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(12) Patent Application Publication (10) Pub. No.: US 2004/0138156A1 Schneider Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2004/0138156A1 Schneider Et Al US 20040138156A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2004/0138156A1 Schneider et al. (43) Pub. Date: Jul. 15, 2004 (54) THERAPEUTIC REGULATION OF Publication Classification DEOXYRIBONUCLEASE-1-LIKE-3 ACTIVITY (51) Int. Cl." ......................... A61K 48/00; A61K 9/127; C12N 15/88; A61K 38/21 (52) U.S. Cl. ............................ 514/44; 435/458; 424/450; (76) Inventors: Michael C. Schneider, Springfield, IL 424/85.6 (US); Andrew Wilber, Springfield, IL (57) ABSTRACT (US) Deoxyribonuclase 1-like 3 (D3) hydrolyzes lipid-complexed Correspondence Address: DNA and decreases transfection efficiency in liposomal FITCH EVEN TABN AND FLANNERY transfection (lipofection) Systems. Accordingly, D1 L3 pro 120 SOUTH LASALLE STREET vides a more accurate test of the efficiency of lipid/liposomal SUTE 1600 based gene therapy than current Standards using deoxyribo CHICAGO, IL 60603-3406 (US) nuclease 1 (D1). Moreover, it has been found that mice with Systemic lupus erythematosus (lupus) have lowered D1 L3 (21) Appl. No.: 10/378,098 activity. Therefore, differing therapeutic benefits may result from either the upward or downward therapeutic regulation (22) Filed: Feb. 26, 2003 of D1 L3 activity. For example, blocking D1 L3 activity enhances liposomal transfection for gene therapy, while Related U.S. Application Data increasing D1 L3 activity may enhance destruction of patho genic DNA, whether viral, bacterial or endogenous. (60) Provisional application No. 60/359,619, filed on Feb. Destruction of pathogenic DNA may provide treatment for 26, 2002. lupus, or viral and oncogenic diseases. Patent Application Publication Jul. 15, 2004 Sheet 1 of 23 US 2004/0138156A1 DNASE1L3 MSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKQEDKNAMDVIVKWIKRCDIILVM DNAS1L2 MGGPRALLAALWALEAA. GTAALRIGAFNIQSFGDSKVSDPACGSIIAKIPAGYDLALVQ DNAS1L1 MHYPTALLFLILANGAOAFRICAFNAORLTLAKVAREQVMDTLVRILARCDIMVLO HDNASE1 MRGMKLLGALLALAALLQGAVSLKIAAFNIQTFGETKMSNATLVSYIVOILSRYDIALVQ EIKDSNNRICPILMEKLNRNSRRGITYNYWISSRLGRNTYKEOYAFLYKEKLWSWKRSYH EVRDPDLSAVSALMEQINSWSEHE.. YSFVSSQPLGRDQYKEMYLFWYRKDAVSVVDTYL EVVDSSGSAIPLLLREINRFDDSGP. YSTLSSPOLGRSTYMETYWYFYRSHKTOVLSSYW EVRDSHLTAVGKLLDNLNQ. DAPD. TYHYVVSEPLGRNSYKERYLFWYRPDQVSAVDSYY YNDE. DDWFAREPFWAOFSLPSNWLP. SLWLWPLHTT YHDYQDG.DADVFSREPFVVWFQSPHTAVK. DFVIIPLHTT YPDP, , , , , EDWFSREPFVVKFSAPGTGERAPPLPSRRALTPPPLPAAAQNLVLIPLHAA YDDGCEPCGNDTFNREPAIWRFFSRFTEVR . EFAIWPLHAA PETSWKEIDELVEWYTDWKHRWKAENFIFMGDFNAGCSYWPKKAWKNIRLRTDPRFWWLI PHQAVAEIDALYDVYLDWIDKWGTDDMLFLGDFNADCSYVRAQDWAAIRLRSSEVFKWLI PKAVEKELNALYDVFLEVSQHWQSKDWILLGDFNADCASLTKKRLDKLELRTEPSFHWVI PGDAVAEIDALYDVYLDVOEKWGLEDWMLMGDFNAGCSYVRPSOWSSIRLWTSPTFOWLI GDQEDTTWKKSTNCAYDRIVLRGQEIVSSVWPKSNSWFDFOKAYKL. TEEEALDVSDHFP PDSADTTW.GNSDCAYDRIVACGARLRRSLKPOSATVHDFQEEFGL. DOTOALAISDHFP ADGEDTTVRASTHCTYDRVVLHGERCR. SLL.HTAAA.FDFPTSFQLLTEEEALNISDHYP PDSADTTAT. PTHCAYDRIVVAGMLLRGAVWPDSALPFNFQAAYGLSDQL.AQAISDHYP WEFKLQSSRAFTNSKKSVTLRKKTKSKRSk ( SEQ ID NO : 1) WEWTLKFHRk (SEO ID NO: 2) WEVELKLSQAHSVQPLSLTVLLLLSLLSPQLCPAA* (SEQ ID NO : 3) WEWMLK ( SEQ ID NO : 4) LOCUSORGANSO OF EXPRESSION UNIOUE STRUCTURAL FEATURES DNASE1 16p13|PANCREAS, KIDNEY, GUT IMAJOR SERUMDNASE, ACTIN INHIBITED DNAS1L1 Xq28 SKELETAL&S CARDIAC MUSCLEHYDROPHOBICC-TERMINUS MEMBRANE BOUND D W DNASE133 SPLEEN THYMUS. 8 LIVER ASIC C-TERMINUS DNAS1L4 190 Patent Application Publication Jul. 15, 2004 Sheet 2 of 23 US 2004/0138156A1 Fig. 2 NORTHERN ANALYSIS OF DNASEIL3 EXPRESSION ADULT Fig. 3 MusD3 SDHFPVEFKLOSSRAFTNNRKSVSLKKRKKGNRS (SEQIDN0:5) RatD3 SDHFPVEFKLQSSRAFTNSRKSVSLKKKKKGSRS (SEQID NO:6) HumD3 SDHFPVEFKLQSSRAFTNSKKSVTLRKKTKSKRS (SEQID NO:7) XenD3 SDHFPIEVRLKESKRPT SRR RKYFKRK (SEQID NO:8) BOVD1 SDHYPVEVTL (SEQID NO:9) hDL1 SDHYPVEVELKLSOAHSVOPLSLTVLLLLSLLSPOLCPAA (SEQDN0:10) BTASSAY Patent Application Publication Jul. 15, 2004 Sheet 3 of 23 US 2004/0138156A1 Fig. 5 A) DNASE RADIAL DIFFUSIONASSAYS CONTROL D1 B ) NUCLEASE ZYMOGRAMOG ANTI-D3C) MOUSE WESTERN SERUM MOUSE SERUM D3 24 HRS 48 HRS 14 CONTROL D1 D3 D3ACTD3-196K Fig. 6B app. Patent Application Publication Jul. 15, 2004 Sheet 4 of 23 US 2004/0138156A1 Fig. 7A Fig. 7B D1-CM D3-CM .4 2 O LaR-FA O11 .8.0.2 5 3 2 WA O .6 O 0. 4 5 3 W I 0. 2 3 || 311 O 3 3 W W DNASE1 DNASE1L3Hill Fig. 9 Patent Application Publication Jul. 15, 2004 Sheet 5 of 23 US 2004/0138156A1 Fig. 10 Fig. 11A CTRL D1 D3 FUGENE-6 GENESHUTTLE-20 GENESHUTTLE-4 LT LT2 Fig. 11B Readent Source Composition BT-present Fugene-6 ROChe Blend of Lipid & Components Yes Effectene Qiagen Non-Liposomal Lipid Yes Superfect Qiagen Activated Dendrimer NO LT-1, LT-2 Mirus Cationic Lipopolyamines Yes Transfectam Promega Dioctadecylamidoglycyl Yes Spermine (DOGS) Gene-Limo Super Q-Biogene Poly-cationic Lipid & Partial Neutral Component Gene Shuttle CPG-Biotech Poly-cationic Lipids Partial 20& 40 Patent Application Publication Jul. 15, 2004 Sheet 6 of 23 US 2004/0138156A1 Fig. 12A M S L. 121 TCAGTGAGCCAGGCACTGTCTTCATCCAGCCTGAAGTCCCAGGAGTGCAAAGATGTCCCTG H P A S P R L. A. S. L. L. L. F I L. A. L H D T 180 CACCCAGCTTCCCCACGCCTGGCCTCCCTGCTGCTCTTCATCCTTGCCCTCCATGACACC L. A L R L C S F N W R S F. G A S K K E N 240 CTGGCCCTAAGGCTCTGCTCCTTCAATGTGAGGTCCTTTGGAGCGAGCAAGAAGGAAAAC H E A M D I I W K I I K R C D L I L. L. M 300 CATGAAGCCATGGATATCATTGTGAAGATCATCAAACGCTGTGACCTTATACTGTTGATG E I K D S S N N I C P M L M E K L N G N 360 GAAATCAAGGACAGCAGCAACAACATCTGTCCCATGCTGATGGAGAAGCTGAATGGAAAT S R R S T T Y N Y V I S S R L G R N T Y 420 TCACGAAGAAGCACAACATACAACTATGTGATTAGTTCTCGACTTGGAAGAAACACGTAC K E Q Y A F W Y K E K L V S V K T K Y H 480 AAAGAGCAGTATGCCTTCGTCTACAAGGAGAAGCTGGTGTCTGTGAAGACAAAATACCAC Y H D Y Q D G D T D V F S R E P F W W W 540 TACCATGACTATCAGGATGGAGACACAGACGTGTTTTCCAGGGAGCCCTTTGTGGTTTGG F H S P F T A V K D F W I W P L H T T P 600 TTCCATTCCCCCTTTACTGCTGTCAAGGACTTCGTGATTGTCCCCTTGCACACAACTCCC E T S W K E I D E L W D W Y T D W R S Q 660 GAGACCTCCGTTAAAGAGATAGATGAGCTGGTCGATGTCTACACGGATGTGAGAAGCCAG W. K. T E N F I F M G D F N A G C S Y V P 720 TGGAAGACAGAGAATTTCATCTTCATGGGTGATTTCAACGCCGGCTGTAGCTATGTCCCC (SEQ ID NO: 13) GGTGATTTCAAgGCCGGCTGTAGCTA (SEQ ID NO: 14) G D F K A K. K. A. W. O N I R L R T D P K F W W L I G AAGAAGGCCTGGCAGAACATTCGTTTGAGGACGGACCCCAAGTTTGTTTGGCTGATTGGG D Q E D T T V K K S T S C A Y D R I V L 840 GACCAAGAGGACACTACGGTCAAGAAGAGTACCAGCTGTGCCTATGACAGGATTGTGCTT C G O E I V N S V V P R S S G V F D F Q 900 TGTGGACAAGAGATAGTCAACTCCGTGGTTCCCCGTTCCAGTGGCGTCTTTGACTTTCAG K. A Y D L S E E E A L D W S D H F P W E 960 AAAGCTTATGACTTGTCTGAGGAGGAGGCCCTGGATGTCAGTGATCACTTTCCAGTTGAG F K L Q S S R A F T N N R K S W S L K K 1021 TTTAAGCTACAGTCTTCAAGGGCCTTCACCAACAACAGAAAATCTGTTTCTCTCAAAAAG R. K. K. G. N. R. S (SEQ ID NO : 12) 108 O AGAAAAAAAGGCAATCGCTCCTAGGTATCACGCTCCG (SEO ID NO : 11) Patent Application Publication Jul. 15, 2004 Sheet 7 of 23 US 2004/0138156A1 ANTI-hDNASL3 IMMUNOBLOT SER Fis 12B a sea Fig. 13 F K L Q S S R A F T (SEQ ID NO: 17) 1021 GTTTAAGCTACAGTCTTCAAGGGCCTTCACC (SEQ ID NO: 16) AGCTACAGTCT"Ta AAGGGCCTTC (SEQ ID NO: 15) Patent Application Publication Jul. 15, 2004 Sheet 9 of 23 US 2004/0138156A1 Fig. 14C Fusion cDNA clone D1+D3CT (SEQ ID NO: 2O) 1 CtgCtgCagCCgtCtCagattggCtttCaggatgCggtaCaCagggCtaatgggaaCact 61 ctCaCCttggtCaaCCtgCtgCagctggCtgggaCtctgagaattgCagCCttCaaCatt 121 CGgaCttttggggagaCtaagatgtCCaatgctaCCCtCtctgtataCtttgttgaaaatC 181 CtgagtCGCtatgaCatCCCtgttatCCaagaggtCagagaCtCCCaCCtggttgctgtt 241 gaagCtCCtggatgaactCaatCgggaCaaaCCtgaCaCCtaCCgctatgtagtCagt 301 gagCCGCtgggCCgCaaaagctaCaaggaaCagtaCCtttttgtgtaCaggCCtgaCCag 361 gtgtCtattotggaCagctatoaatatgatgatggCtgtgaaCCCtgtggaaatgaCaCC 421 ttCagCagagagCCagCCattgttaagttcttttCCCCataCactgaggtCCaagaattt 481 gCatCgtgCCCttgCatgCagCCCCaaCagaagctgtgagtgagatCgaCgCCCtCtaC 541 gatgtttaCCtagatgtCtggCaaaagtggggCCtggaggaCatCatgttCatgggagaC 601 ttcaatgctogctgCagctacgtCaCttCCtCCCagtggtCCtCCattCgCCttCggaCa 661 agcCCCatCttCCagtggCt9atcCCtgaCagtgcggaCaCCaCagtCaCatCaaCaCaC 721 ttgCttatgaCaggattgtggttgctggagCtctgctCCaggCtgctgttgttCCCaaC 781 toggCtgttCCttttgatttCCaagCagaatacggaCtttCCaaCCagCtggCtgaagcC 841 atCagtgaCCattaCCCagtggaggtgaCactCagaaa/gatCCGGGCCTTCACCAACAA 901 CAGAAAATCTGTTTCTCTCAAAAAGAGAAAAAAAGGCAATCGCTCCTAGGTATCACGCTC 961 CGGAATTC Fig. 15A NUCLEASERDAS OF CONDITIONEDMEDIA CONTROL D1 D3 D1+D3CT WESTERNS OF GFP-TRANSFECTED CELLS CONTROL D1 D3 D1+D3CT Patent Application Publication Jul. 15, 2004 Sheet 10 of 23 US 2004/0138156A1 Fig. 15B Murine N40 Site = Nishikawa's ASN18 (SEQ ID NO. 23) LRIAAFNIRTFGETKMSNATLSVYFWKILSRYDIAVIQEVRDSHLVAVGKLLDELNRDK (SEQ ID NO. 24) likiaafnirtfgetkmsnatlasyivrivrydivligevrdshlvavgklldyingdd Murine N128 site = Nishikawa's ASN 106 (SEQ ID NO: 25) YVVSEPLGRKSYKEOYLFWYRPDOVSILDSYOYDDGCEPCGNDTFSREPAI (SEQID NO: 26) yVVSeplgnsykerylflfrpnkVSvldtycyddgCeSCgndsfsrepav Fig. 15C (SEQID NO:27). MUSF-D1-N4OS 5 ACTAAGATGTCCAGTGCTACCCTC (SEQID NO:28) MUSR-D1-N4OS 5 GAGGGTAGCACTGGACATCTTAGT (SEQID NO:29). MUSF-D1-N128S 5' GAACCCTGTGGAAGTGACACCTTC (SEQID NO:30) MUSR-D1-N128S 5 GAAGGTGTCACTTCCACAGGGTTC 2 ESNASE1 3 D1-N4OS 4 D-N128S 5 6 7 SNS #1 8 9 Patent Application Publication Jul. 15, 2004 Sheet 11 of 23 US 2004/0138156A1 D1 D3 D3ACT N196K NICKED - LINEA 3-2 SUPER COILED r (SEQID NO:31) CACAATATACAACTATG Fig. 17A MRL 89 SEGUENCE TRANSLATION CT SEOD NO: 34 R R S T Y N CGAAGA AGCACA ATA TAC AAC SEOD NO: 33 H. H. H. - m Fig. 17C ALIGNMENT OF DNASE1L3S 8 HUMAN DNASE1 MOUSE (SEQID NO:35) GNSRRRSTTYNYVISSRLGR 89ALLELE (SEQID NO:36) GNSRRRSTIYNYVISSRLGR RAT SEOD NO: 37 GNSRRRSTTYNYVISSRLGR HUMAN SEOD NO: 38 RNSRRRGITYNYVSSRIGR hDNASE1 (SEQID NO:39 DAPD. TYHYVVSE ACTIN-BINDING DNA-BINDING Patent Application Publication Jul. 15, 2004 Sheet
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