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(12) United States Patent (10) Patent No.: US 6,664,105 B1 Pecker Et Al

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(12) United States Patent (10) Patent No.: US 6,664,105 B1 Pecker Et Al 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 FIG 2 peak peak || Fraction U.S. Patent Dec. 16, 2003 Sheet 3 of 34 US 6,664,105 B1 11 OO S O d t- 9 (S E CQB 550 O) O 9. SV S CO O Fraction FIG. 3B E O. C st- 9 (US E O CB O) -O cu D a-1 St. S O?) O 1 O 2O 3 O 40 5 O Fraction U.S. Patent Dec. 16, 2003 Sheet 4 of 34 US 6,664,105 B1 FTG 4 1100 S50 O 1 O 2 O 3 O 4 O 5 O Fraction U.S. Patent Dec. 16, 2003 Sheet 5 of 34 US 6,664,105 B1 FIG. 5A 1 OO E Ol d s CS E QO 550 CD O c 9 Su S OO O Fraction FIG. 5B 100 E Cl () d (US E 550 ob O c 92 St. S CO Fraction U.S. Patent Dec. 16, 2003 Sheet 6 of 34 US 6,664,105 B1 FIG. 6A peak II 600 E Ol O st- 1200 9 (VS E O C2 800 d O c 92 s 400 S (?) O O 1 O 20 30 4 O 50 Fraction FIG. 6B 400 E Cl C s 5 c E 700 c O c SD S CO O O 1 O 2 O 30 40 5 O Fraction U.S. Patent Dec. 16, 2003 Sheet 7 of 34 US 6,664,105 B1 FIG 7A 1 400 700 O O 2 O 30 40 5 O Fraction FIG 7B 8OO 400 Fraction U.S. Patent Dec. 16, 2003 Sheet 8 of 34 US 6,664,105 B1 FIG. 8A 1200 peak II E C O t e E C O) O 9 s S Fraction FIG. 8B peak II E O. O g s E O O c 92 St. S O 1 O 2O 3 O 4. O 5 O Fraction U.S. Patent Dec. 16, 2003 Sheet 9 of 34 US 6,664,105 B1 FIG. 9A 700 O i---------------------vivi s w :-- y w O 5 O 5 20 2 3 O 35 4 O 45 Fraction FIG 9B o O 2O 3 O 4 O 5 O Fraction U.S. Patent US 6,664,105 B1 ---wr------a ---------- war-------- ---------&-was-------- *~*~~~~--~~~~¤·············---···········---···---······· 8 : F:30 t33 : w 33 8:338 it 3-8 ; ; U.S. Patent Dec. 16, 2003 Sheet 11 of 34 US 6,664,105 B1 8 & s & 3:... k33. 8 ::: 8.E. E. --M. Ski:::::::$3:... :38: s :... :: 3 : s praisier : 3 4 5 & 7 8 & 3 is 13 * * U.S. Patent Dec. 16, 2003 Sheet 12 of 34 US 6,664,105 B1 : 3: . 88. U.S. Patent Dec. 16, 2003 Sheet 13 of 34 US 6,664,105 B1 Fig. 13 Inouse CTGGCAAGAAGGTCTGGTTGGGAGAGACGAGCTCAGCTTACGGTGGCGGT 50 | | | | | | | | human CTGGCAAGAAGGTCTGGTTAGGAGAAACAAGCTCTGCATATGGAGGCGGA lll:5 Inouse GCACCCTTGCGTCCAACACCTTTGCAGCTGGCTTTATGTGGCTGGATAA OO | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | human GCGCCCTTGCTATCCGACACCTTTGCAGCTGGCTTTATGTGGCTGGATAA 1165 CS ATTGGGCCTGTCAGCCCAGATGGGCATAGAAGTCGTGATGAGGCAGGTGT 50 | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | human ATTGGGCCTGTCAGCCCGAATGGGAATAGAAGTGGTGATGAGGCAAGTAT 125 no use TCTCGGAGCAGGCA ACACCACTAGTGGATGAAAACTTTGAGCCTTA. 2 OO | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | human TCTTTGGAGCAGGAAACTACCATTTAGTGGATGAAAACTTCGATCCTTTA. 265 Iose CCTGATTACTGGCTCTCTCTTCGTCAAGAAACTGGTAGGTCCCAGGGT 25 O | | | | | | | | | | | | | | | | | | | | | | | | human CCTGATTATTGGCTATCTCTTCTGTTCAAGAAATTGGTGGGCACCAAGGT 315 Icouse GTTACTGTCAAGAGTGAAAGGCCCAGACAGGAGCAAACTCCGAGTGTATC 300 1 human GTTAATGGCAAGCGTGCAAGGTTCAAAGAGAAGGAAGCTTCGAGTATACC, 365 IIouse TCCACTGCACTAACGTCTATCACCCACGATATCAGGAAGGAGATCTAACT 350 | | | | | | | | | | | | | | | | | | | | hunan TTCATTGCACAAACACTGACAATCCAAGGTATAAAGAAGGAGATTTAACT 4.5 Inouse CTGTATGTCCTGAACCTCCATAATGT CACCAAGCACTTGAAGGTACCGCC 400 | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | human CTGTATGCCATAAACCTCCATAACGTCACCAAGTACTTGCGGTTACCCTA 1465 mouse TCCGTTGTTCAGGAAACCAGTGGATACGTACCTTCTGAAGCCTTCGGGGC 450 | | | | | | | | | | | | | | | | | | | | | | human TCCTTTTTCTAACAAGCAAGTGGATAAATACCTTCTAAGACCTTTGGGAC 1515 Inouse CGGATGGATTACTTTCCAAATCTGTCCAACTGAACGGTCAAATTCTGAAG 500 | | | | | | | | | | | | | | | | | | | | | | | | | | human CTCATGGATTACTTTCCAAATCTGTCCAACT CAATGGTCTAACTCTAAAG 1565 Thouse ATGGTGGATGAGCAGACCCTGCCAGCTTTGACAGAAAAACCTCTCCCCGC 550 | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | human ATGGTGGATGATCAAACCTTGCCACCTTTAATGGAAAAACCTCTCCGGCC le5 OS e AGGAAGTGCACTAAGCCTGCCTGCCTTTTCCTATGGTTTTTTTGTCATA 600 | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | human AGGAAGTTCACTGGGCTTGCCAGCTTTCTCATATAGTTTTTTTGTGATAA 1665 Inouse GAAATGCCAAAATCGCTGCTTGTATATGAAAATAAAA 637 | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | human GAAATGCCAAAGTTGCTGCTTGCATCTGAAAATAAAA l702 U.S. Patent Dec. 16, 2003 Sheet 14 of 34 US 6,664,105 B1 E. : 3 S S. 3 4 5 - T S & S S S U.S. Patent Dec. 16, 2003 Sheet 15 of 34 US 6,664,105 B1 I 2 3 4 IS-7 8 9 10 11 Ef E2 E3 E4 E5-8 E9 E10 E 11 E U D kb Lambda 8 Plasmid nine Lambda 3 Fig. 15 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 Fig. 16 p U.S. Patent Dec. 16, 2003 Sheet 33 of 34 US 6,664,105 B1 :: 8: 383 8 x 8: 8 8s 83 is : ---------------------------------------wx------- 8. W axis----------------------------------------------------....'...----- Fig. 18 U.S. Patent Dec. 16, 2003 Sheet 34 of 34 US 6,664,105 B1 MIRSKIA, I.M.I.L.E.GPLGPS PAI.PRPAAQOWW) LDFFTQEPLHLWSPSFLSWT , ) PHD EEEEE HE EEEE EEE IDANLATDPRFLILEGSPKLRTLARGLSPAYLRFGGTKTDFLl FDPKKESTFEERSYWOS 120 PEIL) EEE EEEEE HHHHHH HHIE EEEEE HHHHHH QVNQDICKYGS PPDWEEKLRLEWPYOEQI.L.I.REHYQKKFKNSTYSRSSVOVLYTFANCS BO PID HHHHHHHH HHHHHH HHHHHHHHHHHEEH EEEEEEEEEEEE GLDE, FG.NA, RADI.QWNS SNAOLLLDycsSKGYNISWELGNPNSFLKKADI FINGS 240 PHD HHHHHHHHHHHHHHHHHHHHHHHHHHHHHH EEEEE HHHHHHH EEEE QLGEDYIQLHKLLRKSTFKNAKLYGPDWGQPRRKTAKMLKSFLKAGGEVIDSWIWHHYYL 300 PHD HHHHHHHHHHHHHHHHH HHHHHHHHHHHHH EEEEEEEEEEE |NGRTATREDFINPDV LiDJ FISSVQKVFQVVESTRPGKKWWLGETSSAYGGGAPLLSDTFA 360 (i) ; HEEEEEEEEEEEE EEEEEE HHHHHHH AGFMWLDKLGLSARMGIEVVMROVFFGAGNYHVIDENFDPLPDYWLSLLFKKLVGTKVLM 420 PHI) HHHHHHHH HHHH HHHHHHHHH EEEEE HHHHHHHHHHHH EEEEE ASVQGSKRRKLRVYLHCTNTDNPRY KEGDI.T.I.YAINLHNW'KYIRI.JPYPFSNKQVDKYI, 480 PHD EEE E. EEEEEEEE EEEEEE, EEEEE Hili HHHHH RPLGPHGLLSKSWQLNGLTLKMVDDQI'll PLMEKPLRPGSSLGLPAFSYSFFV RNAKVA 540 PHE) HH EEEEEEE EEEEF, EEEEEEEE EE ACT 43 PHD Fig.
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