(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

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

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

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 12 of 23 US 2004/0138156A1

Fig. 18AaatggaaatcacgaagaagcacAACITATtCaactatgtgattagttctg F - V R46-SSP NORMAL89T = SSP () MUTANT891 = SSP (+) C57BL MRL-Mp NZBWF1 DBA2 ZW DIGEST BXSB NZBIBin N wila - + SSP

89T 89 89T 89

ANT-DNASE 13 WESTERN

Fig. DNA-SDS-PAGE Fig. 19B ZYMOGRAM

89T 89 89T 89 ANTI-DNASE 1 L3

Fig. 19C As

DNA-SDS-PAGE Fig. 19Ds ZYMOGRAM Patent Application Publication Jul. 15, 2004 Sheet 13 of 23 US 2004/0138156A1 Fig. 20

1211 +/- 84 690 +/- 10

34 28 19 IMMUNE PREMMUNE IMMUNE ANT-MUS + 50 ug/ml PEPTIDE

Patent Application Publication Jul. 15, 2004 Sheet 14 of 23 US 2004/0138156A1

fn: NONE fin- Cy fn-y

B6 Mr. B6 Mr. B6 Mr.

GFP EXPRESSION LIPOFECTED HeLa

HeLa-CM MACROPHAGE-CM Patent Application Publication Jul. 15, 2004 Sheet 15 of 23 US 2004/0138156A1 Fig. 23 MICE SERUM D3 Levels RDA C57BL 125-11 246 20 2406.65 130+11 3418 118 NZBW 1118t 219 + 12 3385. 143 MRL 142+12 266 16 3052, 130 SPLENOCYTES MACROPHAGES Cellular RDA Cellular RDA D3 Levels Conditioned Media D3 Levels Conditioned Media C57BL 83.22. 346+34 303: 31433S BXSB 422-53 290+25 351 + 25 NZBW463 + 128 326+23 256 32 MRL 50364 286-23 251 - 20 * p < 0.01 for serum RDA activity of BXSB compared to all other mouse strains using Wilcoxon rank sum test. fp = 0.04-0.05 for serum D1 L3 levels in NZB/W F1 mice vs MRL by student's t-test. it p < 0.01 for both macrophage and splenocyte D1 L3 levels of C57BL compared to all lupus mice using Wilcoxon rank sum test. S p < 0.01 for C57BL macrophage RDA activity vs NZ and MRL (but not BXSB) mice. In addition, p < 0.01 was found for the comparison between BXSB and both NZ and MRL. Both Calculations used WilcoxOn rank Sumtest.

Fig. 24A

Fig. 24B Patent Application Publication Jul. 15, 2004 Sheet 16 of 23 US 2004/0138156A1

C57BL NZB/W BXSB MRL Fl8 2 5 ANTI-D1 L3 SS7 Qiy SE

e ANTI-ACTIN 29 kDa

DNASE-RDA

Fl8 2 5 B NUCLEASEZYMOGRAM MOUSE SERUM

(kDa) D1 D3 24hrs 48 hrs 52 - Patent Application Publication Jul. 15, 2004 Sheet 17 of 23 US 2004/0138156A1 Fig. 26

SPLENOCYTES

A. DNASE1L3 WESTERN 29kDa. B. ACTIN WESTERN

C. ZYMOGRAM

Fig. 27 MACROPHAGES

A. DNASE1L3 WESTERN 29kDa

B. ACTIN

WESTERN 46kDa

C. ZYMOGRAM Patent Application Publication Jul. 15, 2004 Sheet 18 of 23 US 2004/0138156A1 Fig. 28 SERUM URNE DNAS13 DNASE-RDA DNASE-RDA 16 MMUNOBLOTS6OO ACTIVITY ACTIVITY 500 4OO 3OO

SPLENOCYTES MACROPHAGES DNAS13 DNASE-RDA IoStars DNASE-RDA g|MNOlig ACTIVITY 12O 400 ACTIVITY 300 1 OO

Fig. 29A

GFP EXPRESSION LIPOFECTED HeLa HeLa-CM MACROPHAGE-CM

Patent Application Publication Jul. 15, 2004 Sheet 19 of 23 US 2004/0138156A1

N CONFESNED & MACROPHAGES

fn Y (U/ml) O 100 500 1000

RDA-DNASE OF CONDITIONED MEDIA

GFP EXPRESSION

IN LIPOFECTED HeLat

RDA-DNASE ZYMOGRAM 5u CONDITIONED 20 CONDITIONED MEDIA MEDIA

HeLa : :. . . .SS .

Fig. 29D ANTI-GFP IMMUNOBLOTSLIPOFECTED HeLa HELA BL1 NZ1 BL2 NZ2 BL NZ1 BL2 NZ2

GFP

PRIMARY ANTI-GFP 1:7000, SECONDARY ANTI-MUS 1:7000 Patent Application Publication Jul. 15, 2004 Sheet 20 of 23 US 2004/0138156A1 Fig. 30

D13 CONDITIONEDMEDIA DILUTIONS 89 89T

100 101 10 -3 100 10

10 -3

Patent Application Publication Jul. 15, 2004 Sheet 21 of 23 US 2004/0138156A1 Fig. 31B

IMMUNOBLOT OF D13 NHUMAN SERUM

Fig. 32

A. Ctrl 89T 89l B. DLUTIONS ANTID3 CC 1:10 1:50 ACTIN 89T 89 89T 89 GFP is ACTINEE EE

ACTIN E E E E

R. East a Patent Application Publication Jul. 15, 2004 Sheet 22 of 23 US 2004/0138156A1

Fig. 33 B) CLONED SEQUENCES DERVED FROM HETERO A) HETEROZYGOUS ZYGOUSPATIENT GENOMIC MUTANT 852G SECUENCE V

(SEQID NO:44) AAGCTTGCAAGCTG (SEQID NOS. 42AND43) AAGCTTAGCAAGCTG NORMA 852A

(SEQID NO: 45) AAGCTTACAAGCTG Fig. 34 DNASE13-HA C-HA 26Y 261C A) ANTIHA

B)ZYMOGRAM c) FD-NUCLEASE (RDA) E. D) ANTIGFP (BTASSAY) E) ACTIN

MOUSE (SEQID NO:46) RAT (SEQ ID NO:47) XENOPUS (SEQ ID NO: 48) HUMAN (SEQ ID NO:49) HUMAN (SEQID NO: 50) HUMAN (SEQID NO:51)

US 2004/0138156A1 Jul. 15, 2004

THERAPEUTIC REGULATION OF directed towards improving the ability to transduce eXog DEOXYRIBONUCLEASE-1-LIKE-3 ACTIVITY enous genetic material into cells or tissues for treatment of diseases. Once a therapeutically effective gene is engineered RELATED APPLICATION into an expression vector, its efficacy still depends on the 0001 Pursuant to 35 U.S.C. S 119(e), this application is ability to productively incorporate the engineered gene into based on, and claims the benefit of, U.S. Provisional Appli the patient's cells in the tissues. Viral transduction and cation Serial No. 60/359,619, filed on Feb. 26, 2002, which lipofection are the two most commonly used methods of in is hereby incorporated by reference in its entirety. vivo gene therapy in trials; other methods include electropo FIELD OF THE INVENTION ration, non-lipid based transduction, and particle transfer 0002) Deoxyribonuclease 1-like 3 (D1L3 or D3) hydro (e.g., a gene gun). At present, of over 600 human gene lyzes DNA. The enzyme, when added to the media bathing therapy trials (http://www.wiley.co.uk/genetherapy/clinical/ cultured cells, blocks or decreases liposomal gene transfec ), about 70% of the trials utilized viral delivery systems and tion (lipofection) efficiency; in other words it confers a 13% use liposomal gene techniques. barrier to liposomal gene transfection (henceforth this is also 0007 Lipofection was first coined for the use of synthetic called BT, or the function will referred to as BT activity or cationic lipids, which form unilamellar liposomes in trans BT effect). This enzyme’s BT activity has varied but sig fection (Feigner et al., (1987) Proc. Natl. Acad. Sci. USA 84, nificant potency against a variety of cationic lipid transfec 7413-7). Liposome self-assembly appears to occur via elec tion reagents. In addition, the Scope of this effect may not trostatic interaction between the lipids and anionic DNA. only apply to lipofection reagents since D1 L3 is also shown Newer liposome preparations may include monocationic to be active in vitro in blocking adenoviral infection (FIG. lipids, polycationic lipids, or neutral lipids, including cho 30). lesterol, gangliosidosis, PEG-polymerized lipids, and fuSO 0003) Liposomal gene therapy in vitro is highly efficient, genic compounds to enhance Stability or uptake by cells but there are significant barriers to in Vivo efficiency. These (Gao et al., (1995) Gene. Ther. 2, 710-22). For example, experiments predict that macrophage-Secreted D1 L3 activity FuGENETM-6, a highly efficient, proprietary, non-liposomal is likely one of the main tissue and Serum barriers for lipid preparation, is available from Roche Diagnostics Corp. liposomal transfection in vivo. This would predict that (Indianapolis, Ind.). blocking or decreasing Such activity, directly or indirectly, 0008 Liposomal transfection is a complex process. Typi would enhance liposomal transfection as used in in Vivo cally lipid-linked polycationic moieties condense around a gene therapy, while increasing D1 L3 activity may enhance negatively charged DNA core and form lamellar-Surrounded destruction of pathogenic DNA, whether viral, bacterial or vesicular structures. These vesicles appear to enter cells endogenous, thereby providing treatment for lupus, or viral easily and abundantly via endocytosis (Gao et al., (1995) and oncogenic diseases. Gene. Ther. 2, 710-22; Godbey et al., (1999) Proc. Natl. 0004) Serum contains at least two deoxyribonucleases Acad. Sci. USA 96, 5177-81). The rate-limiting step for (DNASES): DNASE1L3 and DNASE I. The latter very nuclear localization in vitro appears to be the exodus of the likely contributes towards most of the serum DNASE activ DNA from the endosomes and into the cytoplasm (Xu et al., ity against free DNA. The former enzyme can be detected in (1996) Biochemistry 35, 5616-23; Lechardeur et al., (1999) serum by immunoblot, and likely forms part of the actin Gene. Ther: 6, 482-97; Zabner et al., (1995) J. Biol. Chem. resistant DNASE component. The presence of this enzyme 270, 18997-9007). in serum suggests that it could play a role in the in Vivo 0009. The efficiency of lipofection in vivo is lower than barriers to liposomal gene transfection in many tissues. in vivo viral transduction (infection) with retrovirus, aden 0005 Systemic lupus erythematosus (SLE or lupus) is an ovirus, or adeno-associated virus (Crystal, (1995) Science autoimmune disorder characterized by the presence of 270, 404-10). This is not surprising, since viruses are autoantibodies against nuclear components, Such as DNA selected by evolution for Successful replication. Thus, Viral and chromatin. Macrophages from a model of murine SLE, transduction, which is highly efficient, has become the pre-symptomatic NZB/W F1, cannot provide HeLa cells method of choice for delivering genetic material into an with a barrier to liposomal transfection, while BT activity is organism. However, viral transduction is prone, not unex present in media conditioned by non-lupus prone C57BL pectedly, to induce greater inflammation than non-viral gene mice. In addition, evaluation of DNASE1L3 sequences in transduction. Accordingly, a need exists to improve the African American patients with SLE identifies a patient efficiency of lipofection as a viable alternative to viral heterozygous for a complete loss of function mutation in this transduction. In some ways the goal of gene therapy vector gene; and many patients with an intronic mutation (IVS6+ development is to design carrier agents that possess the 5G>T) that is predicted to affect splicing efficiency, hence as evolutionarily refined efficiency of viral agents with the a hypomorphic allele, lead to decreased DNASE1L3 activ simplicity and non-inflammatory characteristics of liposo ity. This predicts the polygenic human SLE will also be mal reagents. Part of the answer lies in deciphering the characterized by an absence of a macrophage-Secreted bar pathways and mechanisms by which virus-derived gene rier activity. Sequencing assays and/or assays of therapy reagents evade the barriers to gene expression and DNASE1L3 activity can be used as a diagnostic test for efficiently introduce their material into cells. In addition, predisposition to disease, and to identify a target activity that there is a need to decipher the tissue and cellular barriers to could either decrease the risk or progression to SLE. liposomal transfection. BACKGROUND 0010) The barriers to liposomal and viral gene transfec 0006 Relevance of Discovery to Liposomal Gene Trans tion are roughly understood. Cells from the reticuloendot duction and Viral Infection. Significant research today is helial lineage (for example, tissue macrophages) play a role US 2004/0138156A1 Jul. 15, 2004 in scavenging of transduced DNA (Takakura et al., (1999) more accurately reflects the barrier to liposomal gene trans Pharm. Res. 16, 503-8; Takagi et al., (1998) Biochem. fection, and thus define agents that may better evade Such a Biophys. Res. Commun. 245, 729-33). Cellular and serum barrier. nucleases also play a role, though nearly all the attention has focused on DNASE I or D1 and the non-homologous 0014 Relevance of Discovery to Systemic Lupus Erythe lysosomal enzyme “acid” DNASE II, which is active in matosus (SLE). acidic milieu in the absence of divalent cations (Barry et al., 0015 These observations are relevant to systemic lupus (1999) Hum. Gene. Ther: 10, 2461-80; Ross et al., (1998) erythematosus (SLE or lupus), an autoimmune disorder Gene. Ther. 5, 1244-50). characterized by autoantibodies against nuclear compo 0011 Deoxyribonucleases (DNases or also called nents, Such as DNA and chromatin. It has been hypothesized DNAases) are enzymes that catalyze the hydrolysis and that predisposition to SLE includes defects in the clearance breakdown of polymeric Strands of deoxyribonucleic acid. of DNA-associated antigens. These antigens trigger the They can be Subdivided, for example, into endo- and eXo autoimmune response. The importance of antigens in SLE nucleases, or site-specific endonucleases. This invention pathogenesis is Supported by the affinity maturation of will henceforth refer to endonucleases homologous to anti-DNA antibodies, a cardinal feature of the disease (Shlo DNASE I (or DNASE1 or DNase I or D1, the product of the mchik et al., (1990).J. Exp. Med. 171,265-92; Burlingame HUMDNASEI locus, accession if AAA63170; also acces et al., (1994).J. Clin. Invest. 94, 184-92; Mohan et al., (1993) sion M55983.1) as DNASEs. The only exception to this J. Exp. Med. 177, 1367-81). Affinity maturation shows that nomenclature in eukaryotes is the existence of a DNASE II as the disease progresses, continued exposure to DNA-allied (or DNASE2, the product of DRN2 HUMAN locus, acces antigens, Selects for cells expressing autoantibodies of sion # 000115); this is a non-homologous acid DNASE. The higher affinity. The observation that Anti-DNA antibodies main focus of this invention is an activity of a member of the show this progressive increase in high-affinity mutations DNASE1 gene family (DNASE1L3, or DNase-1-like-3, or Strongly Suggests that antigen drives in large part the dis D3-the product in humans of the DNASE1L3 locus on ease. The present model is that DNASEs are pivotal in this 3p21 (accession # NM 004944) and in mouse of the process of degradation, and that deficiency of this process, syntenic DNASE1L3 locus on chromosome 14 (sequence is specifically D1 L3 activity, predisposes to SLE susceptibil represented by the locus and accession # NM 007870, and ity. in rats by locus and accession # NM 053907, U75689 0016. The defects in the innate immunity system seen in (DNASE gamma), or AF039852 (DNase Y); and in Xenopus SLE Strongly imply that inability to clear Specific antigen or by accession # AF059612). The experiments contained antigens bound in immune-complexes, underlies SLE. In herein mainly utilize the murine Sequences, but are almost many previous models of SLE, the broad diversity of certainly applicable to all homologous mammalian antigenic targets Suggested no unifying pathogenic antigens. DNASE1L3s, which are highly conserved. The experiments do not clearly fully predict the character 0012. In humans, there are at least 4 DNASE loci: and components of the antigenic material, other than it is DNASE I, DNASE1 L1, DNASE1 L2, and DNASE1L3 associated with DNA, and in fact the material may in fact be (FIG. 1). They are named as homologues (“DNASE1-like” a constellation of complex debris that includes intracellular enzymes) of the cardinal member of the family, DNASE1. nuclear elements, DNA, and phospholipid membranes, all All share similar core nuclease domains and structure, targets found in the SLE autoantibody repertoire, and likely requiring divalent cations (calcium and magnesium) for all targets exposed, for example, in apoptotic debris. activity and inhibited by zinc. Unlike DNASE 2, a lysoso DNASEs are not predicted to be the sole determinants of the mal endonuclease active at acidic pH, DNASES are active degradation of these complex antigens, but certainly one of at neutral pH (Shiokawa et al., (2001) Biochemistry 40, them. Chromosomal DNA, by virtue of its size, represent a 143-52). All are predicted to derive from an ancestral gene, formidable molecule, which may make the degradation of and in fact, domains exist with affinities to bacterial enzymes other associated elements more difficult. DNA may be of Such as exonuclease III, Suggesting a Superfamily of endogenous or exogenous (infectious) origin. nuclease proteins. In humans, the distinct DNASE loci 0017 SLE pathogenesis may be subdivided into different encode proteins with generally distinct tissue expression Stages, where different predispositions play a role. For patterns, thus likely non-redundant functions in Vivo. This is example, while the pathways leading to the original dyS also supported by the observation that DNASE1L3, unlike regulation and production of pathogenic autoantibodies or DNASE I and DNASE1L2, has a distinctive and longer immune complexes may be shared by large number of C-terminal extension. patients, the organ distribution and nature of complications, 0013. It has been postulated that one or more of these that is terminal manifestations, may be highly individual extra- and/or intracellular nucleases are responsible for the ized. These “inciting mechanisms may not be identical in barriers to efficient liposomal transfection of DNA. Because all patients but are likely to have Some shared features. For most current studies attribute serum DNase activity to D1, example, all patients with lupus may have a defect, either most studies of non-viral DNA transfection (e.g., lipofec Secondary or primary in Some arm of the antigen clearance tion) efficiency utilize D1 as the standard barrier (Xu et al., System, for example complement (possibly C1, C4, or C2) (1996) Biochemistry 35,5616-23; Crook et al., (1998) Gene. or Surface or Serum proteins associating with chromatin, Ther. 5, 137-43; Mullen et al., (2000) Biochim. Biophys. such as serum amyloid-P (SAP) (Bickerstaff et al., (1999) Acta 1523, 103-10; Nidome et al., (1997) J. Biol. Chem. Nat. Med. 5, 694-7) or Scavenging receptors (Takagi et al., 272, 15307-12). However, in vitro studies reveal that D1 has (1998) Biochem. Biophys. Res. Commun. 245, 729-33), or little effect on transfection efficiency. Accordingly, a need finally one of the DNASEs, either DNASE1 or DNASE1L3. exists to provide an assay to investigate the DNASE that They may in addition or else have defects governing the US 2004/0138156A1 Jul. 15, 2004 general activation or responsiveness of leukocytes, includ marrow derived macrophages, and this activity correlates in ing possibly Suppressor and helper T-cell Subsets, dendritic normal macrophages with D1 L3 levels and DNASE activity. cells, and B-cells. These defects may affect, among others, The in vitro experiments establish that D1 L3 conditioned cytokines, cytokine receptors, transcriptional modifiers, or media can convey BT activity, and Suggest macrophage intracellular messengers. Finally the inciting factors may conditioned BT activity is equivalent to D1 L3 activity. include non-genetic modifiers, for example lectins or drug Finally, a defect in BT activity is present in macrophages exposures (Miyasaka, (1996) Intern. Med. 35, 527-8) or from the NZB/W F1 mouse, which has both a D1 L3 SeX-limited modifiers, or genetic modifiers that are Sex missense mutation and defects in macrophage-Secreted limited. The model for a polygenic disease is that each DNASE, also has deficient BT activity. Hence a defect in BT individual inherits or encounters a Set of predispositions in activity, primary or Secondary, and almost certainly due to overlapping or distinct categories. DNASE1L3 deficiency is D1L3 deficiency, commonly underlies polygenic SLE. one of the predispositions, and perhaps a common one, Since 0020. In a broader perspective, a biologic role for D1 L3 there may be primary defects in the enzyme (Suggested by is to protect of cell nuclei and other intracellular compart the 89I mutation in NZ and MRL strains) and secondary ments from exogenous DNA. DNA-containing apoptotic defects, Such as a failure of Ifn-Y induced macrophage BT debris may resemble to liposomal particles containing DNA. activity. Macrophage D1 L3 likely helps degrade these apoptotic 0018 Deficiencies of DNASE1 have long been postu remnants in Vivo and preventing their conversion, in an lated to predispose to SLE (Fros et al., (1968) Clin. Exp. SLE-Susceptible immune System, into inciting autoantigens. Imm. 3, 447-455) by interfering with the clearance or In addition, D1 L3 may be protecting cellular genomes from processing of DNA-associated antigens (Walport, (2000) “autotransfection', which could lead to oncogenesis Nat. Genet. 25, 135-6). In this model, nuclear material is (Holmgren et al., (1999) Blood 93, 3956-63; Bergsmedh et Viewed antigenic debris, perhaps mostly the residua from al., (2001) Proc. Natl. Acad. Sci. USA 98,6407-11). D1 L3 apoptosis that requires clearance by a janitorial DNASE may cooperate with other agent of innate immunity, Such as function. The characteristic presence of the anti-DNA and complement, to clear of DNA-associated material. Finally, it anti-nucleoSomal antibodies Suggested that DNA-associated is likely aiding in the protection against Viral infection. In antigens might be hastening disease development or pro this invention, an effect of D1 L3 on adenoviral infection is gression, and that, correspondingly, antigen degradation demonstrated. This activity is predicted to possibly have could hamper this predisposition. DNASE I has long been wider applications in antiviral (for example, both DNA and identified and partially purified from bovine Sources, and RNA viruses including hepatitis viruses, herpes viruses, was predicted to be present in Serum, thus this enzyme was adeno-associated viruses, and retroviruses) or anti-microbial the initial and prime candidate for the nuclease defective in therapy for intracellular pathogens, including Listeria, SLE. Serum DNASE activity by RDA has been reported to mycobacteria, and others. be low in NZB/W (Macanovic et al., (1997) Clin. Exp. Immunol. 108, 220-6) and human SLE (Chitrabamrung et SUMMARY OF THE INVENTION al., (1981) Rheumatol. Int. 1, 55-60; Tew et al., (2001) Arthritis Rheum. 44, 2446-7), and almost absent from the D1 0021. The present invention provides a method to test the -/- mice (Napirei et al., (2000) Nat. Genet. 25, 177-81); efficiency of liposomal reagents in vitro and their resistance these studies attribute this serum activity solely to the D1 against a composition comprising D1 L3 added to cell cul enzyme. Deoxyribonuclease 1 (D1) -/- mice have been ture media or Supernatant. The Success of a liposomal found to develop a phenotype Similar to SLE (Napirei et al., composition can be measured by transduction efficiency (2000) Nat. Genet. 25, 177-81). Recently, a null allele in D1 following exposure of a transfection composition to a target has been found in two Japanese pedigrees with SLE asso cell in the presence of D1 L3. ciated with elevated IgG anti-nucleosomal titers (Yasutomo 0022. The present invention also provides a method of et al., (2001) Nat. Genet. 28,313-4). The findings to date do protecting against, treating, or reversing the progression of Suggest that DNASE1 deficiency can exacerbate or predis lupus in a mammal, Said method comprising increasing pose to SLE, yet, D1 defects are unusual in human and D1 L3 activity in the mammal. The invention describes an murine SLE (Tew et al., (2001) Arthritis Rheum. 44, 2446 abnormality of D1 L3 in a polygenic model of murine lupus 7). In addition, therapeutic trials with D1 in NZ mice and explains how this could exacerbate the phenotype. (Macanovic et al., (1996) Clin. Exp. Immunol. 106, 243-52; Verthelyi et al., (1998) Lupus 7, 223-30) and a human trial 0023 The present invention also provides a method of with human DNase I, albeit phase I and well-tolerated, destroying a pathogenic encapsulated membrane bound or (Davis et al., (1999) Lupus 8, 68-76) had mixed, if any, micelle-bound DNA comprising increasing D1 L3 activity. Success in ameliorating the progression of disease. This would include endogenous and non-endogenous infec 0019. The role of DNASE1 deficiency in human or tious particles containing DNA, and even possibly RNA. murine polygenic SLE and its potential for SLE protection The material can be present as non-expressible or express appear limited. The enzyme may contribute to a portion of ible endogenous chromatin, or engineered DNA constructs, SLE-protection. DNASE I appears to target endocytosed or pathogenic viral, rickettsial, mycobacterial, mycoplasma, DNA heading towards lySOSomes by virtue of its glycosy yeast, or bacterial genomes. lation. DNA-associated material entering the cell by other 0024. The present invention also provides a method of pathways may to have more relevance to antigenic detec treating a disease in a mammal by gene therapy, Said method tion. The non-overlapping functions of D1 and D1L3 may comprising the administration of a gene therapy composition explain why D1 alone does not suffice for SLE protection to the mammal, wherein the gene therapy composition nor is an effective treatment for lupus. The data presented comprises a recombinant gene to affect the gene therapy, a shows an Ifn-Y inducible BT activity is secreted by bone lipofection reagent, and a D1 L3-activity-reducing agent. US 2004/0138156A1 Jul. 15, 2004

0.025 The present invention provides a gene therapy 0034. Thus, in vivo activation of D1 L3 activity may be composition Suitable for use in mammals, including accomplished by administering effective amounts of Ifn-Y to humans, comprising a recombinant gene to affect the gene induce expression of D1 L3 by macrophages. It is also therapy, a lipofection reagent, and a D3-activity-reducing known that lipopolysaccharides (LPS), phrobol myristate agent. Suitable lipofection reagents include monocationic acetate (PMA), and Ifn-Y (all macrophage activators) also lipids, polycationic lipids, DEAE, dextran, lipo-polyamines, induce D1 L3 levels. and cholesterol. Suitable D3-activity-reducing agents include antibodies, peptides, DNA fragments, and chemi BRIEF DESCRIPTION OF THE FIGURES cals, which reduces or moderates D1 L3 activity in vivo. 0035 FIG.1. Alignment of DNASE I family of proteins. More specifically, Such D3-activity-reducing agents Selec Panel A: Alignment of human DNase 1 L3 and other DNase tively inhibits D1 L3 expression, inhibits D1 L3 nuclease family members. The alignment demonstrates conserved activity, inhibits C-terminus activity, complexes D1 L3, and/ motifs and residues in the core nuclease domains, while or degrades D1 L3. there is divergence at C-termini. Panel B: Summary of 0026. The utility of such an agent that inhibits BT activity information for the 4 DNASEs. is apparent from the present discovery; Such agents would 0036 FIG. 2. Nothern analysis of DNASE1L3 expres enhance gene therapy using either liposomal or adenoviral Sion in normal tissues. or other vectors. Agents that might be of utility in this matter 0037 Methods described in Rodriguez et al. (1997) include: Genomics 42, 507-13). 0027 (1) Peptide reagents that bind D3 and inhibit its activity, identified through combinatorial libraries or other 0038 Human cDNA probe was used to probe multiple methods. tissue northerns from Clontech. Note highly Specific expres Sion in liver. 0028 (2) Monoclonal or other antibodies that bind D1 L3 0039 FIG. 3. Alignment of C-termini of DNASE1L3s protein and inhibit its BT activity, either directly or by from various species with both D1 and D1L1. Alignment interfering with its biologic cellular enhancement of BT begins at highly conserved SDH motif. All D1 L3-CTs have activity. a predominance of basic residues, even Xenopus D1 L3 with 0029 (3) Chemicals based on reagents which are able to a shorter C-terminal extension. block the activity of the enzyme in vitro, including Zinc and chelating agents Such as EDTA. 0040 FIG. 4. Depiction of BT assay. 0041 FIG. 5. Comparison of Nuclease activity. D1-con 0030 Lower D1 L3 activity may predispose to polygenic tains more nuclease activity against free DNA than D3-me SLE. Increasing D1 L3 levels may benefit SLE patients. A dia. Panel A. RDA results show representative nuclease partial loss of function mutation (T891) is common to two activities of equivalent Volumes of media conditioned by independent mice models of polygenic lupus (the NZB/W cells transfected with 2 mg of either vector pcDNA, D1, D3, hybrid and the MRL strain). D3DCT, or N196K-D3. Nearly no clearing is visible in 0.031) Using GFP-tagged adenovirus methods parallel to control and N196K-D3 media, while D1 and D3DCT-media liposomal studies, D1 L3 and D1-D3 CT were shown to consistently showed greater RDA activity than D3. Panel B: afford a barrier to infection, i.e., block in vitro adenoviral Zymogram of equivalent volumes (5 ml/lane) of pcDNA-, infection of HeLa cells (FIG. 30). This demonstrates that, in D1-, D3-media, and serum show the broader band of activity addition to blocking transfection of liposome bound DNA, produced by media containing glycosylated D1 (33-36 kDA) these enzymes may also block viral DNA. This also impli versus the Smaller sharper 28-29 kDa band of D3 activity. cates other pathogenic viruses, Such as retroviruses (e.g., Serum Zymograms appear to show a doublet of activities, HIV), hepadnaviruses (HBV), hepatitis virus (e.g., Hepatitis migration of Serum proteins may be altered relative to media C), Small pox, measles and herpes virus and are believed to due to differences in albumin concentration. Panel C: Par provide a novel, broad-based in vivo antiviral activity. Thus, allel SDS-PAGE lanes of murine serum (5 ml) used for increasing D1 L3 expression may also useful in resisting zymogram (left) and anti-DNASE1L3 peptide immunoblot Viral or oncogenic diseases, by targeting nucleoSomal DNA. demonstrate the presence of D3 in the Serum, and the Further, the ability of these enzymes to block human aden possible relationship to Serum Zymogram activities; how Oviral infection may result in methods to make gene therapy ever, it is not clear if the visible Serum nuclease bands reflect more efficient and less toxic. For example, anti-D3 antibod D3. ies or D1 L3 inhibitors may enhance adenoviral transfection. 0042 FIG. 6. D3-media confers a barrier to liposomal 0.032 Recombinant human D1 L3 can also synthesize by transfection (BT). Panel A: Western of D3-transfected cells. baculovirus or eukaryotic cells. Such D1 L3 is administered Anti-D3 immunoblots show similar levels of protein in cell intravenously to augment native D1 L3 that is already in the lysates from D3, D3DCT, and N196K-D3 expressing cells. serum. To increase D1 L3 or D1+D3 CT activity, intravenous This expression level is derived for the cells conditioning infusion or expression can lead to increased tissue and/or media in FIG. 6B. Panel B: Western of GFP-transfected circulating levels of these enzymes in an effective amount to cells. Anti-GFP immunoblots at 1:5000 (Clontech) of trans fected HeLa cells demonstrate that D3-media prevents GFP prevent or treat viral infections. expression, presumably by blocking gene transduction. 0.033 Media conditioned with Ifn-Y treated macrophages Expression of GFP in D1, D3DCT, and N196K-D3 treated from the C57BL strain also blocks lipofection. This is not cells did not differ significantly from controls. Immunoblots Surprising, Since it is known that Ifn-Y induces macrophage using anti-b-actin demonstrate near equal loading of cell secretion of D1 L3 and increases D1 L3 activity. lysates. US 2004/0138156A1 Jul. 15, 2004

0043 FIG. 7. Fluorescent microscopy of GFP activity in mismatched Sequence. Panel B: A Similar experiment using cells. GFP fluorescence can be seen by microscopy in cells N191K (human homologous mutation), wherein the GST exposed to D1-media but not D3-media. Hela cells trans fusion is shown to have no activity. First four lanes are fected with n1-eGFP in the presence of conditioned media anti-D3 immunoblot with lane 1 control, lane 2 GST cDNA, with DNASE1 (D1-CM) or DNASE1L3 (D3-CM) show that lane 3 N191K shows expected 56 kDa band just as lane 4 GFP expression by fluorescent microscopy only in cells which is wild-type human GST fusion. In lane 5-7 are the exposed to DNASE1. HeLa cells are rinsed in PBS, then Zymographic results of cell lysates of E. Coli induced to fixed in 4% paraformaldehyde dissolved in PBS for 30 express GEX fusions with lane 5 (no insert), lane 6 N191K minutes, then reSuspended in PBS and photographed human D3, and lane 7 wild type GST-human D3. No activity through a FITC high power scope. is seen in N191K lane. Western analysis performed using 0044 FIG. 8. Comparison of RDA-DNASE activity and antisera #497 at 1:4000. In preliminary experiments, addi GFP-transfection efficiency. The brown columns represent tion of ZnCl2 to induction media at a concentration 2 mM, RDA-DNASE units per 5 ml conditioned media for D3DCT enhances the levels of the 56 kDa nuclease. (n=16), D1 (n=24), and D1D3CT (n=4) media. Mean stan 0049 FIG. 13. Mutagenesis of pcDNA-D1L3 to create dard deviations are noted. Both N196K-D3 (n=2) and con D3DCT. Using the forward primer depicted under the nucle trol media (n=24) had similar negligible background RDA otide Sequence, the wild type murine D1 L3 was activity. Green columns represent the mean ratio of GFP mutagenized to create a stop codon (TAA) at codon 289. plasmid transfection efficiency (as deduced from GFP expression measured by anti-GFP western blot) relative to 0050 FIG. 14. Creation of chimeric fusion of N-terminal that seen for parallel control transfection (i.e., pCDNA) for D1 to C-terminal D1 L3 (289-end). Panel A: Sequence of D3DCT (n=9), N196K-D3 (n=2), D1 (n=20), and D1D3CT MusID1 F128. Panel B: Sequence of fusion protein (n=4). D3- and D1D3CT-media respectfully abolished or D1+D3CT. Panel C: Sequence of fusion cDNA clone Significantly reduced transfection efficiency in exposed D1+D3CT. To synthesize D1D3CT, the murine DNASE1 cDNA was obtained as follows: (1) Mus musculus mRNA cells. Despite greater RDA-nuclease activity for D3DCT for deoxyribonuclease I, Sequence locus D83038, accession and D1-media, there was no significant decrease in trans # D83038 with superimposed primers (F128 and R1029) fection efficiency relative to control-media. used for RT-PCR (reverse transcription followed by PCR). 004.5 FIG. 9. BT activity of diluted D3-media. Immu The resulting clone encoding the full-length enzyme was noblots of transfected HeLa cells demonstrate that D3-media cloned into pcDNA3.1, then mutagenized with the primer and ten-fold dilutions appear to impair transfection of plas DNASE1-BGL2F: CACTCAGAAAgATCTGATGT mid. No GFP expression is clearly detectable in ten fold CATTG and its reverse complement. Stop codon is under dilutions of D3 media. Similar results were obtained with lined in previous forward primer. Primer creates a Bgl2 site dilutions of D3 with D3-N196K. Ratio of GFP expression just upstream of Stop codon. This Step was followed by the per lane relative to control are depicted in the lower register. Silent mutagenesis of the remaining Single Bgl2 Site in the 0046 FIG. 10. Dilutions of D3-media maintain BT activ vector pcDNA 3.1 (data not shown). The C-terminus of ity. Immunoblots of GFP transfected HeLa cells demonstrate D1 L3 (cloned previously in pBlueScript plasmid) was ampli that D3-media when diluted ten fold with media conditioned fied with M13F and the D3-CT-Bam Forward primer GCG by N196K-D1L3 transduced HeLa cells has BT activity. No GATCCGGGCCTTCACCAACAACAGAA 3". This frag GFP expression is clearly detectable in five and ten fold ment was cloned as a BamH1-KPN1 cut fragment into dilutions of D1L3 media. Lane 1 (control); lane 2 (N196K); BGL2-KPN cut pcDNA3.1(-Bgl2) containing DNASE1 lane 3 D1 L3-CM, lane 4-5 are 1:5 and 1:10 dilutions of +Bgl2). The resulting fusion sequence depicted in Panel B D1 L3 with N196K media. Actin controls in lower register with DNASE I encoded amino acids in Small case while are from the GFP-transduced cells. The addition of N196K D1 L3 is in capital letters. The resulting chimeric cDNA is at ten-fold dilution does not allow for transduction, Suggest depicted in Panel C with DNASE1 in small case unbolded, ing there is no dominant negative effect of an inactive fusion link marked with slash mark, DNASE1L3 CT capi protein. talized, and a 5 base linker Sequence (gatcc) in bold letters. 0047 FIG. 11. BT activity occurs with multiple liposo 0051 FIG. 15. D1+D3CT enzyme has BT activity. Panel mal reagents. Panel A. The following figures show anti-GFP A: Top portion provides a representative RDA activity from immunoblots of GFP-transduced cells in a variety of con one plate for the labeled clone. Bottom portion provides a ditioned media. The secondary GFP transduction (as GFP immunoblot of HeLa lysates that demonstrate gene opposed to the DNASE-transduction) is carried out in the transduction levels using FuGene-6 in control, D1, D3, and labeled liposomal reagent. Panel B: Scope of BT effect on D1+D3CT-media. Mean ratio of transfection efficiency and various reagents. Table Summarizes the results to date; activity of D1+D3CT is graphically depicted in FIG. 8. however this table is not meant to be the definitive descrip Panel B: Two putative N-linked glycosylation sites present tion of the resistance or sensitivity of the BT activity, but in two mammalian enzymes (mouse top and bovine bottom) implicates the activity as present with a variety of reagents. (Nishikawa et al., (1999) J. Biol. Chem. 274, 19309-15). N-linked glycosylation Sites in bovine and murine high 0048 FIG. 12. Murine mutagenesis primers for N196K lighted. Panel C: Sequence for the mutagenesis primers used primers and human D1 L3 N191K. Panel A. The N196K-D3 to Sequentially alter the N-linked glycosylation Sites. The cDNA clone was created by point mutagenesis of the murine asparagine codon underlined in each foward primer. Panel clone using primer GGTGATTTCAAgGCCGGCTG D: Zymograms of media conditioned by HeLa cells trans TAGCTA 3' and its complement. The mutation was con fected with pcDNA-expressing clones (from left to right): firmed by the creation of a novel Hae3 site in the cDNA. The (1) pcDNA; (2) DNASE1; (3) D1-N40S; (4) D1-N128S; (5) encoded change is bolded below with the primer under the D1-(N4OS+N128S) clone #1; (6) D1-(N4OS+N128S) clone US 2004/0138156A1 Jul. 15, 2004

#2; (7) D1+D3CT; (8) D1+D3CT-N40S; (9) D1+D3CT in E. coli, and purified by GST-columns. In row A, anti-D3 N128S; (10) D1+D3CT-(N4OS+N128S) clone #1; (11) immunoblots performed on column-purified 56 kDa GST D1+D3CT-(N4OS+N128S) clone #2. The (N4OS+N128S) fusions; while row B represents the parallel DNA-SDS clones are double mutants with both asparagines altered. On Zymograms performed on equal loads of the respective the left are DNASE1-derived clones, those on right half are sample. While protein levels are nearly equal (p<0.01) D1+D3CT-derived. As is evident, loss of glycosylation leads between 89I and 89T, the 89I enzyme. Rows C and D to smaller, sharper bands of activity. Bottom part of the represent two representative experiments of immunoblots of figure are the GFP immunoblot of HeLa Cell lysates that cell lysates and Zymograms of conditioned media for D3 were exposed to the media conditioned by the clones enzymes expressed by transduced HeLa. Row C provides a directly above in the Zymogram. Hence, pcDNA (control)- representative experiment where the anti-D3 immunoblot conditioned media does not establish a barrier to liposomal detecting the 29 kDa D1 L3 in cell lysates of HeLa cells transfection (lane 1); however, the loss of glycosylation sites transfected with pcDNA3.1-D3; while row D provides the enhances the BT activity of both D1 and D1+D3CT clones DNA-SDS-zymograms performed on equal loads of the against FuGene-6 transfected N1-eGFP. Also notable is that respective conditioned media. This demonstrates the 50% despite lower levels of activity on Zymogram and RDA (data decreased activity found in the 89I enzyme relative to not shown) the de-glycosylated D1+D3CT show complete protein levels. BT activity, while the glycosylated D1+D3CT still allows 0056 FIG.20. Graph of nuclease activity comparisons of Some transfection to occur. 89T. and 89I-GST-D3 and pcDNA-D3-enzymes. Bars rep 0.052 FIG. 16. DNASE-conditioned media do not fully resent the pixel volumes (X 103) for the normalized mean degrade Fugene-coated plasmid. Representative experiment nuclease activities on Zymograms for both the GST-fusions (n=3) of exposure of 2 mg free (-) or liposomal-bound expressed in bacteria, then GST-column purified, and of the (+FuGene) plasmid to control media with 10% fetal calf HeLa-expressed enzymes. The pixel volumes (areaxinten serum (lanes 1-2), D1 (lanes 3-4), D1 L3 (lanes 5-6), D3DCT sity) are normalized relative to levels of protein. The (lanes 7-8), and N196K-D3 (lanes 9-10). observed nuclease activity is versus free DNA. This graph summarizes the data from the experiments depicted in FIG. 0.053 FIG. 17. Sequence and alignment of mutant and 19. The table Summarizes the mean normalized activity normal DNASEs. Panel A. Wild-type (top) and mutant 89I values and Standard deviation. (lower) sequence traces from C57BL and MRL respectively. Arrows point to where 89 mutation is caused by a T to C 0057 FIG. 21. Anti-D1L3 western of normal mouse transition at base pair 438. Panel B demonstrates that ATA to tissues. (A) Polyclonal anti-D3 immune western blot of ACA codon change due to the transition at bp 438 substitutes murine whole organ protein lysates, (b) control pre-immune isoleucine for threonine in the protein. Panel C illustrates western blot, and (c) added peptide blocked westernblot. where the amino acid change occurs relative to other D1 L3 Two bands of activity in the liver sample are thought to (rat and human) and bovine DNASE 1. Also depicted are reflect cleaved and uncleaved enzyme (34 kDa); while the residues in DNASE I, that are thought by crystallography to 29 kDa bands in spleen and thymus reflects cleaved be involved in actin and DNA binding respectfully. Full enzyme. Anti-mus DNASE1L3 peptide rabbit antisera, alignments of DNASEs can be found in Rodriguez et al. detects expected the 34 and 29 kDa proteins in murine ((1997) Genomics 42, 507-13). Spleen, liver, thymus and faintly in lung. Negative pre immune and peptide blocking controls shown. Primary 0054 FIG. 18. Genotyping of murine strains. Panel A. antibody used at 1:5000 in powdered milk with 0.05% The PCR genotyping Strategy to determine which Strains are Tween and 1% goat serum; HRP-linked 2 antibodies at 89T and which are 89I is depicted; it uses mismatched 1:4000 (Amersham ECL), and exposed on radiographs. To reverse primer to detect mutant amplimerS as SSP1-digest derive protein extracts, whole organs were homogenized and ible (AATATT), while the 48 bp fragment amplified from boiled in 2% SDS-PAGE loading buffer without DTT, wild-type Strains is SSP1-resistant. Results from genotyping Protein load per lane equalized by Bradford assay, confirmed are arranged below each Strain showing both original PCR by visual inspection of Ponceau Red Stained Immobilon (-) and after addition of SSP1 (+) amplimer products. Both blots (Biorad mini-Protean II apparatus). Negative lanes of C57BL and BXSB strains contain the wild-type allele, while brain, heart, Small gut and kidney are not shown. all other mice depicted are 89I, hence SSP1 digestible. The forward genotyping primer was F413-mus)3 5'-AATG 0.058 FIG. 22. Normal and induced expression of D1 L3 GAAATTCACGAAGAAGCAC, while the mismatched in murine macrophages. This figure demonstrates the immu primer R461-musD3SSP was 5'-CGAGAACTAATCACAT. nological detection of DNASE1L3 by anti-D1L3 peptide AGTTGaAT, where the non-capitalized a is a mismatched immunoblot of cell lysates from macrophages or adherent base pair. Panel B illustrates use of the Strategy to evaluate Splenocytes presumed to be of macrophage lineage. In further strains. The same strategy was used on the BXD anti-D1 L3 blots of cell lysates, the protein is seen both as a Strains, whose parents are discrepant, to map the position of 34 kDa uncleaved molecule and a 28 kDa cleaved mol D1L3 to its expected Syntenic position on murine 14 by ecule. In addition the figures demonstrate higher levels of comparing the transmission to the markers referred to in the expression of D1 L3 in bone marrow macrophages from text. For analysis 3 ul of 25 ul PCR reaction digested with MRL, NZB, and BXSB mice compared to C57BL mice. Ssp I for 1 hour at 37 C. and run a 8% acrylamide/1x TAE Finally, the figure shows that the D1 L3 levels in macroph gel w/o EtBr at 100 volts. Stained in EtBr bath 10 mg/mL. ages are inducible by Ifn-y in C57BL, as well as RDA nuclease activity. This was shown to be correlated with 0055 FIG. 19. Comparisons of 89I and 89T enzymes. increased BT activity. Panel A: Demonstrating an anti-D1 L3 ROWS A and B represent two representative experiments of immunoblot of cell lyStates from murine Splenocytes. Cells immunoblots and zymograms of D3-GST fusions expressed were plated on Serum-coated tissue culture dishes and incu US 2004/0138156A1 Jul. 15, 2004 bated overnight, the next morning, non-adherent cells (N) in macrophage conditioned media than C57BL. See text for were rinsed vigorously with PBS 1.x, and both adeherent (A) isolation and culture conditions. and non-adherent cells (N) from C57BL, NZB/W, BXSB, and MRL were lysed and immunoblotted with anti-D1L3. 0061 FIG. 24. Zymograms and RDAs of murine urine. While the 34 kDa size band consistent with uncleaved This figure shows similar zymographic (Panel A) and urine D1 L3 is present in both adherent and non-adherent, the RDA-DNase (Panel B) levels for NZB/W, MRL, BXSB, and cleaved form (28 kDa) is present almost exclusively in the C57BL mice. adherent population of presumed macrophages. Also noti 0062 FIG. 25. Zymograms and anti-D3 westerns of cable are the higher levels of expression of D1 L3 in the 3 murine sera. Panel A: Top row depicts anti-DNAS1L3 latter lupus strains. Panel B: Untreated bone marrow mac western of Sera from various murine Strains. The Second row, rophages from NZB/WF1 mice have a much higher baseline Zymograms of Same Samples, which shows not only the expression expression of D1 L3 than C57BL macrophages. decreased activity of nucleases in 89I Sera, but also the loSS 0059) However, treatment of the cells with 100 U/ml of of the upper band of DNASE activity. The bottom row Ifn-y leads to significant induction of D1 L3 levels. Induction depicts a representative RDA for the Samples. 3 mice were is present in macrophages from both Strain though the bled 4 times each over a course of 4 dayS. Data represented absolute level of induction appears greater in C57BL than in in table form in FIGS. 23 and 28. Panel B: Parallel the SLE-prone NZB/W F1 strain. NZB/W protein loads in zymogram of normal serum and DNASE-transfected HeLa Ifn-y-negative lane is slightly underloaded relative to actin. cells demonstrates the relationship of the Serum activities to Panel C: Bone marrow macrophages isolated from bone D1 and D1 L3, and suggests that most of the serum DNASE marrow (via adherence) from MRL and C57BL mice, and activity is D1. cultured for 2 days in vitro, were induced by Ifn-y. In the top 0063 FIG. 26. Anti-D3 westerns of splenocyte cell register, an anti-D1 L3 immunoblot demonstrates induction lysates and RDAS of Splenocyte-conditioned media. Equiva of D1 L3 expression as both 34 and 28 kDa fractions. More lent numbers and concentrations of Splenocytes (counted significantly, no DNASE activity is detected by Zymogram with hemochromocytometer and incubating both adherent pre-induction, but a 28 kDa band is detectable after induc and non-adherent) were cultured for 24-36 hours to condi tion with interferon gamma. The level of RDA nuclease tion media. Panel is representative of experiments using 3 activity is also increased after interferon gamma induction in mice for each Strain: the register in row a is a western using both MRL and B6 mice. No induction of D1 L3, RDA anti-D3 peptide antisera. The Western demonstrates the DNASE, or zymographic DNASE activity is evident upon induced level of D1 L3 in splenocytes from all lupus-prone tratement with 100 U/ml of Ifn-alpha. Bottom register shows Strains. Row b provides an immunoblot detecting b-actin actin levels in macrophage cell lysates by immunoblot in expression, which is used as a control for levels of cellular order to demonstrate equal loading. Panel D: Bone marrow protein. In row c, intraexperimental (same plate) RDAS of macrophages from C57BL and MRL mice cultured for 7 conditioned media are reproduced for conditioned media days in the presence of GM-CSF were induced with Ifn-y, Samples. The results indicate that despite the induced levels again showing the higher baseline levels of 28 kDa enzyme of cellular D1 L3, there is no parallel increase of RDA in the lupus prone Strain, and in C57BL Showing induction nuclease activity. Mean values were derived for all three of levels aftern Ifn-y. The baseline level of D1 L3 expression experiments by pixel analysis of Scanned images and rep in C57BL is higher, and the degree of induction is lower than resented in table II and FIG. 6. The differences in levels of that observed in macrophages grown in vitro for Shorter protein expression were significant for expression levels culture times (<48 hours) in the absence of GM-CSF. Panel between C57BL and all lupus mice (P<0.05), while DNASE E: HeLa cells exposed to conditioned media from HeLa cells activity was not significant. 100 U/ml of Ifn-y do not have a barrier to liposomal transfection. HeLa cells treated with the same dose of Ifn-y 0064 FIG. 27. Anti-D3 westerns of macrophage cell did not have BT activity either (data not shown). lysates and RDAS of macrophage-conditioned media. Mac 0060 FIG. 23. Table of RDA-DNASE and D1 L3 levels rophages were derived from bone marrow. Row a is repre in SLE and C57BL mice. Table shows pixel values (volumes Sentative of experiments using 3 mice for each Strain: the =areaxintensity) of D3 levels by anti-D1L3 immuno and register in row a is a western using anti-D3 peptide antisera. nuclease activity by RDA, along with Standard errors and The western demonstrates the induced level of D1 L3 in significance. Measurements were performed on C57BL, macrophages from lupus-prone Strains. Row b provides an BXSB, NZB/W, and MRL mice. Often, more than one RDA immunoblot detecting b-actin expression, which is used as a assay on a Specific Sample were conducted. The mean and control for levels of cellular protein. In row c, intraexperi Standard error of measurements is cited. All RDA assays mental (same plate) RDAS of conditioned media are repro were incubated for an equivalent time under the same duced for conditioned media Samples. The results indicate conditions. All immunoblots were performed under Similar that despite the induced levels of cellular D1 L3, there is no conditions. No D1 L3 immunoblot was performed in urine. parallel increase of RDA nuclease activity. Mean values D1 L3 levels and DNASE activity in samples from three were derived for expression and activity by pixel analysis of lupus models (BXSB, NZB/W F1, and MRL) and one scanned images and are represented in FIGS. 23 and 28. control strain (C57BL) are shown. All values are expressed The differences in levels of protein expression were signifi in pixels. All Scans were analyzed by Scion Image using 250 cant between C57BL and all lupus mice (P<0.01); while pixels per inch, 100% size image. The most notable results RDA-DNASE activity was slightly greater in the 89T strains are the high levels of D1 L3 protein induction in C57BL than in the 891 strains. Splenocytes and bone-marrow derived macrophages, despite 0065 FIG. 28. Graphs depicting D1 L3 levels and absence of parallel induction of DNASE activity. In addi DNASE activity in murine samples. The right column set in tion, both NZ and MRL mice had significantly less activity each display shows the mean D1 L3 levels by immunoblot of US 2004/0138156A1 Jul. 15, 2004

Serum and Splenocyte/macrophage cell lysates for different 0069 FIG. 32. Anti-human D1 L3-peptide immunoblot mice Strains and models. All graphs display from left to of human peripheral leukocytes. Panel A: Protein lysates right: C57BL, BXSB, NZB/W F1, and MRL results. No were prepared from histopaque purified peripheral mono anti-D1L3 immunoblot was performed for urine. The left nuclear cells from human blood, and run on a 14% SDS column sets documents DNASE RDA activity measured in PAGE and immunoblotted with anti-human D1 L3 peptide Serum, urine, and Splenoctye and macrophage-conditioned antisera, and Secondary anti-Rabbit. Both cleaved and media. Mean values and Standard deviations for all obser uncleaved bands are seen. Panel B: Immunoblot of D1 L3 in vations in FIG. 23. The results demonstrare that despite the human Serum. D1L3 protein induction in Splenocytes and macrophages is 0070 FIG. 33. Comparison of 89I and 89T BT activity. ineffectual in raising RDA-nuclease levels. Compared by BT assay, the 89I enzyme is significantly 0.066 FIG. 29. Media conditioned by Ifn-Y treated mac defective. Normalized to immunoblotted D3 protein levels rophages from C57BI, but not NZB/W F1 mice is able to in pcDNA3.1-D3 transfected cells, 89I had a 8 fold defect confer an in vitro barrier to liposomal gene transduction in BT activity (n=6, p-value =0.03 T-test). Panel A. FD (BT). Panel A. Media conditioned by interferon-g (Ifn-y) nuclease activity in the conditioned media was similar in 89 treated bone marrow-derived macrophages from C57BL and 89T, because of a nearly 2-fold higher level of 89I in confers a barrier to liposomal transfection to HeLa. These transfected cell lysates. Panel B: a ten-fold dilution of 89T results utilize the C57BL cells and media analyzed for FIG. nearly completely blocks GFP transduction, while liposomal 22C. Panel B: As interferon dose increased, the ability by gene transfection is possible in Similarly diluted 89I-con C57BL macrophages to confer a barrier to transfection taining media. Panel C. Relative levels of GFP expression. increased. In the absence of Ifn-y, macrophages confer a Undiluted conditioned media in these experiments, which weaker barrier. Panel C: Interferon gamma induces both achieves higher levels of FD-nuclease activity than Seen in DNASE activity by RDA and a DNASE band compatible Ifn-Y induced macrophage-conditioned media, conferred with DNASE1L3 at 28 kD, plus induces a BT activity. saturated BT activity with both 89I and 89T. These results Samples were from macrophages from two C57BL mice (B1 establish that a significant primary defect in D3-BT activity and B2). In the left register is pre- and post interferon RDAs, is present in both the NZ and MRL strains of mice, and this in the middle register, pre and post interferon, and in the finding in independent SLE models Suggests a role in their right register are the BT assays (GFP immunoblots of Susceptibility to the disease. tranfected HeLa cells) on HeLa cells, including HeLa cells 0071 FIG. 34. Y261C human sequence traces. Sequence treated directly with interferon gamma for 48 hours. Media traces of Y261 C mutation and demonstration of heterozy conditioned for 48 hours. Panel D: BT activity in macroph gozity. a) Sequence trace of the original heterozygous age-conditioned media from NZB/W F1 (NZ) or C57BL genomic amplicon. Similar heterozygoZity was observed on (BL). Two mice from each class were Sacrificed, and bone reverse Strand. b) Sample was re-amplified from original marrow derived cells were Segregated into two wells each. stock, and cloned into TA vector. Six individual clones were Media was conditioned for 48 hours in the absence of isolated and Sequenced; two of the clones depicting either GM-CSF, and then overlaid over HeLa cells. Wild-type mutant (n=4) or normal (n=2) sequence are shown. macrophages are able to Secrete or condition media with a barrier to liposomal transfection, while SLE mice are not. 0072 FIG.35. Y261 C mutation causes complete loss of Interferon-gamma did not alter the results. D3 function Ahuman DNASE1L3 clone (derived from EST 82269, (31), fused in frame to hemagglutinnin tag (HA) was 0067 FIG. 30. D3-media confers an in vitro barrier to used for this analysis. Mutagenesis of the clone was per adenoviral gene transduction (BT). This figure provides formed using QuikChange kit (Stratagene) and the anti-GFP immunoblots at 1:5000 (Clontech) of Ad.V.GFP Sequence-verified pcDNA-3.1 derived HA-tagged clone was infected (MO 100: 1) of 100,000 HeLa cells. Infection of expressed in HeLa a) Anti-HA blots with 12AC5 (1:200) HeLa cells with Ad5.CMV-GFP (a 1st generation (DE1/ showed no difference in expression of the DNASE clones DE3) adenovirus serotype 5 expressing GFP under the (data not shown). C-HA control lane was transfected with control of CMV-IE promoter (Qbiogene)) was performed in parental vector. b) The mutant 261 C showed no activity by the presence of D1, D3, D1+D3CT, or control conditioned Zymogram, and only faint, if any, activity by RDA (c), and media. Infected cells were lysed at 72 hours and tested for BT assay (anti-GFP) showed an absence of barrier activity GFP expression (n=3 expts). The immunoblots (n=2) dem (d). e)Actin lane shows equivalent protein loads for BT onstrate that D3- and D1+D3CT conditioned media prevents asSay. GFP expression, presumably by blocking infection. Thus DNASE1L3 or DNASE enzymes linked to the D3CT dimin 0073 FIG. 36. Alignment focused on DNASE1L3 resi ish adenoviral gene transfer. The increased expression of dues at 261 showing high degree of conservation GFP in the D1 lane remains to be explained. Actin levels 0074 FIG. 37. IVS6+5 homozygotes and heterozygotes. were generally equivalent in infected cells. a) Sequence traces of (left to right): wild-type GG homozy gote, GT heterozygote, and TT homozygote. Arrows illus 0068 FIG. 31.89I D1 L3 differs from 89T by at least 10 trate polymorphic IVS6+5 nucleotide. The 3' end of exon 6 fold in BT activity. When dilutions are made of D1 L3 is shaded gray. b) Array represents consensus splice donor containing media, the ten-fold dilution conditioned media region. Numbers below are the percent of verterbrate introns containing the wild-type 89T enzyme shows no GFP trans with a given residue at that position; thus, the +5 nucleotide fection efficiency, while the 89I shows transfection. Ample is G in 85% of exon junctions. transfection is seen with a 1000-fold dilution of either, non with undiluted samples. RDA nuclease levels are show DETAILED DESCRIPTION alongside. Results Suggest a possible 10-fold difference in 0075) DNASE-1-like 3 (DNASE1L3 or D1L3). The BT activity. human DNase gene family consists of 4 homologous, yet US 2004/0138156A1 Jul. 15, 2004 distinct loci, with different tissue expression patterns 0078 Studies of in vivo Effects of D1 L3: (Shiokawa et al., (2001) Biochemistry 40, 143-52; Rod 0079 D1 L3 Is a Barrier to Liposomal Gene Transfection: riguez et al., (1997) Genomics 42, 507-13) (FIGS. 1A, B). Definition of BT Activity. Liposomal transfection is less D1L3 expression by northern was prominent in liver (FIG. efficient in vivo than in vitro (Crystal, (1995) Science 270, 2). The protein was immunodetected in liver, spleen, and 404-10). While DNase 1 (D1) has been implicated as a prime thymus, where it is mostly present in macrophage-derived candidate for imparting barrier to transfection, this nuclease (adherent) populations (FIG.22). By western, D1 L3 expres has little in vitro activity on liposome-complexed DNA. Sion is found in Spleen, liver, and thymus. In addition, the More significantly, D1-conditioned media has little effect on enzyme is found by western blot mainly in adherent cell in vitro transfection efficiency. On the other hand, media populations from Spleen (presumably macrophages) (FIG. conditioned with D1 L3 provides a potent barrier to in vitro 22A and Baron, 1998 #4) and serum (FIG.25). The enzyme liposomal transfection and may be the in Vivo basis for may also be produced by other thymocytes and non-adherent barrier to transfection (BT) in wild-type organisms. Splenocytes. The enzyme is Secreted and concurrent Zymo grams of cell lysates and conditioned media of transfected 0080. The experiments summarized in the figures reveal that D1 L3-conditioned media (CM) generally ablated cells confirm D1 L3’s Signal peptide is cleaved, as are the expression of GFP in the lipofection studies, while GFP other DNASEs. expression did not differ greatly between cells incubated in 0076) The full biologic functions of D1 L3 are unclear. either control of D1 conditioned media. In all, D1 condi Apoptotic endonucleic “laddering of lymphocytes to intra tioned media had more nuclease activity against free DNA cellular DNAS1 L3 (Shiokawa et al., (2001) Biochemistry by RDA than D1 L3 conditioned media. By densitometry of 40, 143-52) has been attributed to this enzyme. In cultured RDAs, D1 media contained a mean of 2.7 fold (SD=0.8; macrophages, the enzyme and the activity in conditioned n=24 assays, p<0.001) more activity by RDA than D1 L3. In FIG. 8, the values were obtained from scanned tiff figures media are inducible by Ifn-y, LPS, and phorbol myristate used to calculate pixel “volumes” of RDA activity (areax acetate, all agents that promote monocyte differentiation and pixel darkness). D1 and D1 L3 conditioned media were activation. Lipofection-associated DNA does show Some assayed in parallel on the same agarose dish. Inter-experi laddering with D1 L3 exposure (FIG.16). If D1 L3 is crucial mental incubation conditions were nearly identical (24 for oligosomal degradation of apoptotic DNA, the absence hours, 37 degrees, same concentration DNA and agarose). of this macrophage-Secreted nuclease from cultures of most D1 media contained a mean of 28.2 Units of activity cultured cell lines may explain why apoptotic DNA ladders (SD=0.7) versus only 12.0 (SD=0.2) for D1 L3; n=24 assays; are less consistently observed there verSuS primary cell p<0.001). The mean ratio of GFP expression in D1-exposed cultures. These Studies Suggest this apoptotic hallmark, cells versus control was 0.8+0.2 (n=20 assays). Actin immu thought to reflect only intrinsic enzyme activation, could be noblots did not differ among Samples, showing equal protein acquired via D3-entry into cells from Surrounding milieu. loading (FIG. 6). No GFP expression was seen in D1 L3 D1 L3 is easily detectable in serum by immunoblot; there exposed cells. Fluorescent microscopy at 24 hours con fore, the BT effect is likely distributed throughout tissues. firmed the absence of GFP fluorescence in cells incubated in Since the nuclease levels by RDA and the levels of immu D1 L3 conditioned media, while abundant GFP can be visu nodetectable D1 L3 in serum are similar or lower to those alized in D1 conditioned media exposed cells (FIG. 7). generated in the conditioned media of the described experi Zymograms of the respective CM demonstrate the expected ments; an in vitro D3-BT effect is likely to be present in bands of activity for both the DNases, with glycosylated D1 Serum in Vivo. showing a larger, more intense, and diffuse band than D1 L3. 0081. In the main assay, an eukaryotic marker such as 0077. The human D1 L3 gene is located on human chro green fluorescent protein (GFP) expression plasmid com moSome 3p14-3p21 and in the Syntenic location on mouse plexed with a lipid reagent is transfected into HeLa cells 14. Herein, D1 L3 may refer to native D1 L3, or natural, incubated in control media, D1 L3-conditioned media, or Synthetic or recombinant variants thereof that retain the D1-conditioned media (FIG. 4). The assay is not limited to D1 L3 activity. Unless otherwise specified, D1 L3 herein the use of GFP as a marker, but could use any of many refers to D1 L3 that is found in any Source, including murine, expressed biomarkers encoded by nucleotides that can be bovine, Ovine, porcine or human. Unlike D1, native Serum assayed either immunochemistry, fluorescent detection, or D1 L3 has a basic C-terminal extension and does not have enzymatic detectection, and these include but are not cir glycosylation sites. D1 L3 is described in more detail in cumscribed to luciferase, chloramphenicol acetyl-trans Genomics 42:507-513 (1997), which is hereby incorporated ferase, antibiotic or chemical resistance genes, Beta-galac by reference. Murine D1 L3 extends 21 residues (289-310) tosidase, FLAG-, myc-hemaglutinnin-, or other epitope further than the aligned D1 enzyme (nearly half these amino tagged molecules, as well as other markers of gene or acids are basic (arginine or lysine). FIG.3 aligns murine D1 nucleotide transduction. The endpoint of the assay is the and D1 L3 enzymes from mouse, human and Xenopus, detection of a Synthesized protein, but could easily have respectively, Starting at the highly conserved motif, SDH, been the detection of a transcribed gene product, Since the and shows the conserved basic character of this stretch. assay ultimately measures the ability of D1 L3 added the Although the core D1 L3 is highly homologous to D1 and media/milieu overlaying the cells to block the ultimate DNase 1-like 1 (D1L1), D1 L3 activity differs from D1 asSociation of an expressible plasmid with the transcrip activity in that D1 L3 effectively blocks liposomal transfec tional machinery. tion (lipofection), while D1 does not. This novel DNase 1 L3 activity is an in vitro BT, and in vivo likely forms part of a 0082) The ability of an agent to overcome the BT effect cellular shield to the nuclear acquisition of exogenous DNA. reflects the ability of the agent to overcome the barrier to the US 2004/0138156A1 Jul. 15, 2004

passage of the transduced agent from outside of the cell into distribute this protective effect throughout tissues. Thus, an intact and active transcriptional Site, presumably nuclear. these observations of an in vitro BT effect are likely to be The exact site of action of D1 L3 (or D1+D3CT) is unknown; true also in Vivo, where D1 L3 shares the Serum compartment the data Suggests that glycosylation leads to the targeting of with D1. a less effective site, likely lySOSomal. The experiments also indicate interaction with cells appears to be required. The 0086 Therefore, blocking D1 L3 activity may increase present invention provides a gene therapy composition com effectiveness of certain gene therapies. Peptide or mono prising a recombinant gene expressing D1 L3-like barrier to clonal blocking agents against D1 L3 may protect lipid/ transfection and a lipofection reagent. Media conditioned liposome based transfection Systems. This is useful, particu with Such enzymes will prevent liposomal transfection, and larly near areas of circulation, because non-viral gene can be used to test, which transfection reagents overcome transduction is believed to be leSS toxic than viral transduc this barrier. The enzyme can be provided to the media by tion. either expression of a gene containing the full-length 0087 BT Activity Occurs with Various Liposomal Trans Sequence of D1 L3 Such that the enzyme can be Secreted in fection Reagents. Most of the experiments were performed eukaryotic cells. In addition, the enzyme can be Synthesized using FuGENETM-6 (Roche Diagnostics Corp.; Indianapo and purified from other Systems. Such as bacteria, yeast, lis, Ind.). The studies show that D1 has no effect on the tissues, and cell culture, and isolated in a glycerol-Stabilized transfection efficiency of liposomal transfection Systems format. Another endpoint that could be used in assays to such as FuGene TM-6, while D1 L3 effectively blocks trans overcome the BT effect could also be the ability to provide fection efficiency in FuGene TM-6. However, using accom distinctive capacity to a cell line Via transient or permanent panying protocols, a variety of transfection reagents (FIG. modification of the genome, for example ability to Survive 11B, ne2 for each reagent) were tested. Examples include or permit Selection in a specific milieu. The goal of Such the non-liposomal lipid-based Effectene (Qiagen); the cat assays would be the derivation of more effective liposomal ionic lipopolyamines-based LT-1 and LT-2 (Mirus); the transfection reagents. dioctadecylamidoglycyl spermine (DOGS)-based Trans 0083) Deoxyribonuclease 1-like 3 (D1L3) hydrolyzes fectam (Promega); the polycationic lipid/lipid mixture of lipid-complexed DNA and decreases transfection efficiency Gene-Limo Super (Q-Biogene); and the polycationic lipid in liposomal transfection (lipofection) Systems. AS Such, based GeneShuttle 20 and 40 (CPG-Biotech). A BT effect D1 L3 provides a better test for the efficiency of lipid/ was observed with all polycationic lipid reagents. With lipOSomal based gene therapy than current Standards using GeneShuttle and GeneLimo reagents (n=2 experiments), the deoxyribonuclease 1 (D1). Moreover, blocking D1 L3 activ effect of D1 L3 conditioned media was partial; however, the ity enhances liposomal transfection for gene therapy, while pattern was still similar (D1L3 GFP expression

0108) D1 L3 Levels and DNASE Activity in Lupus and D14Eyu1 and D14Mit 99, and the expected syntenic location Normal Macrophages. on proximal chromosome 14 (data not shown). 0109) Deficiency of deoxyribonuclease (DNASE) activ 0112 Three a priori observations suggest 89I impairs ity is postulated to predispose to the polygenic disease of D1L3’s function: first, D3-89T is conserved across species Systemic lupus erythematosus (SLE or lupus). SLE is a (FIG. 17), thus, the 89I allele is described as mutant. multifactorial disease characterized by autoantibodies Second, the mutation is adjacent to a highly conserved against nucleosomal components, including DNA (Her tyrosine involved in DNA-D1 contact (Jones et al., (1996).J. rmann et al., (2000) Immunol. Today 21, 424-6). Among the Mol. Biol 264, 1154–63) a potential PKC phosphorylation hereditary factorS Suggested to predispose to SLE are defects site (SRR) present in both D1 L3 and D1. Finally, the residue nestles in a region of D1 involved in nuclease-actin inter in the clearance of DNA-associated antigens or immune action (FIG. 17) (Suck, (1994) J. Mol. Recognit. 7, 65-70). complexed-antigens. For example, mutations in early The NZ, MRL, C3H, and DBA/2 strains likely inherited this complement factors, most prominently C1, are highly asso mutation from a common Castle-derived ancestor (Becket ciated with SLE development in humans and mice (Walport, al., (2000) Nat. Genet. 24, 23-5). However, the NZ and MRL (2000) Nat. Genet. 25, 135-6; Botto et al., (1998) Nat. Strains are regarded as independent models, with generally Genet. 19, 56-9). Complement forms is described as part of non-overlapping susceptibility loci (Kono, (1999) in Genes the “innate immunity System. In addition, to aiding in the and Genetics of Autoimmunity, Vol. 1 (AN., T., ed.), clearance of apoptotic remnants and immune complexes, the Karger). While no murine SLE susceptibility allele matches complement cascades play a role in inflammation and in the this locus on mouse chromosome 14, a significant associa bodies response to pathogenic infectious agents. tion was found for Some human SLE patients to 3p14 region 0110. This invention details the role in murine SLE of containing the D1 L3 locus (Moser et al., (1998) Proc. Natl. defects in “macrophage DNASE’, D1 L3. The deficient Acad. Sci. USA 95, 14869-74). Ultimately, the selection of activity of macrophage DNASE in the NZB/W F1 strain is two “independent’ SLE strains with the 89 mutation is asSociated with a defect in macrophage-conditioned BT highly Suggestive by itself of a role for this allele in their activity associated with a paradoxical induction of D1 L3 genetic Susceptibility to SLE. levels. Normally, this induction would be associated with a 0113) To determine if the 891- and 89T-D3 differed in high Ifn-y State, increased in macrophage-Secreted DNASE nuclease activity after prokaryotic (E. Coli) and eukaryotic activity and BT activity. The latter two are absent from SLE (HeLa) expression. First, DNASE activity in media condi mice. The invention proposes a defect of D1 L3 characterizes tioned by D1 L3-transfected HeLa cells was measured by a model of polygenic SLE (NZB/W F1 mice); and suggests both zymograms (28 kDa activity) and radial diffusion similar defects will be found in man. The invention assays (RDAs) (FIG. 19). Media conditioned by pcDNA3.1 describes a primary defect in D1 L3 and a Secondary defect vector has nearly undetectable activity. Nuclease activity on in macrophage-secreted DNASE, (presumably D1L3). The Zymograms was normalized relative to the immunoblotted invention asserts that the above defect is not due to DNASE1 D1 L3 levels in cell lysates. 891 and 89T protein levels were deficiency, Since there is no primary Sequence variance in equivalent. Zymograms from 891-transfected HeLa cells the coding Sequence in SLE mice, and only mild defects of had 44% of 89T-transfected cells (n=16; p=0.0004 by Stu serum nuclease activity. In addition, DNASE levels in the dent's t-test) (FIG. 20). Similar results were observed when urine, a compartment reflecting D1 expression, are equal comparing RDAS of media conditioned by transfected cells (FIG. 25). and by similar assays transfecting D3-eGFP fusions immu 0111 Primary Defect of D1 L3 is Present in NZ and MRL noblotted with anti-GFP (N1-eGFP vector, Clontech) Strains. The coding sequence of DNASE1 (D1) and the (unpublished observations). In conclusion, expression stud distinct macrophage-secreted homologue DNASE1L3 (D3) ies find partial loss of D1 L3 function is present in the 89I were analyzed in lupus-prone mice (MRL, NZ Strains, and enzyme. BXSB) and in non-SLE prone BALB-C and C57BL strains. 0114. In summary, when the activities of both wild type The cDNAS Spanning the protein coding Sequence were (89T) and variant (89I) D1 L3 were assayed in vitro, the 89I Sequenced and analyzed. No alterations that change amino D1 L3 has about half the RDA-nuclease activity of the 89T acids were uncovered for DNASE1. A C to T transition at enzyme (p<0.001). Thus a primary RDA-defect, albeit a base pair 438 of murine D1 L3 was present in both the NZ limited one, of D1 L3 is present in Some models of polygenic and MRL sequences (FIG. 17), and confirmed by analysis of SLE. Since the variant is also present in Strains not know to amplified genomic DNA (FIG. 18). Thus, MRL and NZ be predisposed to SLE, it can be only one Susceptibility models are homozygous for D1 L3 alleles leading Substitut factor, and additional Secondary predispositions, which ing threonine at residue 89 for isoleucine. The mutation Serve to decrease D1 L3 activity may exists. Initial Studies encodes the non-conservative Substitution of threonine at had shown undiluted conditioned media containing either amino acid 89 by isoleucine (T89I) (FIG. 17), and will 89I and 89T enzymes were no different in their ability to henceforth referred to as the 89I or mutant allele. The block liposomal transfection. However, when dilutions of C57BL allele is referred to as the D1 L3-89T or 89T or the the media were made, indications are that there may be a wild-type allele. Similar terminology will be used to refer to ten-fold difference between these two enzymes in BT activ the protein. By genomic PCR and/or sequencing, the 89I ity (FIG. 31). This would be a powerful indictment of a allele was also present in DBA/2, C3H, and Astrains, and significant primary role of D1 L3 in 89-associated murine absent from the SM, AKR, LG, 129, BALB/C, BXSB, and SLE C57BL.B10 strains. Genotyping of DNA from the BXD Strains, obtained from Jackson laboratories, using the above 0115 Additionally, FIG. 33 provides comparison of 89I PCR Strategy showed complete concordance with markers and 89TBT activity. Compared by BT assay, the 89I enzyme US 2004/0138156A1 Jul. 15, 2004

is significantly defective. Normalized to immunoblotted D3 0118. The DNASE activity in urine, which expresses protein levels in pcDNA3.1-D3 transfected cells, 89I had a high levels of DNASE I, was analyzed (Takeshita et al., 8 fold defect in BT activity (n=6, p-value=0.03 T-test). As (1995) J. Biochem. (Tokyo) 118, 932-8; Nakajima et al., shown in FIG. 33A, FD-nuclease activity in the conditioned (2000) Exp. Clin. Immunogenet. 17, 71-6), and no differ media was similar in 89I and 89T., because of a nearly 2-fold ences among the strains/models studied was found (FIGS. higher level of 89I in transfected cell lysates. As shown in 24 and 23). Urine had higher concentration of DNASE of all FIG. 33B, a ten-fold dilution of 89T nearly completely the compartments examined. In urine, there appeared to be blocks GFP transduction, while liposomal gene transfection few discrepancies in DNASE activity between lupus mice is possible in similarly diluted 89I-containing media. FIG. and C57BL. This supports the contention that DNASE I 33C provides relative levels of GFP expression for the 89T likely does not play an important role in polygenic SLE. and 89I enzymes. Undiluted conditioned media in these 0119) DNASE I appears to be the main serum DNASE. experiments, which achieves higher levels of FD-nuclease Previous mouse studies found serum DNASE deficiency in activity than Seen in Ifn-yinduced macrophage-conditioned NZ. mice, and attributed this to D1 defect (Macanovic et al., media, conferred Saturated BT activity with both 89 and (1997) Clin. Exp. Immunol. 108, 220-6). In addition, D1 -/- 89T. These results establish that a significant primary defect mice ablate nearly all DNASE zymographic activity in D3-BT activity is present in both the NZ and MRL strains (Napirei et al., (2000) Nat. Genet. 25, 177-81). While D1 L3 of mice, and this finding in independent SLE models Sug can be detected in serum by immunoblot in both mice and gests a role in their Susceptibility to the disease. humans, Zymograms i bands of DNASE activity are likely 0116 Normal D1 L3 Expression in Tissues. Normal D1 L3 DNASE I-related (>30 kDa) (FIG. 25). There are differ protein is detected by anti-D1L3 immunoblot in spleen, ences of uncertain significance in the patterns of Serum liver, and thymus (FIG. 21). In both spleen and bone DNASE activity by Zymogram and levels of activity marrow derived cells, expression of the 28 kDa cleaved between SLE and wild-type mice. The 89T lupus-prone product is present almost eXclusively in adherent popula BXSB mice had more activity by RDA and zymogram (FIG. tions, especially cultured macrophages (FIG.22A). This is 23) than either NZBW or MRL (p<0.001). While serum consistent with other published observations (Baron et al., D1 L3 levels in NZ mice were less than those in MRL (1998) Gene 215, 291-301). The enzyme is inducible in (p>0.05), serum RDA activity in NZBW was not signifi macrophages by activating agents Such as lipopolysaccaride cantly different than wild-type (Macanovic et al., (1997) (LPS), phorbol myristate acetate (PMA), and Ifn-y (FIGS. Clin. Exp. Immunol. 108, 220-6). 22B-D). Treatment of C57BL macrophages induces both 0120) The most striking observation was D1 L3 induction levels of D1 L3 in cell lysates but even more the levels of in splenocytes (4-5 fold) (FIG. 26) and macrophages (2-3 secreted DNASE activity (FIG. 22C). By Zymogram, only fold) (FIG. 27) of all lupus models relative to C57BL a sharp 28 kDa band of activity consistent with D1 L3 is (p<0.01) (FIGS. 23 and 28). Similarly induced levels were observed and only after Ifn-y stimulation. seen in control (C57BL) macrophages after treatment with 0117. Abnormalities in D1 L3 Levels and DNASE Activ PMA or Ifn-y, but unlike lupus mice, induction led a parallel ity in SLE Mice. In cultured macrophages and leukocytes increase in secreted DNASE activity. In contrast, RDAs of from C57BL mice, secreted nuclease activity correlates with media conditioned by SLE Splenocytes and bone-marrow the expression levels of the Ifn-y-inducible D1 L3. Therefore, derived macrophages failed to show increased activity. initially the nuclease activity by RDAs and the levels D1 L3 Thus, relative to expression levels, macrophages and Sple enzyme by western in SLE and C57BL mice were examined. nocytes from both 89 and 89TSLE strains show a defect in Young mice (<12 weeks old) were studied in all cases. While DNASE activity. In macrophage-conditioned media, the the DNASE activity expressed by cultured leukocytes from RDA activity of the 891 strains was significantly lower than lupus mice is similar to that found in C57BL mice, this is C57BL (p<0.01). An additional observation was that the 89T only achieved by a 4-5 fold level of D1 L3 protein induction BXSB mouse, while still relatively deficient, showed sig in Splenocytes and 2-3 fold induction in macrophages. In the nificantly more activity than the 89I lupus mice (p<0.01) MRL and NZ models, this deficiency likely reflects in part (FIG. 27). the presence of the hypomorphic 89I allele, but secondary 0121 The data supports hypothesis in which murine defects are likely involved in these models and in the BXSB models and patients with SLE and normal D1 enzymes, the strain, which has a wild-type D1 L3 allele. Deficiency in lupus-predisposing deficiency of DNASE activity is caused D1L3 activity is predicted to predispose mice to lupus-like by defects in macrophage D1 L3. Despite induction, the disease by interfering in degradation of DNA-containing lupus mice fail either in secretion or have inhibitors of the antigenic material. Finally the results in this Section only enzyme. On fresh isolation, lupus macrophages appear to be refer to RDA-nuclease activity. The RDA nuclease activity in an Ifn-Y induced but D1 L3 impotent state. It is possible describes the enzymes activity against “free' DNA. D1 only that the secondary defect in DNASE activity seen in SLE shows RDA nuclease activity and minimal, if any, BT mice is due to aberrant cytokine milieu, leading to macroph activity. D1 L3 has both, though appears to have less RDA age malfunction. It is also possible that D1 L3 malfunction is activity than DNASE1. In a following section, a defect in BT crucial in dendritic (antigen-presenting cells) or lympho activity in media conditioned by macrophages from SLE cytes. While D1 L3 is clearly expressed by macrophages, mice is described. That activity is dependent but not iden hence a D1 L3 defect in this lineage is the prime candidate tical with RDA activity as shown in prior sections. The table for causing the Susceptibility, the defect could be due to the in FIG. 23, represented in graphic format in FIG. 28, defect in another cell line. summarizes the pixel values observed for RDAs and immu noblots of the various compartments. All the measurements 0122) While 89 lupus strains have significantly lower described in this section refer to this table. macrophage DNASE activity than 89T BXSB and C57BL, US 2004/0138156A1 Jul. 15, 2004

all lupus strains, including the 89T BXSB, have defects of cellular levels of D1 L3 protein in 89 strains not prone to similar magnitude relative to the protein levels (FIGS. 23 spontaneous lupus, such as DBA/2 and C3H. Finally, based and 28). Thus, the 89I mutation is likely not the only cause on linkage data in human SLE (Moser et al., (1998) Proc. for the DNASE defect in SLE mice. In addition, 89I is Natl. Acad. Sci. USA 95, 14869-74), human patients with present in Strains not prone to Spontaneous SLE Such as SLE are predicted to also have primary or secondary D1 L3 DBA/2, A, and C3H. At a molecular level, the observed in abnormalities. Vivo defect in relative activity is greater than that expected 0127. In conclusion, primary and secondary defects in the from 89I allele. Ultimately, the relative DNASE deficiency activity of this leukocyte DNASE are associated with poly in SLE mice must have a secondary (non-allelic) compo genic lupus-prone strains of murine SLE. DNASE1L3 may nent. have the foremost role among nucleases in Suppressing SLE. 0123. The full cause of the relative D1 L3 activity defect Serum RDA activity may not be the most important require in SLE macrophages is unknown, especially in the 89T ment for lupus-prevention, and may explain why Systemi BXSB Strain. The induced cellular expression Suggests a cally administered D1 appeared to be ineffective as therapy failure of Secretion or the presence of inhibitors. No D1 L3 of the disease in the NZB/W F1 model and humans. D1 may inhibitors were apparent in RDAS from serum mixing only be part of the organism's defense against nucleosomal experiments between normal and SLE mice (data not antigen Stimulation, and D1 L3, a circulating actin-resistant shown). Previous studies found anti-D1 antibodies capable macrophage-produced DNASE may be the relevant enzyme of inhibiting D1 activity in MRL mice (Madaio et al., (1996) to Supplement in lupus. Eur: J. Immunol. 26, 3035-41); however, this would not 0128 BT Activity in Lupus and Normal Macrophages. explain a deficiency in media conditioned by macrophages. Ifn-y-dependant induction of D1 L3 levels and secreted 0.124 Macrophage numbers appear expanded in SLE DNase in normal macrophages was demonstrated above (Muller et al., (1991) Eur: J. Immunol. 21, 22.11-7). The (FIG. 22); as expected, this induction is associated with cellular correlate of macrophage DNASE defects in SLE is increased BT activity. Media conditioned by Ifn-y-stimu hypothesized to be deficient degradation of phagocytosed lated C57BL bone marrow-derived macrophagesis confers a material. Defects in macrophage phagocytosis have been barrier to liposomal transfection to HeLa (FIG. 29). described (Licht et al., (2001) Lupus 10, 102-7; Laderach et Increased BT activity was obtained with higher doses of al., D., (1998).J. Leukoc, Biol. 64, 774-80), but these results Ifn-Y (1000 U/ml vs 100U/ml) (FIG. 2B). In the absence of are controversial (Russell et al., (1986) J. Leukoc, Biol. 39, Ifn-y stimulation, macrophages from C57BL, which have 49-62). Clearance of immune complexes, defective in C1q. low baseline D1 L3 levels, had weaker BT activity. Anti-D3 deficient mice, may be a better assay of SLE (Nash et al., immunoblots on cell lysates for this experiment did not (2001) Clin. Exp. Immunol. 123, 196-202; Davies et al., show as a parallel linear induction (FIG. 22C), Suggesting (1992) J. Clin. Invest. 90, 2075-83). Ultimately, the BT that part of the increased DNASE activity is due to secretion assay may be even better at determining who is predisposed or other enhancements of already Synthesized protein. to SLE. In SLE mice, macrophages appear activated due to 0129. If SLE mice were deficient in D1 L3 activity and abnormal levels of circulating cytokines (Alleva et al., D1 L3 was the sole source of BT activity, macrophage (1997).J. Immunol. 159,5610-9); this state could be respon conditioned media from SLE mice would have deficient BT Sible for the induced levels of D1 L3 in SLE mice. D1 L3 is activity. Preliminary experiments comparing MRL and inducible in normal macrophages by activatorS Such as Ifn-y. C57BL macrophages show no difference in BT activity, yet these results were obtained after 7 day in vitro culture in 0.125 Splenocyte and macrophage D1 L3 protein levels GM-CSF (data not show). These experiments suggest that are induced in polygenic murine lupus relative to C57BL. In MRL macrophages can recover from the D1 L3 defect. When addition, the 89I DBA/2 strain, and not the 89T C57BL, SLE can be experimentally induced by the exposure to an extrin media is conditioned for 36 hours by freshly isolated mac sic anti-idiotype manipulation (Mozes et al., (1997) Clin. rophages from NZB/W F1 mice versus C57BL, a qualitative Immunol. Immunopathol. 85, 28-34). Thus likely other sus difference in BT activity is observed (FIG.29). Conditioned ceptibility factors in 89I SLE mice either increase the media from C57BL macrophages, but not NZB/W F1 production or circulation of DNA-associated auto-antigens, hybrids blocks transfection. Serum from these mice did not perhaps by overactive apoptosis, or that additional factors differ in BT activity; 10% fresh serum has BT activity for decrease nuclease activity further. For example, inhibitory both mice, despite the lower RDA-nuclease activity found in autoantibodies to D1 may play this role in MRL mice NZB/W F1 serum (FIG. 28). (Madaio et al., (1996) Eur: J. Immunol. 26, 3035-41). 0.130. In conclusion, macrophages from SLE-prone NZB/W mice are deficient in BT activity (FIG. 29). It 0.126 These observations suggest that introduction of the appears that this paradigm is common to polygenic SLE in 89I allele on to the 30) genetic background of lupus-prone humans, Since in all murine models there are macrophage congenics derived from NZxC57BI crosses (Morel et al., DNASE1L3 defects both in the presence or absence of a (2000) Proc. Natl. Acad. Sci. USA 97, 6670-5) may exac primary defect in DNASE1L3. The following predictions erbate disease. In addition, the 89I allele may have syner would follow from this discovery: gistic lupus-promoting effects with other defects that poten tially alter antigen clearance Such as murine D1 (Napirei et 0131 (1) Primary defects in DNASE1L3 are likely al., (2000) Nat. Genet. 25, 177-81), C1q (Botto et al., (1998) present in some humans with SLE and form part of their Nat. Genet. 19, 56-9), If n-Y receptor knockouts, or SAP null Susceptibility to the disease (Bickerstaff et al., (1999) Nat. Med. 5, 694-7) mice. Experi 0132) (2) Secondary defects in DNASE1L3 BT activity mental induction of nucleosomal autoimmunity with pris are present in some humans with SLE and form part of their tane or other agents may be associated with increased Susceptibility to the disease US 2004/0138156A1 Jul. 15, 2004 16

0133 (3) Enhancement of DNASE1L3 activity in the Vivo, or in vitro functional assays, or by immunodetection. appropriate compartment may prevent development, block For example, a protein or hormone which can transiently progression, or possibly treat SLE. It may be possible to alter metabolic or biochemical parameters can be adminis address the above goals by increasing Serum concentrations tered, and the measure of BT becomes the ability of the of active enzyme by providing this or other enzyme com transduced gene to alter the predicted biochemical param binations with BT activity by either intravenous or other eter. The present model predicts that individuals predisposed delivery Systems. In mice, this may be achieved via intra to SLE because of D1 L3 defects will allow greater levels of peritoneal injection or by other Systemic or local delivery gene transduction to occur. Systems. The enzyme may also need to be delivered to 0137 Analysis of DNASE1L3 sequences in human SLE. appropriate tissue sites that are normally addressed by To determine if mutations potentially leading to D3 defi macrophage Secretion. ciency were present in human SLE, DNA samples were 0134) (4) If D1 L3 defects predispose to SLE and a major obtained from the Lupus Multiplex Registry & Repository biologic role of D1 L3 is a BT activity, then a potential (LMRR) (http://omrf.uokhsc.edu/lupus/). This NIH-spon Side-effect of repeated courses of in Vivo liposomal gene Sored Study maintains data, Serum, and DNA from families therapy could be SLE-like autoimmunity. that have at least two (2) well-characterized members that 0135 (5) Finally, testing for a defect in BT activity may have been diagnosed with lupus. These Samples were also of predict which individuals are at risk for SLE. In mice, the interest because they had shown a significant multipoint assay was described using bone marrow macrophages, these linkage to the 3p21 D3S1766 microsatellite marker with a are not easily obtained from humans. However, human LOD score of 1.68 using a recessive model (Moser et al., D1 L3 is detectable by anti-D1L3 immunoblot in human (1998) Proc. Natl. Acad. Sci. USA 95, 14869-74). Dr. Harley, Serum and peripheral mononuclear cells from normal and LMRR director at the OMRF (Oklahoma Medical Research SLE patients. One possible assay would be the in vitro Foundation), kindly enriched the DNA samples in our pos culture of Such a Sample derived via peripheral phlebotomy, Session with individuals, mostly African-Americans, Show in Similar culture media to that used above, and Sampling of ing linkage. D3S,1766 and DNASE1L3 showed no recom the ability of the Ifn-Y induced conditioned media to confer bination in radiation hybrids. a barrier to liposomal transfection. It may not be necesarry 0.138. The genomic sequence contained in locus to obtain using a purified Sample of macrophages, and the NM 004944 and our previous studies suggest that the gene monocyte-derived populations from the periphery may Suf encoding the 1kb D3 transcript spans approximately 15-20 fice. kilobases. Genomic fragments encompassing all eight D3 0136 (6) An alternative assay may test the in vivo ability exons and the immediate (10-12 bp) peri-exonic sequence of individuals to block an administered liposomal transfec (using the primers in Table 1) were amplified using PFU/ tion “load” in vivo. The proposed test of the in vivo TAO polymerase mixture for greater fidelity. The fragments transfection Susceptibility would require the administration were purified with MinFlute PCR purification spin columns to a live Subject of a D1 L3-Susceptible marker plus reagent, (Amersham), and Subjected to bi-allelic dideoxyfluorescent for example, but not limited to a plasmid marker plus Sequencing with the same primers used for amplification. liposomal reagent or an adenoviral gene transduction Forward and reverse Sequencing was performed using a 96 reagent. The measured variable would be either the distal well format at the SIU Soybean Genome Center at Carbon recovery of the administered agent or a measure of the dale in 96 well format (www.siu.edu/pbgc/). Sequences expression of the transduced agent. For example, it would be were analyzed and edited with DNASTAR SeqmanII (Laser predicted that a Systemic D1 L3 defect would be accompa gene). nied by an increased ability to transduce liposome-mediated 0139. Over 95% of the stated D3 sequence for 50 affected DNA markers. In the present example, a non-allergenic individuals and 33 unaffected relatives (7500 bp) from 29 Soluble and Secreted gene product could be used as a DNA independent pedigrees has been obtained with over 90-95% marker, for example, an individual would be administered certainty. EXOn 4 is being resequenced with internal primers by intravenous route with a plasmid encoding a foreign but due to Sequence ambiguities over a portion of the eXon. non-immunostimulatory peptide or protein. The protein DNA is available for an additional 48 unaffected relatives could be an allelic variant of a normal protein, which does from these pedigrees. Heterozygous changes were ascer not elicit an immune response and yet can be distinguished tained only when the alteration was evident on both Strands. from the endogenous product by either ELISA, or in vivo, ex To date, analysis has identified seven variants (Table 2).

TABLE 1. Human genomic DNASE1L3 sequence and primers

NMOO 4944 NT 005 670 Forward primer Reverse primer

1 1-15

2 15-213 2236 1 49-223 6345 CAGCACTCCAAGCACTGCTGTC AAGTCTGCAGACAGGAGAGAGG

3 2 14-300 22.38805-2238893 TCTGTGGTAAATGACCTCAAAC TGTGTGAACTGGTGGTCAAGTG

4, 299-391 2241551-22416 40 GAATGTTTACTCCAAGATGCAGT, CGATTTATTGGGGCCATGTTCC

Internal primers (exon 4) TACTCTCAAGAGCAGTGT ATTGGTGGCCATGTTCCAGG US 2004/0138156A1 Jul. 15, 2004 17

TABLE 1-continued Human genomic DNASE1L3 sequence and primers NM OO 4944 NT 005 670 Forward primer Reverse primer

5 391-502 2242230-2242342 GCTGACTCAGCTGGGAGGACTT TTAATGGGCTCATGCTCAAGGCT

6 5 O3- 615 22 4 6 002-224 61.14 AGTGGTTTATGAGACCTTGACAG TGCTAGATGGGAATTCGTCTGAC

7 6.15-771 22 49 133-224 9290 CAGTGTGAAAGCATTCTGCAGCA GCACACCAAGCACTGTGGTGAG

8 772-869 22.53677-225.3775 CTTGGTTTGATCTCAGCAATCAC ATCTTTCCAGGACAATGGCATAG

9 870-1079 225 4310-22544 63 GGCATCTCCTAATTTCCATGTCT CCTTCCAATTTGGCTCAAGTCAG

0140 0143. The biologic significance of the mutation (or poly morphism) at IVS6+5 G>T (or G t T at the +5 nucleotide of TABLE 2 the splice donor consensus just after exon 6) has not been established (FIG. 35). It was remarkably common in our Nucleotide African-American pool of SLE (41% of alleles and present NM 004944 Alteration Presumed effect in 55% of patients (FIG. 14). The +5G of the intron is 1. 22.46063 C > G Missense-Ile166Met conserved in >85% of splice donor sites; however, this does 2 22.46069 G > A Silent-Glu168Glu 3 2241596 G > C Silent-Arg92Arg (exon 4) not imply that a splice donor site with +5G mutates in 15% 4 225375.4 A > G Missense-Tyr > Cys at aa 261 (Y261C) of alleles; it states up to 15% exons are not +5G. The most 5 2254337 T> G Missense-Val277Gly common allele in our population, and likely normative in 6 2246119 G > T Affects splice donor consensus IVS6 + 5 other populations, contains a G at this site; thus the Tallele is very likely to be a mutation, and hence could be delete rious. The final effect of intronic mutations in residues other 0.141. The missense changes and the splice donor muta than the immediate 2 residues to the junction (+/-1 and 2) tion were further evaluated. All missense mutations were are difficult to predict a priori. Numerous disease-related first confirmed by re-amplification from original DNA mutations in diverse genes affect residues at a similar Sample, cloning of amplicons, and Sequencing of 6 indepen intronic position, including for diseases Such as, but not dent clones (FIG. 34). In addition, when possible, the limited to: thalassemias (Danckwardt et al., (2002) Blood genotype of other members of the pedigree was examined. 99, 1811-6), Usher syndrome (Bolz et al., (2002) Nat. Genet. The I166M mutation is not being evaluated further since 27, 108-12), phenylketonuria (PKU) http://data.mch.m- pedigree analysis shows that it is part of the haplotype cgill.ca/pahdb new/about.html, 2002), and cystic fibrosis containing the Y261C and Glu 168Glu variants. All three (Highsmith, et al., (1997) Hum. Mutat. 9, 332-8; Bisceglia, variants are present in two unrelated individuals, one et al., (1994) Hum. Mutat. 4, 136-40). The alterations usually affected and the other unaffected. The affected woman with result in intron Skipping, with attendant frame-shifted or the Y261C mutation inherited the mutant IVS6+5 Tallele on insertion product or alternatively lead to a decrease or her other allele from her mother, and the Y261 C from her absence of expression. The compact and highly folded father. Thus she is a compound heterozygote for two differ DNASE proteins are unlikely to tolerate any significant ent mutations, while the unaffected woman carrying the Stretch of amino acids. The association and linkage of the Y261 mutation is homozygous for the more common and mutation to SLE will be further studied by examining the consensus IVS6+5G allele. The effect on activity of the enzyme levels and D3 activity in leukocytes from IVS6+5T Val277Gly variant found in one patient remains to be homozygotes, and by examining RNA from Such patients for analyzed. abnormal transcripts or expression. 0142. To analyze the Y261 C mutation, this mutation was 0144) When control populations are compared for the inserted by mutagenesis into a pcDNA-D3-HA vector, prevalence of the intronic +5 Tallele, the following preva which expresses the full-length human DNASE1L3 fused to lence data is found for the Tallele: 41% of alleles in the hemagglutinin at the C-terminus. The Y261 C enzyme has Studied SLE population; 7% of alleles in a random Sampling complete loSS of function by Zymogram, RDA, and BTassay of Springfield, Ill. (90% Caucasian); 25% of alleles in (FIG. 35). This was not unexpected: the mutation alters a random African American controls. This Suggests that the T highly conserved tyrosine and likely distorts the Structure by allele is more frequent in the African-American population. creating a novel uncoupled cysteine in an extracellular This may be consistent with the LMRR linkage data for protein predicted to contain two disulfide bonds. Since the 3p21 and imply that this is a Strong Susceptibility at least in affected individual is a compound heterozygote for Y261C/ this population. IVS6+5 G>T, this patient may be a complete loss of func tion. However, it serves to recall that the only reported 0145 Since our studied SLE population derived from 26 DNASE1 mutations in human SLE are heterozygous. The pedigrees, analysis of the Samples in the following fashion finding of an independent loSS of function mutation in D3 in showed these results: If each pedigree was counted as a asSociation with the Same phenotype in man and mouse “proband', the allele frequency of T was 41%; when com Serves as compelling evidence that D3 deficiency increases pared to the random African-American value of 25%. A SLE susceptibility. p-value of <0.5 would require a chi-square value of 3.7 for US 2004/0138156A1 Jul. 15, 2004

1 degree of freedom, yet a value of 2.75 is obtained. If the GAG 3' and after initial subcloning into a TA vector, Xho1 above numbers are converted to alleles, then a chi Square of and Kpn1 flanked construct was cloned into pcDNA3.1. 5.6 with a p value of 0.02 is obtained. This strongly suggests that if the observed pattern of allele distribution is found in 0153. The sequences of both the D1 L3 and D1 clones a larger population of affecteds, then significance would be were identical to reference Sequences for the mice genes (for achieved without recourse to pedigree assignment. DNASE1L3 accession if NM 007870 and for DNASE1 accession if NP 034191. 0146 Ultimately, these findings confirm the hypothesis that loSS of function mutations or polymorphisms of 0154) The D3 pCT enzyme was made by mutagenizing Ser289 (TCA to TAA) with a stop codon to truncate D1 L3 DNASE1L3 are present in human SLE. C-terminus (QuikChange XL, Stratagene) using primer 0147 They suggest that in polygenic human SLE, these D3p289-310 AGCTACAGTCTTAAAGGGCCTTC 3' and mutations may be common. These findings Suggest that its reverse-complement, and change confirmed the mutation sequencing or determination of in vivo DNASE1L3 activity by Sequencing. in humans may help diagnose or predict Susceptibility to SLE. Finally, if deficiency is associated with SLE, the 0155 The N196K-D3 cDNA clone was created by point restoration of DNASE1L3 activity in human SLE may mutagenesis using primer GGTGATTTCAAgGCCGGCT. mitigate or prevent the disease. GTAGCTA 3' and its complement. 0156 The mutation was confirmed by the creation of a 0148 Experimental Materials and Methods: novel Hae3 site in the cDNA. The human N191K clone was 0149 Murine Stocks and Genotyping. Mice that were created by mutagenesis of the wild-type (wt) EST 82269 8-weeks-old of C3H, DBA/2, BXSB, C57BL/6, and MRL (Rodriguez et al., (1997) Genomics 42,507-13). Both wt and strains, as well as the NZB/W F1 hybrid were studied. mutant (N191 K) cDNAs (starting at bp 53) were cloned in Purified DNA for PCR based genotyping was obtained from frame to C-terminus of GST protein (PGEX4T3). The LG, AKR, SM, 129, A, BALB/c, and from 25 BXD (C57BL/ N191K mutation was PCR amplified with a primer carrying 6xDBA/2) lines (Jackson Labs). Strain characteristics, an internal Single bp change, and recloning fragment into the including BXD marker data, were obtained from the MGI wt-cDNA. 2.6 database at http://www.informatics.jax.org/. 0157 To create the fusion of N-terminal D1 to the 0150. For sequencing of D1 L3 cDNAs, total liver RNA C-terminal (289-305) D1 L3, the pcDNA3.1-D1 plasmid was was isolated using a guanidinium-phenol method (TRIZOL, mutagenized using primer DNase 1-BGL2F/R to create an Lifetech), and PCR-amplified first with D3F95' GCACT artifactual BGL2 site centered at the next to last codon. The GTCTTCATCCAGCCTG and D3R1O 5' CTTAAGGC Single BGL2 Site located in a non-functional Sector of CTCGCACTCTGGAT, then sequenced with D3F10 5' pcDNA was mutagenized to abolish the restriction site. CCACCACTGCAAAGATGTCC and D3R95' CTTCTGA Next, the terminal ~60 bps (289 to past poly-A tail) of the CATCGAATTTGAGT. For D1, cDNA first amplified from D1 L3 were amplified with D3-CT-Bam F and M13F, creat kidney total RNA with D1F1285' CTGCTGCAGCCGTCT ing a novel upstream BAMH1 site. This Bam-Kpn fragment CAGATTG and D1R1O295' AAGCAGTATGGCTGAACT was cloned into Bgl2-Kpn digested pcDNA3.1-D1-BGL2 GCTC; and sequenced with the same primers. The CDNAS (FIG. 14). were sequenced by ABI sequencers at a core facility (Iowa University Sequencing Facility), and analyzed initially by 0158 To compare the in vitro activity of 89I and 89T BLAST comparisons with Genebank Sequences. enzymes, D1 L3 cDNA (AA 11-305) was cloned in-frame downstream of a prokaryotic GST cassette (pGEX4T) 0151. The genotype at residue 438 was analyzed in (Amersham Pharmacia). Oligonucleotide site directed multiple Strains by amplifying a 48bp fragment Surrounding mutagenesis was used to Synthesize the mutant 89I allele this residue from genomic DNA with the following primers, using the QuikChange kit (Stratagene) and the primers F413 and R461SSP, which mutates residue 441 from A to T MRL-F and MRL-R. For cloning into pcDNA3.1, the prim (FIG. 18). The primers flank residue 438 (italicized in the erS F575-GFP GCAGAGCTGGTTTAGTGAACCGTC 3' underlined SSP1 site AATATT)(FIG. 18). The amplified and D3R 13-Kpn GAGCGTGGTACCTAGGAGCGATTG 3' mutant fragment is susceptible to SSP1, while the normal were used to amplify D1 L3 and introduced into pcDNA3.1 Sequence is resistant to cleavage. Products were analyzed by (Invitrogen) after Nhe 1 and Kpn1 digestion. Mutant coun 1xTAE 8% gel (acrylamide/bis 30:1) electrophoresis using terparts of the pcDNA clone were created with QuikChange mini-Protean II gel apparatus (Bio-Rad). kit. Vector inserts were Subjected to Single Strand Sequencing for confirmation. 0152 Plasmid Constructs. Full-length mice D1 L3 cDNA was amplified from C57BL liver RNA with the SuperScript 0159 Transfection Reagents and Resistance Assays. II system (Life Tech) with addition of 0.1 UPfu polymerase Lipofection of respective pcDNA3.1-D1 or -D3 (2 jgs (Stratagene) using primers F575 GCAGAGCTGGTT. purified with Promega Wizard Plus DNA system) was TAGTGAACCGTC 3' and D3-Kpn GAGCGTGGTAC performed independently into 1-2 million HeLa cells, which CTAGGAGCGATTG 3'. The amplified cDNA was cloned produce a low background of Secreted nuclease activity, into BamH1/Kpn 1 digested eukaryotic expression vector when grown in DMEM with 10% FCS (LifeTech) in 6 well pcDNA3.1 (Invitrogen). Both strands of the amplified prod culture plates (Becton-Dickinson). No difference was ucts were directly Sequenced using dideoxy ABI sequencers. observed in cell morphology between control, D1 and D3 The full-length D1 cDNA was similarly amplified from transfected or exposed cells. Media of the transfected cells kidney RNA using D1F128 CTGCTGCAGCCGTCTCA were conditioned for 36-48 hours and served as Source of the GATTG 3' and D1R1029 TTCGTCATACCGACTTGAC respective enzymes. Nuclease activity in conditioned Super US 2004/0138156A1 Jul. 15, 2004 natant was measured by RDAS, Zymograms and by testing 0.167 Secondary HRP-linked antibodies were used as per activity against Supercoiled p3lueScript (Stratagene). ECL protocol (Amersham Pharmacia). Cell lysates for immunoblots were obtained by boiling in 2% SDS buffer. 0160 To test for the ability of the nucleases to block Per well, 100 ug of protein was electrophoretically separated transfection, the media in individual wells of naive HeLa by 12% SDS-PAGE gel (acrylamide/bis 30:1) at room cells were replaced with conditioned media, and cells trans temperature and 100 V for 3 hours using mini-Protean II gel fected with 2 gs of N1-eGFP plasmid (Clontech) using apparatus, followed by electrotransfer to nitrocellulose various transfection reagents (FIG. 11) and their respective (Sigma) in tissue studies, or Immobilon-P membranes (Mil protocols. GFP expression by immunoblot directly corre lipore) for cell expression studies. Equal loading of protein lated with transfection efficiency in present experiments. samples was determined by BCA Protein Assay (Pierce) Thus, transfection efficiency was compared by densitometric and/or immunoblot of cellular actin expression levels. Mem analysis of GFP immunoblots. branes were blocked for 2 hours in PBS-0.1% Tween-20 01.61 GFP expression was also examined by fluorescent (PBS-T) containing 5% non-fat dry milk, 4% goat serum, microScopy using a FITC filter. For microScopy, cells were and then incubated overnight at 4 C. with PBS-T containing fixed for 5 minutes in 4% paraformaldehyde dissolved into 5% dry milk and either anti-D3 antisera at 1:4000 or the 1% PBS. anti-GFP at 1:2000. Following one 30-minute and three 15-minute washes in PBS-T, membranes were incubated 0162 Reagents. Recombinant mouse GM-CSF and IL-4 with peroxidase-conjugated anti-rabbit antibody at 1:5000 were obtained from R&D Systems. Ifn-y (Genzyme) was (Amersham Pharmacia) for 1 hour at room temperature. kindly donated by Dr. Dennis Crouse (SIUSOM). Chemicals After three 15-minute-washes with PBS-T, ECL detection and enzymes, including Taq polymerase, were from Fisher was performed with X-ray films (Fisher Scientific). Images Scientific (St. Louis) and Promega (Madison). Deionized and photographs were Scanned as 600 dpi Tiff images, and water further purified with a Millipore Milli-Q system analyzed by measuring pixel area and intensity of traced (Millipore) was used. bands, using Scion Image 4.02 software for PC (http:// 0163 Antibody Reagents and Immunoblotting. After www.Scioncorp.com/). 24-36 hours, cells transfected with N1-eGFP were lysed in 0168 Nuclease Assays. Nuclease activity against SDS-buffer and immunoblotted for expression with anti “naked’ or free DNA in CM was quantified by densitometry GFP at 1:5000 dilution (Clontech). To confirm nearly equal of RDAS. For assays of media by murine cells, RDAs were protein loading per lane, anti-f-actin monoclonal antibody performed using conditioned media, after 36 hour incuba at 1:7,000 dilution (Sigma) were prepared. For immunob tion of equal numbers of target cell population. Each con lots, 100 mg protein was eletrophoretically Separated by ditioned media was sampled in triplicate. RDAs for all four 12% SDS-PAGE gel, followed by electrotransfer to Immo mice were performed on a single dish. For RDAS, 2-5 ul of bilon-P membranes (Millipore) for expression studies. conditioned Supernatant was dotted on 100 mm dishes Immunoblotting was performed using ECL detection containing 2% agarose, 250 ug/ml DNA, 0.001% EtBr, 0.5% (Amersham). filtered milk, 10 mM Ca"/MgCl; and 12.5ug/mlkanamy cin. Multiple observations were generated for every Sample 0164. Transfection efficiency was compared, wherever obtained. After 24 hours of 37 incubation, plates are possible, by pixel densitometry of the Scanned images using photographed under UV transillumination; and Scanned Scion Image 4.02 software. photos were analyzed with NIH image. For RDAs, the 0.165 Student's T-test was used to compare paired activity was quantified as the product of mean density and results. area (traced on image) of Zones of clearing. For observations of Serum/urine, 2-5 ul of Serum were dotted on agarose 0166 Rabbit antisera were raised against the KLH-linked plates. mice D3 peptide sequence: KAYDLSEEEALD (Sigma Genosys). The immunizing peptide represent hydrophilic 01.69 Zymograms are in-gel renaturation assays per regions specific for DNAS1L3, and not other DNAS1LS. formed by SDS-PAGE through a gel impregnated with 200 The hD3 peptide KAYDLSEEEALD was used to develop g/ml DNA and ethidium bromide, eluting away SDS with rabbit anti-human D1 L3 Specific antisera, the Sera was tested repeated washes in 25 mM Tris-HCl pH 7.5, then incubating by the ability to recognize by western the expected 34 and for 12-16 hours at 37° C. in 25 mM Tris-HCl pH 7.5 with 29 (signal peptide cleaved) kDa bands in both Serum, 10 mM Ca"/Mg" buffer. Activity is measured as dark peripheral leukocytes, and human D1 L3 transfected cell “bands” of DNA digestion (Rosenthal et al., (1977) Anal. lines (FIG. 32), the absence of this recognition in pre Biochem. 80, 76-90) and quantified using scanned images immune Sera, and the ability of the above peptide to block using NIH Image. Photographed RDA results were scanned the reaction (FIG. 12B) has been found to be useful in 600 dpi Tiff images, and activity was measured as pixel area developing antisera in rabbits against the human protein. times density of traced Zones of clearing (n=3 for each The Specificity of the polyclonal antisera was demonstrated Zone). Student's T-test was used to compare paired results. by the ability to recognize by western the expected 34 and Nuclease activity ratioS were obtained by comparing intra 29 (signal peptide cleaved) kDa bands in both tissues and D3 plate results. RDA activity of control and N196K media was transfected cell lines, the absence of this recognition in faint and comparable to the activity of unconditioned culture pre-immune Sera, and the ability of the above peptide to medium. block the reaction (data not shown). Secondary HRP-linked 0170 Cell-Free Nuclease Activity Assay. Free plasmid antibodies were used as per ECL protocol (Amersham DNA or plasmid complexed with lipofection reagent was Pharmacia). Monoclonal anti-GFP were obtained from incubated with conditioned at 37 C. for 1 hour. The Clontech and anti-b-actin antibodies from Sigma. incubation was terminated with 50 mgM EDTA, and DNA US 2004/0138156A1 Jul. 15, 2004 extracted using Promega Wizard DNA Purification System, compare paired means from expression Studies, while activ eluted in 40 l water, and electrophoresed on a 1% agarose ity assays for mouse-derived Samples were compared using gel. Wilcoxon rank Sum tests. 0171 Prokaryotic Expression and GST Purification. 0173 Therapeutic Uses. The experimental data indicates Human D1 L3-GST expression was performed by growing that D1 and D1 L3 function as non-overlapping nucleases transformed BL21 Cells (Stratagene) at log phase were that protect tissues against exogenous DNA. D1 protects induced in 50 mg/ml IPTG for 5-7 hours at 32°. Bacterial against free DNA, while D1 L3 targets membrane-bound pellets were lysed in 1xSDS-PAGE buffer and loaded on DNA SDS-12% PAGE. 0172 Assays of Murine Cells. For assays of tissues and 0.174. In Lupus, deficient clearance of apoptotic debris by cells, mice were Sacrificed a week after arrival. Three mice DNases and complement has been speculated to lead to were Sacrificed for each Strain or model to obtain the results autoimmunity against nucleosomal antigens. Recently, apo in FIGS. 22 and 28. Serum samples were obtained by tail ptotic bodies themselves, much like DNA-coated liposomes, bleeds and repeated (n=3 per mouse). Splenocytes were have been found to horizontally transduce DNA in vitro. filtered through cell Strainers (Becton Dickinson), and Circulating apoptotic debris or infectious agents, for treated with red cell lysing buffer (Sigma). Splenocytes were example, may resemble the liposomal units targeted by counted using a hemochromocytometer, and plated at 2 D1L3 in these experiments. Accordingly, increasing D1 L3 million cells/ml on plastic 6-well Falcon tissue culture activity by administration of D1 L3 or compounds that dishes (Becton Dickinson) in RPMI with 10% FCS. Bone induce D1 L3 activity may provide treatment for Lupus. marrow-cultures were isolated by flushing femoral and tibial 0.175. The observations also have a number of implica marrow into culture, filtering through cell Strainer, followed tions for gene therapy. Initially, D1 L3 should be the standard by red cell lysis, and then plating population on plastic nuclease to evaluate the in Vivo potential of transfecting 6-well Falcon tissue culture dishes in 2 ml RPMI containing agents. Moreover, blocking this macrophage-Secreted activ 10% FBS, 2 mM HEPES, 50 mM b-mercaptoethanol, 20 ity by administration of agents that prevent expression of ng/mL GM-CSF, and 1 ng/mL IL-4. The cells were incu this enzyme or inhibit its activity may also enhance gene bated at 37 C. in a humidified 5% CO atmosphere with therapy. media changes and removal of non-adherent cells every 3 days. After 7 days of culture, a homogeneous population of 0176 Finally, administration of D1 L3 or D1 L3 inducing adherent cells was obtained. Splenocytes were lysed for agents may provide treatment against membrane bound immunoblots either on isolation or after 36 hours in culture; pathogenic DNA. Suitable D1 L3 inducing agents include, results were similar. Immunoblots for each Sample were for example, interferon-gamma, LPS, Phorbol-myristate performed in duplicate. The Student's t-test was used to acetate and the like.

SEQUENCE LISTING

<160> NUMBER OF SEQ ID NOS: 85

<21 Oc SEQ ID NO 1 <211 LENGTH 301 <212> TYPE PRT ORGANISM: Homo sapiens <400 SEQUENCE: 1

Met Ser Arg Glu Lieu Ala Pro Leu Lleu. Teu Telu Teu Lleu. Ser Ile His 1 5 10 15

Ser Ala Teu Ala Met Arg Ile Ser Phe Asn Wall Arg Ser Phe Gly 25 30

Glu Ser Lys Glin Glu Asp Lys Asn Ala Met Asp Wall Ile Wall Wall 35 40 45

Ile Lys Arg Ile Ile Leu Wal Met Glu Ile Lys Ser Asn 5 O 55 60

Asn Arg Ile Pro Ile Teu Met Glu Lys Lieu. Asn Asn Ser Arg 65 70 75

Arg Gly Ile Thr Tyr Asn Tyr Wall Ile Ser Ser Arg Telu Gly Arg Asn 85 90 95

Thr Tyr Glu Glin Tyr Ala Phe Leu Tyr Glu Lys Leu Wal Ser 100 105 110 Val Lys Arg Ser Tyr His Asn Asp Glu Asp Asp Wal Phe Ala Arg US 2004/0138156A1 Jul. 15, 2004 21

-continued

115 120 125

Glu Pro Phe Wall Ala Glin Phe Ser Leu Pro Ser Asn. Wall Leu Pro Ser 130 135 1 4 0 Leu Val Leu Val Pro Leu. His Thr Thr Pro Glu Thir Ser Val Lys Glu 145 15 O 155 160 Ile Asp Glu Lieu Val Glu Val Tyr Thr Asp Wall Lys His Arg Trip Lys 1.65 170 175 Ala Glu Asin Phe Ile Phe Met Gly Asp Phe Asn Ala Gly Cys Ser Tyr 18O 185 19 O Val Pro Lys Lys Ala Trp Lys Asn. Ile Arg Lieu Arg Thr Asp Pro Arg 195 200 2O5 Phe Val Trp Leu Ile Gly Asp Glin Glu Asp Thir Thr Val Lys Lys Ser 210 215 220 Thr Asn. Cys Ala Tyr Asp Arg Ile Val Lieu Arg Gly Glin Glu Ile Val 225 230 235 240 Ser Ser Val Val Pro Lys Ser Asn Ser Val Phe Asp Phe Glin Lys Ala 245 250 255 Tyr Lys Lieu. Thr Glu Glu Glu Ala Lieu. Asp Val Ser Asp His Phe Pro 260 265 27 O Val Glu Phe Lys Lieu Glin Ser Ser Arg Ala Phe Thr Asn. Ser Lys Lys 275 280 285 Ser Val Thr Leu Arg Lys Lys Thr Lys Ser Lys Arg Ser 29 O 295 3OO

<210> SEQ ID NO 2 &2 11s LENGTH 2.83 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 2 Met Gly Gly Pro Arg Ala Leu Lieu Ala Ala Leu Trp Ala Leu Glu Ala 1 5 10 15 Ala Gly Thr Ala Ala Lieu Arg Ile Gly Ala Phe Asn. Ile Glin Ser Phe 2O 25 30 Gly Asp Ser Lys Val Ser Asp Pro Ala Cys Gly Ser Ile Ile Ala Lys 35 40 45 Ile Pro Ala Gly Tyr Asp Leu Ala Lieu Val Glin Glu Val Arg Asp Pro 50 55 60 Asp Leu Ser Ala Val Ser Ala Lieu Met Glu Glin Ile Asn. Ser Val Ser 65 70 75 8O Glu His Glu Tyr Ser Phe Val Ser Ser Glin Pro Leu Gly Arg Asp Gln 85 90 95 Tyr Lys Glu Met Tyr Leu Phe Val Tyr Arg Lys Asp Ala Val Ser Val 100 105 110 Val Asp Thr Tyr Lieu. Tyr His Asp Tyr Glin Asp Gly Asp Ala Asp Val 115 120 125 Phe Ser Arg Glu Pro Phe Val Val Trp Phe Glin Ser Pro His Thr Ala 130 135 1 4 0 Val Lys Asp Phe Val Ile Ile Pro Leu. His Thir Thr Pro His Glin Ala 145 15 O 155 160 Val Ala Glu Ile Asp Ala Leu Tyr Asp Val Tyr Lieu. Asp Val Ile Asp 1.65 170 175 US 2004/0138156A1 Jul. 15, 2004 22

-continued Lys Trp Gly Thr Asp Asp Met Leu Phe Leu Gly Asp Phe Asn Ala Asp 18O 185 19 O Cys Ser Tyr Val Arg Ala Glin Asp Trp Ala Ala Ile Arg Lieu Arg Ser 195 200 2O5 Ser Glu Val Phe Lys Trp Leu Ile Pro Asp Ser Ala Asp Thr Thr Val 210 215 220 Gly Asn. Ser Asp Cys Ala Tyr Asp Arg Ile Val Ala Cys Gly Ala Arg 225 230 235 240 Leu Arg Arg Ser Lieu Lys Pro Glin Ser Ala Thr Val His Asp Phe Glin 245 250 255 Glu Glu Phe Gly Lieu. Asp Glin Thr Glin Ala Lieu Ala Ile Ser Asp His 260 265 27 O Phe Pro Val Glu Val Thr Leu Lys Phe His Arg 275 280

<210> SEQ ID NO 3 &2 11s LENGTH 323 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 3 Met His Tyr Pro Thr Ala Leu Lleu Phe Lieu. Ile Leu Ala Asn Gly Ala 1 5 10 15 Gln Ala Phe Arg Ile Cys Ala Phe Asn Ala Glin Arg Lieu. Thr Leu Ala 2O 25 30 Lys Wall Ala Arg Glu Glin Wal Met Asp Thir Lieu Val Arg Ile Leu Ala 35 40 45 Arg Cys Asp Ile Met Val Lieu Glin Glu Val Val Asp Ser Ser Gly Ser 50 55 60 Ala Ile Pro Leu Lleu Lleu Arg Glu Ile Asin Arg Phe Asp Asp Ser Gly 65 70 75 8O Pro Tyr Ser Thr Leu Ser Ser Pro Gln Leu Gly Arg Ser Thr Tyr Met 85 90 95 Glu Thr Tyr Val Tyr Phe Tyr Arg Ser His Lys Thr Glin Val Leu Ser 100 105 110 Ser Tyr Val Tyr Pro Asp Pro Glu Asp Val Phe Ser Arg Glu Pro Phe 115 120 125 Val Val Lys Phe Ser Ala Pro Gly Thr Gly Glu Arg Ala Pro Pro Leu 130 135 1 4 0 Pro Ser Arg Arg Ala Leu Thr Pro Pro Pro Leu Pro Ala Ala Ala Glin 145 15 O 155 160 Asn Lieu Val Lieu. Ile Pro Leu. His Ala Ala Pro Lys Ala Val Glu Lys 1.65 170 175 Glu Lieu. Asn Ala Leu Tyr Asp Val Phe Leu Glu Val Ser Glin His Trp 18O 185 19 O Glin Ser Lys Asp Val Ile Leu Lieu Gly Asp Phe Asn Ala Asp Cys Ala 195 200 2O5 Ser Lieu. Thir Lys Lys Arg Lieu. Asp Llys Lieu Glu Lieu Arg Thr Glu Pro 210 215 220 Ser Phe His Trp Val Ile Ala Asp Gly Glu Asp Thr Thr Val Arg Ala 225 230 235 240 Ser Thr His Cys Thr Tyr Asp Arg Val Val Leu. His Gly Glu Arg Cys 245 250 255 US 2004/0138156A1 Jul. 15, 2004 23

-continued

Arg Ser Leu Leu. His Thr Ala Ala Ala Phe Asp Phe Pro Thr Ser Phe 260 265 27 O Glin Leu Lieu. Thr Glu Glu Glu Ala Lieu. Asn. Ile Ser Asp His Tyr Pro 275 280 285 Val Glu Val Glu Lieu Lys Lieu Ser Glin Ala His Ser Val Glin Pro Leu 29 O 295 3OO Ser Lieu. Thr Val Lieu Lleu Lleu Lleu Ser Lieu Lleu Ser Pro Glin Lieu. Cys 305 310 315 320

Pro Ala Ala

<210> SEQ ID NO 4 &2 11s LENGTH 2.82 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 4 Met Arg Gly Met Lys Lieu Lleu Gly Ala Lieu Lieu Ala Leu Ala Ala Lieu 1 5 10 15 Leu Glin Gly Ala Val Ser Lieu Lys Ile Ala Ala Phe Asn. Ile Glin Thr 2O 25 30 Phe Gly Glu Thr Lys Met Ser Asn Ala Thr Leu Val Ser Tyr Ile Val 35 40 45 Gln Ile Leu Ser Arg Tyr Asp Ile Ala Leu Val Glin Glu Val Arg Asp 50 55 60 Ser His Lieu. Thir Ala Val Gly Lys Lieu Lieu. Asp Asn Lieu. Asn. Glin Asp 65 70 75 8O Ala Pro Asp Thr Tyr His Tyr Val Val Ser Glu Pro Leu Gly Arg Asn 85 90 95 Ser Tyr Lys Glu Arg Tyr Leu Phe Val Tyr Arg Pro Asp Glin Val Ser 100 105 110 Ala Val Asp Ser Tyr Tyr Tyr Asp Asp Gly Cys Glu Pro Cys Gly Asn 115 120 125 Asp Thr Phe Asin Arg Glu Pro Ala Ile Val Arg Phe Phe Ser Arg Phe 130 135 1 4 0 Thr Glu Val Arg Glu Phe Ala Ile Val Pro Leu. His Ala Ala Pro Gly 145 15 O 155 160 Asp Ala Wall Ala Glu Ile Asp Ala Leu Tyr Asp Val Tyr Lieu. Asp Val 1.65 170 175 Glin Glu Lys Trp Gly Lieu Glu Asp Wal Met Leu Met Gly Asp Phe Asn 18O 185 19 O Ala Gly Cys Ser Tyr Val Arg Pro Ser Gln Trp Ser Ser Ile Arg Leu 195 200 2O5

Trp h Ser Pro Thr Phe Gln Trp Leu Ile Pro Asp Ser Ala Asp Thr O 215 220 Thr Ala Thr Pro Thr His Cys Ala Tyr Asp Arg Ile Val Val Ala Gly 225 230 235 240 Met Leu Lieu Arg Gly Ala Val Val Pro Asp Ser Ala Leu Pro Phe Asn 245 250 255 Phe Glin Ala Ala Tyr Gly Lieu Ser Asp Gln Leu Ala Glin Ala Ile Ser 260 265 27 O Asp His Tyr Pro Val Glu Val Met Leu Lys 275 280 US 2004/0138156A1 Jul. 15, 2004 24

-continued

<210 SEQ ID NO 5 &2 11s LENGTH 34 &212> TYPE PRT <213> ORGANISM: Mus musculus

<400 SEQUENCE: 5 Ser Asp His Phe Pro Val Glu Phe Lys Leu Gln Ser Ser Arg Ala Phe 1 5 10 15 Thr Asn. Asn Arg Lys Ser Val Ser Lieu Lys Lys Arg Lys Lys Gly Asn 2O 25 30

Arg Ser

<210> SEQ ID NO 6 &2 11s LENGTH 34 &212> TYPE PRT <213> ORGANISM Rat

<400 SEQUENCE: 6 Ser Asp His Phe Pro Val Glu Phe Lys Leu Gln Ser Ser Arg Ala Phe 1 5 10 15 Thr Asn. Ser Arg Lys Ser Val Ser Lieu Lys Lys Lys Lys Lys Gly Ser 2O 25 30

Arg Ser

<210 SEQ ID NO 7 &2 11s LENGTH 34 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 7 Ser Asp His Phe Pro Val Glu Phe Lys Leu Gln Ser Ser Arg Ala Phe 1 5 10 15 Thr Asn. Ser Lys Lys Ser Val Thr Lieu Arg Lys Lys Thr Lys Ser Lys 2O 25 30

Arg Ser

<210 SEQ ID NO 8 &2 11s LENGTH 27 &212> TYPE PRT <213> ORGANISM: Xenopus sp. <400 SEQUENCE: 8 Ser Asp His Phe Pro Ile Glu Val Arg Lieu Lys Glu Ser Lys Arg Pro 1 5 10 15 Thir Ser Arg Arg Arg Lys Tyr Phe Lys Arg Lys 2O 25

<210 SEQ ID NO 9 &2 11s LENGTH 10 &212> TYPE PRT <213> ORGANISM: Bovine

<400 SEQUENCE: 9 Ser Asp His Tyr Pro Val Glu Val Thr Leu 1 5 10

<210> SEQ ID NO 10 US 2004/0138156A1 Jul. 15, 2004 25

-continued

<211& LENGTH: 40 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 10 Ser Asp His Tyr Pro Val Glu Val Glu Lieu Lys Lieu Ser Glin Ala His 1 5 10 15

Ser Wall Glin Pro Leu Ser Lieu. Thir Wall Leu Lleu Lleu Lleu Ser Leu Lieu 2O 25 30 Ser Pro Glin Lieu. Cys Pro Ala Ala 35 40

<210> SEQ ID NO 11 &2 11s LENGTH 998 &212> TYPE DNA <213> ORGANISM: Murine

<400 SEQUENCE: 11 toagtgagcc agg cactgtc. titcatccago citgaagttccc aggagtgcaa agatgtc.cct 60 gcaccoagct tcc.ccacgcc td.gc.citccct gctgctottc atccttgccc tocatgacac 120 cctggcc.cta aggctotgct cottcaatgt gaggtoctitt gag.cgagca agaaggaaaa 18O ccatgaagcc atggatat cattgttgaagat catcaaacgc tigt gaccitta tactgttgat 240 ggaaatcaag gacago agca acaa.catctg. tcc catgctd atggagaagc tigaatggaaa ttcacgaaga agcacaac at acaactatot gattagttct c gacittggaa gaalacacgta 360 caaagagcag tatgcctt.cg totacaagga gaagctdgto tctgtgaaga caaaatacca 420 citaccatgac tat caggatg gag acacaga C gtgtttitcc agg gag.ccct ttgttggtttg 480 gttccattcc ccctttact.g. citgtcaagga cittcgtgatt gtc.cccttgc acacaactcc 540 c gag acctico gttaaagaga tagatgagct g g to gatgtc. tacacggatg tdagaagcca 600 gtggaagaca gagaattitca tottcatggg tatttcaac gocggct gta gctatotic cc 660 caagaaggcc togg cagaa.ca titcgtttgag gacgg accoc aagtttgttt goctoattgg 720 ggaccalagag gacactacgg to aagaagag taccagotgt gcc tatgaca ggattgttgct ttgtggacaa gagatagtica acticcgtggit toccc.gttcc agtgg.cgtot ttgactittca 840 gaaagctitat gacittgtctg aggaggaggc cct ggatgtc. agtgatcact titccagttga 9 OO gtttaa.gcta cagtc.ttcaa gogcc titcac caacaacaga aaatctgttt citctoaaaaa 96.O gagaaaaaaa gocaatc.gct cottaggitatc acgcticcg 998

<210> SEQ ID NO 12 &2 11s LENGTH 310 &212> TYPE PRT <213> ORGANISM: Murine

<400 SEQUENCE: 12 Met Ser Lieu. His Pro Ala Ser Pro Arg Lieu Ala Ser Lieu Lleu Lieu Phe 1 5 10 15 Ile Leu Ala Lieu. His Asp Thr Lieu Ala Lieu Arg Lieu. Cys Ser Phe Asn 2O 25 30 Val Arg Ser Phe Gly Ala Ser Lys Lys Glu Asn His Glu Ala Met Asp 35 40 45 Ile Ile Val Lys Ile Ile Lys Arg Cys Asp Lieu. Ile Leu Lieu Met Glu 50 55 60 US 2004/0138156A1 Jul. 15, 2004 26

-continued

Ile Asp Ser Ser Asn Asn Ile Cys Pro Met Teu Met Glu Lys Telu 65 70 75 8O

Asn Gly Asn Ser Ser Thr Thr Tyr Asn Wall Ile Ser Ser 85 90 95

Arg Telu Gly Arg Asn Thr Glu Glin Phe Wall 100 105 110

Glu Telu Wall Ser Wall Thr Lys His His Asp Glin 115 120 125

Asp Gly Asp Thr Asp Wall Phe Ser Glu Pro Phe Wall Wall Trp Phe 130 135 1 4 0

His Ser Pro Phe Thr Ala Wall Phe Wall Ile Wall Pro Telu His 145 15 O 155 160

Thr Thr Pro Glu Thr Ser Wall Glu Ile Asp Glu Teu Wall Asp Wall 1.65 170 175

Thr Asp Wall Arg Ser Glin Trp Lys Thr Glu Asn Phe Ile Phe Met 18O 185 19 O

Gly Asp Phe Asn Ala Gly Cys Ser Tyr Wall Pro Lys Ala Trp Glin 195 200

Asn Ile Arg Telu Arg Thr Asp Pro Lys Phe Wall Trp Teu Ile Gly Asp 210 215 220

Glin Glu Asp Thr Thr Wall Ser Thr Ser Ala Asp Arg 225 230 235 240

Ile Wall Telu Gly Glin Glu Wall Asn Ser Wall Wall Pro Arg Ser 245 250 255

Ser Gly Wall Phe Asp Phe Glin Ala Asp Teu Ser Glu Glu Glu 260 265 27 O

Ala Telu Asp Wall Ser Asp His Phe Pro Wall Glu Phe Lys Telu Glin Ser 275 280 285

Ser Arg Ala Phe Thr Asn Asn Arg Lys Ser Wall Ser Teu Arg 29 O 295 3OO

Lys Gly Asn Arg Ser 305 310

SEQ ID NO 13 LENGTH 26 TYPE DNA ORGANISM: Artificial FEATURE: OTHER INFORMATION: Mutagenesis Primer <400 SEQUENCE: 13 ggtgatttica aggcc.ggctg. tagcta

SEQ ID NO 14 LENGTH: 31 O TYPE ORGANISM: Artificial FEATURE: OTHER INFORMATION IN196K-D3 cDNA

<400 SEQUENCE: 14 Met Ser Lieu. His Pro Ala Ser Pro Arg Lieu Ala Ser Lieu Lleu Lieu Phe 1 5 10 15 Ile Leu Ala Lieu. His Asp Thr Lieu Ala Lieu Arg Lieu. Cys Ser Phe Asn 25 30 US 2004/0138156A1 Jul. 15, 2004 27

-continued

Val Arg Ser Phe Gly Ala Ser Lys Lys Glu Asn His Glu Ala Met Asp 35 40 45 Ile Ile Val Lys Ile Ile Lys Arg Cys Asp Lieu. Ile Leu Lieu Met Glu 50 55 60 Ile Lys Asp Ser Ser Asn. Asn. Ile Cys Pro Met Leu Met Glu Lys Lieu 65 70 75 8O Asn Gly Asn Ser Arg Arg Ser Thr Thr Tyr Asn Tyr Val Ile Ser Ser 85 90 95 Arg Lieu Gly Arg Asn. Thir Tyr Lys Glu Glin Tyr Ala Phe Val Tyr Lys 100 105 110 Glu Lys Leu Val Ser Val Lys Thr Lys Tyr His Tyr His Asp Tyr Glin 115 120 125 Asp Gly Asp Thr Asp Val Phe Ser Arg Glu Pro Phe Val Val Trp Phe 130 135 1 4 0 His Ser Pro Phe Thr Ala Val Lys Asp Phe Val Ile Val Pro Leu. His 145 15 O 155 160 Thir Thr Pro Glu Thir Ser Val Lys Glu Ile Asp Glu Leu Val Asp Val 1.65 170 175 Tyr Thr Asp Val Arg Ser Gln Trp Llys Thr Glu Asn Phe Ile Phe Met 18O 185 19 O Gly Asp Phe Lys Ala Gly Cys Ser Tyr Val Pro Lys Lys Ala Trp Glin 195 200 2O5 Asn. Ile Arg Lieu Arg Thr Asp Pro Llys Phe Val Trp Lieu. Ile Gly Asp 210 215 220 Glin Glu Asp Thir Thr Val Lys Lys Ser Thir Ser Cys Ala Tyr Asp Arg 225 230 235 240 Ile Val Leu Cys Gly Glin Glu Ile Val Asin Ser Val Val Pro Arg Ser 245 250 255 Ser Gly Val Phe Asp Phe Glin Lys Ala Tyr Asp Leu Ser Glu Glu Glu 260 265 27 O Ala Lieu. Asp Val Ser Asp His Phe Pro Val Glu Phe Lys Lieu Glin Ser 275 280 285 Ser Arg Ala Phe Thr Asn. Asn Arg Lys Ser Val Ser Lieu Lys Lys Arg 29 O 295 3OO Lys Lys Gly Asn Arg Ser 305 310

<210 SEQ ID NO 15 &2 11s LENGTH 23 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: Mutagenesis Primer <400 SEQUENCE: 15 agctacagtc. ttaaagggcc titc 23

<210> SEQ ID NO 16 &2 11s LENGTH: 31 &212> TYPE DNA <213> ORGANISM: Murine

<400 SEQUENCE: 16 gtttaa.gcta cagtc.ttcaa gogcc titcac c 31 US 2004/0138156A1 Jul. 15, 2004 28

-contin ued

<210 SEQ ID NO 17 &2 11s LENGTH 10 &212> TYPE PRT <213> ORGANISM: Murine

<400 SEQUENCE: 17 Phe Lys Leu Glin Ser Ser Arg Ala Phe Thr 1 5 10

<210> SEQ ID NO 18 &2 11s LENGTH 871 &212> TYPE DNA <213> ORGANISM: Mus musculus

<400 SEQUENCE: 18 gctttcagga tgcggtacac agggctaatg ggaac actoc tdaccittggit caacctgctg 60 cagotggctg ggacitctgag aattgcagoc ttcaa.cattc ggactitttgg ggagactaag 120 atgtccaatg citacccticitc. tgtatactitt gtgaaaatcc tgagtc.gcta tgacatcgct 18O gttatccaag aggtoagaga citcccaccitg gttgctgttg ggaagcticct ggatgaactic 240 aatcgggaca aacct gacac citaccgctat gtagt cagtg agcc.gctggg cc.gcaaaag.c tacaaggaac agtaccttitt tgtgtacagg cct gaccagg tgtctattot ggacagotat 360

Caatatgatg atggctgtga accCtgtgga aatgacacct tdag cagaga gccagccatt 420 gttaagttct titt.ccc.cata cactgaggto caagaatttg cgatcgtgcc cittgcatgca 480 gcc.cca acag aagctgtgag tgagatcgac gcc ct citacg atgtttacct agatgtctgg 540

Caaaagtggg gCCt99agga catcatgttc atgggagact totaatgctgg citgcago tac 600 gtoactitcct cc.cagtggto citccattc.gc citt.cggacaa gcc.ccatctt ccagtggctg 660 atccctgaca gtgcggacac cacagtcaca toalacacact gtgct tatga caggattgtg 720 gttgctggag citctgctoca ggctgctgtt gttcc caact cggctgttcc ttittgattitc caag cagaat acggacttitc calaccagotg gctgaagcca totagt gacca ttacccagtg 840 gaggtgacac to agaaaaat citgatgtcat t 871

<210 SEQ ID NO 19 &2 11s LENGTH 303 &212> TYPE PRT <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: Fusion Protein

<400 SEQUENCE: 19 Tyr Thr Gly Leu Met Gl y Thr Leu Lieu. Thir Lieu Wall Asn Lieu Leu Glin 1 5 10 15

Leu Ala Gly Thir Lieu Arg Ile Ala Ala Phe Asn Ile Arg Thr Phe Gly 25 30

Glu Thr Lys Met Ser As in Ala Thr Leu Ser Wall Tyr Phe Val Lys Ile 35 40 45

Leu Ser Arg Tyr Asp Il e Ala Wall Ile Glin Glu Val Arg Asp Ser His 50 55 60

Leu Wall Ala Val Gly Lys Lieu Lieu Asp Glu Lieu Asn Arg Asp Lys Pro 65 70 75

Asp Thr Tyr Arg Tyr Val Val Ser Glu Pro Leu Gly Arg Lys Ser Tyr US 2004/0138156A1 Jul. 15, 2004 29

-continued

85 90 95 Lys Glu Gln Tyr Leu Phe Val Tyr Arg Pro Asp Glin Val Ser Ile Leu 100 105 110 Asp Ser Tyr Glin Tyr Asp Asp Gly Cys Glu Pro Cys Gly Asn Asp Thr 115 120 125 Phe Ser Arg Glu Pro Ala Ile Val Lys Phe Phe Ser Pro Tyr Thr Glu 130 135 1 4 0

Wall Glin Glu Phe Ala Ile Val Pro Leu. His Ala Ala Pro Thr Glu Ala 145 15 O 155 160 Val Ser Glu Ile Asp Ala Leu Tyr Asp Val Tyr Lieu. Asp Val Trp Glin 1.65 170 175 Lys Trp Gly Lieu Glu Asp Ile Met Phe Met Gly Asp Phe Asn Ala Gly 18O 185 19 O Cys Ser Tyr Val Thr Ser Ser Gln Trp Ser Ser Ile Arg Leu Arg Thr 195 200 2O5 Ser Pro Ile Phe Gln Trp Leu Ile Pro Asp Ser Ala Asp Thr Thr Val 210 215 220 Thr Ser Thr His Cys Ala Tyr Asp Arg Ile Val Val Val Arg Ala Leu 225 230 235 240 Leu Glin Ala Ala Val Val Pro Asn Ser Ala Val Pro Phe Asp Phe Glin 245 250 255 Ala Glu Tyr Gly Leu Ser Asn Gln Leu Ala Glu Ala Ile Ser Asp His 260 265 27 O Tyr Pro Val Glu Val Thr Leu Arg Lys Ile Arg Ala Phe Thr Asn Asn 275 280 285 Arg Lys Ser Val Ser Lieu Lys Lys Arg Lys Lys Gly Asn Arg Ser 29 O 295 3OO

<210> SEQ ID NO 20 &2 11s LENGTH 967 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: Fusion Clone

<400 SEQUENCE: 20 citgctgcago C gtc.to agat tdgctttcag gatgcgg tac acagggctaa toggaac act 60 citcaccittgg to aacctgct gcagotggct g g g actotga gaattgcago cittcaac att 120 cgg acttittg g g gagacitaa gatgtccaat gct accotct citgtatactt totgaaaatc 18O citgagtc.gct atgacatcgc tigittatccaa gaggtoa gag acticccacct g gttgctgtt 240 gggaagcticc toggatgaact caatcgggac aaacct gaca cct accgcta totagtcagt 3OO gag.ccgctgg gcc.gcaaaag citacaaggaa cagtaccttt ttgttgtacag goctoaccag 360 gtgtctatto togacagota totaatatgat gatggctgtg aaccotgtgg aaatgacacc 420 ttcagoagag agc.ca.gc.cat tdttaagttc tttitc.cc cat acact gaggit coaagaattit 480 gc gatcgtgc ccttgcatgc agc.cccaa.ca gaagctdtga gtgagat.cga C goccitctac 540 gatgtttacc tagatgtctg goaaaagtgg ggcctggagg acatcatgtt catgg gagac 600 ttcaatgctg gctgcagota cqt cactitcc toccagtggit cotcc attcg cctitcggaca 660 agc.cccatct tccagtggct gatcc ctdac agtgcggaca ccacagt cac atcaacacac 720 tgtgct tatg acaggattgt ggttgctgga gctctgctoc aggctgctgttgttc.ccaac 78O US 2004/0138156A1 Jul. 15, 2004 30

-continued toggctgttc cittittgattt coaag cagaa tacgg actitt coalaccagot goctogaa.gc.c 840 atcagtgacc attacccagt ggaggtgaca citcagaaaga toc gg gcctt caccaacaac 9 OO agaaaatctg tittctotcaa aaagagaaaa aaaggcaatc gct cotaggt atcacgcto c 96.O ggaattic 967

<210> SEQ ID NO 21 &2 11s LENGTH 25 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: Mutagenesis Primer <400 SEQUENCE: 21 cacticagaaa gatctgatgt cattg 25

<210> SEQ ID NO 22 &2 11s LENGTH 29 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: Primer

<400 SEQUENCE: 22 gc ggat.ccgg gcc titcacca acaacagaa 29

<210> SEQ ID NO 23 &2 11s LENGTH 59 &212> TYPE PRT <213> ORGANISM: Murine

<400 SEQUENCE: 23 Leu Arg Ile Ala Ala Phe Asn Ile Arg Thr Phe Gly Glu Thr Lys Met 1 5 10 15 Ser Asn Ala Thr Leu Ser Val Tyr Phe Val Lys Ile Leu Ser Arg Tyr 2O 25 30 Asp Ile Ala Val Ile Glin Glu Val Arg Asp Ser His Leu Val Ala Val 35 40 45 Gly Lys Lieu Lieu. Asp Glu Lieu. Asn Arg Asp Lys 50 55

<210> SEQ ID NO 24 &2 11s LENGTH 59 &212> TYPE PRT <213> ORGANISM: Bovine

<400 SEQUENCE: 24 Leu Lys Ile Ala Ala Phe Asn. Ile Arg Thr Phe Gly Glu Thir Lys Met 1 5 10 15 Ser Asn Ala Thr Lieu Ala Ser Tyr Ile Val Arg Ile Val Arg Arg Tyr 2O 25 30 Asp Ile Val Lieu. Ile Glin Glu Val Arg Asp Ser His Leu Val Ala Val 35 40 45 Gly Lys Lieu Lieu. Asp Tyr Lieu. Asn Glin Asp Asp 50 55

<210> SEQ ID NO 25 &2 11s LENGTH 51 US 2004/0138156A1 Jul. 15, 2004 31

-continued

&212> TYPE PRT <213> ORGANISM: Murine

<400 SEQUENCE: 25 Tyr Val Val Ser Glu Pro Leu Gly Arg Lys Ser Tyr Lys Glu Glin Tyr 1 5 10 15 Leu Phe Val Tyr Arg Pro Asp Glin Val Ser Ile Leu Asp Ser Tyr Glin 2O 25 30 Tyr Asp Asp Gly Cys Glu Pro Cys Gly Asn Asp Thr Phe Ser Arg Glu 35 40 45

Pro Ala Ile 50

<210> SEQ ID NO 26 &2 11s LENGTH 51 &212> TYPE PRT <213> ORGANISM: Bovine

<400 SEQUENCE: 26 Tyr Val Val Ser Glu Pro Leu Gly Arg Asn Ser Tyr Lys Glu Arg Tyr 1 5 10 15 Leu Phe Leu Phe Arg Pro Asn Lys Val Ser Val Leu Asp Thr Tyr Glin 2O 25 30 Tyr Asp Asp Gly Cys Glu Ser Cys Gly Asn Asp Ser Phe Ser Arg Glu 35 40 45

Pro Ala Wall 50

<210 SEQ ID NO 27 <211& LENGTH 24 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: Primer

<400 SEQUENCE: 27 actaagatgt coagtgctac cotc 24

<210> SEQ ID NO 28 <211& LENGTH 24 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: Primer

<400 SEQUENCE: 28 gagggtag ca citggacatct tagt 24

<210 SEQ ID NO 29 <211& LENGTH 24 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: Primer

<400 SEQUENCE: 29 gaac cotgtg gaagtgacac ctitc 24

<210 SEQ ID NO 30 <211& LENGTH 24 US 2004/0138156A1 Jul. 15, 2004 32

-continued

&212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: Primer

<400 SEQUENCE: 30 galaggtgtca citt.ccacagg gttc 24

<210> SEQ ID NO 31 &2 11s LENGTH 18 &212> TYPE DNA <213> ORGANISM: Murine

<400 SEQUENCE: 31 cacalatatac alactatoct 18

<210> SEQ ID NO 32 &2 11s LENGTH 18 &212> TYPE DNA <213> ORGANISM: Murine

<400 SEQUENCE: 32 caca acatac alactatot 18

<210 SEQ ID NO 33 <211& LENGTH 21 &212> TYPE DNA <213> ORGANISM: Murine

<400 SEQUENCE: 33 cgaagaag.ca caatatacaa c 21

<210> SEQ ID NO 34 &2 11s LENGTH 7 &212> TYPE PRT <213> ORGANISM: Murine

<400 SEQUENCE: 34 Arg Arg Ser Thr Ile Tyr Asn 1 5

<210 SEQ ID NO 35 &2 11s LENGTH 2.0 &212> TYPE PRT <213> ORGANISM: Mouse

<400 SEQUENCE: 35 Gly Asin Ser Arg Arg Arg Ser Thr Thr Tyr Asn Tyr Val Ile Ser Ser 1 5 10 15 Arg Lieu Gly Arg 2O

<210 SEQ ID NO 36 &2 11s LENGTH 2.0 &212> TYPE PRT <213> ORGANISM: Mouse

<400 SEQUENCE: 36 Gly Asin Ser Arg Arg Arg Ser Thr Ile Tyr Asn Tyr Val Ile Ser Ser 1 5 10 15 Arg Lieu Gly Arg US 2004/0138156A1 Jul. 15, 2004 33

-continued

<210 SEQ ID NO 37 &2 11s LENGTH 2.0 &212> TYPE PRT <213> ORGANISM Rat

<400 SEQUENCE: 37 Gly Asin Ser Arg Arg Arg Ser Thr Thr Tyr Asn Tyr Val Ile Ser Ser 1 5 10 15 Arg Lieu Gly Arg 2O

<210 SEQ ID NO 38 &2 11s LENGTH 2.0 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 38 Arg Asn. Ser Arg Arg Arg Gly Ile Thr Tyr Asn Tyr Val Ile Ser Ser 1 5 10 15 Arg Lieu Gly Arg 2O

<210 SEQ ID NO 39 &2 11s LENGTH 16 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 39 Asp Ala Pro Asp Thr Tyr His Tyr Val Val Ser Glu Pro Leu Gly Arg 1 5 10 15

<210> SEQ ID NO 40 &2 11s LENGTH 23 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: Primer

<400 SEQUENCE: 40 aatggaaatt cacgaagaag cac 23

<210> SEQ ID NO 41 <211& LENGTH 24 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: Primer

<400 SEQUENCE: 41 cgagaactaa toacatagitt gaat 24

<210> SEQ ID NO 42 <211& LENGTH: 14 &212> TYPE DNA <213> ORGANISM: Homo sapiens <400 SEQUENCE: 42 aagcttacaa gotg 14 US 2004/0138156A1 Jul. 15, 2004 34

-continued <210> SEQ ID NO 43 <211& LENGTH: 14 &212> TYPE DNA <213> ORGANISM: Homo sapiens <400 SEQUENCE: 43 aagcttgcaa gotg 14

<210> SEQ ID NO 44 <211& LENGTH: 14 &212> TYPE DNA <213> ORGANISM: Homo sapiens <400 SEQUENCE: 44 aagcttgcaa gotg 14

<210> SEQ ID NO 45 <211& LENGTH: 14 &212> TYPE DNA <213> ORGANISM: Homo sapiens <400 SEQUENCE: 45 aagcttacaa gotg 14

<210> SEQ ID NO 46 &2 11s LENGTH 16 &212> TYPE PRT <213> ORGANISM: Mouse

<400 SEQUENCE: 46 Phe Glin Lys Ala Tyr Asp Leu Ser Glu Glu Glu Ala Lieu. Asp Wal Ser 1 5 10 15

<210> SEQ ID NO 47 &2 11s LENGTH 16 &212> TYPE PRT <213> ORGANISM Rat

<400 SEQUENCE: 47 Phe Glin Lys Ala Tyr Glu Lieu Ser Glu Glu Glu Ala Lieu. Asp Wal Ser 1 5 10 15

<210> SEQ ID NO 48 &2 11s LENGTH 16 &212> TYPE PRT <213> ORGANISM: Xenopus sp. <400 SEQUENCE: 48 Phe Glin Lys Ala Tyr Lys Lieu. Thr Glu Glu Glu Ala Lieu. Asp Wal Ser 1 5 10 15

<210 SEQ ID NO 49 &2 11s LENGTH 16 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 49 Phe Met Val Ala Tyr Gly Leu Thr Glu Glu Glin Ala Leu Glu Val Ser 1 5 10 15

<210 SEQ ID NO 50 &2 11s LENGTH 16 US 2004/0138156A1 Jul. 15, 2004 35

-continued

&212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 50 Phe Glin Ala Ala Tyr Gly Lieu Ser Asp Gln Leu Ala Glin Ala Ile Ser 1 5 10 15

<210 SEQ ID NO 51 &2 11s LENGTH 16 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 51 Phe Glin Glu Glu Phe Gly Lieu. Asp Glin Thr Glin Ala Lieu Ala Ile Ser 1 5 10 15

<210> SEQ ID NO 52 <211& LENGTH: 12 &212> TYPE DNA <213> ORGANISM: Homo sapiens <400 SEQUENCE: 52 gaggtgagga ct 12

<210 SEQ ID NO 53 <211& LENGTH: 12 &212> TYPE DNA <213> ORGANISM: Homo sapiens <400 SEQUENCE: 53 gaggtgakga ct 12

<210> SSEQ ID NO 54 <211& LLENGTH: 12 &212> TYPE DNA <213> ORGANISM: Homo sapiens <400 SEQUENCE: 54 gaggtgatga ct 12

<210 SEQ ID NO 55 <211& LENGTH 22 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: Primer

<400 SEQUENCE: 55 cago actoca agc actgctg. tc 22

<210 SEQ ID NO 56 <211& LENGTH 22 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: Primer

<400 SEQUENCE: 56 aagttctgcag acaggagaga gg 22

<210 SEQ ID NO 57 <211& LENGTH 22 US 2004/0138156A1 Jul. 15, 2004 36

-continued

&212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: Primer

<400 SEQUENCE: 57 totgtggtaa atgaccitcaa ac 22

<210 SEQ ID NO 58 <211& LENGTH 22 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: Primer

<400 SEQUENCE: 58 tgtgtgaact ggtggtoaag to 22

<210 SEQ ID NO 59 &2 11s LENGTH 25 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: Primer

<400 SEQUENCE: 59 gaatgtttac tottcaagat gcagt 25

<210 SEQ ID NO 60 &2 11s LENGTH 23 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: Primer

<400 SEQUENCE: 60 cgatttattg gtggcc atgt to c 23

<210> SEQ ID NO 61 <211& LENGTH 21 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: Primer

<400 SEQUENCE: 61 tact cittcaa gatgcagtgt t 21

<210> SEQ ID NO 62 &2 11s LENGTH 2.0 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: Primer

<400 SEQUENCE: 62 attggtggcc atgttcCagg 20

<210 SEQ ID NO 63 <211& LENGTH 22 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: Primer US 2004/0138156A1 Jul. 15, 2004 37

-continued

<400 SEQUENCE: 63 gctgacticag citgg gaggac tt 22

<210> SEQ ID NO 64 &2 11s LENGTH 23 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: Primer

<400 SEQUENCE: 64 ttaatgggct catgctcaag got 23

<210 SEQ ID NO 65 &2 11s LENGTH 23 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: Primer

<400 SEQUENCE: 65 agtggtttat gag accittga cag 23

<210 SEQ ID NO 66 &2 11s LENGTH 23 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: Primer

<400 SEQUENCE: 66 tgctagatgg gaattcgtct gac 23

<210 SEQ ID NO 67 &2 11s LENGTH 23 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: Primer

<400 SEQUENCE: 67 cagtgtgaaa goattctgca gca 23

<210 SEQ ID NO 68 <211& LENGTH 22 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: Primer

<400 SEQUENCE: 68 gcacaccalag cactgtggtg ag 22

<210 SEQ ID NO 69 &2 11s LENGTH 23 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: Primer

<400 SEQUENCE: 69 cittggitttga totcagdaat cac 23 US 2004/0138156A1 Jul. 15, 2004 38

-continued

<210 SEQ ID NO 70 &2 11s LENGTH 23 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: Primer

<400 SEQUENCE: 70 atctitt.ccag gacaatggca tag 23

<210 SEQ ID NO 71 &2 11s LENGTH 23 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: Primer

<400 SEQUENCE: 71 ggcatctoct aattitccatg tot 23

<210 SEQ ID NO 72 &2 11s LENGTH 23 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: Primer

<400 SEQUENCE: 72 cct tccaatt toggctdaagt cag 23

<210 SEQ ID NO 73 <211& LENGTH 21 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: Primer

<400 SEQUENCE: 73 gcactgtc.tt catccago: ct g 21

<210> SEQ ID NO 74 <211& LENGTH 22 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: Primer

<400 SEQUENCE: 74 citta aggcct c go actctgg at 22

<210 SEQ ID NO 75 &2 11s LENGTH 2.0 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: Primer

<400 SEQUENCE: 75 ccaccactgc aaagatgtc.c 20

<210 SEQ ID NO 76 <211& LENGTH 21 US 2004/0138156A1 Jul. 15, 2004 39

-continued

&212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: Primer

<400 SEQUENCE: 76 cittctgacat cqaatttgag t 21

<210 SEQ ID NO 77 <211& LENGTH 22 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OOTHER INFORMATION: Primer

<400 SEQUENCE: 77 citgctgcago C gtc.to agat to 22

<210 SEQ ID NO 78 <211& LENGTH 22 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OOTHER INFORMATION: Primer

<400 SEQUENCE: 78 aag cagtatg gctgaactgc tic 22

<210 SEQ ID NO 79 <211& LENGTH 24 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: Primer

<400 SEQUENCE: 79 gcagagctgg tittagtgaac cqtc 24

<210 SEQ ID NO 80 <211& LENGTH 24 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: Primer

<400 SEQUENCE: 80 gag.cgtggta Cctaggagcg attg 24

<210> SEQ ID NO 81 <211& LENGTH 22 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: Primer

<400 SEQUENCE: 81 titcgtoatac cqacttgacg ag 22

<210> SEQ ID NO 82 &2 11s LENGTH 23 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: Mutagenesis Primer US 2004/0138156A1 Jul. 15, 2004 40

-continued

<400 SEQUENCE: 82 agctacagtc. ttaaagggcc titc 23

<210 SEQ ID NO 83 <211& LENGTH: 12 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 83 Lys Ala Tyr Asp Leu Ser Glu Glu Glu Ala Lieu. Asp 1 5 10

<210> SEQ ID NO 84 <211& LENGTH 22 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: Primer

<400 SEQUENCE: 84 citgctgcago C gtc.to agat to 22

<210 SEQ ID NO 85 <211& LENGTH 22 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: Primer

<400 SEQUENCE: 85 gag cagttca gccatact gc tit 22

1. A gene therapy composition, comprising a recombinant 9. A method of testing lipofection efficiency comprising gene to affect the gene therapy, a lipofection reagent, and a exposing a transfection composition to a target cell in the D3-activity-reducing agent. presence of D1 L3. 2. The gene therapy composition according to claim 1, 10. A method of protecting against, treating, or reversing wherein the lipofection reagent is Selected from the group the progression of lupus in a mammal, Said method com consisting of monocationic lipids, polycationic lipids, prising increasing D1 L3 activity in the mammal. DEAE, dextran, lipo-polyamines, and cholesterol. 11. The method of claim 10, wherein D1 L3 activity is 3. The gene therapy composition according to claim 2, increased by intravenous administration of D1 L3 or a D1 L3 wherein the D3-activity-reducing agent is Selected from the inducing agent. group consisting of antibodies, peptides, DNA fragments, 12. The method of claim 11, wherein the D1 L3 inducing and chemicals. agent is interferon-gamma. 4. The gene therapy composition according to claim 2, 13. A method of destroying a pathogenic encapsulated, wherein the D3-activity-reducing agent is Selected from the membrane bound or micellar bound DNA comprising group consisting of monoclonal antibodies, enzymatic increasing D1 L3 activity. inhibitors, and enzymes that target D1L3. 14. The method of claim 13, wherein D1 L3 activity is 5. The gene therapy composition according to claim 4, increased by intravenous administration of D1 L3 or a D1 L3 wherein the D3-activity-reducing agent Selectively inhibits inducing agent. D1 L3 expression, inhibits D1 L3 nuclease activity, inhibits 15. The method of claim 14, wherein the D1 L3 inducing C-terminus activity, complexes D1 L3, or degrades D1 L3. agent is interferon-gamma. 6. An antiviral composition comprising an effective 16. The method of claim 14, wherein the pathogenic amount of D1 L3 or a D1 L3 inducing agent selected from encapsulated, membrane bound or micellar bound DNA is interferon-gamma or LPS. Selected from the group consisting of infectious, mostly viral 7. A chimeric DNase comprising a wild-type DNase particles, oncogenic/mutagenic DNA in apoptotic debris, attached to a D1 L3 C-terminus Selected from Sequences A, and intracellular microbacterial pathogens. B, and C in FIG. 14. 17. A method of treating a disease in a mammal by gene 8. Alipofection efficiency testing composition comprising therapy, Said method comprising the administration of a D1L3. gene therapy composition to the mammal, wherein the gene US 2004/0138156A1 Jul. 15, 2004 therapy composition comprises a recombinant gene to affect 21. The method of treating a disease according to claim the gene therapy, a lipofection reagent, and a D3-activity 17, wherein the disease is located in a tissue directly bathed reducing agent. by circulation with or without a blood-brain barrier. 18. The method of treating a disease according to claim 17, wherein the gene therapy targets vascular organs con 22. A method of identifying an individual having a taining large amounts of reticuloendothelial or immunologic mutation associated with lupus comprising: cells. 19. The method of treating a disease according to claim a) providing a nucleic acid sample from the individual, 18, wherein the reticuloendothelial or immunologic cells are wherein the nucleic acid Sample comprises a nucleic Selected from the group consisting of lymphocytes, mono acid sequence encoding DNASE1L3 or DNASE3; and cyte-related cells and monocyte-derived cells. 20. The method of treating a disease according to claim b) detecting a mutation in the nucleic acid Sequence, 18, wherein the vascular organs are Selected from the group wherein the presence of the mutation identifies the consisting of liver, Spleen thymus, bone marrow, lymphoid individual as having a mutation associated with lupus. tissues, lung, pancreas, gut, kidney, and Vascular endothe lium.