatcto atacattttg cccag catcc ccatttcc.gc cgaatctgtt 38940 gcttgctaat toctitccago titcatttcat citgaaatttg acaaacatct totattitc.tt 39 OOO tgtcgtcatg ttattgacitt cagaatataa aataaaa.cac taitacccalaa. ttaaa.ccc.ca 39 O60 ccctcattgc ccagoctoat gtgaaaataa to agcataca ttaagcttac ccttgatata 39 120 tgtgtag cat cittittagata aatatacago tgattaa.gca atatagoct atggtataat 39 18O atcttgcc.ca tgtacctdat cittatctoca gcaggattaa ttcacagtga toagatttac 39240 ctittaaactt tgtagcaaaa taticcitc.tcc. aaaag catat citaaaactitt tgtgtgtact 393OO cittgcaagtt tottaattitc atgcagaa.ca ggctottacc actgttagot ggagatattt 3936 O tdaag accita ttitttgtttg tggitttcctg atgatggtoa tgg catttcc cc citt cactic 39420 catctaaaaa. ttgaggtgat acaggcttitt a.a.a. Casaac C. aacticatata gactgagtac 394.80 aactgcaatg caggcatgct aacctctgct acaat catgg gcqtgctatt gatatgtctt 3954 O aagttacaga acacagggct gag.cgtotca ttaggtoaaa atgtaalacca gtttittctg.c 396 OO to actogatgc ttaatgagga Cagggtgttga gag atttctt taaggaaaac aaatatataa. 39 660 taatgctaca tggaaaaata totalacatta gagaattaag taaataaact aatatactica 39.720 caccatggaa tottgtgcag acattaaaat tatgtag togg atggatgttt aatggtgttga 3978O gaaaaagtta ggatgtgctg gggtgggggg aagaatcaag ttittaagaaa atacagtata 3984 O cc catacitta agtaaaaaaa aaaaaaaagg tatgtacagt catgttgttgc ttaatgatgg 399 OO ggatacattc cgagaaatgt gtogataggit gattitcatcc ttgttgttgaac atcatagagt 3996 O gaacttacac aaaccitagat ggtotagoct actatgttatc taggctatat gactagoctd 40 O20 ttgctoctag gctacaaacc tgtaaag cat gttactgtag c gaatataca aatacittaac 40080 acaatggcaa. gctatoattg tgttalagtag ttgttgttatct aaa.catatoct aaaacataga aaactaatgt gttgttgctac aatgttacaa tgacitat gac attgctaggc aatagga att 40200 attaattittat ccttittatgg alaccacactt atatatgcgg to catggtgg accalaalacat 4.0260 ccittatgtgg catatgacitg tatacatgita cacaaaaaat agatgaaaga atgaatatac 40320 atcaaaatat ttaaaatggit tataatgact taggittactt titatittatct tagtaataat 40.380 US 6,664,105 B1 113 114

-continued aatgatgata gataatacitt ttatagtgtt tactatataa. aag acactot tataagttgtt 4 04 40 citacatacitt tacatgitatt acctaaatga tataaatata actctgacag talactaatct 40500 tatacgttct cittittct titt tittitttittitt cittitttittag acagaatctt gct citaccag 4.0560 gctggagtgc agggtgcaat citcggctdac tgcaaccitcc gcc toccagg ttcaaacgat 40 620 totcatgtct cagoctocto agtagctggg actacaggca cacaccacca tgc.ccggcta 4 O 680 attitttgtat ttittggg tag agatggagtt ttgccatgtt ggcCaggctg atcttgaact 40740 cctggcctica agtgatctgc citgccitcago citcccaaagt gctgggatta caggtgttgaa 40800 ccactgtgct cggcctaatc ttacaagttt tdaatattta aag agtgcta actttgttga 4 O 860 calatataaaa. catatttgag aaaaagagat ataag catct tatttagaat tatgaaaata tdaatagacc tacago.cgac taaagcttitt cittcataag.c tottgccitat attgattc.gc toctgttgaat atgcattaat ttgatttaaa taataagitat gtataagaaa talacacttitt 41040 ccittaattitt taagaacgtt caacagttitt taatttgaat to caatagtg aaatacatag 41100 aaaatataaa. attittctgta gtttagccala attgtttittg titt caccaca gcattctacc 41160 aaaattitctt aataa.cagta agaaaatgaa tgcataccto Ctgcagg gag aggggagitta 41220 ggcagttitat ggg catagitt acaagtgaga aattitcattg gctaccattt acgctaaatt 41280 cataaaaact gcattcaatt citatatatoct attittctitta cataaaaaag gtttcaatta 41340 ttggcc atta aataaaatag ccaccattcc agaagttgtg tdatgttitat ccitttittata 41 4 00 ccaccatcat attgccitatt atatagattg tgttgttgttcc attittctgta atgggcc aga 41 460 cagtaagtat ttctggctitt ggagtccata tggtotcitat catalactact catctotgcc 41520 attgtag citt aaagattatc taggtoaaat gcc talagtoga tatagtgttg aaatacaagt 41580 tatataatat aggctgccac aaaaaaaaat ttatttggto taaaaaagat ttcatgactt 416 40 ttgtag cago atgggtgggg catgcaccac ttggittaact cggtgitatct ttctoctittg 417 OO cagatctgtc caactcaatg gtotaactict aaagatggtg gatgatcaaa ccttgccacc 4.1760 tittaatggaa aalacct citcc ggcCaggaag ttcactgggc ttgccag citt totcatatag 4.1820 titttitttgttg ataagaaatg ccaaagttgc tgcttgcatc tgaaaataaa atatactagt 4.1880 cctgacactg aatttittcaa. gtatactaag agtaaag caa citcaagttat aggaaaggaa 41940 gcagatacct tgcaaagcaa Ctagtgggtg citt gagagac actgggacac tgtcagtgct agatttagca cagtattittg atctogctag gtagaac act gctaataata atagotaata 42060 ataccttgtt ccaaatactg cittag cattt tgcatgttitt acttittatct aaagttttgt 42120 tttgttittat tatttattta tittatttatt ttgagacaga atctostctict gto acco agg 421.80 citggagtgcc atggtgcg at cittggctoac tgcaactitta agcaattcto citgccitcago 42240 titcctgagta gctgggatta tagg.cgtgtg ccaccacgcc cagotactitt citatatttitt 42300 tgtagagatg gagtttcgcc atattggc.ca agctggtotc gaacticcitgt cctcgaactic 42360 citgtc.citcaa gtgatccacc cgc.cticagoc totcaaagtg citgggattac aggttgagc 4242 O caccacacco agcagtgttt tatttittgag acagggitatc attctgttgc ccaggcttga 424.80 gtgcagtggit gcaatcatag atcactgcag ccittittaact cctgggcto a agtcatcctc 42540 citgcttagcc toccaagtag citaggaccac agacacatgc catcacactit ggctatttitt 42600 aaaaaattitt ttgtagagat ggggtotcgc tatgttacco aaactggtoc tgaactcctg 42 660 gactica attg atccitcc.cac cittggcctitc Caggtgctgg gatttctittg ggagtacagc 42.720 US 6,664,105 B1 115 116

-continued atggtacago aggagatcat ttgatgttac citctgtgcag tgttgctagt cagogaaaga 42780 citataatacc tgtggggaca gcq attagcc accaccalacca gtotttattt aaagttatta 428 40 aaaatggctg ggcgcagtgg citcacaccitg taatcctago actittgg gag gcc gagg Cag 429 OO atggat cacc tgacgtgagg aatttgagac cagoctogcc aacatggtoga aa.ccc.catct 42960 citactaaaaa. attacaaaaat tagctgggtg tggtoctota gtoccagcta Cttgggaggc 43020 tggggcagga gaattacittg aac coaggag gCagaggttg Cagtgagcc.g agattgttgcc actgcacticc agCCtgggtg acagagagag atticcatcto aaaaaaaaa. gttattaaaa 431 40 atgtatatga atgctoctaa tatggtoagg aa.gcaaggaa gCgaaggata tattatgagt 43200 tittaagaagg tgcttagctg tatattitatic tittcaaaatg tattagalaga ttittagaatt 4326 O cittitcc titca tgttgccatct citacaggcac ccatcagaaa aag catactg cc.gttaccgt 4.3320 gaaactggitt gtaaaagaga alactatoctat ttgcaccitta aaaga cagot agattittgct 4.3380 gattittctitc tittcggittitt citttgtcago aataatatgt gagagga Cag attgttagat 434 40 atgatagitat aaaaaatggit taatgacaat to a gaggcga. ggagattctg taalactitaaa. 43500 attactataa. atgaaattga tttgttcaaga ggataaattit tagaaaacac ccalatacctt 43560 ataactgtct gttaatgctt gctttittcto tacctitt citt ccttgtttca gttgggaagic 43620 ttittggctg.c aagtaacaga aacticcitaat toaaatggct taagcaataa ggaaatgitat 43 680 attic ccacat aactagacgt. toaaacaggc caggctc.cag cactitcagta cgtcaccagg 43740 gatctgggitt cittcccagot citctgctotg ccatctittag cgctggcttic attctdagac 4.3800 totggtagca tgatggctgt agctgtttca tgggc.cccitt caaacct cat agcaaccaga 4.386 O ggaagaaaat gag coattitt ttgagtctoc ttcatag act tgaataactic tttittcagag 4392 O cittctoacag caaacctcitc citcatgtcto citcatgtctt attgttcaga aatgggtaat 43980 gtggcc attt caccagtcac tgccaacaac aac gaggttc citataattgt citctgagtaa 44040 cc ctittggaa tggagagggit gttggtoagt citacaaactg aac acto cag ttctg.cgctt 44 100 tttaccagtg aaaaaatgta attattitt.cc cct cittaagg attaatatto ttcaaatgta 4 416 O tgcctgttat ggatatagta totttaaaat tittittattitt aatag ctitta ggggtacaca 4 4220 citttittgctt acaggggtga attgttgtagt ggtgaag act cggcttittaa tgtacttgtc 4 428O acctgagtga tgtacattgt accoaatagg taattittitca to cattaccc toctitcc.gc.c 4 4340 citct tcc citt citgagtctoc aa.catcc.citt ataccactgt gtatgttctt gtgtacctac 4 4400 agctaagctt ccactitataa. gtgagaac at gcagtatttg gtttitccatt cctgagttac 4 4460 titcc cittagg atalacagocc ccagttcc.gt ccaagttgct gcaaaataca titattottct 44520 titatggctga gtaatagtico atggtacata taitaccacat tittctititatic cacttatcag 44580 ttgatggaca cittaggittaa titccattcaa. titt cattcaa. tittaagtata tttgtaagga 44 640 gctaaagctg aaaattalaat tittagatctt tdaatactict talaattittat atgtaagtgg 4400 tittittatatt ttcacatttg aaataaagta atttittataa. ccttgatatt gtatgacitat 44760 tottttagta atgtaaag.cc tacagacticc tacatttgga accactagtg tgttgtttca 4 4820 ccccittgtta tactato agg atcctcga 4 4848

<210> SEQ ID NO 43 &2 11s LENGTH 2396 &212> TYPE DNA <213> ORGANISM: Mus musculus

<400 SEQUENCE: 43

US 6,664,105 B1 119 120

-continued

gtgtggtgtt citctotaaga agaatact gc aggtggtgac agittaatago actgtg 2396

SEQ ID NO 44 LENGTH 535 TYPE PRT ORGANISM: Mus musculus

<400 SEQUENCE: 44

Met Telu Arg Telu Teu Teu Teu Trp Telu Trp Gly Pro Teu Ala Telu 1 5 10 15

Ala Glin Gly Ala Pro Ala Gly Thr Ala Pro Thr Asp Asp Wall Wall Asp 25 30

Teu Glu Phe Thr Arg Pro Telu Arg Ser Wall Ser Pro Ser Phe 35 40 45

Teu Ser Ile Thr Ile Asp Ala Ser Telu Ala Thr Asp Pro Arg Phe Telu 50 55 60

Thr Phe Telu Gly Ser Pro Arg Telu Arg Ala Teu Ala Arg Telu Ser 65 70 75

Pro Ala Telu Arg Phe Gly Gly Thr Lys Thr Asp Phe Teu Ile Phe 85 90 95

Asp Pro Asp Lys Glu Pro Thr Ser Glu Glu Arg Ser Trp Lys Ser 100 105 110

Glin Wall Asn His Asp Ile Arg Ser Glu Pro Wall Ser Ala Ala Wall 115 120 125

Teu Arg Telu Glin Wall Glu Trp Pro Phe Glin Glu Teu Teu Telu Telu 130 135 1 4 0

Arg Glu Glin Glin Lys Glu Phe ASn Ser Thr Ser Arg Ser 145 15 O 155 160

Ser Wall Asp Met Teu Ser Phe Ala Lys Ser Gly Teu Asp Telu 1.65 170 175

Ile Phe Gly Telu Asn Ala Teu Telu Arg Thr Pro Asp Teu Arg Trp Asn 18O 185 190

Ser Ser Asn Ala Glin Teu Teu Telu Asp Ser Ser Gly Tyr 195 200

Asn Ile Ser Trp Glu Teu Gly Asn Glu Pro Asn Ser Phe Trp Lys Lys 210 215 220

Ala His Ile Telu Ile Asp Gly Telu Glin Telu Gly Glu Asp Phe Wall Glu 225 230 235 240

Teu His Telu Teu Glin Arg Ser Ala Phe Glin Asn Ala Telu Tyr 245 250 255

Gly Pro Asp Ile Gly Glin Pro Arg Gly Thr Wall Teu Telu 260 265 27 O

Ser Phe Telu Ala Gly Gly Glu Wall Ile Asp Ser Teu Thr Trp His 275 280 285

His Tyr Telu Asn Gly Arg Ile Ala Thr Glu Asp Phe Telu Ser 29 O 295

Ser Asp Telu Asp Thr Phe Ile Telu Ser Wall Glin Ile Telu Lys 305 310 315 320

Wall Thr Glu Ile Thr Pro Gly Lys Wall Trp Teu Glu Thr 325 330 335

Ser Ser Ala Tyr Gly Gly Gly Ala Pro Telu Teu Ser Asn Thr Phe Ala 340 345 350

Ala Gly Phe Met Trp Teu Asp Lys Telu Gly Teu Ser Ala Glin Met Gly 355 360 365

US 6,664,105 B1 127 128

-continued <222> LOCATION: (507) . . () <223> OTHER INFORMATION: any nucleotide <400 SEQUENCE: 47 aaatcaggac atatoctitca cittatttgcc tottggtoat attggaggca tttgtattoa 60 tttittaataa cccitcaaaat agtgcatgca aagtgctaag cqt catttgc cacatggtgc 120 cattaactgt caccacct gc agtggtotac ttagagaa.ca cc.gcactgga tigtta acact 18O gaag.cgc.gtg cccc.gc.ccitc cc gaggctict g gatccagog titgaagcttg ccc.cgcc citc 240 cc gaggctict g gatccagoa citggagcatg ccc.cgcc citc cc.gaggctot gagcttgct 3OO aaggagtc.cg citc.cct accq citggggttitt gctittattot tatgaatgac accoct acc 360 gcttitcgtct cagggg tact gtaatgccitt ttattittcat atacaagct g c gattittggc 420 atttctitatg acaaaaaacc cataggaaaa gqcgggcacg cittagtgagc titcctg.cggg 480 gag aggttitt totgttagag citggcanggit citgct catcg accatcttca ggc citcgtgc 540 c 541

What is claimed is: 25 3. An antisense nucleic acid construct comprising a pro 1. An antisense oligonucleotide comprising a polynucle moter Sequence operably-linked to a polynucleotide otide of a least 10 bases which Specifically targets and Sequence encoding the antisense oligonucleotide of claim 1. inhibits the expression of the polynucleotide of SEQ ID No: 4. The antisense nucleic acid construct of claim3, wherein 9 or SEQ ID NO: 13, wherein the polynucleotides SEQ ID Said polypeptide having heparanase catalytic activity is as NO: 9 and 13 encode polypeptides having heparanase cata set forth in SEQ ID NOS: 10, or 14. lytic activity. 5. A nucleic acid construct comprising SEQ ID NO: 13, 2. The antisense oligonucleotide of claim 1, wherein Said encoding a polypeptide having heparanase catalytic activity. polypeptide having heparanase catalytic activity is as Set forth in SEO ID NOS: 10 and 14.