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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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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MIRSKIA, I.M.I.L.E.GPLGPS PAI.PRPAAQOWW) LDFFTQEPLHLWSPSFLSWT , ) PHD EEEEE HE EEEE EEE
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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
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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 proteoglycans of the BM in order to migrate out of the vascular compartment. Several cellular enzymes (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 extracellular matrix (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 Heparan Sulfate 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 (0.5heparin that might be of potential nent components of blood vessels (3). In large blood vessels use in preventing extravasation of blood-borne cells was they are concentrated mostly in the intima and inner media, also investigated by the present inventors. Inhibition of whereas in capillaries they are found mainly in the Suben 40 heparanase was best achieved by heparin Species containing dothelial basement membrane where they Support prolifer 16 Sugar units or more and having Sulfate groups at both the ating and migrating endothelial cells and Stabilize the Struc N and O positions. While O-desulfation abolished the ture of the capillary wall. The ability of HSPG to interact heparanase inhibiting effect of heparin, O-Sulfated, with ECM macromolecules Such as collagen, laminin and N-acetylated heparin retained a high inhibitory activity, fibronectin, and with different attachment Sites on plasma 45 provided that the N-substituted molecules had a molecular membranes Suggests a key role for this proteoglycan in the size of about 4,000 daltons or more (7). Treatment of self-assembly and insolubility of ECM components, as well experimental animals with heparanase inhibitors (e.g., non as in cell adhesion and locomotion. Cleavage of the heparan anticoagulant species of heparin) markedly reduced (>90%) sulfate (HS) chains may therefore result in degradation of the incidence of lung metastases induced by B16 melanoma, the subendothelial ECM and hence may play a decisive role 50 Lewis lung carcinoma and mammary adenocarcinoma cells in extravasation of blood-borne cells. HS catabolism is (7, 8, 16). Heparin fractions with high and low affinity to observed in inflammation, wound repair, diabetes, and can anti-thrombin III exhibited a comparable high anti cer metastasis, Suggesting that enzymes which degrade HS metastatic activity, indicating that the heparanase inhibiting play important roles in pathologic processes. Heparanase activity of heparin, rather than its anticoagulant activity, activity has been described in activated immune System cells 55 plays a role in the anti-metastatic properties of the polysac and highly metastatic cancer cells (6-8), but research has charide is (7). been handicapped by the lack of biologic tools to explore Heparanase activity in the urine of cancer.patients: In an potential causative roles of heparanase in disease conditions. attempt to further elucidate the involvement of heparanase in Involvement of Heparanase in Tumor Cell Invasion and tumor progression and its relevance to human cancer, urine Metastasis: Circulating tumor cells arrested in the capillary 60 Samples for heparanase activity were screened (16a). beds of different organs must invade the endothelial cell Heparanase activity was detected in the urine of Some, but lining and degrade its underlying basement membrane (BM) not all, cancer patients. High levels of heparanase activity in order to invade into the extravascular tissue(s) where they were determined in the urine of patients with an aggressive establish metastasis (9, 10). Metastatic tumor cells often metastatic disease and there was no detectable activity in the attach at or near the intercellular junctions between adjacent 65 urine of healthy donors. endothelial cells. Such attachment of the metastatic cells is Heparanase activity was also found in the urine of 20% of followed by rupture of the junctions, retraction of the normal and microalbuminuric insulin dependent diabetes US 6,664,105 B1 3 4 mellitus (IDDM) patients, most likely due to diabetic dation of the ECM HSPG by inflammatory leukocytes and nephropathy, the most important Single disorder leading to malignant cells. renal failure in adults. Second, a large proportion of the platelet heparanase PoSSible involvement of heparanase in tumor angiogen exists in a latent form, probably as a complex with chon esis: Fibroblast growth factors are a family of structurally related polypeptides characterized by high affinity to heparin droitin sulfate. The latent enzyme is activated by tumor (17). They are highly mitogenic for vascular endothelial cell-derived factor(s) and may then facilitate cell invasion cells and are among the most potent inducers of neovascu through the vascular endothelium in the process of tumor larization (17, 18). Basic fibroblast growth factor (bFGF) metastasis. has been extracted from the Subendothelial ECM produced Third, release of the platelet heparanase from C-granules in vitro (19) and from basement membranes of the cornea is induced by a strong Stimulant (i.e., thrombin), but not in (20), Suggesting that ECM may serve as a reservoir for response to platelet activation on ECM. bFGF. Immunohistochemical staining revealed the localiza Fourth, the neutrophil heparanase is preferentially and tion of bFGF in basement membranes of diverse tissues and readily released in response to a threshold activation and blood vessels (21). Despite the ubiquitous presence of bFGF upon incubation of the cells on ECM. in normal tissues, endothelial cell proliferation in these 15 tissues is usually very low, Suggesting that bFGF is Some Fifth, contact of neutrophils with ECM inhibited release how Sequestered from its site of action. Studies on the of noxious enzymes (proteases, lysozyme) and oxygen interaction of bRGF with ECM revealed that bRGF binds to radicals, but not of enzymes (heparanase, gelatinase) which HSPG in the ECM and can be released in an active form by may enable diapedesis. This protective role of the Suben HS degrading enzymes (15, 20, 22). It was demonstrated dothelial ECM was observed when the cells were stimulated that heparanase activity expressed by platelets, mast cells, with soluble factors but not with phagocytosable stimulants. neutrophils, and lymphoma cells is involved in release of Sixth, intracellular heparanase is Secreted within minutes active bFGF from ECM and basement membranes (23), after exposure of T cell lines to Specific antigens. Suggesting that heparanase activity may not only function in Seventh, mitogens (Con A, LPS) induce synthesis and cell migration and invasion, but may also elicit an indirect 25 Secretion of heparanase by normal T and B lymphocytes neovascular response. These results Suggest that the ECM maintained in Vitro. T lymphocyte heparanase is also HSPG provides a natural storage depot for bFGF and possibly other heparin-binding growth promoting factors induced by immunization with antigen in Vivo. (24, 25). Displacement of bFGF from its storage within Eighth, heparanase activity is expressed by pre-B lym basement membranes and ECM may therefore provide a phomas and B-lymphomas, but not by plasmacytomas and novel mechanism for induction of neovascularization in resting normal B lymphocytes. normal and pathological situations. Ninth, heparanase activity is expressed by activated mac Recent studies indicate that heparin and HS are involved rophages during incubation with ECM, but there was little or in binding of bFGF to high affinity cell Surface receptors and no release of the enzyme into the incubation medium. in bFGF cell signaling (26, 27). Moreover, the size of HS 35 Similar results were obtained with human myeloid leukemia required for optimal effect was similar to that of HS frag cells induced to differentiate to mature macrophages. ments released by heparanase (28). Similar results were Tenth, T-cell mediated delayed type hyperSensitivity and obtained with vascular endothelial cells growth factor experimental autoimmunity are Suppressed by low doses of (VEGF) (29), Suggesting the operation of a dual receptor heparanase inhibiting non-anticoagulant species of heparin mechanism involving HS in cell interaction with heparin 40 (30). binding growth factors. It is therefore proposed that restric Eleventh, heparanase activity expressed by platelets, neu tion of endothelial cell growth factors in ECM prevents their trophils and metastatic tumor cells releases active bFGF Systemic action on the vascular endothelium, thus maintain from ECM and basement membranes. Release of bFGF ing a very low rate of endothelial cells turnover and vessel from Storage in ECM may elicit a localized neovascular growth. On the other hand, release of bFGF from storage in 45 response in processes Such as wound healing, inflammation ECM as a complex with HS fragment, may elicit localized and tumor development. endothelial cell proliferation and neovascularization in pro Twelfth, among the breakdown products of the ECM ceSSes Such as wound healing, inflammation and tumor generated by heparanase is a tri-Sulfated disaccharide that development (24, 25). can inhibit T-cell mediated inflammation in vivo (31). This Expression of heparanase by cells of the immune System: 50 inhibition was associated with an inhibitory effect of the Heparanase activity correlates with the ability of activated disaccharide on the production of biologically active TNFC. cells of the immune System to leave the circulation and elicit by activated T cells in vitro (31). both inflammatory and autoimmune responses. Interaction Other potential therapeutic applications: Apart from its of platelets, granulocytes, T and B lymphocytes, macroph involvement in tumor cell metastasis, inflammation and ages and mast cells with the subendothelial ECM is asso 55 autoimmunity, mammalian heparanase may be applied to ciated with degradation of HS by a specific heparanase modulate: bioavailability of heparin-binding growth factors activity (6). The enzyme is released from intracellular com (15); cellular responses to heparin-binding growth factors partments (e.g., lysosomes, Specific granules, etc.) in (e.g., bFGF, VEGF) and cytokines (IL-8) (31a, 29); cell response to various activation signals (e.g., thrombin, cal interaction with plasma lipoproteins (32); cellular Suscepti cium ionophore, immune complexes, antigens, mitogens, 60 bility to certain viral and Some bacterial and protozoa etc.), Suggesting its regulated involvement in inflammation infections (33, 33a, 33b); and disintegration of amyloid and cellular immunity. plaques (34). Heparanase may thus prove useful for condi Some of the observations regarding the heparanase tions Such as wound healing, angiogenesis, restenosis, enzyme were reviewed in reference No. 6 and are listed atherOSclerosis, inflammation, neurodegenerative diseases hereinbelow: 65 and Viral infections. Mammalian heparanase can be used to First, a proteolytic activity (plasminogen activator) and neutralize plasma heparin, as a potential replacement of heparanase participate Synergistically in Sequential degra protamine. Anti-heparanase antibodies may be applied for US 6,664,105 B1 S 6 immunodetection and diagnosis of micrometastases, by the normal immune response; (iii) biochemical autoimmune lesions and renal failure in biopsy Specimens, correction, primarily for the treatment of Single gene defects, plasma Samples, and body fluids. Common use in basic where a normal copy of the gene is added to the affected or research is expected. other cells. The identification of the hpa gene encoding for hepara To allow efficient transfer of the therapeutic genes, a nase enzyme will enable the production of a recombinant variety of gene delivery techniques have been developed enzyme in heterologous expression Systems. Availability of based on viral and non-viral vector Systems. The most the recombinant protein will pave the way for Solving the widely used and most efficient Systems for delivering protein Structure function relationship and will provide a genetic material into target cells are viral vectors. So far, 329 tool for developing new inhibitors. clinical studies (phase I, I/II and II) with over 2,500 patients Viral Infection: The presence of heparan sulfate on cell have been initiated Worldwide since 1989 (50). Surfaces have been shown to be the principal requirement for The approach of gene addition pose Serious barriers. The the binding of Herpes Simplex (33) and Dengue (33a) expression of many genes is tightly regulated and context Viruses to cells and for Subsequent infection of the cells. dependent, So achieving the correct balance and function of Removal of the cell Surface heparan Sulfate by heparanase 15 expression is challenging. The gene itself is often quite may therefore abolish virus infection. In fact, treatment of large, containing many exons and introns. The delivery cells with bacterial heparitinase (degrading heparan Sulfate) vector is usually a virus, which can infect with a high or heparinase (degrading heparan) reduced the binding of efficiency but may, on the other hand, induce immunological two related animal herpes viruses to cells and rendered the response and consequently decreases effectiveness, espe cells at least partially resistant to virus infection (33). There cially upon Secondary administration. Most of the current are Some indications that the cell Surface heparan Sulfate is expression vector-based gene therapy protocols fail to also involved in HIV infection (33b). achieve clinically Significant transgene expression required Neurodegenerative diseases: Heparan Sulfate proteogly for treating genetic diseases. Apparently, it is difficult to cans were identified in the prion protein amyloid plaques of deliver enough virus to the right cell type to elicit an Genstmann-Straussler Syndrome, Creutzfeldt-Jakob disease 25 effective and therapeutic effect (51). and Scrape (34). Heparanase may disintegrate these amyloid Homologous recombination, which was initially consid plaques which are also thought to play a role in the patho ered to be of limited use for gene therapy because of its low genesis of Alzheimer's disease. frequency in mammalian cells, has recently emerged as a Restenosis and Atherosclerosis: Proliferation of arterial potential Strategy for developing gene therapy. Different smooth muscle cells (SMCs) in response to endothelial approaches have been used to study homologous recombi injury and accumulation of cholesterol rich lipoproteins are nation in mammalian cells, Some involve DNA repair basic events in the pathogenesis of atherosclerosis and mechanisms. These Studies aimed at either gene disruption restenosis (35). Apart from its involvement in SMC prolif or gene correction and include RNA/DNA chimeric eration (i.e., low affinity receptors for heparin-binding oligonucleotides, Small or large homologous DNA growth factors), HS is also involved in lipoprotein binding, 35 fragments, or adeno-associated viral vectors. Most of these retention and uptake (36). It was demonstrated that HSPG Studies show a reasonable frequency of homologous and lipoprotein lipase participate in a novel catabolic path recombination, which warrants further in vivo testing (52). way that may allow Substantial cellular and interstitial Homologous recombination-based gene therapy has the accumulation of cholesterol rich lipoproteins (32). The latter potential to develop into a powerful therapeutic modality for pathway is expected to be highly atherogenic by promoting 40 genetic diseases. It can offer permanent expression and accumulation of apoB and apo rich lipoproteins (i.e. LDL, normal regulation of corrected genes in appropriate cells or VLDL, chylomicrons), independent of feedback inhibition organs and probably can be used for treating dominantly by the cellular sterol content. Removal of SMC HS by inherited diseaseS Such as polycystic kidney disease. heparanase is therefore expected to inhibit both SMC pro 45 Genomic Sequencesfunction in regulation of gene expres liferation and lipid accumulation and thus may halt the Sion: progression of restenosis and atherOSclerosis. The efficient expression of therapeutic genes in target Gene therapy: cells or tissues is an important component of efficient and The ultimate goal in the management of inherited as well Safe gene therapy. The expression of genes is driven by the as acquired diseases is a rational therapy with the aim to 50 promoter region upstream of the coding Sequence, although eliminate the underlying biochemical defects associated regulation of expression may be Supplemented by farther with the disease rather then Symptomatic treatment. Gene upstream or downstream DNA sequences or DNA in the therapy is a promising candidate to meet these objectives. introns of the gene. Since this important information is Initially it was developed for treatment of genetic disorders, embedded in the DNA, the description of gene structure is however, the consensus view today is that it offers the 55 crucial to the analysis of gene regulation. Characterization of prospect of providing therapy for a variety of acquired cell Specific or tissue Specific promoters, as well as other diseases, including cancer, Viral infections, vascular diseases tissue specific regulatory elements enables the use of Such and neurodegenerative disorders. Sequences to direct efficient cell Specific, or developmental The gene-based therapeutic can act either intracellularly, Stage specific gene expression. This information provides affecting only the cells to which it is delivered, or 60 the basis for targeting individual genes and for control of extracellularly, using the recipient cells as local endogenous their expression by exogenous agents, Such as drugs. Iden factories for the therapeutic product(s). The application of tification of transcription factors and other regulatory pro gene therapy may follow any of the following Strategies: (i) teins required for proper gene expression will point at new prophylactic gene therapy, Such as using gene transfer to potential targets for modulating gene expression, when So protect cells against Viral infection; (ii) cytotoxic gene 65 desired or required. therapy, Such as cancer therapy, where genes encode cyto Efficient expression of many mammalian genes depends toxic products to render the target cells Vulnerable to attack on the presence of at least one intron. The expression of US 6,664,105 B1 7 8 mouse thymidylate Synthase (TS) gene, for example, is tory elements and trans-acting factors that control alternative greatly influenced by intron Sequences. The addition of Splicing of Specific mRNAS. almost any of the introns from the mouse TS gene to an This mechanism results in the generation of variant iso intronless TS minigene leads to a large increase in expres forms of various proteins from a single gene. These include sion (42). The involvement of intron 1 in the regulation of cell Surface molecules Such as CD44, receptors, cytokines expression was demonstrated for many other genes. In such as VEGF and enzymes. Products of alternatively human factor IX (hFIX), intron 1 is able to increase the Spliced transcripts differ in their expression pattern, Sub expression level about 3 fold mare as compared to that of the Strate Specificity and other biological parameters. HFIX cDNA (43). The expression enhancing activity of The FGF receptor RNA undergoes alternative splicing intron 1 is due to efficient functional Splicing Sequences, which results in the production of several isoforms, which present in the precursor mRNA. By being efficiently exhibit different ligand binding specificities. The alternative assembled into Spliceosome complexes, transcripts with splicing is regulated in a cell Specific manner (53). Splicing Sequences may be better protected in the nucleus Alternative spliced mRNAS are often correlated with from random degradations, than those without Such malignancy. An increase in Specific Splice variant of tyro Sequences (44). 15 Sinase was identified in murine melanomas (54). Multiple A forward-inserted intron1-carrying HFIX expression Splicing variants of estrogen receptor are present in indi cassette Suggested to be useful for directed gene transfer, vidual human breast tumors. CD44 has various isoform, while for retroviral-mediated gene transfer System, Some are characteristic of malignant tissues. reversely-inserted intron 1-carrying HFIX expression cas Identification of tumor specific alternative Splice variants Sette was considered (43). provide new tool for cancer diagnostics. CD44 variants have A highly conserved cis-acting Sequence element was been used for detection of malignancy in urine Samples from identified in the first intron of the mouse and rat c-Ha-ras, patients with urothelial cancer by competitive RT-PCR (55). and in the first eXon of Ha- and Ki-ras genes of human, CD44 eXOn 6 was Suggested as prognostic indicator of mouse and rat. This cis-acting regulatory Sequence confers metastasis in breast cancer (56). Strong transcription enhancer activity that is differentially 25 Different enzymes or polypeptides generated by alterna modulated by Steroid hormones in metastatic and nonmeta tive splicing may have different function or catalytic Speci Static Subpopulations. Perturbations in the regulatory activi ficity. The identification and characterization of the enzyme ties of Such cis-acting Sequences may play an important role forms, which are involved in pathological processes, is in governing oncogenic potency of Ha-ras through transcrip crucial for the design of appropriate and efficient drugs. tional control mechanisms (45). Modulation of gene expression-AntiSense technology: Intron Sequences affect tissue Specific, as well as inducible An antisense oligonucleotide (e.g., antisense gene expression. A 182 bp intron 1 DNA segment of the oligodeoxyribonucleotide) may bind its target nucleic acid mouse Col2a1 gene contains the necessary information to either by Watson-Crick base pairing or Hoogsteen and confer high-level, temporally correct, chondrocyte expres 35 anti-Hoogsteen base pairing (64). According to the Watson Sion on a reporter gene in intact mouse embryos, while Crick base pairing, heterocyclic bases of the antisense Col2a1 promoter Sequences are dispensable for chondrocyte oligonucleotide form hydrogen bonds with the heterocyclic expression (46). In Col1A1 gene the intron plays little or no bases of target single-Stranded nucleic acids (RNA or Single role in constitutive expression of collagen in the Skin, and in Stranded DNA), whereas according to the Hoogsteen base cultured cells derived from the skin, however, in the lungs 40 pairing, the heterocyclic bases of the target nucleic acid are of young mice, intron deletion results in decrease of expres double-stranded DNA, wherein a third strand is accommo sion to less than 50% (47). dated in the major groove of the B-form DNA duplex by A classical enhancer activity was shown in the 2 kb intron Hoogsteen and anti-Hoogsteen base pairing to form a triple fragment in bovine beta-casein gene. The enhancer activity helix Structure. was largely dependent on is the lactogenic hormones, espe 45 According to both the Watson-Crick and the Hoogsteen cially prolactin. It was Suggested that Several elements in the base pairing models, antisense oligonucleotides have the intron-1 of the bovine beta-casein gene cooperatively inter potential to regulate gene expression and to disrupt the act not only with each other but also with its promoter for essential functions of the nucleic acids in cells. Therefore, hormonal induction (48). antisense oligonucleotides have possible uses in modulating Identification and characterization of regulatory elements 50 a wide range of diseases in which gene expression is altered. in genomic non-coding Sequences, Such as introns, provides Since the development of effective methods for chemi a tool for designing and constructing novel vectors for tissue cally Synthesizing oligonucleotides, these molecules have Specific, hormone regulated or any other defined expression been extensively used in biochemistry and biological pattern, for gene therapy. Such an expression cassette was research and have the potential use in medicine, Since developed, utilizing regulatory elements from the human 55 carefully devised oligonucleotides can be used to control cytokeratin 18 (K18) gene, including 5' genomic Sequences gene expression by regulating levels of transcription, tran and one of its introns. This cassette efficiently expresses Scripts and/or translation. reporter genes, as well as the human cystic fibrosis trans Oligodeoxyribonucleotides as long as 100 base pairs (bp) membrane conductance regulator (CFTR) gene, in cultured are routinely Synthesized by Solid phase methods using lung epithelial cells (49). 60 commercially available, fully automated Synthesis Alternative splicing: machines. The chemical Synthesis of oligoribonucleotides, Alternative splicing of pre mRNA is a powerful and however, is far less routine. Oligoribonucleotides are also Versatile regulatory mechanism that can effect quantitative much leSS Stable than oligodeoxyribonucleotides, a fact control of gene expression and functional diversification of which has contributed to the more prevalent use of oligode proteins. It contributes to major developmental decisions 65 oxyribonucleotides in medical and biological research, and also to a fine-tuning of gene function. Genetic and directed at, for example, the regulation of transcription or biochemical approaches have identified cis-acting regula translation levels. US 6,664,105 B1 9 10 Gene expression involves few distinct and well regulated At the transcription level, antisense or Sense oligonucle Steps. The first major Step of gene expression involves otides or analogs that bind to the genomic DNA by strand transcription of a messenger RNA (mRNA) which is an displacement or the formation of a triple helix, may prevent RNA sequence complementary to the antisense (i.e., -) DNA transcription (64). Strand, or, in other words, identical in Sequence to the DNA 5 At the transcript level, antisense oligonucleotides or ana Sense (i.e., +) Strand, composing the gene. In eukaryotes, logs that bind target mRNA molecules lead to the enzymatic transcription occurs in the cell nucleus. cleavage of the hybrid by intracellular RNase H (65). In this The Second major Step of gene expression involves trans case, by hybridizing to the targeted mRNA, the oligonucle lation of a protein (e.g., enzymes, structural proteins, otides or oligonucleotide analogs provide a duplex hybrid Secreted proteins, gene expression factors, etc.) in which the recognized and destroyed by the RNase H enzyme. mRNA interacts with ribosomal RNA complexes Alternatively, such hybrid formation may lead to interfer (ribosomes) and amino acid activated transfer RNAS ence with correct splicing (66). As a result, in both cases, the (tRNAS) to direct the synthesis of the protein coded for by number of the target mRNA intact transcripts ready for the mRNA sequence. translation is reduced or eliminated. Initiation of transcription requires Specific recognition of 15 At the translation level, antisense oligonucleotides or a promoter DNA sequence located upstream to the coding analogs that bind target mRNA molecules prevent, by Steric Sequence of a gene by an RNA-synthesizing enzyme-RNA hindrance, binding of essential translation factors polymerase. This recognition is preceded by Sequence (ribosomes), to the target mRNA, a phenomenon known in Specific binding of one or more transcription factors to the the art as hybridization arrest, disabling the translation of promoter Sequence. Additional proteins which bind at or such mRNAs (67). close to the promoter Sequence may trans upregulate tran Thus, antisense Sequences, which as described herein Scription via cis elements known as enhancer Sequences. above may arrest the expression of any endogenous and/or Other proteins which bind to or close to the promoter, but exogenous gene depending on their Specific Sequence, whose binding prohibits the action of RNA polymerase, are attracted much attention by Scientists and pharmacologists known as repressors. 25 who were devoted at developing the antisense approach into There are also evidence that in Some cases gene expres a new pharmacological tool (68). Sion is downregulated by endogenous antisense RNA repres For example, Several antisense oligonucleotides have Sors that bind a complementary mRNA transcript and been shown to arrest hematopoietic cell proliferation (69), thereby prevent its translation into a functional protein. growth (70), entry into the S phase of the cell cycle (71), Thus, gene expression is typically upregulated by tran reduced Survival (72) and prevent receptor mediated Scription factors and enhancers and downregulated by responses (73). For use of antisense oligonucleotides as repressors. antiviral agents the reader is referred to reference 74. However, in many disease situation gene expression is For efficient in Vivo inhibition of gene expression using impaired. In many cases, Such as different types of cancer, 35 antisense oligonucleotides or analogs, the oligonucleotides for various reasons the expression of a specific endogenous or analogs must fulfill the following requirements (i) Suffi or exogenous (e.g., of a pathogen Such as a virus) gene is cient specificity in binding to the target Sequence; (ii) upregulated. Furthermore, in infectious diseases caused by pathogens Such as parasites, bacteria or viruses, the disease Solubility in water; (iii) Stability against intra- and extracel progression depends on expression of the pathogen genes, lular nucleases; (iv) capability of penetration through the 40 cell membrane; and (v) when used to treat an organism, low this phenomenon may also be considered as far as the patient toxicity. is concerned as upregulation of exogenous genes. Unmodified oligonucleotides are impractical for use as Most conventional drugs function by interaction with and antisense Sequences Since they have Short in Vivo half-lives, modulation of one or more targeted endogenous or exog during which they are degraded rapidly by nucleases. enous proteins, e.g., enzymes. Such drugs, however, typi 45 cally are not specific for targeted proteins but interact with Furthermore, they are difficult to prepare in more than other proteins as well. Thus, a relatively large dose of drug milligram quantities. In addition, Such oligonucleotides are must be used to effectively modulate a targeted protein. poor cell membrane penetraters (75). Thus it is apparent that in order to meet all the above listed Typical daily doses of drugs are from 10-10 milli requirements, oligonucleotide analogs need to be devised in moles per kilogram of body weight or 10-10 millimoles 50 for a 100 kilogram person. If this modulation instead could a Suitable manner. Therefore, an extensive Search for modi be effected by interaction with and inactivation of mRNA, a fied oligonucleotides has been initiated. dramatic reduction in the necessary amount of drug could For example, problems arising in connection with double likely be achieved, along with a corresponding reduction in stranded DNA (dsDNA) recognition through triple helix side effects. Further reductions could be effected if Such 55 formation have been diminished by a clever “switch back” interaction could be rendered site-specific. Given that a chemical linking, whereby a Sequence of polypurine on one functioning gene continually produces mRNA, it would thus Strand is recognized, and by “Switching back', a homopurine be even more advantageous if gene transcription could be Sequence on the other Strand can be recognized. Also, good arrested in its entirety. helix formation has been obtained by using artificial bases, Given these facts, it would be advantageous if gene 60 thereby improving binding conditions with regard to ionic expression could be arrested or downmodulated at the Strength and pH. transcription level. In addition, in order to improve half-life as well as The ability of chemically Synthesizing oligonucleotides membrane penetration, a large number of variations in and analogs thereof having a Selected predetermined polynucleotide backbones have been done, nevertheless Sequence offerS means for downmodulating gene expres 65 with little Success. Sion. Three types of gene expression modulation Strategies Oligonucleotides can be modified either in the base, the may be considered. Sugar or the phosphate moiety. These modifications include, US 6,664,105 B1 11 12 for example, the use of methylphosphonates, RNA oligonucleotides may also be used for antisense monothiophosphates, dithiophosphates, phosphoramidates, inhibition as they form a stable RNA-RNA duplex with the phosphate esters, bridged phosphorothioates, bridged target, Suggesting efficient inhibition. However, due to their phosphoramidates, bridged methylenephosphonates, low Stability RNA oligonucleotides are typically expressed dephospho internucleotide analogs with Siloxane bridges, inside the cells using vectorS designed for this purpose. This carbonate bridges, carboxymethyl ester bridges, carbonate approach is favored when attempting to target a mRNA that bridges, carboxyrnethyl ester bridges, acetamide bridges, carbamate bridges, thioether bridges, Sulfoxy bridges, Sul encodes an abundant and long-lived protein (57). fono bridges, various "plastic' DNAS, C-anomeric bridges Recent Scientific publications have validated the efficacy and borane derivatives. For further details the reader is of antisense compounds in animal models of hepatitis, referred to reference 76. cancers, coronary artery restenosis and other diseases. The International patent application WO 89/12060 discloses first antisense drug was recently approved by the FDA. This various building blocks for Synthesizing oligonucleotide drug FomivirSen, developed by Isis, is indicated for local analogs, as well as oligonucleotide analogs formed by treatment of cytomegalovirus in patients with AIDS who are joining Such building blocks in a defined Sequence. The intolerant of or have a contraindication to other treatments building blocks may be either “rigid' (i.e., containing a ring 15 for CMV retinitis or who were insufficiently responsive to Structure) or “flexible” (i.e., lacking a ring structure). In both previous treatments for CMV retinitis (Pharmacotherapy cases, the building blockS contain a hydroxy group and a News Network). mercapto group, through which the building blocks are said Several antisense compounds are now in clinical trials in to join to form oligonucleotide analogs. The linking moiety the United States. These include locally administered in the oligonucleotide analogs is Selected from the group antivirals, Systemic cancer therapeutics. AntiSense therapeu consisting of sulfide (-S-), sulfoxide (-SO-), and sul ticS has the potential to treat many life-threatening diseases fone (-SO-). However, the application provides no data with a number of advantages over traditional drugs. Tradi Supporting the Specific binding of an oligonucleotide analog tional drugs intervene after a disease-causing protein is to a target oligonucleotide. formed. Antisense therapeutics, however, block mRNA International patent application WO92/20702 describe an 25 acyclic oligonucleotide which includes a peptide backbone transcription/translation and intervene before a protein is on which any Selected chemical nucleobases or analogs are formed, and Since antisense therapeutics target only one Stringed and Serve as coding characters as they do in natural specific mRNA, they should be more effective with fewer DNA or RNA. These new compounds, known as peptide Side effects than current protein-inhibiting therapy. nucleic acids (PNAS), are not only more stable in cells than A Second option for disrupting gene expression at the their natural counterparts, but also bind natural DNA and level of transcription uses Synthetic oligonucleotides RNA 50 to 100 times more tightly than the natural nucleic capable of hybridizing with double stranded DNA. A triple acids cling to each other (77). PNA oligomers can be helix is formed. Such oligonucleotides may prevent binding Synthesized from the four protected monomers containing of transcription factors to the gene's promoter and therefore thymine, cytosine, adenine and guanine by Merrifield Solid inhibit transcription. Alternatively, they may prevent duplex phase peptide Synthesis. In order to increase Solubility in 35 unwinding and, therefore, transcription of genes within the Water and to prevent aggregation, a lysine amide group is triple helical Structure. placed at the C-terminal. Another approach is the use of Specific nucleic acid Thus, antisense technology requires pairing of messenger Sequences to act as decoys for transcription factors. Since RNA with an oligonucleotide to form a double helix that transcription factors bind Specific DNA sequences it is inhibits translation. The concept of antisense-mediated gene 40 possible to Synthesize oligonucleotides that will effectively therapy was already introduced in 1978 for cancer therapy. This approach was based on certain genes that are crucial in compete with the native DNA sequences for available tran cell division and growth of cancer cells. Synthetic fragments Scription factors in Vivo. This approach requires the identi of genetic Substance DNA can achieve this goal. Such fication of gene specific transcription factor (57). molecules bind to the targeted gene molecules in RNA of 45 Indirect inhibition of gene expression was demonstrated tumor cells, thereby inhibiting the translation of the genes for matrix metalloproteinase genes (MMP-1, -3, and -9), and resulting in dysfunctional growth of these cells. Other which are associated with invasive potential of human mechanisms has also been proposed. These Strategies have cancer cells. E1AF is a transcription activator of MMP been used, with Some SucceSS in treatment of cancers, as genes. Expression of E1AF antisense RNA in HSC3AS cells well as other illnesses, including viral and other infectious 50 showed decrease in mRNA and protein levels of MMP-1, -3, diseases. AntiSense oligonucleotides are typically Synthe and -9. Moreover, HSC3AS showed lower invasive potential sized in lengths of 13-30 nucleotides. The life span of in vitro and in vivo. These results imply that transfection of oligonucleotide molecules in blood is rather short. Thus, antisense inhibits tumor invasion by down-regulating MMP they have to be chemically modified to prevent destruction genes (58). by ubiquitous nucleaseS present in the body. Phosphorothio 55 Ribozymes: ates are very widely used modification in antisense oligo Ribozymes are being increasingly used for the Sequence nucleotide ongoing clinical trials (57). A new generation of Specific inhibition of gene expression by the cleavage of antisense molecules consist of hybrid antisense oligonucle mRNAS encoding proteins of interest. The possibility of otide with a central portion of synthetic DNA while four designing ribozymes to cleave any specific target RNA has bases on each end have been modified with 2 O-methyl 60 rendered them valuable tools in both basic research and ribose to resemble RNA. In preclinical studies in laboratory therapeutic applications. In the therapeutics area, ribozymes animals, Such compounds have demonstrated greater Stabil have been exploited to target viral RNAS in infectious ity to metabolism in body tissues and an improved Safety diseases, dominant oncogenes in cancers and Specific profile when compared with the first-generation unmodified Somatic mutations in genetic disorders. Most notably, Sev phosphorothioate (Hybridon Inc. news). Dosens of other 65 eral ribozyme gene therapy protocols for HIV patients are nucleotide analogs have also been tested in antisense tech already in Phase 1 trials (62). More recently, ribozymes have nology. been used for transgenic animal research, gene target vali US 6,664,105 B1 13 14 dation and pathway elucidation. Several ribozymes are in Glycosyl hydrolases: various stages of clinical trials. ANGIOZYME was the first Glycosyl hydrolases are a widespread group of enzymes chemically Synthesized ribozyme to be Studied in human that hydrolyze the o-glycosidic bond between two or more clinical trials. ANGIOZYME specifically inhibits formation carbohydrates or between a carbohydrate and a noncarbo of the VEGF-r (Vascular Endothelial Growth Factor receptor), a key component in the angiogenesis pathway. hydrate moiety. The enzymatic hydrolysis of glycosidic Ribozyme Pharmaceuticals, Inc., as well as other firms have bond occurs by using major one or two mechanisms leading demonstrated the importance of anti-angiogenesis therapeu to overall retention or inversion of the anomeric configura tics in animal models. HEPTAZYME, a ribozyme designed tion. In both mechanisms catalysis involves two residues: a to selectively destroy Hepatitis C Virus (HCV) RNA, was proton donor and a nucleophile. Glycosyl hydrolyses have found effective in decreasing Hepatitis C viral RNA in cell been classified into 58 families based on amino acid simi culture assays (Ribozyme Pharmaceuticals, Incorporated larities. The glycosyl hydrolyses from families 1, 2, 5, 10, WEB home page). 17, 30, 35, 39 and 42 act on a large variety of substrates, Gene disruption in animal models: however, they all hydrolyze the glycosidic bond in a general The emergence of gene inactivation by homologous acid catalysis mechanism, with retention of the anomeric recombination methodology in embryonic Stem cells has 15 configuration. The mechanism involves two glutamic acid revolutionized the field of mouse genetics. The availability residues, which are the proton donors and the nucleophile, of a rapidly growing number of mouse null mutants has with an aspargine always preceding the proton donor. Analy represented an invaluable Source of knowledge on mamma SeS of a set of known 3D Structures from this group revealed lian development, cellular biology and physiology, and has that their catalytic domains, despite the low level of provided many models for human inherited diseases. Animal Sequence identity, adopt a similar (C/B) 8 fold with the models are required for an effective drug delivery develop proton donor and the nucleophile located at the C-terminal ment program and evaluation of gene therapy approach. The ends of strands B4 and B7, respectively. Mutations in the improvement of the original knockout Strategy, as well as functional conserved amino acids of lySOSomal glycosyl exploitation of exogenous enzymatic Systems that are active hydrolases were identified in lySOSomal Storage diseases. in the recombination process, has been considerably 25 extended the range of genetic manipulations that can be LySo Somal glycosyl hydrola Se S including produced. Additional methods have been developed to pro B-glucuronidase, B-manosidase, B-glucocerebrosidase, vide Versatile research tools: Double replacement method, B-galactosidase and C-L iduronidase, are all eXO-glycosyl Sequential gene targeting, conditional cell type specific gene hydrolases, belong to the GH-A clan and share a similar targeting, Single copy integration method, inducible gene catalytic Site. However, many endo-glucanases from various targeting, gene disruption by Viral delivery, replacing one organisms, Such as bacterial and fungal Xylenases and cel gene with another, the So called knock-in method and the lulases share this catalytic domain. induction of Specific balanced chromosomal translocation. It Genomic Sequence of hpa gene and its implications: is now possible to introduce a point mutation as a unique It is well established that heparanase activity is correlated change in the entire genome, therefore allowing very fine 35 with cancer metastasis. This correlation was demonstrated at dissection of gene function in Vivo. Furthermore, the advent the level of enzymatic activity as well as the levels of protein of methods allowing conditional gene targeting opens the and hpa cDNA expression in highly metastatic cancer cells way for analysis of consequence of a particular mutation in as compared with non-metastatic cells. AS Such, inhibition of a defined organ and at a specific time during the life of the heparanase activity is desirable, and has been attempted by experimental animal (59). Several means. The genomic region, encoding the hpa gene DNA vaccination: 40 and the Surrounding, provides a new powerful tool for Observations in the early 1990s that plasmid DNA could directly transfect animal cells in Vivo Sparked exploration of regulation of heparanase activity at the level of gene expres the use of DNA plasmids to induce immune response by Sion. Regulatory Sequences may reside in noncoding regions direct injection into animal of DNA encoding antigenic both upstream and downstream the transcribed region as protein. When a DNA vaccine plasmid enters the eukaryotic 45 well as in intron Sequences. A DNA sequence upstream of cell, the protein it encodes is transcribed and translated the transcription start Site contains the promoter region and within the cell. In the case of pathogens, these proteins are potential regulatory elements. Regulatory factors, which presented to the immune System in their native form, mim interact with the promoter region may be identified and be icking the presentation of antigens during a natural infec used as potential drugs for inhibition of cancer, metastasis tion. DNA vaccination is particularly useful for the induc 50 and inflammation. The promoter region can be used to tion of T cell activation. It was applied for viral and bacterial Screen for inhibitors of heparanase gene expression. infectious diseases, as well as for allergy and for cancer. The Furthermore, the hpa promoter can be used to direct cell central hypothesis behind active specific immunotherapy for Specific, particularly cancer cell Specific, expression of for cancer is that tumor cells express unique antigens that eign genes, Such as cytotoxic or apoptotic genes, in order to should stimulate the immune system. The first DNA vaccine 55 Specifically destroy cancer cells. against tumor was carcino-embrionic antigen (CEA). DNA Cancer and yet unknown related genetic disorders may vaccinated animals expressed immunoprotection and immu involve rearrangements and mutations in the heparanase notherapy of human CEA-expressing Syngeneic mouse gene, either in coding or non-coding regions. Such muta colon and breast carcinoma (61). In a mouse model of tions may affect expression level or enzymatic activity. The neuroblastoma, DNA immunization with HuD resulted in 60 genomic Sequence of hpa enables the amplification of Spe tumor growth inhibition with no neurological disease (60). cific genomic DNA fragments, identification and diagnosis Immunity to the brown locus protein, gp75 tyrosinase of mutations. related protein-1, associated with melanoma, was investi There is thus a widely recognized need for, and it would gated in a Syngeneic mouse model. Priming with human be highly advantageous to have genomic, cDNA and com gp75 DNA broke tolerance to mouse gp75. Immunity 65 posite polynucleotides encoding a polypeptide having against mouse gp75 provided significant tumor protection heparanase activity, Vectors including Same, genetically (60). modified cells expressing heparanase and a recombinant US 6,664,105 B1 15 16 protein having heparanase activity, as well as antisense were identified and characterized. The human heparanase oligonucleotides, constructs and ribozymes which can be promoter has been isolated, identified and positively tested used for down regulation heparanase activity. for activity. The mouse heparanase promoter has been isolated and identified as well. AntiSense heparanase con SUMMARY OF THE INVENTION Structs were prepared and their influence on cells in Vitro Cloning of the human hpa gene which encodes tested. A predicted heparanase active site was identified. And heparanase, and expression of recombinant heparanase by finally, the presence of Sequences hybridizing with human transfected host cells is reported herein, as well as down heparanase Sequences was demonstrated for a variety of regulation of heparanase activity by antisense technology. mammalians and for an avian. A purified preparation of heparanase isolated from human According to one aspect of the present invention there is hepatoma cells was Subjected to tryptic digestion and provided an isolated nucleic acid comprising a genomic, microsequencing. The YGPDVGQPR (SEQ ID NO:8) complementary or composite polynucleotide Sequence Sequence revealed was used to Screen EST databases for encoding a polypeptide having heparanase catalytic activity. homology to the corresponding back translated DNA According to further features in preferred embodiments of Sequence. Two closely related EST sequences were identi 15 the invention described below, the polynucleotide or a fied and were thereafter found to be identical. Both clones portion thereof is hybridizable with SEQ ID NOs: 9, 13, 42, contained an insert of 1020 bp which included an open 43 or a portion thereof at 68° C. in 6xSSC, 1% SDS, reading frame of 973 bp followed by a 27 bp of 3' untrans 5xDenharts, 10% dextran sulfate, 100 lug/ml salmon sperm lated region and a Poly A tail. Translation Start Site was not DNA, and p labeled probe and wash at 68°C. with 3xSSC identified. and 0.1% SDS. Cloning of the missing 5' end of hpa was performed by According to Still further features in the described pre PCR amplification of DNA from placenta Marathon RACE ferred embodiments the polynucleotide or a portion thereof cDNA composite using primerS Selected according to the is at least 60% identical with SEQ ID NOs: 9, 13, 42, 43 or EST clones sequence and the linkers of the composite. A900 portions thereofas determined using the Bestfit procedure of bp PCR fragment, partially overlapping with the identified 3' 25 the DNA sequence analysis Software package developed by encoding EST clones was obtained. The joined cDNA the Genetic Computer Group (GCG) at the university of fragment (hpa), 1721 bp long (SEQ ID NO:9), contained an Wisconsin (gap creation penalty-12, gap extension open reading frame which encodes a polypeptide of 543 penalty-4). amino acids (SEQ ID NO:10) with a calculated molecular According to Still further features in the described pre weight of 61,192 daltons. ferred embodiments the polypeptide is as set forth in SEQID Cloning an extended 5' Sequence was enabled from the NOs: 10, 14, 44 or portions thereof. human SK-hep1 cell line by PCR amplification using the According to Still further features in the described pre Marathon RACE. The 5' extended sequence of the SK-hep1 ferred embodiments the polypeptide is at least 60% homolo hpa cDNA was assembled with the sequence of the hpa gous to SEQ ID NOS: 10, 14, 44 or portions thereof as cDNA isolated from human placenta (SEQ ID NO:9). The 35 determined with the Smith-Waterman algorithm, using the assembled Sequence contained an open reading frame, SEQ Bioaccelerator platform developed by Compugene (gapop: ID NOS: 13 and 15, which encodes, as shown in SEQ ID 10.0, gapext: 0.5, matrix: blosum62). NOs: 14 and 15, a polypeptide of 592 amino acids with a According to additional aspects of the present invention calculated molecular weight of 66,407 daltons. there are provided a nucleic acid construct (vector) com The ability of the hpa gene product to catalyze degrada 40 prising the isolated nucleic acid described herein and a host tion of heparan Sulfate in an in vitro assay was examined by cell comprising the construct. expressing the entire open reading frame of hpa in insect According to a further aspect of the present invention cells, using the Baculovirus expression System. Extracts and there is provided an antisense oligonucleotide comprising a conditioned media of cells infected with virus containing the 45 polynucleotide or a polynucleotide analog of at least 10 hpa gene, demonstrated a high level of heparan Sulfate bases being hybridizable in Vivo, under physiological degradation activity both towards soluble ECM-derived conditions, with a portion of a polynucleotide Strand encod HSPG and intact ECM. This degradation activity was inhib ing a polypeptide having heparanase catalytic activity. ited by heparin, which is another Substrate of heparanase. According to an additional aspect of the present invention Cells infected with a similar construct containing no hpa 50 there is provided a method of in Vivo downregulating gene had no Such activity, nor did non-infected cells. The heparanase activity comprising the Step of in Vivo adminis ability of heparanase expressed from the extended 5' clone tering the antisense oligonucleotide herein described. towards heparin was demonstrated in a mammalian expres According to yet an additional aspect of the present Sion System. invention there is provided a pharmaceutical composition The expression pattern of hpa RNA in various tissues and 55 comprising the antisense oligonucleotide herein described cell lines was investigated using RT-PCR. It was found to be and a pharmaceutically acceptable carrier. expressed only in tissues and cells previously known to have According to Still an additional aspect of the present heparanase activity. invention there is provided a ribozyme comprising the A panel of monochromosomal human/CHO and human/ antisense oligonucleotide described herein and a ribozyme mouse Somatic cell hybrids was used to localize the human 60 Sequence. heparanase gene to human chromosome 4. The newly iso According to a further aspect of the present invention lated heparanase Sequence can be used to identify a chro there is provided an antisense nucleic acid construct com moSome region harboring a human heparanase gene in a prising a promoter Sequence and a polynucleotide Sequence chromosome spread. directing the Synthesis of an antisense RNA sequence of at A human genomic library was Screened and the human 65 least 10 bases being hybridizable in Vivo, under physiologi locus harboring the heparanase gene isolated, Sequenced and cal conditions, with a portion of a polynucleotide Strand characterized. Alternatively Spliced heparanase mRNAS encoding a polypeptide having heparanase catalytic activity. US 6,664,105 B1 17 18 According to further features in preferred embodiments of a nucleic acid construct including a polynucleotide Segment the invention described below, the polynucleotide strand corresponding to at least a portion of SEQ ID NOS:9, 13 or encoding the polypeptide having heparanase catalytic activ 43 and a promoter for directing the expression of Said ity is as set forth in SEQ ID NOs: 9, 13, 42 or 43. polynucleotide Segment in Vivo. Accordingly, there is pro According to Still further features in the described pre vided also a DNA vaccine for in vivo eliciting anti ferred embodiments the polypeptide having heparanase heparanase antibodies comprising a nucleic acid construct catalytic activity is as set forth in SEQID NOS: 10, 14 or 44. including a polynucleotide Segment corresponding to at least According to Still a further aspect of the present invention a portion of SEQ ID NOS:9, 13 or 43 and a promoter for there is provided a method of in Vivo downregulating directing the expression of Said polynucleotide Segment in heparanase activity comprising the Step of in Vivo adminis WVO. tering the antisense nucleic acid construct herein described. The present invention can be used to develop new drugs According to yet a further aspect of the present invention to inhibit tumor cell metastasis, inflammation and autoim there is provided a pharmaceutical composition comprising munity. The identification of the hpa gene encoding for the antisense nucleic acid construct herein described and a heparanase enzyme enables the production of a recombinant 15 enzyme in heterologous expression Systems. Additional pharmaceutically acceptable carrier. features, advantages, uses and applications of the present According to a further aspect of the present invention invention in biological Science and in diagnostic and thera there is provided a nucleic acid construct comprising a polynucleotide Sequence functioning as a promoter, the peutic medicine are described hereinafter. polynucleotide sequence is derived from SEQID NO:42 and BRIEF DESCRIPTION OF THE DRAWINGS includes at least nucleotides 2535-2635 thereof or from The invention herein described, by way of example only, SEO ID NO:43 and includes at least nucleotides 320-420. with reference to the accompanying drawings, wherein: According to a further aspect of the present invention FIG. 1 presents nucleotide Sequence and deduced amino there is provided a method of expressing a polynucleotide acid sequence of hpa cDNA (SEQ ID NO: 11). A single Sequence comprising the Step of ligating the polynucleotide 25 nucleotide difference at position 799 (A to T) between the Sequence into the nucleic acid construct described above, EST (Expressed Sequence Tag) and the PCR amplified downstream of the polynucleotide Sequence derived from cDNA (reverse transcribed RNA) and the resulting amino SEO ID NOS:42 or 43. acid substitution (Tyr to Phe) are indicated above and below According to a further aspect of the present invention the Substituted unit, respectively. Cysteine residues and the there is provided a recombinant protein comprising a poly adenylation consensus Sequence are underlined. The polypeptide having heparanase catalytic activity. asterisk denotes the Stop codon TGA. According to further features in preferred embodiments of FIG. 2 demonstrates degradation of soluble sulfate the invention described below, the polypeptide includes at labeled HSPG substrate by lysates of High Five cells least a portion of SEQ ID NOS:10, 14 or 44. infected with pFhpa2 virus. Lysates of High Five cells that According to Still further features in the described pre 35 were infected with pFhpa2 virus (O) or control pF2 virus ferred embodiments the protein is encoded by a polynucle (O) were incubated (18 h, 37° C) with sulfate labeled otide hybridizable with SEQ ID NOs: 9, 13, 42, 43 or a ECM-derived soluble HSPG (peak I). The incubation portion thereof at 68° C. in 6xSSC, 1% SDS, 5xDenharts, medium was then Subjected to gel filtration on Sepharose 10% dextran sulfate, 100 ug/ml salmon sperm DNA, and 'p 6B. Low molecular weight HS degradation fragments (peak labeled probe and wash at 68° C. with 3xSSC and 0.1% 40 II) were produced only during incubation with the pFhpa2 SDS. infected cells, but there was no degradation of the HSPG According to Still further features in the described pre substrate (()) by lysates of pF2 infected cells. ferred embodiments the protein is encoded by a polynucle FIGS. 3a–b demonstrate degradation of soluble sulfate otide at least 60% identical with SEQ ID NOs: 9, 13, 42, 43 45 labeled HSPG substrate by the culture medium of pFhpa2 or portions thereofas determined using the Bestfit procedure and pFhpa4 infected cells. Culture media of High Five cells of the DNA sequence analysis Software package developed infected with pFhpa2 (3.a) or pFhpa4 (3.b) viruses (O), or by the Genetic Computer Group (GCG) at the university of with control viruses (D) were incubated (18 h, 37° C) with Wisconsin (gap creation penalty-12, gap extension sulfate labeled ECM-derived soluble HSPG (peak I, ()). The penalty-4). 50 incubation media were then Subjected to gel filtration on According to a further aspect of the present invention Sepharose 6B. Low molecular weight HS degradation frag there is provided a pharmaceutical composition comprising, ments (peak II) were produced only during incubation with as an active ingredient, the recombinant protein herein the hpa gene containing viruses. There was no degradation described. of the HSPG Substrate by the culture medium of cells According to a further aspect of the present invention 55 infected with control viruses. there is provided a method of identifying a chromosome FIG. 4 presents size fractionation of heparanase activity region harboring a heparanase gene in a chromosome spread expressed by pFhpa2 infected cells. Culture medium of comprising the steps of (a) hybridizing the chromosome pFhpa2 infected High Five cells was applied onto a 50 kDa Spread with a tagged polynucleotide probe encoding hepara cut-off membrane. Heparanase activity (conversion of the nase; (b) washing the chromosome spread, thereby remov 60 peak I Substrate, (()) into peak II HS degradation fragments) ing excess of non-hybridized probe; and (c) Searching for was found in the high (>50 kDa) (O),but not low (<50 kDa) Signals associated with the hybridized tagged polynucleotide (O) molecular weight compartment. probe, wherein detected Signals being indicative of a chro FIGS. 5a-b demonstrate the effect of heparin on hepara mosome region harboring a heparanase gene. nase activity expressed by pFhpa2 and pFhpa4 infected High According to a further aspect of the present invention 65 Five cells. Culture media of pFhpa2 (5a) and pFhpa4 (5b) there is provided a method of in Vivo eliciting anti infected High Five cells were incubated (18 h, 37° C) with heparanase antibodies comprising the Steps of administering sulfate labeled ECM-derived soluble HSPG (peak I, ()) in US 6,664,105 B1 19 20 the absence (O) or presence (A) of 10 ug/ml heparin. Seen between the appearance of a major protein band Production of low molecular weight HS degradation frag (MW-63,000) in fractions 4-7 and heparanase activity. ments was completely abolished in the presence of heparin, FIGS. 12a-e demonstrate expression of the hpa gene by a potent inhibitor of heparanase activity (6, 7). RT-PCR with total RNA from human embryonal tissues FIGS. 6a–b demonstrate degradation of sulfate labeled (12a), human extra-embryonal tissues (12b) and cell lines intact ECM by virus infected High Five and Sf21 cells. High from different origins (12c-e). RT-PCR products using hpa Five (6a) and Sf21 (6b) cells were plated on sulfate labeled Specific primers (I), primers for GAPDH housekeeping gene ECM and infected (48 h, 28°C.) with pFhpa4 (O) or control (II), and control reactions without reverse transcriptase pF1 (D) viruses. Control non-infected Sf21 cells (A) were demonstrating absence of genomic DNA or other contami plated on the labeled ECM as well. The pH of the cultured nation in RNA samples (III). M-DNA molecular weight medium was adjusted to 6.0–6.2 followed by 24 h incuba marker VI (Boehringer Mannheim). For 12a: lane tion at 37 C. Sulfate labeled material released into the I-neutrophil cells (adult), lane 2-muscle, lane 3-thymus, incubation medium was analyzed by gel filtration on lane 4-heart, lane 5-adrenal. For 12b: lane 1-kidney, Sepharose 6B. HS degradation fragments were produced lane 2-placenta (8 weeks), lane 3-placenta (11 weeks), only by cells infected with the hpa containing virus. 15 lanes 4-7-mole (complete hydatidiform mole), lane FIGS. 7a-b demonstrate degradation of sulfate labeled 8-cytotrophoblast cells (freshly isolated), lane intact ECM by virus infected cells. High Five (7a) and Sf21 9-cytotrophoblast cells (1.5 h in vitro), lane (7b) cells were plated on sulfate labeled ECM and infected 10-cytotrophoblast cells (6 h in vitro), lane (48 h, 28° C) with pFhpaa (O) or control pF1 (O) viruses. 11-cytotrophoblast cells (18 h in vitro), lane Control non-infected Sf21 cells (A) were plate on labeled 12-cytotrophoblast cells (48 h in vitro). For 12c: lane ECM as well. The pH of the cultured medium was adjusted 1-JAR bladder cell line, lane 2-NCITT testicular tumor to 6.0–6.2, followed by 48 h incubation at 28 C. Sulfate cell line, lane 3-SW-480 human hepatoma cell line, lane labeled degradation fragments released into the incubation 4-HTR (cytotrophoblasts transformed by SV40), lane medium was analyzed by gel filtration on Sepharose 6B. HS 5-HPTLP-I hepatocellular carcinoma cell line, lane 6-EJ degradation fragments were produced only by cells infected 25 28 bladder carcinoma cell line. For 12d: lane 1-SK-hep-1 with the hpa containing virus. human hepatoma cell line, lane 2-DAMI human mega FIGS. 8a–b demonstrate degradation of sulfate labeled karyocytic cell line, lane 3-DAMI cell line--PMA, lane intact ECM by the culture medium of pFhpa4 infected cells. 4-CHRF cell line--PMA, lane 5-CHRF cell line. For 12e: Culture media of High Five (8a) and Sf21 (8b) cells that lane 1-ABAEbovine aortic endothelial cells, lane 2-1063 were infected with pFhpa4 (O) or control pF1 (O) viruses human ovarian cell line, lane 3-human breast carcinoma were incubated (48 h, 37 C., pH 6.0) with intact sulfate MDA435 cell line, lane 4-human breast carcinoma labeled ECM. The ECM was also incubated with the culture MDA231 cell line. medium of control non-infected Sf21 cells (A). Sulfate FIG. 13 presents a comparison between nucleotide labeled material released into the reaction mixture was sequences of the human hpa and a mouse EST cDNA Subjected to gel filtration analysis. Heparanase activity was 35 fragment (SEQ ID NO:12) which is 80% homologous to the detected only in the culture medium of pFhpa4 infected 3' end (starting at nucleotide 1066 of SEQ ID NO:9) of the cells. human hpa. The aligned termination codons are underlined. FIGS. 9a–b demonstrate the effect of heparin on hepara FIG. 14 demonstrates the chromosomal localization of the nase activity in the culture medium of pFhpa4 infected cells. 40 hpa gene. PCR products of DNA derived from Somatic cell Sulfate labeled ECM was incubated (24 h, 37° C., pH 6.0) hybrids and of genomic DNA of hamster, mouse and human with culture medium of pFhpa4 infected High Five (9a) and of were separated on 0.7% agarose gel following amplifi Sf21 (9b) cells in the absence (O) or presence (A) of 10 cation with hpa specific primers. Lane 1-Lambda DNA tug/ml heparin. Sulfate labeled material released into the digested with BstEII, lane 2-no DNA control, lanes 3-29, incubation medium was Subjected to gel filtration on PCR amplification products. Lanes 3-5-human, mouse and Sepharose 6B. Heparanase activity (production of peak II 45 hamster genomic DNA, respectively. Lanes 6-29, human HS degradation fragments) was completely inhibited in the monochromosomal Somatic cell hybrids representing chro presence of heparin. mosomes 1-22 and X and Y, respectively. Lane FIGS. 10a–b demonstrate purification of recombinant 30-Lambda DNA digested with BstEII. An amplification heparanase on heparin-Sepharose. Culture medium of Sf21 50 product of approximately 2.8 Kb is observed only in lanes cells infected with pFhpa4 Virus was Subjected to heparin 5 and 9, representing human genomic DNA and DNA Sepharose chromatography. Elution of fractions was per derived from cell hybrid carrying human chromosome 4, formed with 0.35-2 M NaCl gradient (()). Heparanase respectively. These results demonstrate that the hpa gene is activity in the eluted fractions is demonstrated in FIG. 10a localized in human chromosome 4. (O). Fractions 15–28 were subjected to 15% SDS 55 FIG. 15 demonstrates the genomic exon-intron structure polyacrylamide gel electrophoresis followed by Silver of the human hpa locus (top) and the relative positions of the nitrate Staining. A correlation is demonstrated between a lambda clones used as Sequencing templates to Sequence the major protein band (MW-63,000) in fractions 19-24 and locus (below). The vertical rectangles represent exons (E) heparanase activity. and the horizontal lines therebetween represent introns (I), FIGS. 11a–b demonstrate purification of recombinant 60 upstream (U) and downstream (D) regions. Continuous lines heparanase on a SuperdeX 75 gel filtration column. Active represent DNA fragments, which were used for Sequence fractions eluted from heparin-Sepharose (FIG. 10a) were analysis. The discontinuous line in lambda 6 represent a pooled, concentrated and applied onto Superdex 75 FPLC region, which Overlaps with lambda 8 and hence was not column. Fractions were collected and aliquots of each frac analyzed. The plasmid contains a PCR product, which tion were tested for heparanase activity (O, FIG. 11a) and 65 bridges the gap between L3 and L6. analyzed by SDS-polyacrylamide gel electrophoresis fol FIGS. 16a-p presents the nucleotide sequence of the lowed by silver nitrate staining (FIG. 11b). A correlation is genomic region of the hpa gene (SEQ ID NO: 42). Exon US 6,664,105 B1 21 22 Sequences appear in upper case and intron Sequences in corresponding back translated DNA sequences. Two closely lower case. The deduced amino acid Sequence of the exons related EST sequences were identified and were thereafter is printed below the nucleotide Sequence. Two predicted found to be identical. transcription start Sites are shown in bold. Both clones contained an insert of 1020 bp which includes FIG. 17 presents an alignment of the amino acid an open reading frame of 973 bp followed by a 3' untrans sequences of human heparanase (SEQ ID NO: 11), mouse lated region of 27 bp and a Poly A tail, whereas a translation (SEQ ID NOS: 44, 45) and partial sequences of rat homo Start Site was not identified. logues (SEQ ID NOS: 46, 47). The human and the mouse Cloning of the missing 5' end was performed by PCR Sequences were determined by Sequence analysis of the amplification of DNA from placenta Marathon RACE cDNA isolated cDNAS. The rat sequence is derived from two composite using primerS Selected according to the EST different EST clones, which represent two different regions clones Sequence and the linkers of the composite. (5' and 3') of the rathpa cDNA. The human sequence and the A 900 bp PCR fragment, partially overlapping with the amino acids in the mouse and rat homologues, which are identified 3' encoding EST clones was obtained. The joined identical to the human Sequence, appear in bold. cDNA fragment (hpa), 1721 bp long (SEQ ID NO:9), FIG. 18 presents a heparanase Zoo blot. Ten micrograms 15 contained an open reading frame which encodes, as shown of genomic DNA from various Sources were digested with in FIG. 1 and SEQ ID NO:11, a polypeptide of 543 amino EcoRI and separated on 0.7% agarose-TBE gel. Following acids (SEQ ID NO:10) with a calculated molecular weight electrophoresis, the was gel treated with HCl and than with of 61,192 daltons. NaOH and the DNA fragments were downward transferred A single nucleotide difference at position 799 (A to T) to a nylon membrane (Hybond N+, Amersham) with 0.4N between the EST clones and the PCR amplified cDNA was NaOH. The membrane was hybridized with a 1.6 Kb DNA observed. This difference results in a Single amino acid probe that contained the entire hpa cDNA. Lane order: substitution (Tyr to Phe) (FIG. 1). Furthermore, the pub H-Human; M-Mouse; Rt-Rat; P-Pig, Cw-Cow; lished EST Sequences contained an unidentified nucleotide, Hr-Horse; S-Sheep; Rb-Rabbit; D-Dog, which following DNA sequencing of both the EST clones Ch-Chicken; F Fish. Size markers (Lambda Bste.II) are 25 was resolved into two nucleotides (G and C at positions shown on the left 1630 and 1631 in SEQ ID NO:9, respectively). FIG. 19 demonstrates the secondary structure prediction The ability of the hpa gene product to catalyze degrada for heparanase performed using the PHD server-Profile tion of heparan Sulfate in an in vitro assay was examined by network Prediction Heidelberg. H-helix, E-extended expressing the entire open reading frame in insect cells, (beta Strand), The glutamic acid predicted as the proton using the Baculovirus expression System. donor is marked by asterisk and the possible nucleophiles Extracts and conditioned media of cells infected with are underlined. Virus containing the hpa gene, demonstrated a high level of heparan Sulfate degradation activity both towards Soluble DESCRIPTION OF THE PREFERRED ECM-derived HSPG and intact ECM, which was inhibited EMBODIMENTS 35 by heparin, while cells infected with a similar construct The present invention is of a polynucleotide or nucleic containing no hpa gene had no Such activity, nor did acid, referred to hereinbelow interchangeably as hpa, hpa non-infected cells. cDNA or hpa gene or identified by its SEQ ID NOS, The expression pattern of hpa RNA in various tissues and encoding a polypeptide having heparanase activity, Vectors 40 cell lines was investigated using RT-PCR. It was found to be or nucleic acid constructs including Same and which are expressed only in tissues and cells previously known to have used for over-expression or antisense inhibition of heparanase activity. heparanase, genetically modified cells expressing Same, Cloning an extended 5' Sequence was enabled from the recombinant protein having heparanase activity, antisense human SK-hep1 cell line by PCR amplification using the oligonucleotides and ribozymes for heparanase modulation, 45 Marathon RACE. The 5' extended sequence of the SK-hep1 and heparanase promoter Sequences which can be used to hpa cDNA was assembled with the sequence of the hpa direct the expression of desired genes. cDNA isolated from human placenta (SEQ ID NO:9). The Before explaining at least one embodiment of the inven assembled Sequence contained an open reading frame, SEQ tion in detail, it is to be understood that the invention is not ID NOS: 13 and 15, which encodes, as shown in SEQ ID limited in its application to the details of construction and 50 NOs: 14 and 15, a polypeptide of 592 amino acids, with a the arrangement of the components Set forth in the following calculated molecular weight of 66,407 daltons. This open description or illustrated in the drawings. The invention is reading frame was shown to direct the expression of cata capable of other embodiments or of being practiced or lytically active heparanase in a mammalian cell expression carried out in various ways. Also, it is to be understood that System. The expressed heparanase was detectable by anti the phraseology and terminology employed herein is for the 55 heparanase antibodies in Western blot analysis. purpose of description and should not be regarded as lim A panel of monochromosomal human/CHO and human/ iting. mouse Somatic cell hybrids was used to localize the human Cloning of the human and mouse hpa genes, cDNAS and heparanase gene to human chromosome 4. The newly iso genomic Sequence (for human), encoding heparanase and lated heparanase Sequence can therefore be used to identify expressing recombinant heparanase by transfected cells is 60 a chromosome region harboring a human heparanase gene in reported herein. These are the first mammalian heparanase a chromosome spread. genes to be cloned. The hpa cDNA was then used as a probe to screen a A purified preparation of heparanase isolated from human human genomic library. Several phages were positive. These hepatoma cells was Subjected to tryptic digestion and phages were analyzed and were found to cover most of the microSequencing. 65 hpa locus, except for a Small portion which was recovered The YGPDVGQPR (SEQ ID NO:8) sequence revealed by bridging PCR. The hpa locus covers about 50,000 bp. The was used to screen EST databases for homology to the hpa gene includes 12 exons Separated by 11 introns. US 6,664,105 B1 23 24 RT-PCR performed on a variety of cells revealed alter According to another preferred embodiment of the present natively spliced hpa transcripts. invention the polypeptide encoded by the polynucleotide The amino acid Sequence of human heparanase was used sequence is as set forth in SEQ ID NOS:10, 14, 44 or to Search for homologous Sequences in the DNA and protein portions thereof having heparanase catalytic activity. Such databases. Several human ESTs were identified, as well as portions are expected to include amino acids Asp-Glu mouse Sequences highly homologous to human heparanase. 224-225 (SEQID NO:10), which can serve as proton donors The following mouse EST's were identified AA177901, and glutamic acid 343 or 396 which can serve as a nucleo AA674378, AA67997, AAO47943, AA690179, AI122034, phile. all sharing an identical Sequence and correspond to amino According to another preferred embodiment of the present acids 336–543 of the human heparanase sequence. The invention the polypeptide encoded by the polynucleotide entire mouse heparanase cDNA was cloned, based on the sequence is at least 60%, preferably at least 65%, more nucleotide Sequence of the mouse EST's using Marathon preferably at least 70%, more preferably at least 75%, more cDNA libraries. The mouse and the human hpa genes Share preferably at least 80%, more preferably at least 85%, more an average homology of 78% between the nucleotide preferably at least 90%, most preferably, 95-100% homolo Sequences and 81% similarity between the deduced amino 15 gous (both similar and identical acids) to SEQ ID NOS: 10, acid Sequences. hpa homologous Sequences from rat were 14, 44 or portions thereof as determined with the Smith also uncovered (EST's AIO60284 and AI237828). Waterman algorithm, using the Bioaccelerator platform Homology Search of heparanase amino acid Sequence developed by Compugene (gapop: 10.0, gapext: 0.5, matrix: against the DNA and the protein databases and prediction of blosum62, see also the description to FIG. 17). its protein Secondary Structure enabled to identify candidate Further according to the present invention there is pro amino acids that participate in the heparanase active site. Vided a nucleic acid construct comprising the isolated Expression of hpa antisense in mammalian cell lines nucleic acid described herein. The construct may and pref resulted in about five fold decrease in the number of recov erably further include an origin of replication and trans regulatory elements, Such as promoter and enhancer erable cells as compared to controls. 25 Human Hpa cDNA was shown to hybridize with genomic Sequences. DNAS of a variety of mammalian Species and with an avian. The construct or vector can be of any type. It may be a The human and mouse hpa promoters were identified and phage which infects bacteria or a virus which infects eukary the human promoter was tested positive in directing the otic cells. It may also be a plasmid, phagemid, cosmid, expression of a reporter gene. bacmid or an artificial chromosome. Further according to the present invention there is pro Thus, according to the present invention there is provided Vided a host cell comprising the nucleic acid construct an isolated nucleic acid comprising a genomic, complemen described herein. The host cell can be of any type. It may be tary or composite polynucleotide Sequence encoding a a prokaryotic cell, an eukaryotic cell, a cell line, or a cell as polypeptide having heparanase catalytic activity. a portion of an organism. The polynucleotide encoding The phrase “composite polynucleotide Sequence” refers 35 heparanase can be permanently or transiently present in the to a Sequence which includes exonal Sequences required to cell. In other words, genetically modified cells obtained encode the polypeptide having heparanase activity, as well following Stable or transient transfection, transformation or as any number of intronal Sequences. The intronal Sequences transduction are all within the Scope of the present invention. can be of any Source and typically will include conserved The polynucleotide can be present in the cell in low copy Splicing Signal Sequences. Such intronal Sequences may 40 (say 1-5 copies) or high copy number (Say 5-50 copies or further include cis acting expression regulatory elements. more). It may be integrated in one or more chromosomes at The term "heparanase catalytic activity” or its equivalent any location or be present as an extrachromosomal material. term “heparanase activity” both refer to a mammalian The present invention is further directed at providing a endoglycosidase hydrolyzing activity which is specific for heparanase over-expression System which includes a cell heparan or heparan Sulfate proteoglycan Substrates, as 45 overexpressing heparanase catalytic activity. The cell may opposed to the activity of bacterial enzymes (heparinase I, II be a genetically modified host cell transiently or stably and III) which degrade heparin or heparan Sulfate by means transfected or transformed with any suitable vector which of B-elimination (37). includes a polynucleotide Sequence encoding a polypeptide According to a preferred embodiment of the present 50 having heparanase activity and a Suitable promoter and invention the polynucleotide or a portion thereof is hybrid enhancer Sequences to direct over-expression of heparanase. izable with SEQ ID NOs: 9, 13, 42, 43 or a portion thereof However, the overexpressing cell may also be a product of at 68 C. in 6xSSC, 1% SDS, 5xDenharts, 10% dextran an insertion (e.g., via homologous recombination) of a sulfate, 100 ug/ml salmon sperm DNA, and p labeled promoter and/or enhancer Sequence downstream to the probe and wash at 68° C. with 3, 2, 1, 0.5 or 0.1xSSC and 55 endogenous heparanase gene of the expressing cell, which O.1% SDS. will direct over-expression from the endogenous gene. According to another preferred embodiment of the present The term “over-expression' as used herein in the Speci invention the polynucleotide or a portion thereof is at least fication and claims below refers to a level of expression 60%, preferably at least 65%, more preferably at least 70%, which is higher than a basal level of expression typically more preferably at least 75%, more preferably at least 80%, 60 characterizing a given cell under otherwise identical condi more preferably at least 85%, more preferably at least 90%, tions. most preferably, 95-100% identical with SEQ ID NOs: 9, According to another aspect the present invention pro 13, 42, 43 or portions thereofas determined using the Bestfit vides an antisense oligonucleotide comprising a polynucle procedure of the DNA sequence analysis Software package otide or a polynucleotide analog of at least 10, preferably developed by the Genetic Computer Group (GCG) at the 65 11-15, more preferably 16-17, more preferably 18, more university of Wisconsin (gap creation penalty-12, gap preferably 19–25, more preferably 26-35, most preferably extension penalty-4-which are the default parameters). 35-100 bases being hybridizable in vivo, under physiologi US 6,664,105 B1 25 26 cal conditions, with a portion of a polynucleotide Strand lasting from Several days to Several months or until a cure is encoding a polypeptide having heparanase catalytic activity. effected or a diminution of disease State is achieved. Persons The antisense oligonucleotide can be used for downregulat ordinarily skilled in the art can easily determine optimum ing heparanase activity by in Vivo administration thereof to dosages, dosing methodologies and repetition rates. a patient. AS Such, the antisense oligonucleotide according to Further according to the present invention there is pro the present invention can be used to treat types of cancers Vided a nucleic acid construct comprising a polynucleotide which are characterized by impaired (over) expression of Sequence functioning as a promoter, the polynucleotide heparanase, and are dependent on the expression of hepara sequence is derived from SEQ ID NO:42 and includes at nase for proliferating or forming metastases. least nucleotides 2135-2635, preferably 2235-2635, more The antisense oligonucleotide can be DNA or RNA or preferably 2335–2635, more preferably 2435-2635, most even include nucleotide analogs, examples of which are preferably 2535-2635 thereof, or SEQ ID NO:43 and provided in the Background Section hereinabove. The anti includes at least nucleotides 1-420, preferably 120-420, Sense oligonucleotide according to the present invention can more preferably 220-420, most preferably 320-420, thereof be Synthetic and is preferably prepared by Solid phase These nucleotides are shown in the example Section that 15 follows to direct the Synthesis of a reporter gene in trans Synthesis. In addition, it can be of any desired length which formed cells. Thus, further according to the present inven Still provides specific base pairing (e.g., 8 or 10, preferably tion there is provided a method of expressing a polynucle more, nucleotides long) and it can include mismatches that otide Sequence comprising the Step of ligating the do not hamper base pairing under physiological conditions. polynucleotide Sequence downstream to either of the pro Further according to the present invention there is pro moter Sequences described herein. Heparanase promoters Vided a pharmaceutical composition comprising the anti can be isolated from a variety of mammalian an other Sense oligonucleotide herein described and a pharmaceuti Species by cloning genomic regions present 5' to the coding cally acceptable carrier. The carrier can be, for example, a Sequence thereof. This can be readily achievable by one lipoSome loadable with the antisense oligonucleotide. ordinarily Skilled in the art using the heparanase polynucle According to a preferred embodiment of the present 25 otides described herein, which are shown in the Examples invention the antisense oligonucleofide further includes a Section that follows to participate in efficient croSS Species ribozyme Sequence. The ribozyme Sequence Serves to cleave hybridization. a heparanase RNA molecule to which the antisense oligo Further according to the present invention there is pro nucleotide binds, to thereby downregulate heparanase Vided a recombinant protein comprising a polypeptide hav expression. ing heparanase catalytic activity. The protein according to Further according to the present invention there is pro the present invention include modifications known as post Vided an anfisense nucleic acid construct comprising a translational modifications, including, but not limited to, promoter Sequence and a polynucleotide Sequence directing proteolysis (e.g., removal of a signal peptide and of a pro the Synthesis of an antisense RNA sequence of at least 10 or preprotein sequence), methionine modification, bases being hybridizable in Vivo, under physiological 35 glycosylation, alkylation (e.g., methylation), acetylation, conditions, with a portion of a polynucleotide Strand encod etc. According to preferred embodiments the polypeptide ing a polypeptide having heparanase catalytic activity. Like includes at least a portion of SEQ ID NOS: 10, 14 or 44, the the antisense oligonucleotide, the antisense construct can be portion has heparanase catalytic activity. According to pre used for downregulating heparanase activity by in vivo ferred embodiments of the present invention the protein is administration thereof to a patient. AS Such, the antisense 40 encoded by any of the above described isolated nucleic construct, like the antisense oligonucleotide, according to acids. Further according to the present invention there is the present invention can be used to treat types of cancers provided a pharmaceutical composition comprising, as an which are characterized by impaired (over) expression of active ingredient, the recombinant protein described herein. heparanase, and are dependent on the expression of hepara The recombinant protein may be purified by any conven nase for proliferating or forming metastases. 45 tional protein purification procedure close to homogeneity Thus, further according to the present invention there is and/or be mixed with additives. The recombinant protein provided a pharmaceutical composition comprising the anti may be manufactured using any of the genetically modified Sense construct herein described and a pharmaceutically cells described above, which include any of the expression acceptable carrier. The carrier can be, for example, a lipo nucleic acid constructs described herein. The recombinant Some loadable with the antisense construct. 50 protein may be in any form. It may be in a crystallized form, Formulations for topical administration may include, but a dehydrated powder form or in solution. The recombinant are not limited to, lotions, ointments, gels, creams, protein may be useful in obtaining pure heparanase, which Suppositories, drops, liquids, Sprays and powders. Conven in turn may be useful in eliciting anti-heparanase antibodies, tional pharmaceutical carriers, aqueous, powder or oily either poly or monoclonal antibodies, and as a Screening bases, thickenerS and the like may be necessary or desirable. 55 active ingredient in an anti-heparanase inhibitors or drugs Coated condoms, Stents, active pads, and other medical Screening assay or System. devices may also be useful. Compositions for oral admin Further according to the present invention there is pro istration include powders or granules, Suspensions or Solu Vided a method of identifying a chromosome region harbor tions in water or non-aqueous media, Sachets, capsules or ing a human heparanase gene in a chromosome spread the tablets. Thickeners, diluents, flavorings, dispersing aids, 60 method is executed implementing the following method emulsifiers or binders may be desirable. Formulations for Steps, in which in a first step the chromosome spread (either parenteral administration may include, but are not limited to, interphase or metaphase spread) is hybridized with a tagged Sterile aqueous Solutions which may also contain buffers, polynucleotide probe encoding heparanase. The tag is pref diluents and other Suitable additives. erably a fluorescent tag. In a Second Step according to the Dosing is dependent on Severity and responsiveness of the 65 method the chromosome spread is washed, thereby excess of condition to be treated, but will normally be one or more non-hybridized probe is removed. Finally, Signals associated doses per day, week or month with course of treatment with the hybridized tagged polynucleotide probe are US 6,664,105 B1 27 28 Searched for, wherein detected Signals being indicative of a childbirth, including injuries Sustained during medical pro chromosome region harboring the human heparanase gene. cedures Such as episiotomies, trauma-induced injuries One ordinarily skilled in the art would know how to use the including cuts, those injuries Sustained in automobile and Sequences disclosed herein in Suitable labeling reactions and other mechanical accidents, and those caused by bullets, how to use the tagged probes to detect, using in Situ knives and other weapons, and post-Surgical injuries, as well hybridization, a chromosome region harboring a human as chronic conditions Such as pressure Sores, bedsores, heparanase gene. conditions related to diabetes and poor circulation, and all Further according to the present invention there is pro types of acne, etc. Vided a method of in Vivo eliciting anti-heparanase antibod Anti-heparanase antibodies, raised against the recombi ies comprising the Steps of administering a nucleic acid nant enzyme, would be useful for immunodetection and construct including a polynucleotide Segment corresponding diagnosis of micrometastases, autoimmune lesions and renal to at least a portion of SEQ ID NOS:9, 13 or 43 and a failure in biopsy Specimens, plasma Samples, and body promoter for directing the expression of Said polynucleotide fluids. Such antibodies may also serve as neutralizing agents Segment in Vivo. Accordingly, there is provided also a DNA for heparanase activity. vaccine for in Vivo eliciting anti-heparanase antibodies 15 The genomic heparanase Sequences described herein can comprising a nucleic acid construct including a polynucle be used to construct knock-in and knock-out constructs. otide Segment corresponding to at least a portion of SEQ ID Such constructs include a fragment of 10-20 Kb of a NOs:9, 13 or 43 and a promoter for directing the expression heparanase locus and a negative and a positive Selection of Said polynucleotide Segment in Vivo. The vaccine option markers and can be used to provide heparanase knock-in and ally further includes a pharmaceutically acceptable carrier, knock-out animal models by methods known to the skilled Such as a virus, liposome or an antigen presenting cell. artisan. Such animal models can be used for Studying the Alternatively, the vaccine is employed as a naked DNA function of heparanase in developmental processes, and in Vaccine. normal as well as pathological processes. They can also The present invention can be used to develop treatments Serve as an experimental model for testing drugs and gene for various diseases, to develop diagnostic assays for these 25 therapy protocols. The complementary heparanase Sequence diseases and to provide new tools for basic research espe (cDNA) can be used to derive transgenic animals, overex cially in the fields of medicine and biology. pressing heparanase for Same. Alternatively, if cloned in the Specifically, the present invention can be used to develop antisense orientation, the complementary heparanase new drugs to inhibit tumor cell metastasis, inflammation and Sequence (cDNA) can be used to derive transgenic animals autoimmunity. The identification of the hpa gene encoding under-expressing heparanase for Same. for the heparanase enzyme enables the production of a The heparanase promoter Sequences described herein and recombinant enzyme in heterologous expression Systems. other cis regulatory elements linked to the heparanase locus Furthermore, the present invention can be used to modu can be used to regulated the expression of genes. For late bioavailability of heparin-binding growth factors, cel example, these promoters can be used to direct the expres lular responses to heparin-binding growth factors (e.g., 35 sion of a cytotoxic protein, such as TNF, in tumor cells. It bFGF, VEGF) and cytokines (e.g., IL-8), cell interaction will be appreciated that heparanase itself is abnormally with plasma lipoproteins, cellular Susceptibility to viral, expressed under the control of its own promoter and other protozoa and Some bacterial infections, and disintegration of cis acting elements in a variety of tumors, and its expression neurodegenerative plaques. Recombinant heparanase offers is correlated with metastasis. It is also abnormally highly a potential treatment for wound healing, angiogenesis, 40 expressed in inflammatory cells. The introns of the hepara restenosis, atherosclerosis, inflammation, neurodegenerative nase gene can be used for the same purpose, as it is known diseases (such as, for example, Genstmann-Straussler that introns, especially upstream introns include cis acting Syndrome, Creutzfeldt-Jakob disease, Scrape and Alzhe element which affect expression. A heparanase promoter fused to a reporter protein can be used to Study/monitor its imer's disease) and certain viral and Some bacterial and 45 protozoa infections. Recombinant heparanase can be used to activity. neutralize plasma heparin, as a potential replacement of The polynucleotide Sequences described herein can also protamine. be used to provide DNA vaccines which will elicit in vivo AS used herein, the term "modulate' includes Substan antiheparanase antibodies. Such vaccines can therefore be tially inhibiting, slowing or reversing the progression of a 50 used to combat inflammatory and cancer. disease, Substantially ameliorating clinical Symptoms of a AntiSense oligonucleotides derived according to the disease or condition, or Substantially preventing the appear heparanase Sequences described herein, especially Such oli ance of clinical Symptoms of a disease or condition. A gonucleotides Supplemented with ribozyme activity, can be "modulator' therefore includes an agent which may modu used to modulate heparanase expression. Such oligonucle late a disease or condition. Modulation of Viral, protozoa and 55 otides can be from the coding region, from the introns or bacterial infections includes any effect which substantially promoter Specific. AntiSense heparanase nucleic acid con interrupts, prevents or reduces any viral, bacterial or proto Structs can Similarly function, as well known in the art. Zoa activity and/or stage of the virus, bacterium or protozoon The heparanase Sequences described herein can be used to life cycle, or which reduces or prevents infection by the Study the catalytic mechanism of heparanase. Carefully Virus, bacterium or protozoon in a Subject, Such as a human 60 Selected Site directed mutagenesis can be employed to or lower animal. provide modified heparanase proteins having modified char AS used herein, the term "wound” includes any injury to acteristics in terms of, for example, Substrate Specificity, any portion of the body of a Subject including, but not Sensitivity to inhibitors, etc. limited to, acute conditions Such as thermal burns, chemical While Studying heparanase expression in a variety of cell bums, radiation burns, bums caused by exceSS exposure to 65 types alternatively Spliced transcripts were identified. Such ultraViolet radiation Such as Sunburn, damage to bodily transcripts if found characteristic of certain pathological tissueS Such as the perineum as a result of labor and conditions can be used as markers for Such conditions. Such US 6,664,105 B1 29 30 transcripts are expected to direct the Synthesis of hepara calf serum and 5% FCS. bFGF (1 ng/ml) was added every nases with altered functions. other day during the phase of active cell growth (13, 14). Additional objects, advantages, and novel features of the Preparation of dishes coated with ECM: BCECs (second present invention will become apparent to one ordinarily to fifth passage) were plated into 4-well plates at an initial skilled in the art upon examination of the following density of 2x10 cells/ml, and cultured in sulfate-free Fisher examples, which are not intended to be limiting. medium plus 5% dextran T-40 for 12 days. NaSO (25 Additionally, each of the various embodiments and aspects puCi/ml) was added on day 1 and 5 after Seeding and the of the present invention as delineated hereinabove and as cultures were incubated with the label without medium claimed in the claims Section below finds experimental change. The subendothelial ECM was exposed by dissolving Support in the following examples. (5 min., room temperature) the cell layer with PBS contain ing 0.5% Triton X-100 and 20 mM NHOH, followed by EXAMPLES four washes with PBS. The ECM remained intact, free of Generally, the nomenclature used herein and the labora cellular debris and firmly attached to the entire area of the tory procedures in recombinant DNA technology described tissue culture dish (19, 22). below are those well known and commonly employed in the 15 To prepare Soluble Sulfate labeled proteoglycans (peak I art. Standard techniques are used for cloning, DNA and material), the ECM was digested with trypsin (25 ug/ml, 6 RNA isolation, amplification and purification. Generally h, 37 C.), the digest was concentrated by reverse dialysis enzymatic reactions involving DNA ligase, DNA and the concentrated material was applied onto a Sepharose polymerase, restriction endonucleases and the like are per 6B gel filtration column. The resulting high molecular formed according to the manufacturers’ Specifications. weight material (Kav-0.2, peak I) was collected. More than These techniques and various other techniques are generally 80% of the labeled material was shown to be composed of performed according to Sambrook et al., Molecular heparan Sulfate proteoglycans (11,39). Cloning-A Laboratory Manual, Cold Spring Harbor Heparanase activity: Cells (1x10/35-mm dish), cell Laboratory, Cold Spring Harbor, N.Y. (1989), which is lysates or conditioned media were incubated on top of incorporated herein by reference. Other general references 25 S-labeled ECM (18 h, 37° C.) in the presence of 20 mM are provided throughout this document. The procedures phosphate buffer (pH 6.2). Cell lysates and conditioned therein are believed to be well known in the art and are media were also incubated with sulfate labeled peak I provided for the convenience of the reader. All the infor material (10–20 ul). The incubation medium was collected, mation contained therein is incorporated herein by refer centrifuged (18,000xg, 4 C., 3 min.), and sulfate labeled CCC. material analyzed by gel filtration on a Sepharose CL-6B The following protocols and experimental details are column (0.9x30 cm). Fractions (0.2 ml) were eluted with referenced in the Examples that follow: PBS at a flow rate of 5 ml/h and counted for radioactivity Purification and characterization of heparanase from a using Bio-fluor scintillation fluid. The excluded volume (V) human hepatoma cell line and human placenta: A human was marked by blue 20 dextran and the total included hepatoma cell line (Sk-hep-1) was chosen as a Source for 35 volume (V) by phenol red. The latter was shown to comi purification of a human tumor-derived heparanase. Purifi grate with free Sulfate (7, 11, 23). Degradation fragments of cation was essentially as described in U.S. Pat. No. 5,362, HS side chains were eluted from Sepharose 6B at 641 to Fuks, which is incorporated by reference as if fully 0.5-Kav-0.8 (peak II) (7, 11, 23). A nearly intact HSPG set forth herein. Briefly, 500 liter, 5x10" cells were grown released from ECM by trypsin-and, to a lower extent, in Suspension and the heparanase enzyme was purified about 40 during incubation with PBS alone-was eluted next to V. 240,000 fold by applying the following steps: (i) cation (Kav-0.2, peak I). Recoveries of labeled material applied on exchange (CM-Sephadex) chromatography performed at pH the columns ranged from 85 to 95% in different experiments 6.0, 0.3-1.4 M NaCl gradient; (ii) cation exchange (CM (11). Each experiment was performed at least three times Sephadex) chromatography performed at pH 7.4 in the and the variation of elution positions (Kav Values) did not presence of 0.1% CHAPS, 0.3–1.1 M NaCl gradient; (iii) 45 exceed +/-15%. heparin-Sepharose chromatography performed at pH 7.4 in Cloning of hpa cDNA. cDNA clones 257548 and 260138 the presence of 0.1% CHAPS, 0.35–1.1 M NaCl gradient; were obtained from the I.M.A.G.E Consortium (2130 (iv) ConA-Sepharose chromatography performed at pH 6.0 Memorial Parkway SW, Hunstville, Ala. 35801). The in buffer containing 0.1% CHAPS and 1 M NaCl, elution cDNAs were originally cloned in EcoRI and Not cloning with 0.25 M C.-methyl mannoside; and (v) HPLC cation 50 sites in the plasmid vector pT3T7D-Pac. Although these exchange (Mono-S) chromatography performed at pH 7.4 in clones are reported to be Somewhat different, DNA sequenc the presence of 0.1% CHAPS, 0.25-1 M NaCl gradient. ing demonstrated that these clones are identical to one Active fractions were pooled, precipitated with TCA and another. Marathon RACE (rapid amplification of cDNA the precipitate Subjected to SDS polyacrylamide gel elec 55 ends) human placenta (poly-A) cDNA composite was a gift trophoresis and/or tryptic digestion and reverse phase of Prof. Yossi Shiloh of Tel Aviv University. This composite HPLC. Tryptic peptides of the purified protein were sepa is vector free, as it includes reverse transcribed cDNA rated by reverse phase HPLC (C8 column) and homoge fragments to which double, partially Single Stranded adapt neous peaks were Subjected to amino acid Sequence analy ers are attached on both sides. The construction of the SS. 60 Specific composite employed is described in reference 39a. The purified enzyme was applied to reverse phase HPLC Amplification of hp3 PCR fragment was performed and Subjected to N-terminal amino acid Sequencing using according to the protocol provided by Clontech laboratories. the amino acid sequencer (Applied BioSystems). The template used for amplification was a Sample taken Cells: Cultures of bovine corneal endothelial cells from the above composite. The primers used for amplifica (BCECs) were established from steer eyes as previously 65 tion were: described (19, 38). Stock cultures were maintained in First Step: 5'-primer: AP1 : DMEM (1 g glucose/liter) supplemented with 10% newborn 5'-CCATCCTAATACGACTCACTATAGGGC-3', SEQ ID US 6,664,105 B1 31 32 NO:1; 3'-primer: HPL229: (GibcoBRL). The transfer vector pFastBac was digested 5'-GTAGTGATGCCATGTAACTGAATC-3', SEQ ID with Sal and Not and ligated with a 1.7 kb fragment of NO:2. phpa2 digested with XhoI and Not. The resulting plasmid Second step: nested 5'-primer: A P2: was designated pFasthpa2. An identical plasmid designated 5'-ACTCACTATAGGGCTCGAGCGGC-3', SEQ ID NO:3: pFasthpaa was prepared as a duplicate and both indepen nested 3'- primer: H P L 171: dently served for further experimentations. Recombinant bacmid was generated according to the instructions of the 5'-GCATCTTAGCCGTCTTTCTTCG-3', SEQ ID NO:4. manufacturer with pFasthpa2, pFasthpa4 and with pFastBac. The HPL229 and HPL171 were selected according to the The latter Served as a negative control. Recombinant bacmid sequence of the EST clones. They include nucleotides DNAS were transfected into Sf21 insect cells. Five days after 933–956 and 876–897 of SEQ ID NO:9, respectively. transfection recombinant Viruses were harvested and used to PCR program was 94 C-4 min., followed by 30 cycles infect High Five insect cells, 3x10 cells in T-25 flasks. Cells of 94 C-40 sec., 62 C-1 min.., 72 C-2.5 min. were harvested 2–3 days after infection. 4x10" cells were Amplification was performed with Expand High Fidelity centrifuged and resuspended in a reaction buffer containing (Boehringer Mannheim). The resulting ca. 900 bp hp3 PCR 20 mM phosphate citrate buffer, 50 mM NaCl. Cells under product was digested with BfrI and PvulI. Clone 257548 15 went three cycles of freeze and thaw and lysates were Stored (phpa1) was digested with EcoRI, followed by end filling at -80 C. Conditioned medium was stored at 4 C. and was then further digested with BfrI. Thereafter the Partial purification of recombinant heparanase: Partial PvulI-BfrI fragment of the hp3 PCR product was cloned into purification of recombinant heparanase was performed by the blunt end-BfrI end of clone phpa1 which resulted in heparin-Sepharose column chromatography followed by having the entire cDNA cloned in pT3T7-pac vector, des Superdex 75 column gel filtration. Culture medium (150 ml) ignated phpa2. of Sf21 cells infected with pFhpa4 virus was subjected to RT-PCR: RNA was prepared using TRI-Reagent heparin-Sepharose chromatography. Elution of 1 ml frac (Molecular research center Inc.) according to the manufac tions was performed with 0.35-2 M NaCl gradient in turer instructions. 1.25 ug were taken for reverse transcrip presence of 0.1% CHAPS and 1 mM DTT in 10 mM sodium tion reaction using MuMLV Reverse transcriptase (Gibco 25 acetate buffer, pH 5.0. A 25 ul sample of each fraction was BRL) and Oligo (dT) primer, SEQ ID NO:5, (Promega). tested for heparanase activity. Heparanase activity was Amplification of the resultant first strand cDNA was per eluted at the range of 0.65-1.1 M NaCl (fractions 18-26, formed with Taq polymerase (Promega). The following FIG. 10a). 5 ul of each fraction was subjected to 15% primers were used: SDS-polyacrylamide gel electrophoresis followed by silver nitrate Staining. Active fractions eluted from heparin HPU-355: 5'-TTCGATCCCAAGAAGGAATCAAC-3', Sepharose (FIG. 10a) were pooled and concentrated (x6) on SEQ ID NO:6, nucleotides 372-394 in SEQ ID NOs:9 YM3 cut-off membrane. 0.5 ml of the concentrated material or 11. was applied onto 30 ml Superdex 75 FPLC column equili HPL-229: 5'-GTAGTGATGCCATGTAACTGAATC-3', brated with 10 mMSodium acetate buffer, pH 5.0, containing SEQ ID NO:7, nucleotides 933–956 in SEQ ID NOs:9 35 0.8 M NaCl, 1 mM DTT and 0.1% CHAPS. Fractions (0.56 or 11. ml) were collected at a flow rate of 0.75 ml/min. Aliquots of PCR program: 94 C-4 min., followed by 30 cycles of each fraction were tested for heparanase activity and were 94 C-40 sec., 62 C-1 min.., 72 C-1 min. Subjected to SDS-polyacrylamide gel electrophoresis fol Alternatively, total RNA was prepared from cell cultures lowed by silver nitrate staining (FIG. 11b). using Tri-reagent (Molecular Research Center, Inc.) accord 40 PCR amplification of genomic DNA: 94 C. 3 minutes, ing to the manufacturer recommendation. Poly A+ RNA was isolated from total RNA using mRNA separator (Clontech). followed by 32 cycles of 94 C. 45 seconds, 64 C. 1 minute, Reverse transcription was performed with total RNA using 68 C. 5 minutes, and one cycle at 72 C., 7 minutes. Primers Superscript II (GibcoBRL). PCR was performed with used for amplification of genomic DNA included: Expand high fidelity (Boehringer Mannheim). Primers used GHpu-L3 5'-AGGCACCCTAGAGATGTTCCAG-3', for amplification were as follows: 45 SEO ID NO:30 GHpl-L6 5'-GAAGATTTCTGTTTCCATGACGTG-3', Hpu-685, 5'-GAGCAGCCAGGTGAGCCCAAGAT-3', SEO ID NO:31. SEO ID NO:24 Screening of genomic libraries: A human genomic library Hpu-355, 5'-TTCGATCCCAAGAAGGAATCAAC-3', SEO ID NO:25 in Lambda phage EMBLE3 SP6/T7 (Clontech, Paulo Alto, 50 CA) was screened. 5x10 plaques were plated at 5x10" Hpu 565, 5'-AGCTCTGTAGATGTGCTATACAC-3', pfu/plate on NZCYM agar/top agarose plates. Phages were SEO ID NO:26 absorbed on nylon membranes in duplicates (Qiagen). Hpl 967, 5'-TCAGATGCAAGCAGCAACTTTGGC-3', Hybridization was performed at 65 C. in 5xSSC, SEO ID NO:27 5xDenharts, 10% dextran sulfate, 100 lug/ml Salmon sperm, Hpl 171, 5'-GCATCTTAGCCGTCTTTCTTCG-3', SEQ 55 p labeled probe (10 cpm/ml). A 1.6 kb fragment, con ID NO:28 taining the entire hpa cDNA was labeled by random priming Hpl 229, 5'-GTAGTGATGCCATGTAACTGAATC-3', (Boehringer Mannheim). Following hybridization mem SEO ID NO:29 branes were washed once with 2xSSC, 0.1% SDS at 65° C. PCR reaction was performed as follows: 94 C.3 minutes, for 20 minutes, and twice with 0.2xSSC, 0.1% SDS at 65 followed by 32 cycles of 94 C. 40 seconds, 64 C. 1 minute, 60 C. for 15 minutes. Hybridizing plaques were picked, and 72 C. 3 minutes, and one cycle 72 C., 7 minutes. plated at 100 pfu/plate. Hybridization was performed as Expression of recombinant heparanase in insect cells: above and Single isolated positive plaques were picked. Cells, High Five and Sf21 insect cell lines were maintained Phage DNA was extracted using a Lambda DNA extrac as monolayer cultures in SF900II-SFM medium tion kit (Qiagen). DNA was digested with XhoI and EcoRI, (GibcoBRL). 65 Separated on 0.7% agarose gel and transferred to nylon Recombinant Baculovirus: Recombinant Virus containing membrane Hybond N+ (Amersham). Hybridization and the hpa gene was constructed using the Bac to Bac System washes were performed as above. US 6,664,105 B1 33 34 cDNA Sequence analysis: Sequence determinations were Search was performed using the basic Search option of the performed with vector Specific and gene Specific primers, NCBI Server. Sequence analysis and alignments were done using an automated DNA sequencer (Applied BioSystems, using the DNA sequence analysis Software package devel model 373A). Each nucleotide was read from at least two oped by the Genetic Computer Group (GCG) at the univer independent primers. sity of Wisconsin. Alignments of two Sequences were per Genomic Sequence analysis: Large-Scale Sequencing was formed using Bestfit (gap creation penalty-12, gap performed by Commonwealth Biotechnology Incorporation. extension penalty-4). Protein homology Search was per Isolation of mouse hpa: Mouse hpa cDNA was amplified formed with the Smith-Waterman algorithm, using the Bio from either Marathon ready cDNA library of mouse embryo accelerator platform developed by Compugene. The protein or from mRNA isolated from mouse melanoma cell line database Swplus was Searched using the following param BL6, using the Marathon RACE kit from Clontech. Both eters: gapop: 10.0, gapext: 0.5, matrix: bloSum62. BlockS procedures were performed according to the manufacturer's homology was performed using the Blocks WWW server recommendation. developed at Fred Hutchinson Cancer Research Center in Primers used for PCR amplification of mouse hpa: Seattle, Wash., USA. Secondary structure prediction was Mhpl773 5'-CCACACTGAATGTAATACTGAAGTG-3', 15 performed using the PHD server-Profile network Predic SEO ID NO:32 tion Heidelberg. Fold recognition (threading) was performed using the UCLA-DOE structure prediction server. The MHp1736 5'-CGAAGCTCTGGAACTCGGCAAG-3', method used for prediction was gonnet-predss. Alignment SEO ID NO:33 of three Sequences was performed using the pileup applica MHpl83 5'-GCCAGCTGCAAAGGTGTTGGAC-3', tion (gap creation penalty-5, gap extension penalty—1). SEO ID NO:34 Promoter analysis was performed using TSSW and TSSG Mhpl152 5'-AACACCTGCCTCATCACGACTTC-3', programs (BCM Search Launcher Human Genome Center, SEO ID NO:35 Baylor College of Medicine, Houston Tex.). Mhp1114 5'-GCCAGGCTGGCGTCGATGGTGA-3', SEO ID NO:36 25 Example 1 MHpl103 5'-GTCGATGGTGATGGACAGGAAC-3', SEO ID NO:37 Cloning of Human hpa cDNA Ap1 5'-GTAATACGACTCACTATAGGGC-3', SEQ ID Purified fraction of heparanase isolated from human NO:38-(Genome walker) hepatoma cells (SK-hep-1) was Subjected to tryptic diges Ap2 5'-ACTATAGGGCACGCGTGGT-3', SEQ ID tion and microSequencing. EST (Expressed Sequence Tag) databases were Screened for homology to the back translated NO:39-(Genome walker) DNA sequences corresponding to the obtained peptides. Ap1 5'-CCATCCTAATACGACTCACTATAGGGC-3', Two EST sequences (accession Nos. N41349 and N45367) SEQ ID NO:40–(Marathon RACE) contained a DNA sequence encoding the peptide YGPD Ap2 5'-ACTCACTATAGGGCTCGAGCGGC-3', SEQ 35 VGQPR (SEQID NO:8). These two sequences were derived ID NO:41–(Marathon RACE) from clones 257548 and 260138 (I.M.A.G.E Consortium) Southern analysis of genomic DNA: Genomic DNA was prepared from 8 to 9 weeks placenta cDNA library (Soares). extracted from animal or from human blood using Blood and Both clones which were found to be identical contained an cell culture DNA maxi kit (Qiagene). DNA was digested insert of 1020 bp which included an open reading frame with EcoRI, Separated by gel electrophoresis and transferred 40 (ORF) of 973 bp followed by a 3' untranslated region of 27 to a nylon membrane Hybond N+ (Amersham). Hybridiza bp and a Poly A tail. No translation start site (AUG) was tion was performed at 68° C. in 6xSSC, 1% SDS, identified at the 5' end of these clones. 5xDenharts, 10% dextran sulfate, 100 lug/ml salmon sperm Cloning of the missing 5' end was performed by PCR DNA, and p labeled probe. A 1.6 kb fragment, containing amplification of DNA from a placenta Marathon RACE the entire hpa cDNA was used as a probe. Following 45 hybridization, the membrane was washed with 3xSSC, 0.1% cDNA composite. A 900 bp fragment (designated hp3), SDS, at 68 C. and exposed to X-ray film for 3 days. partially overlapping with the identified 3' encoding EST Membranes were then washed with 1xSSC, 0.1% SDS, at clones was obtained. 68 C. and were reexposed for 5 days. The joined cDNA fragment, 1721 bp long (SEQ ID Construction of hpa promoter-GFP expression vector: 50 NO:9), contained an open reading frame which encodes, as Lambda DNA of phage L3, was digested with SacI and shown in FIG. 1 and SEQ IDs NO:11, a polypeptide of 543 BglII, resulting in a 1712 bp fragment which contained the amino acids (SEQ ID NO:10) with a calculated molecular hpa promoter (877–2688 of SEQ ID NO:42). The pEGFP-1 weight of 61,192 daltons. The 3' end of the partial cl)NA plasmid (Clontech) was digested with BglII and SacI and inserts contained in clones 257548 and 260138 started at ligated with the 1712 bp fragment of the hpa promoter 55 nucleotide G7 of SEQ ID NO:9 and FIG. 1. Sequence. The resulting plasmid was designated phpBGL. A AS further shown in FIG. 1, there was a Single Sequence Second hpa promoter-GFP plasmid was constructed contain discrepancy between the EST clones and the PCR amplified ing a shorter fragment of the hpa promoter region: phpBGL Sequence, which led to an amino acid Substitution from was digested with HindIII, and the resulting 1095 bp frag Tyr' in the EST to Phe" in the amplified cDNA. The ment (nucleotides 1593–2688 of SEQ ID NO:42) was 60 nucleotide sequence of the PCR amplified cDNA fragment ligated with HindIII digested pEGFP-1. The resulting plas was verified from two independent amplification products. mid was designated phpBGS. The new gene was designated hpa. Computer analysis of Sequences: Homology Searches As stated above, the 3' end of the partial cDNA inserts were performed using Several computer Servers, and various contained in EST clones 257548 and 2601.38 Started at databases. Blast 2.0 service, at the NCBI server was used to 65 nucleotide 721 of hpa (SEQ ID NO:9). The ability of the hpa Screen the protein database Swplus and DNA databaseS Such cDNA to form Stable Secondary Structures, Such as Stem and as GenBank, EMBL, and the EST databases. Blast 2.0 loop Structures involving nucleotide Stretches in the vicinity US 6,664,105 B1 35 36 of position 721 was investigated using computer modeling. estimation of the molecular weight of the recombinant It was found that Stable Stem and loop Structures are likely heparanase enzyme. AS demonstrated in FIG. 4, all the to be formed involving nucleotides 698–724 (SEQ ID enzymatic activity was retained in the upper compartment NO:9). In addition, a high GC content, up to 70%, charac and there was no activity in the flow through (<50 kDa) terizes the 5' end region of the hpa gene, as compared to material. This result is consistent with the expected molecu about only 40% in the 3' region. These findings may explain lar weight of the hpa gene product. the immature termination and therefore lack of 5' ends in the In order to further characterize the hpa product the inhibi EST clones. tory effect of heparin, a potent inhibitor of heparanase To examine the ability of the hpa gene product to catalyze mediated HS degradation (40) was examined. As demonstrated in FIGS. 5a-b, conversion of the peak I degradation of heparan Sulfate in an in vitro assay the entire Substrate into peak II HS degradation fragments was com open reading frame was expressed in insect cells, using the pletely abolished in the presence of heparin. Baculovirus expression System. Extracts of cells, infected Altogether, these results indicate that the heparanase with Virus containing the hpa gene, demonstrated a high enzyme is expressed in an active form by insect cells level of heparan Sulfate degradation activity, while cells infected with Baculovirus containing the newly identified infected with a similar construct containing no hpa gene had 15 human hpa gene. no Such activity, nor did non-infected cells. These results are further demonstrated in the following Examples. Example 3 Example 2 Degradation of HSPG in Intact ECM Next, the ability of intact infected insect cells to degrade Degradation of Soluble ECM-derived HSPG HS in intact, naturally produced ECM was investigated. For this purpose, High Five or Sf21 cells were seeded on Monolayer cultures of High Five cells were infected (72 metabolically sulfate labeled ECM followed by infection (48 h, 28 C.) with recombinant Bacoluvirus containing the h, 28°C.) with either the pFhpa4 or control pF2 viruses. The pFasthpa plasmid or with control virus containing an insert pH of the medium was then adjusted to pH 6.2–6.4 and the free plasmid. The cells were harvested and lysed in hepara 25 cells further incubated with the labeled ECM for another 48 nase reaction buffer by three cycles of freezing and thawing. h at 28°C. or 24h at 37° C. Sulfate labeled material released The cell lysates were then incubated (18 h, 37° C.) with into the incubation medium was analyzed by gel filtration on sulfate labeled, ECM-derived HSPG (peak I), followed by Sepharose 6B. gel filtration analysis (Sepharose 6B) of the reaction mix As shown in FIGS. 6a-b and 7a-b, incubation of the ture. ECM with cells infected with the control pF2 virus resulted As shown in FIG. 2, the Substrate alone included almost in a constant release of labeled material that consisted entirely high molecular weight (Mr) material eluted next to almost entirely (>90%) of high Mr fragments (peak I) eluted V (peak I, fractions 5-20, Kav-0.35). A similar elution with or next to V. It was previously shown that a proteolytic pattern was obtained when the HSPG substrate was incu activity residing in the ECM itself and/or expressed by cells 35 is responsible for release of the high Mr material (6). This bated with lysates of cells that were infected with control nearly intact HSPG provides a soluble Substrate for Subse virus. In contrast, incubation of the HSPG Substrate with quent degradation by heparanase, as also indicated by the lysates of cells infected with the hpa containing virus relatively large amount of peak I material accumulating resulted in a complete conversion of the high Mr Substrate when the heparanase enzyme is inhibited by heparin (6, 7, into low Mr labeled degradation fragments (peak II, frac 12, FIG. 9). On the other hand, incubation of the labeled tions 22–35, 0.5Expression 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-Xylanase 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-amylase from Barley, 1ecea The following mouse EST's were identified AA177901, endocellulase from Acidothermus Cellulolyticus and 1 qbc AA674378, AA67997, AAO47943, AA690179, A122034, hexosaminidase alpha chain, glycosyl hydrolase. 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, lactase 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 cellulase 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 cellulases 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. 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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.