US 2011 001 6543A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2011/0016543 A1 Weinstein et al. (43) Pub. Date: Jan. 20, 2011

(54) GENOMIC EDITING OF INVOLVED Feb. 25, 2010, provisional application No. 61/336,000, IN filed on Jan. 14, 2010, provisional application No. 61/263.904, filed on Nov. 24, 2009, provisional appli (75) Inventors: Edward Weinstein, St. Louis, MO cation No. 61/263,696, filed on Nov. 23, 2009, provi (US); Xiaoxia Cui, St. Louis, MO sional application No. 61/245,877, filed on Sep. 25, (US); Phil Simmons, St. Louis, MO 2009, provisional application No. 61/232,620, filed on (US) Aug. 10, 2009, provisional application No. 61/228, 419, filed on Jul. 24, 2009, provisional application No. Correspondence Address: 61/200.985, filed on Dec. 4, 2008, provisional appli POLSNELLISHUGHART PC cation No. 61/205,970, filed on Jan. 26, 2009. 700 W. 47TH STREET, SUITE 1000 KANSAS CITY, MO 64112-1802 (US) Publication Classification (73) Assignee: SIGMA-ALDRICH CO., St. (51) Int. Cl. Louis, MO (US) GOIN 33/00 (2006.01) AOIK 67/00 (2006.01) (21) Appl. No.: 12/842,999 CI2N 5/10 (2006.01) (52) U.S. Cl...... 800/3; 800/13:800/15; 800/17; (22) Filed: Jul. 23, 2010 800/16; 800/14; 435/325; 435/351; 435/350; Related U.S. Application Data 435/352:435/363; 435/353 (63) Continuation-in-part of application No. 12/592,852, (57) ABSTRACT filed on Dec. 3, 2009. The present invention provides genetically modified animals (60) Provisional application No. 61/343.287, filed on Apr. and cells comprising edited chromosomal sequences encod 26, 2010, provisional application No. 61/323,702, ing inflammation-related . In particular, the animals filed on Apr. 13, 2010, provisional application No. or cells are generated using a nuclease-mediated 61/323,719, filed on Apr. 13, 2010, provisional appli editing process. Also provided are methods of assessing the cation No. 61/323,698, filed on Apr. 13, 2010, provi effects of agents in genetically modified animals and cells sional application No. 61/309.729, filed on Mar. 2, comprising edited chromosomal sequences encoding inflam 2010, provisional application No. 61/308,089, filed on mation-related proteins. Patent Application Publication Jan. 20, 2011 Sheet 1 of 2 US 2011/OO16543 A1

Patent Application Publication Jan. 20, 2011 Sheet 2 of 2 US 2011/OO16543 A1

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GENOMIC EDITING OF GENES INVOLVED nuclease that recognizes a chromosomal sequence encoding IN INFLAMMATION an inflammation-related , and, optionally, at least one donor polynucleotide comprising a sequence encoding an CROSS-REFERENCE TO RELATED inflammation related protein. APPLICATIONS 0006 Yet an additional aspect encompasses a method for 0001. This application claims the priority of U.S. provi assessing the effect of mutant inflammation-related proteins sional application No. 61/343,287, filed Apr. 26, 2010, U.S. on the progression or symptoms of a disease state associated provisional application No. 61/323,702, filed Apr. 13, 2010, with inflammation-related proteins in an animal. The method U.S. provisional application No. 61/323,719, filed Apr. 13, comprises comparing a wild type animal to a genetically 2010, U.S. provisional application No. 61/323,698, filed Apr. modified animal comprising at least one edited chromosomal 13, 2010, U.S. provisional application No. 61/309,729, filed sequence encoding an inflammation-related protein, and Mar. 2, 2010, U.S. provisional application No. 61/308,089, measuring a phenotype associated with the disease state. filed Feb. 25, 2010, U.S. provisional application No. 61/336, 0007 Another aspect encompasses a method for assessing 000, filed Jan. 14, 2010, U.S. provisional application No. the effect of an agent on progression or symptoms of inflam 61/263.904, filed Nov. 24, 2009, U.S. provisional application mation. The method comprises (a) contacting a genetically No. 61/263,696, filed Nov. 23, 2009, U.S. provisional appli modified animal comprising at least one edited chromosomal cation No. 61/245,877, filed Sep. 25, 2009, U.S. provisional sequence encoding an inflammation-related protein with the application No. 61/232,620, filed Aug. 10, 2009, U.S. provi agent, measuring an inflammation-related phenotype, and (c) sional application No. 61/228,419, filed Jul. 24, 2009, and is comparing results of the inflammation-related phenotype in a continuation in part of U.S. non-provisional application Ser. (b) to results obtained from a control genetically modified No. 12/592,852, filed Dec. 3, 2009, which claims priority to animal comprising said edited chromosomal sequence encod U.S. provisional 61/200,985, filed Dec. 4, 2008 and U.S. ing an inflammation-related protein not contacted with the provisional application 61/205,970, filed Jan. 26, 2009, all of agent. which are hereby incorporated by reference in their entirety. 0008. Other aspects and features of the disclosure are described more thoroughly below. FIELD OF THE INVENTION REFERENCE TO COLOR FIGURES 0002 The invention generally relates to genetically modi fied animals or cells comprising at least one edited chromo 0009. The application file contains at least one figure Somal sequence encoding inflammation-related proteins. In executed in color. Copies of this patent application publica particular, the invention relates to the use of a Zinc finger tion with color figures will be provided by the Office upon nuclease-mediated process to edit chromosomal sequences request and payment of the necessary fee. encoding inflammation-related proteins. BRIEF DESCRIPTION OF THE FIGURES BACKGROUND OF THE INVENTION 0010 FIG. 1 presents the DNA sequences of edited Ragl 0003 Inflammation is part of the complex biological loci in two animals. The upper sequence (SEQID NO:5) has response of vascular tissues to harmful stimuli. Such as patho a 808 bp deletion in exon 2, and the lower sequence (SEQID gens, damaged cells, or irritants. Inflammation is a protective NO:6) has a 29 bp deletion in exon 2. The exon sequence is attempt by the organism to remove the injurious stimuli and to shown in green; the target site is presented in yellow, and the initiate the healing process. A large variety of proteins are deletions are shown in dark blue. involved in inflammation, and any one of them is open to a 0011 FIG. 2 presents the DNA sequences of edited Rag2 genetic mutation which impairs or otherwise dysregulates the loci in two animals. The upper sequence (SEQID NO:25) has normal function and expression of that protein. Without a 13 bp deletion in the target sequence in exon3, and the lower inflammation, wounds and infections would never heal. How sequence (SEQ ID NO:26) has a 2 bp deletion in the target ever, chronic inflammation can also lead to a host of diseases. sequence in exon 2. The exon sequence is shown in green; the Examples of disorders associated with inflammation include: target site is presented in yellow, and the deletions are shown acne Vulgaris, asthma, hay fever, atheroscloris, autoimmune in dark blue. diseases, chronic inflammation, chronic prostatitis, glomeru lonephritis, hyperSensitivities, inflammatory bowel diseases, DETAILED DESCRIPTION OF THE INVENTION pelvic inflammatory disease, reperfusion injury, rheumatoid 0012. The present disclosure provides a genetically modi , transplant rejection, Vasculitis, interstitial cystitis. It fied animal or animal cell comprising at least one edited is for that reason that inflammation is normally closely regu chromosomal sequence encoding a protein associated with lated by the body. What are needed are animal models with inflammation. The edited chromosomal sequence may be (1) these proteins genetically modified to provide research tools inactivated, (2) modified, or (3) comprise an integrated that allow the elucidation of mechanisms underlying devel sequence. An inactivated chromosomal sequence is altered opment and progression of inflammation. Such that a functional protein is not made. Thus, a genetically modified animal comprising an inactivated chromosomal SUMMARY OF THE INVENTION sequence may be termed a "knock out' or a “conditional 0004 One aspect of the present disclosure encompasses a knockout.” Similarly, a genetically modified animal compris genetically modified animal comprising at least one edited ing an integrated sequence may be termed a “knockin' or a chromosomal sequence encoding an inflammation-related “conditional knock in.” As detailed below, a knock in animal protein. may be a humanized animal. Furthermore, a genetically 0005. A further aspect provides a non-human embryo modified animal comprising a modified chromosomal comprising at least one RNA molecule encoding a Zinc finger sequence may comprise a targeted point mutation(s) or other US 2011/001 6543 A1 Jan. 20, 2011

modification Such that an altered protein product is produced. is inactivated and a functional inflammation-related protein is The chromosomal sequence encoding the protein associated not produced. The inactivated chromosomal sequence com with inflammation generally is edited using a Zinc finger prises no exogenously introduced sequence. Such an animal nuclease-mediated process. Briefly, the process comprises may be termed a “knockout.” Also included herein are geneti introducing into an embryo or cell at least one RNA molecule cally modified animals in which two, three, or more chromo encoding a targeted Zinc finger nuclease and, optionally, at Somal sequences encoding inflammation-related proteins are least one accessory polynucleotide. The method further com inactivated. prises incubating the embryo or cell to allow expression of the 0015. In another embodiment, the genetically modified Zinc finger nuclease, wherein a double-stranded break intro animal may comprise at least one edited chromosomal duced into the targeted chromosomal sequence by the Zinc sequence encoding an inflammation-related protein such that finger nuclease is repaired by an error-prone non-homolo the sequence is over-expressed and a functional inflamma gous end-joining DNA repair process or a homology-directed tion-related protein is over-produced. For example, the regu DNA repair process. The method of editing chromosomal latory regions controlling the expression of the inflammation sequences encoding a protein associated with inflammation related protein may be altered such that the inflammation using targeted Zinc finger nuclease technology is rapid, pre related protein is over-expressed. cise, and highly efficient. 0016. In yet another embodiment, the genetically modi fied animal may comprise at least one chromosomally inte (I) Genetically Modified Animals. grated sequence encoding an inflammation-related protein. 0013. One aspect of the present disclosure provides a For example, an exogenous sequence encoding an ortholo genetically modified animal in which at least one chromo gous or an endogenous inflammation-related protein may be Somal sequence encoding an inflammation-related protein integrated into a chromosomal sequence encoding an inflam has been edited. For example, the edited chromosomal mation-related protein such that the chromosomal sequence sequence may be inactivated Such that the sequence is not is inactivated, but wherein the exogenous sequence encoding transcribed and/or a functional inflammation-related protein the orthologous or endogenous inflammation-related protein is not produced. Alternatively, the chromosomal sequence may be expressed or over-expressed. In such a case, the may be edited Such that the sequence is over-expressed and a sequence encoding the orthologous or endogenous inflam functional inflammation-related protein is over-produced. mation-related protein may be operably linked to a promoter The edited chromosomal sequence may also be modified Such control sequence. Alternatively, an exogenous sequence that it codes for an altered inflammation-related protein. For encoding an orthologous or endogenous inflammation-re example, the chromosomal sequence may be modified Such lated protein may be integrated into a chromosomal sequence that at least one nucleotide is changed and the expressed without affecting expression of a chromosomal sequence. For inflammation-related protein comprises at least one changed example, an exogenous sequence encoding an inflammation amino acid residue (missense mutation). The chromosomal related protein may be integrated into a “safe harbor , sequence may be modified to comprise more than one mis such as the Rosa26 locus, HPRT locus, or AAV locus, wherein sense mutation Such that more than one amino acid is the exogenous sequence encoding the orthologous or endog changed. Additionally, the chromosomal sequence may be enous inflammation-related protein may be expressed or modified to have a three nucleotide deletion or insertion such over-expressed. In one iteration of the disclosure, an animal that the expressed inflammation-related protein comprises a comprising a chromosomally integrated sequence encoding single amino acid deletion or insertion, provided Such a pro an inflammation-related protein may be called a “knock-in'. tein is functional. The modified inflammation-related protein and it should be understood that in such an iteration of the may have altered substrate specificity, altered enzyme activ animal, no selectable marker is present. The present disclo ity, altered kinetic rates, and so forth. Furthermore, the edited Sure also encompasses genetically modified animals in which chromosomal sequence encoding an inflammation-related 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, protein may comprise a sequence encoding an inflammation 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or more related protein integrated into the genome of the animal. The sequences encoding inflammation-related proteins are inte chromosomally integrated sequence may encode an endog grated into the genome. enous inflammation-related protein normally found in the 0017. The chromosomally integrated sequence encoding animal, or the integrated sequence may encode an ortholo an inflammation-related protein may encode the wild type gous inflammation-related protein, or combinations of both. form of the inflammation-related protein. Alternatively, the The genetically modified animal disclosed herein may be chromosomally integrated sequence encoding an inflamma heterozygous for the edited chromosomal sequence encoding tion-related protein may comprise at least one modification an inflammation-related protein. Alternatively, the geneti such that an altered version of the inflammation-related pro cally modified animal may be homozygous for the edited tein is produced. In some embodiments, the chromosomally chromosomal sequence encoding an inflammation-related integrated sequence encoding an inflammation-related pro protein. tein comprises at least one modification Such that the altered 0014. In one embodiment, the genetically modified animal version of the protein causes inflammation. In other embodi may comprise at least one inactivated chromosomal sequence ments, the chromosomally integrated sequence encoding an encoding an inflammation-related protein. The inactivated inflammation-related protein comprises at least one modifi chromosomal sequence may include a deletion mutation (i.e., cation such that the altered version of the inflammation deletion of one or more nucleotides), an insertion mutation related protein protects against inflammation. (i.e., insertion of one or more nucleotides), or a nonsense 0018. In an additional embodiment, the genetically modi mutation (i.e., Substitution of a single nucleotide for another fied animal may be a "humanized animal comprising at least nucleotide Such that a stop codon is introduced). As a conse one chromosomally integrated sequence encoding a func quence of the mutation, the targeted chromosomal sequence tional human inflammation-related protein. The functional US 2011/001 6543 A1 Jan. 20, 2011

human inflammation-related protein may have no corre the inflammation-related proteins may be identified by sponding ortholog in the genetically modified animal. Alter obtaining expression profiles of the genes encoding the natively, the wild-type animal from which the genetically proteins using genomic techniques including but not limited modified animal is derived may comprise an ortholog corre to DNA microarray analysis, serial analysis of gene expres sponding to the functional human inflammation-related pro sion (SAGE), and quantitative real-time polymerase chain tein. In this case, the orthologous sequence in the “human reaction (Q-PCR). ized animal is inactivated Such that no functional protein is made and the “humanized animal comprises at least one 0021. By way of non-limiting example, inflammation-re chromosomally integrated sequence encoding the human lated proteins include but are not limited to the proteins listed inflammation-related protein. For example, a humanized ani in Table A. mal may comprise an inactivated abat sequence and a chro mosomally integrated human ABAT sequence. Those of skill TABLE A in the art appreciate that “humanized animals may be gen Edited Chromosomal erated by crossing a knockout animal with a knock in animal Sequence Encoded Protein comprising the chromosomally integrated sequence. A4GALT CD77 0019. In yet another embodiment, the genetically modi ABL1. ABL1 fied animal may comprise at least one edited chromosomal ACE angiotensin converting sequence encoding an inflammation-related protein such that enzyme, CD143 the expression pattern of the protein is altered. For example, ACIN1 Acinus ADAM17 CD156b, TNFa. converting regulatory regions controlling the expression of the protein, enzyme Such as a promoter or binding site, may be ADAM8 CD156a, ADAM8 altered such that the inflammation-related protein is over ADCY1 adenylyl cyclase I ADCY2 adenylyl cyclase II produced, or the tissue-specific or temporal expression of the ADCY4 adenylyl cyclase IV protein is altered, or a combination thereof. Alternatively, the ADCYS adenylyl cyclase V expression pattern of the inflammation-related protein may ADCY6 adenylyl cyclase VI be altered using a conditional knockout system. A non-limit ADORA1 adenosine A1 ADORA2A adenosine receptor A2A ing example of a conditional knockout system includes a ADORA2B adenosine receptor A2B Cre-lox recombination system. A Cre-lox recombination sys ADORA3 adenosine receptor A3 tem comprises a Cre recombinase enzyme, a site-specific ADRA2A alpha2-adrenergic receptor A DNA recombinase that can catalyze the recombination of a ADRA2C alpha2-adrenergic receptor C ADRB2 beta2-adrenergic receptor nucleic acid sequence between specific sites (lox sites) in a ADRBK1 GRK2, G-protein receptor nucleic acid molecule. Methods of using this system to pro kinase 2 duce temporal and tissue specific expression are known in the AGER RAGE art. In general, a genetically modified animal is generated AKAPS AKAPS AKR1C3 PGFS, F-prostanoid with loX sites flanking a chromosomal sequence, such as a synthase chromosomal sequence encoding an inflammation-related AKT1 AKT protein. The genetically modified animal comprising the loX AKT2 AKT flanked chromosomal sequence encoding an inflammation AKT3 AKT ALCAM CD166, activated leukocyte related protein may then be crossed with another genetically cell adhesion molecule modified animal expressing Cre recombinase. Progeny ani ALOX12 ALOX12 mals comprising the loX-flanked chromosomal sequence and ALOX12B ALOX13 the Cre recombinase are then produced, and the lox-flanked ALOX15 ALOX15 ALOX1SB ALOX16 chromosomal sequence encoding an inflammation-related ALOX5 ALOX5 protein is recombined, leading to deletion or inversion of the ALOXSAP ALOXSAP chromosomal sequence encoding the protein. Expression of ALS2 ALS2 Cre recombinase may be temporally and conditionally regu ANPEP CD13 lated to effect temporally and conditionally regulated recom APAF1 Apaf1 ARAF A-Raf bination of the chromosomal sequence encoding an inflam ARHGAP1 Cdc42 mation-related protein. ATF1 ATF-1 ATF2 ATF-2 ATF3 ATF3 (a) Inflammation-Related Proteins ATF4 ATF4 0020. The present disclosure comprises editing of any B3GAT1 CD57 BAD BAD chromosomal sequences that encode proteins associated with BAK1 BAK inflammation. The inflammation-related proteins are typi BAX BAX cally selected based on an experimental association of the BBC3 BBC3 inflammation-related protein to an inflammation disorder. BCAR1 CAS For example, the production rate or circulating concentration BCL10 BCL10 of an inflammation-related protein may be elevated or BCL2 Bcl-2 depressed in a population having an inflammation disorder relative to a population lacking the inflammation disorder. BCL2L10 BCL2L10 Differences in protein levels may be assessed using pro BCL2L11 BCL2L11 teomic techniques including but not limited to Western blot, BCL2L12 BCL2L12 immunohistochemical staining, enzyme linked immunosor bent assay (ELISA), and mass spectrometry. Alternatively,

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TABLE A-continued TABLE A-continued

Edited Chromosomal Edited Chromosomal Sequence Encoded Protein Sequence Encoded Protein CXCL11 CXCL11 CXCL12 CXCL12 CXCL13 CXCL13 D86 CXCL14 CXCL14 CXCL16 CXCL16 CXCL2 CXCL2 CXCL3 CXCL3 D96 CXCLS CXCLS D97 CXCL9 CXCL9 D99 CXCR6 CXCR6 DC2 CYBB cytochrome b-245, beta DC37 CYCS Cytochrome C DHS CYSLTR1 CYSLTR DKN1A CYSLTR2 CYSLTR DKN1B DAP DAP EACAM1 DAP3 EACAM3 DAPK1 EACAMS DAPP1 EACAM6 Duffy blood group, EACAM8 receptor EBPB Daxx CHOP FD RIG CAD FI CAD FLAR I Diablo HUK fk Kalpha DPEP AS1 AS1 DPEP ITA ITA C E C 3 DPEP SH S CD26 TED2 bp/p300-interacting ansactivator MKPS CKLF PAC1 CMA1 CMA1 COP1 COP1 MKP1,2,3,4 COX1 cytochrome c oxidase 1 or MKP1,2,3,4 cyclooxygenase 1 (COX1) 27 COX2 cytochrome c oxidase 2 endothelin receptor type A (COX2) endothelin receptor type B CPAMD8 CPAMD8 eEF2K CR CR Epidermal CR2 CR2 receptor CRADD RAIDD EGF receptor CREB1 CRE PKR. CREBBP CBP/p300 CRE CREM CRK CRK CRP CRP EN CRSP2 DRIP1SO CSF1 CSF1R M-CSF Receptor CSF GM-CSF CSF2RB GM-CSF receptor, beta, low-affinity D D FADD G-CSF Receptor Fas associated factor 1 Csk FAIM CK2 Fas CK2 FasL CK2 gA receptor cystatin F gE receptor I, high affinity Cytotoxic T-Lymphocyte FCER1 g (Fc epsilon R1g) Antigen 4 (CTLA4, CD152) gE receptor II, CD23 CTNNB1 beta-catenin CTNND1 catenin delta 1 CD64c, Fc gamma receptor CTSS cathespin S C CTTN cortactin Low affinity CX3CL1 Chemokine (C-X3–C motif) immunoglobulingamma Fc ligand 1 (CX3CL1) region receptor II-a Chemokine (C-X3–C motif) FCGR2 receptor 1 (CX3CR1) gG receptor IIB, (FCGR2B CXCL1 CXCL10 CXCL10

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TABLE A-continued TABLE A-continued

Edited Chromosomal Edited Chromosomal Encoded Protein Sequence Encoded Protein interleukin 1 family, RF8 RF8 member 6 (epsilon) SGF3G RF9 IL1R7 TGA1 egrin A1, CD49a IL1E8 TGA2 egrin A2, CD49b interleukin 1 family, TGA2B egrin, alpha2b member 9 TGA3 egrin A3, CD49c interleukin 1 receptor, type I TGA4 D49D, VLA-4 IL-1RTLR TGAS egrin 5, CD49e TGA6 egrin A6, CD49f TGAD D 1 1 C TGAE egrin alpha E, CD103 TGAL D11A TGAM TGAV egrin AV, CD51 TGAX D11c interleukin 21 receptor TGB1 D29, fibronectin receptor TGB2 D18 interleukin 22 receptor, TGB3 egrin B3, CD61 TGB4 egrin B4, CD104 TGB7 integrin B7, CD103b interleukin 23 receptor TK L2-inducible T-cell kinase AK1 AK1 AK2 AK2 AK3 AK3 receptor, AM2 iunctional adhesion molecule 2 interleukin 28A (, AM3 iunctional adhesion lambda 2) molecule 3 interleukin 28B (interferon, UN lambda 3) UND L29 (interferon, KCNH8 lambda 1) AA1271 interleukin 2 receptor, R2DS4 KIR2DS4 R3DL2 KIR3DL2 interleukin 2 receptor, beta R3DL3 KIR3DL3 interleukin 2 receptor, TLG Kit ligand CD161 s

CD94 IL3Ralpha NKG2D Interleukin-4 (IL-4) karyopherin alpha 1 receptor (importin alpha 5) interleukin 5 Ras interleukin 5 receptor, alpha LAG-3 LAIR1 Interleukin-6 (IL-6) LAT interleukin 6 receptor GP130 LCK SLP-76 interleukin 7 receptor leukocyte cell-derived chemotaxin 2 leukemia inhibitory factor leukemia inhibitory factor receptor LILRA1 CD85i, LIR-6 LILRA2 CD85h, ILT1 LILRA3 CD85c, ILT6 LILRA4 CD85g. ILT7 LILRAS CD85f, ILT11 LILRA6 CD85b, ILT8 CD85, ILT2 CD85d, ILT4 CD85a, ILT5 LILRB4 CD85k, ILT3 CD85K, ILT3 CD85m, ILT10 PINCH1 Lamin A LRPB7 -alpha US 2011/001 6543 A1 Jan. 20, 2011

TABLE A-continued TABLE A-continued

Edited Chromosomal Edited Chromosomal Sequence Encoded Protein Sequence Encoded Protein MUC1 CD227, mucin-1 LTBR lymphotoxin-beta receptor c-Myc MYCN N-Myc LY96 MD-2 MYD88 MYD88 LYN Lyn MYH10 myosin lib MADD MADD MYH4 beta-catenin MAL MAL MYH9 myosin lia MALT1 MALT1 MYL6 myosin, light polypeptide 6 MAP2K1 MEK1,2 NALP1 CARD7 MAP2K2 MEK1,2 NALP12 NALP12 MAP2K3 MKK3 NALP2 NALP2 MAP2K4 MKK4 NALP6 NALP6 MAP2K6 MKK6 NCAM1 CDS6 MAP2K7 MKK7 NCF2 neutrophil cytosolic factor 2 MAP3K1 MEKK1 NCOA1 NCOA1 MAP3K14 NIK NCOA2 MAP3K3 MEKK3 coactivator 2 MAP3KS ASK1 NCR1 NKP46 MAP3K6 ASK2, MAP3K6 NCR2 NKP44 MAP3K7 TAK1 MAP3K7IP1 TAB1 NFAT5 NFAT5 MAP3K7IP2 TAB2 NFATC1 NFATC1 MAP3K8 Cot NFATC2 NFATC2 MAP4K1 HPK1, hematopoietic NFATC3 NFATC3 progenitor kinase 1 NFATC4 NFATC4 MAPK1 ERK1 NFIL3 NF-IL-3 MAPK11 p38 MAPK beta NFKB1 NF-kB p105 MAPK12 p38 MAPK gamma MAPK13 p38 MAPK delta NFKBIA IkappaB alpha MAPK14 p38 MAPK alpha NFKBIB IkappaB beta MAPK3 ERK2 NFKBIE IkappaB-epsilon MAPK8 JNK1 NFRKB NFRKB MAPK9 MAPK9 MAPKAPK2 MAPKAPK2 NMI NMI MAPKAPK3 MAPKAPK3 NOD2 (CARD15) nucleotide-binding MAPKAPKS PRAK oligomerization domain MARCO MARCO containing 2 (NOD2) MASP1 MASP1 NOS2A INOS MASP2 MASP2 NOS3 eNOS MAX Max MBL2 MBL NR3C1 nuclear receptor 3C, MCL Mc1 MDM2 MDM2 NR4A1 MEF2A MEF2A NR4A2 Nuclear receptor 4A2 MEF2B MEF2B NRAS Ras MEF2C MEF2C NRIP1 NRIP1 MEF2D MEF2D NTSE CD73 MEF MEFV OAS1 OAS1 MENA ENAH OAS2 OAS2 MGST2 microsomal glutathione S OPRD1 delta opioid receptor transferase 2 (DOR) MGST3 microsomal glutathione S OPRK1 kappa opioid receptor transferase 3 (KOR) MICA MIC-A OPRM1 mu opioid receptor (MOR) MICB MIC-B OSM MIF MIF OSMR MKNK1 MINK1 PAG1 PAG MLCK MLCK PAK1 PAK MMP1 MMP1 PARP1 PARP MMP10 MMP10 PAX5 PAX5 MMP12 MMP12 PBEF1 Pre- enhancing MMP14 MMP14 actor MMP19 MMP19 MMP2 MMP2 PDE1A phosphodiesterase 1A MMP7 MMP7 PDE1B phosphodiesterase 1B MMP9 MMP9 PDE1C phosphodiesterase 1C PDE2A phosphodiesterase 2A MS4A2 IgE receptor I, beta PDE3A phosphodiesterase 3A Subunit PDE3B phosphodiesterase 3B MSR1 scavenger PDE4A phosphodiesterase 4A receptor 1 PDE4B phosphodiesterase 4B MST1R macrophage stimulating 1 PDE4C phosphodiesterase 4C receptor, CDw136 PDE4D phosphodiesterase 4D US 2011/001 6543 A1 Jan. 20, 2011

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Edited Chromosomal Edited Chromosomal Sequence Encoded Protein Sequence Encoded Protein PDGFB Platelet-derived growth PRF1 Perforin-1 actor B PRG2 proteoglycan 2 PDGFRA Platelet-derived growth PRKACA PKA catalytic subunit actor receptor A alpha PDGFRB Platelet-derived growth PRKACB PKA catalytic subunit beta actor receptor B PRKACG PKA catalytic subunit PDPK1 PDK1 gamma PECAM1 PECAM PRKAR1A PKA regulatory subunit 1 PFC Factor P alpha PGDS PGDS PRKAR2A PKA regulatory subunit 2 PGLYRP1 PGLYRP1 alpha PGLYRP2 Peptidoglycan recognition PRKAR2B PKA regulatory subunit 2 protein 2 beta PGLYRP3 Peptidoglycan recognition PRKCA PKCalpha protein 3 PRKCB1 PKCbeta PGLYRP4 Peptidoglycan recognition PRKCD PKCdelta protein 4 PRKCE PKCepsilon PLAS1 PIAS PRKCQ PKCtheta PIAS2 PIAS2 PRKCZ PKCaeta PLAS3 PIAS3 PRKDC Protein kinase, DNA PLAS4 PIAS4 activated, catalytic PIGR Poly-Ig receptor polypeptide1 PIK3AP1 PI3KAP1, BCAP PRKRA PRKRA PIK3CA PI3K 110 PRSS16 protease, serine, 16 PIK3CB PI3K 110 PRTN3 Proteinase 3 PIK3CD PI3K 110 PSMA1 PSMA1 PIK3R1 PI3K 85 PSMB5 PSMB5 PIK3R2 PI3K 85 PSMB9 PSMB9 PIK3R3 PI3K 85 PSME PSME1 PIK3RS PI3K 101 PSME PSME2 PILRB Paired immunoglobulin PTAFR platelet-activating factor ike receptor B receptor PLA2G2A PLA3 PTEN PTEN PLA2G2D phospholipase A2 G2D PTGDR PTGDR PLA2G4A cPLA2 PTGDS PGDS PLAUR CD87 PTGER1 PTGER1 PLCB1 phospholipase B1 PTGER2 PTGER2 PLCB2 phospholipase B2 PTGER3 PTGER3 PLCB3 phospholipase B3 PTGER4 PTGER4 PLCB4 phospholipase B4 PTGES PGES PLCG1 PLC PTGES2 PGES PPARA Peroxisome proliferator PTGES3 prostaglandin E synthase 3 activated receptor alpha PTGFR PTGFR (PPAR-alpha), or nuclear PTGIR PTGIR receptor subfamily 1, PTGIS PGIS group C, member 1 PTK2 FAK (NR1C1) PPARBP PPAR binding protein PTPN PTP1B PPARG PPAR PTPN11 SHP2 PPARGC1A PPARGC1A PTPN13 Fas-associated PPBP CXC chemokine ligand 7 phosphatase-1 PPM1A protein phosphatase 1A, PTPN2 TC-PTP Mg2+ PTPN22 Protein tyrosine XX P1 CA C P1 phosphatase, non XX P1CB C P1 receptor type 22 XX P1CC C P1 (lymphoid), (PTPN22) PPP1R 7 PP1 PP2A PTPN6 SHP XX P2CA C P2 PTPN7 PTPN7 XX P2CB C P2 PTPNS1 PTPNS1 XX P2R1A C P2 PTP-PEST PTPN12 PPP2R1B protein phosphatase 2 R1 PTPRC CD45 beta PTPRT PTPRT PPP2R2B protein phosphatase 2 R2 PTPRK Protein tyrosine beta phosphatase receptor type K PPP2R3A PP1 PP2A PTPRU Protein tyrosine PPP3CA Calcineurin phosphatase receptor type U PPP3CB Calcineurin PTX3 pentraxin-3, TNFAIP5 PPP3CC Calcineurin PXN Paxillin PPP3R1 Calcineurin PYCARD ASCCARDS PPP3R2 Calcineurin RAC1 Rac PRDX1 peroxiredoxin 1 RAC2 Rac PRDX2 peroxiredoxin 2 RAET1E ULBP4. PRDX4 peroxiredoxin 4 RAF1 c-Raf US 2011/001 6543 A1 Jan. 20, 2011 10

TABLE A-continued TABLE A-continued

Edited Chromosomal Edited Chromosomal Sequence Encoded Protein Sequence Encoded Protein RAG1 Rag-1 SIGLEC10 SIGLEC10 RAG2 Rag-2 SIGLECS Siglec-5, CD170 RAP1A Rap1a. SIGLECT AIRM1 RAP1GAP Rap1GAP SIPA1 SIPA1 RAPGEF1 C3G SIRPB1 CD172b RAPGEF3 EPAC SIRPG CD172g RASA1 Ras GAP SITPEC SITPEC RASGRP1 Ras GRP SLA2 Src-like adapter protein-2 RASSFS RAPL SLAMF1 SLAMF1 REL c-REL SLAMF6 SLAMF6 RELA NF-kB p65 SLAMF7 SLAMF7 RELB ReB SLAMF9 SLAMF9 RFX1 regulatory factor X, 1 SLC22A1 (OCT1) Solute carrier family 22 RFX4 regulatory factor X, 4 member 1 (SLC22A1) RFXANK regulatory factor X- SLC3A2 CD98 associated ankyrin- SLC7AS CD98 containing protein SOCS1 SOCS1 RGS1 RGS1 SOCS2 SOCS2 RHEB Rheb SOCS3 SOCS3 RHOA RhoA SOCS4 SOCS4 RHOH RhoEH SOCS5 SOCS5 RIPK1 RIP SOCS6 SOCS6 RIPK2 RIPK2 SOD1 SOD1 RIPK3 RIPK3 SOD2 SOD2 ROCK1 ROCK1 SOS1 SOS ROCK2 ROCK2 SOS2 SOS RPS6KA1 p90RSK SPN CD43, leukosialin RPS6KA4 MSK1 SPTAN1 Fodrin RPS6KAS MSK2 SRC Src. RPS6KB1 p70S6K STAT1 STAT1 RPS6KB2 p70S6K STAT2 STAT2 S100A12 S100A12 STAT3 STAT3 S100A8 S1OOA8 STAT4 STAT4 S100A9 S100A9 STATSA STATSA SARM1 SARM1 STATSB STATSB SCARF1 Scavenger receptor class STAT6 STAT6 F, member 1 SYK Syk SCARF2 Scavenger receptor class SYNGAP1 Syn GAP F, member 2 TACR1 Tachykinin receptor 1 SCGB1A1 Unteroglobulin TANK TANK SCGB3A1 secretoglobin 3A1 TAP1 TAP1 SCN9A Sodium channel, voltage- TAP2 TAP2 gated, type X, alpha TAPBP TAP binding protein (SCN9A) TBK1 NAK SDPR. serum deprivation TBX21 T-box response (TBX21) (phosphatidylserine TBXA2R TBXA2R binding protein) TBXAS1 TXS SECTM1 Secreted and TCF7 Transcription factor 7 transmembrane 1 TCF8 Transcription factor 8 SELE E-selectin TCIRG1 T-cell immune regulator 1 SELL L-selectin, CD62L TEC Tec SELP P-selectin, CD62P TFEB TFEB SELPLG P-selectin Ligand TGFA TGF-alpha SEMA4D CD100 TGFB1 TGF-beta1 SERPINA1 SERPINA1 TGFB2 TGF-beta2 SERPINAS SERPINAS TGFB3 TGF-beta SERPINC1 SERPINC1 TGFBR1 Type I Receptor SERPIND1 SERPIND1 TGFBR2 Type II Receptor SERPINE1 SERPINE1 TGIF TGIF SERPINF2 SERPINF2 TGIF2 TGFB-induced factor 2 SERPING1 SERPING1 THEM4 CTMP SGK serum glucocorticoid THPO thrombopoietin regulated kinase THY1 CD7 SH2B1 SH2-B PH domain TICAM1 TICAM1, TRIF containing signaling TICAM2 TICAM2 mediator 1 (SH2BPSM1) TIRAP TIRAP SH2D1A SAP TLN1 Talin SH2D1B EAT-2 TLN2 Talin

SHC1 SHC TLR2 TLR2 SIGLEC1 CD169, Siglec-1 TLR3 TLR3 US 2011/001 6543 A1 Jan. 20, 2011

TABLE A-continued TABLE A-continued

Edited Chromosomal Edited Chromosomal Sequence Encoded Protein Sequence Encoded Protein

TLR4 XCR1 XCR1 TLRS YWHAB 14-3-3beta TLR6 YWHAG 14-3-3gamma TLR7 YWHAH 14-3-3theta TLR8 YWHAQ 14-3-3eta TLR9 YWHAZ 14-3-3Zeta FA ZAP70 Zap70 alpha (TNF-alpha) FAIP3 A2O FAIP6 TNFAIP6 0022. The identity of the inflammation-related protein FRSF1 OA DR4, TRAIL-R2 FRSF1 OB DR5, TRAIL-R1 whose chromosomal sequence is edited can and will vary. In FRSF10C DCR1, TRAIL-R3 preferred embodiments, the inflammation-related proteins FRSF1 OD DCR2, TRAIL-R4 whose chromosomal sequence is edited may be the FRSF11A FR4, TRANCE FRSF11B FRSF11B chemoattractant protein-1 (MCP1) encoded by the Cer2 FRSF12A FRSF12A, TWEAK-R gene, the C-C chemokine receptor type 5 (CCR5) encoded by FRSF13B FRSF13B, CD267 the CerS gene, the IgG receptor IIB (FCGR2b, also termed FRSF13C FRSF13C, BAFF-R CD32) encoded by the Fcgr2b gene, the Fc epsilon R1g FRSF14 FRSF14, LIGHT-R FRSF17 FSF17, BCM, CD269 (FCER1g) protein encoded by the Fcerlg gene, the forkhead FRSF19 FRSF19 box N1 transcription factor (FOXN1) encoded by the FOXN1 FRSF1A -R1, CD120a gene, Interferon-gamma (IFN-Y) encoded by the IFNg gene, FRSF1B F-R2, CD120b interleukin 4 (IL-4) encoded by the IL-4 gene, perforin-1 FRSF21 6 FRSF25 RR3, APO-3 encoded by the PRF-1 gene, the cyclooxygenase 1 protein TNFRSF4, OX40 (COX1) encoded by the COX1 gene, the cyclooxygenase 2 CD27, TNFRSF7 protein (COX2) encoded by the COX2 gene, the T-box tran TNFRSF9, 4-1BB scription factor (TBX21) protein encoded by the TBX21 APO-2L, TRAIL RANKL gene, the SH2-B PH domain containing signaling mediator 1 APO-3L, TWEAK, DR3L protein (SH2BPSM1) encoded by the SH2B1 gene (also BAFF termed SH2BPSM1), the fibroblast growth factor receptor 2 FSF14, LIGHT, CD258 (FGFR2) protein encoded by the FGFR2 gene, the solute FSF15, TL1A FSF18 carrier family 22 member 1 (SLC22A1) protein encoded by FSF4, OX40L, CD252 the OCT1 gene (also termed SLC22A1), the peroxisome FSF7, CD70, CD27L proliferator-activated receptor alpha protein (PPAR-alpha, FSF8, CD153, CD3OL also termed the nuclear receptor Subfamily 1, group C, mem FSF9, 4-1BB-L FAIP3 interacting ber 1: NR1C1) encoded by the PPARA gene, the phosphatase ein 1 and tensin homolog protein (PTEN) encoded by the PTEN LLIP gene, interleukin 1 alpha (IL-1C...) encoded by the IL-1A gene, . s interleukin 1 beta (IL-1B) encoded by the IL-1B gene, inter TRADD TRAF1 leukin 6 (IL-6) encoded by the IL-6 gene, TRAF2 (IL-10) encoded by the IL-10 gene, alpha TRAF3 (IL-12C.) encoded by the IL-12A gene, interleukin 12 beta TRAFS TRAF6 (IL-12B) encoded by the IL-12B gene, (IL-13) TREM1 encoded by the IL-13 gene, interleukin 17A(IL-17A, also TREM2 termed CTLA8) encoded by the IL-17A gene, interleukin TCRgamma variable 9 17B(IL-17B) encoded by the IL-17B gene, interleukin 17C TSC TSC2 (IL-17C) encoded by the IL-17C gene interleukin 17D (IL TTRAP 17D) encoded by the IL-17D gene interleukin 17F (IL-17F) TXK tyrosine kinase encoded by the IL-17F gene, interleukin 23 (IL-23) encoded interleukin 14, taxilin alpha by the IL-23 gene, the chemokine (C-X3-C motif) receptor 1 TYK2 protein (CX3CR1) encoded by the CX3CR1 gene, the DAP12 chemokine (C-X3-C motif) ligand 1 protein (CX3CL1) UBE2IN encoded by the CX3CL1 gene, the recombination activating ULBP3 gene 1 protein (RAG1) encoded by the RAG1 gene, the WASP recombination activating gene 2 protein (RAG2) encoded by Waw WCAM1 the RAG2 gene, the protein kinase, DNA-activated, catalytic Vinculin polypeptide1 (PRKDC) encoded by the PRKDC (DNAPK) VEGF gene, the protein tyrosine phosphatase non-receptor type 22 villin 2 (eZrin) wPreB protein (PTPN22) encoded by the PTPN22 gene, tumor B7-E4 necrosis factor alpha (TNFC) encoded by the TNFA gene, the X-box binding protein 1 nucleotide-binding oligomerization domain containing 2 Lymphotactin protein (NOD2) encoded by the NOD2 gene (also termed CARD15), or the cytotoxic T-lymphocyte antigen 4 protein US 2011/001 6543 A1 Jan. 20, 2011

(CTLA4, also termed CD152) encoded by the CTLA4 gene. 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more disrupted In an exemplary embodiment, the genetically modified ani chromosomal sequences encoding an inflammation-related mal is a rat, and the edited chromosomal sequence encoding protein and Zero, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, the inflammation-related protein is as listed in Table B. 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more chromosomally integrated sequences TABLE B encoding the disrupted inflammation-related protein. 0024. The edited or integrated chromosomal sequence Edited Chromosomal NCBI Reference may be modified to encode an altered inflammation-related Sequence Encoded Protein Sequence protein. A number of mutations in inflammation-related chro Ccr2 monocyte chemoattractant NM 021866 mosomal sequences have been associated with inflammation. protein-1 (MCP1) For instance, the Delta 32 mutation in CCR5 results in the CcrS C-C chemokine receptor type NM 053960 5 (CCR5) genetic deletion of the CCR5 gene, which plays a role in COX1 cytochrome c oxidase 1 or NM 017.043 inflammatory responses to infection. Homozygous carriers of cyclooxygenase 1 (COX1) this mutation are resistant to HIV-1 infection. Missense and COX2 cytochrome c oxidase 2 NM 017232 truncating mutations in perforin-1, Such as W374stop (i.e. (COX2) CTLA4 Cytotoxic T-Lymphocyte NM O31674 tryptophan at position 219 is changed to stop codon produc Antigen 4 (CTLA4, CD152) ing a truncated polypeptide), V50M (i.e. valine at position 50 CX3CL1 Chemokine (C-X3–C NM 134455 is changed to a methionine) and I224D (i.e. isoleucine at motif) ligand 1 (CX3CL1) position 224 is changed to aspartate), have been shown to CX3CR1 Chemokine (C-X3–C NM 133534 motif) receptor 1 (CX3CR1) cause familial hemophagocytic lymphohistiocytosis (FHL). FCER1G FCER1g (Fe epsilon R1g) NM 00131 OO1 Missense mutations or copy numbergains of FGFR2 gene are FCGR2B gG receptor IIB, (FCGR2b or NM 175756 associated with Crouzon syndrome, Pfeiffer syndrome, Cran CD32) iosynostosis, Apert Syndrome, Jackson-Weiss syndrome, FGFR2 Fibroblast growth factor NW 001084773 receptor 2 (FGFR2) Beare-Stevenson cutis gyrata syndrome, Saethre-Chotzen FNG interferon-gamma (IFN-) NM 13888O syndrome, and syndromic craniosynostosis. Other associa L-10 interleukin-10 (IL-10 or NM O12854 tions of genetic variants in inflammation-associated genes L10), also known as human synthesis inhibitory and disease are known are known in the art. See, for example, actor (CSIF) Loza et al. (2007) PLoS One 10:e1035, the disclosure of L-12A Subunit alpha of interleukin NM O53390 which is incorporated by reference herein in its entirety. 2 L-12B Subunit beta of interleukin 12 NM 022611 (b) Animals L-13 interleukin 13 (IL-13) NM 053828 L17A interleukin 17A NM 001106897 0025. The term “animal,” as used herein, refers to a non L17B interleukin 17B NM 053789 human animal. The animal may be an embryo, a juvenile, or L-17C interleukin 17C XM 002725399, an adult. Suitable animals include vertebrates such as mam XM 001078615 L-17D interleukin 17D XM 001079675 mals, birds, reptiles, amphibians, and fish. Examples of Suit L-17F interleukin 17F NM 001015O11 able mammals include without limit rodents, companion ani L1A interleukin 1, alpha NM 017019 mals, livestock, and primates. Non-limiting examples of L-1B interleukin-1 beta (IL-1beta) NM 031512 L-23 interleukin 23 NM 130410 rodents include mice, rats, hamsters, gerbils, and guinea pigs. L-4 interleukin-4 (IL-4) NM 201270 Suitable companion animals include but are not limited to L-6 interleukin-6 (IL-6) NM 012589 cats, dogs, rabbits, hedgehogs, and ferrets. Non-limiting NOD2 (CARD15) nucleotide-binding NM 001106172 examples of livestock include horses, goats, sheep, Swine, oligomerization domain containing 2 (NOD2) cattle, llamas, and alpacas. Suitable primates include but are PPARA Peroxisome proliferator- NM 013196 not limited to capuchin monkeys, chimpanzees, lemurs, activated receptor alpha macaques, marmosets, tamarins, spider monkeys, Squirrel (PPAR-alpha), or nuclear monkeys, and Vervet monkeys. Non-limiting examples of receptor Subfamily 1, group C, member 1 (NR1C1) birds include chickens, turkeys, ducks, and geese. Alterna PRF1 Perforin-1 NM tively, the animal may be an invertebrate Such as an insect, a PTEN phosphatase and tensin NM 031606 nematode, and the like. Non-limiting examples of insects homolog (PTEN) include Drosophila and mosquitoes. An exemplary animal is PTPN22 Protein tyrosine NM 001106460 phosphatase, non-receptor a rat. Non-limiting examples of Suitable rat Strains include type 22 (lymphoid), Dahl Salt-Sensitive, Fischer 344, Lewis, Long Evans (PTPN22) Hooded, Sprague-Dawley, and Wistar. Non-limiting RAG1 recombination activating XM 00107.9242 examples of commonly used rat Strains suitable for genetic gene 1 (RAG1) SH2B1 SH2-B PH domain containing NM 001048180, manipulation include Dahl Salt-Sensitive, Fischer 344, signaling mediator 1 NM 134456 Lewis, Long Evans Hooded, Sprague-Dawley and Wistar. In (SH2BPSM1) another iteration of the invention, the animal does not com SLC22A1 (OCT1) Solute carrier family 22 NM 012697 prise a genetically modified mouse. In each of the foregoing member 1 (SLC22A1) TBX21 T-box transcription factor NM 001107043 iterations of suitable animals for the invention, the animal (TBX21) does not include exogenously introduced, randomly inte TNFA tumor necrosis factor-alpha NM 012675 grated transposon sequences. (TNF-alpha) (c) Inflammation-Related Proteins 0023 The animal or cell may comprise 1, 2, 3, 4, 5, 6, 7, 8, 0026. The inflammation-related protein may be from any 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, of the animals listed above. Furthermore, the inflammation US 2011/001 6543 A1 Jan. 20, 2011

related protein may be a human inflammation-related protein. 0030. When mammalian cell lines are used, the cell line Additionally, the inflammation-related protein may be a bac may be any established cell line or a primary cell line that is terial, fungal, or plant protein. The type of animal and the not yet described. The cell line may be adherent or non Source of the protein can and will vary. As an example, the adherent, or the cell line may be grown under conditions that genetically modified animal may be a rat, cat, dog, or pig, and encourage adherent, non-adherent or organotypic growth the inflammation-related protein may be human. Alterna using standard techniques known to individuals skilled in the tively, the genetically modified animal may be a rat, cat, or art. Non-limiting examples of Suitable mammalian cell lines pig, and the inflammation-related protein may be canine. One include Chinese hamster ovary (CHO) cells, monkey kidney of skill in the art will readily appreciate that numerous com CVI line transformed by SV40 (COS7), human embryonic binations are possible and are encompassed by the present kidney line 293, baby hamster kidney cells (BHK), mouse invention. In an exemplary embodiment, the genetically Sertoli cells (TM4), monkey kidney cells (CVI-76), African modified animal is a rat, and the inflammation-related protein green monkey kidney cells (VERO), human cervical carci is human. noma cells (HelLa), canine kidney cells (MDCK), buffalo rat 0027. Additionally, the inflammation-related gene may be liver cells (BRL 3A), human lung cells (W138), human liver modified to include a tag or reporter gene or genes as are cells (Hep G2), mouse mammary tumor cells (MMT), rat well-known. Reporter genes include those encoding select hepatoma cells (HTC), HIH/3T3 cells, the human U2-OS able markers such as chloramphenicol acetyltransferase osteosarcoma cell line, the human A549 cell line, the human (CAT) and neomycin phosphotransferase (neo), and those K562 cell line, the human HEK293 cell lines, the human encoding a fluorescent protein such as green fluorescent pro HEK293T cell line, and TRI cells. For an extensive list of tein (GFP), red fluorescent protein, or any genetically engi mammalian cell lines, those of ordinary skill in the art may neered variant thereof that improves the reporter perfor refer to the American Type Culture Collection catalog mance. Non-limiting examples of known such FP variants (ATCC(R), Mamassas, Va.). include EGFP, blue fluorescent protein (EBFP, EBFP2, AZur 0031. In still other embodiments, the cell may be a stem ite, mKalama1), cyan fluorescent protein (ECFP, Cerulean, cell. Suitable stem cells include without limit embryonic stem CyPet) and yellow fluorescent protein derivatives (YFP Cit cells, ES-like stem cells, fetal stem cells, adult stem cells, rine, Venus, YPet). For example, in a genetic construct con pluripotent stem cells, induced pluripotent stem cells, multi taining a reporter gene, the reporter gene sequence can be potent stem cells, oligopotent stem cells, and unipotent stem fused directly to the targeted gene to create a gene fusion. A cells. reporter sequence can be integrated in a targeted manner in the targeted gene, for example the reporter sequences may be (III) Zinc Finger-Mediated Genomic Editing integrated specifically at the 5' or 3' end of the targeted gene. The two genes are thus under the control of the same promoter 0032. In general, the genetically modified animal or cell elements and are transcribed into a single messenger RNA detailed above in sections (I) and (II), respectively, is gener molecule. Alternatively, the reporter gene may be used to ated using a Zinc finger nuclease-mediated genome editing monitor the activity of a promoter in a genetic construct, for process. The process for editing a chromosomal sequence example by placing the reporter sequence downstream of the comprises: (a) introducing into an embryo or cell at least one target promoter Such that expression of the reporter gene is nucleic acid encoding a Zinc finger nuclease that recognizes a under the control of the target promoter, and activity of the target sequence in the chromosomal sequence and is able to reporter gene can be directly and quantitatively measured, cleave a site in the chromosomal sequence, and, optionally, (i) typically in comparison to activity observed under a strong at least one donor polynucleotide comprising a sequence for consensus promoter. It will be understood that doing so may integration flanked by an upstream sequence and a down or may not lead to destruction of the targeted gene. stream sequence that share substantial sequence identity with either side of the cleavage site, or (ii) at least one exchange (II) Genetically Modified Cells polynucleotide comprising a sequence that is substantially identical to a portion of the chromosomal sequence at the 0028. A further aspect of the present disclosure provides cleavage site and which further comprises at least one nucle genetically modified cells or cell lines comprising at least one otide change; and (b) culturing the embryo or cell to allow edited chromosomal sequence encoding an inflammation expression of the Zinc finger nuclease Such that the Zinc finger related protein. The genetically modified cell or cell line may nuclease introduces a double-stranded break into the chromo be derived from any of the genetically modified animals dis Somal sequence, and wherein the double-stranded break is closed herein. Alternatively, the chromosomal sequence cod repaired by (i) a non-homologous end-joining repair process ing an inflammation-related protein may be edited in a cell as Such that an inactivating mutation is introduced into the chro detailed below. The disclosure also encompasses a lysate of mosomal sequence, or (ii) a homology-directed repair pro said cells or cell lines. cess such that the sequence in the donor polynucleotide is 0029. In general, the cells will be eukaryotic cells. Suit integrated into the chromosomal sequence or the sequence in able host cells include fungi or yeast, such as Pichia, Saccha the exchange polynucleotide is exchanged with the portion of romyces, or Schizosaccharomyces; insect cells, such as SF9 the chromosomal sequence. cells from Spodoptera frugiperda or S2 cells from Drosophila 0033 Components of the zinc finger nuclease-mediated melanogaster, and animal cells, such as mouse, rat, hamster, method are described in more detail below. non-human primate, or human cells. Exemplary cells are mammalian. The mammalian cells may be primary cells. In (a) Zinc Finger Nuclease general, any primary cell that is sensitive to double strand breaks may be used. The cells may be of a variety of cell 0034. The method comprises, in part, introducing into an types, e.g., fibroblast, myoblast, T or B cell, macrophage, embryo or cell at least one nucleic acid encoding a Zinc finger epithelial cell, and so forth. nuclease. Typically, a Zinc finger nuclease comprises a DNA US 2011/001 6543 A1 Jan. 20, 2011

binding domain (i.e., Zinc finger) and a cleavage domain (i.e., for example, U.S. Pat. Nos. 6,607,882; 6,534.261 and 6,453, nuclease). The DNA binding and cleavage domains are 242, the disclosures of which are incorporated by reference described below. The nucleic acid encoding a Zinc finger herein in their entireties. nuclease may comprise DNA or RNA. For example, the 0038 Exemplary methods of selecting a zinc finger rec nucleic acid encoding a Zinc finger nuclease may comprise ognition region may include phage display and two-hybrid mRNA. When the nucleic acid encoding a zinc finger systems, and are disclosed in U.S. Pat. Nos. 5,789,538; 5,925, nuclease comprises mRNA, the mRNA molecule may be 5' 523: 6,007,988: 6,013,453; 6,410,248; 6,140,466; 6,200,759; capped. Similarly, when the nucleic acid encoding a Zinc and 6,242,568; as well as WO 98/37186: WO98/53057; WO finger nuclease comprises mRNA, the mRNA molecule may 00/27878; WOO1/88.197 and GB 2,338,237, each of which is be polyadenylated. An exemplary nucleic acid according to incorporated by reference herein in its entirety. In addition, the method is a capped and polyadenylated mRNA molecule enhancement of binding specificity for Zinc finger binding encoding a Zinc finger nuclease. Methods for capping and domains has been described, for example, in WO 02/077227. polyadenylating mRNA is known in the art. 0039 Zinc finger binding domains and methods for design 0035 (i) Zinc Finger Binding Domain and construction of fusion proteins (and polynucleotides 0036 Zinc finger binding domains may be engineered to encoding same) are known to those of skill in the art and are recognize and bind to any nucleic acid sequence of choice. described in detail in U.S. Patent Application Publication Nos. 20050064474 and 2006018.8987, each incorporated by See, for example, Beerli et al. (2002) Nat. Biotechnol. reference herein in its entirety. Zinc finger recognition 20:135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70:313 regions and/or multi-fingered Zinc finger proteins may be 340; Isalan et al. (2001) Nat. Biotechnol. 19:656-660; Segal linked together using Suitable linker sequences, including for et al. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al. example, linkers of five or more amino acids in length. See, (2000) Curr. Opin. Struct. Biol. 10:411-416: Zhang et al. U.S. Pat. Nos. 6,479,626; 6,903,185; and 7,153,949, the dis (2000) J. Biol. Chem. 275(43):33.850-33860; Doyon et al. closures of which are incorporated by reference herein in their (2008) Nat. Biotechnol. 26:702-708; and Santiago et al. entireties, for non-limiting examples of linker sequences of (2008) Proc. Natl. Acad. Sci. USA 105:5809-5814. An engi six or more amino acids in length. The Zinc finger binding neered Zinc finger binding domain may have a novel binding domain described herein may include a combination of Suit specificity compared to a naturally-occurring Zinc finger pro able linkers between the individual zinc fingers of the protein. tein. Engineering methods include, but are not limited to, 0040. In some embodiments, the zinc finger nuclease may rational design and various types of selection. Rational design further comprise a nuclear localization signal or sequence includes, for example, using databases comprising doublet, (NLS). A NLS is an amino acid sequence, which facilitates triplet, and/or quadruplet nucleotide sequences and indi targeting the Zinc finger nuclease protein into the nucleus to vidual Zinc finger amino acid sequences, in which each dou introduce a double Stranded break at the target sequence in the blet, triplet or quadruplet nucleotide sequence is associated . Nuclear localization signals are known in the with one or more amino acid sequences of Zinc fingers which art. See, for example, Makkerhet al. (1996) Current Biology bind the particular triplet or quadruplet sequence. See, for 6:1025-1027. example, U.S. Pat. Nos. 6,453,242 and 6,534.261, the disclo 0041 (ii) Cleavage Domain sures of which are incorporated by reference herein in their 0042 A. Zinc finger nuclease also includes a cleavage entireties. As an example, the algorithm of described in U.S. domain. The cleavage domain portion of the Zinc finger Pat. No. 6,453.242 may be used to design a zinc finger bind nucleases disclosed herein may be obtained from any endo ing domain to target a preselected sequence. Alternative nuclease or exonuclease. Non-limiting examples of endonu methods, such as rational design using a nondegenerate rec cleases from which a cleavage domain may be derived ognition code table may also be used to design a Zinc finger include, but are not limited to, restriction endonucleases and binding domain to target a specific sequence (Sera et al. homing endonucleases. See, for example, 2002-2003 Cata (2002) 41:7074-7081). Publically available log, New England Biolabs, Beverly, Mass.; and Belfort et al. web-based tools for identifying potential target sites in DNA (1997) Nucleic Acids Res. 25:3379-3388 or www.neb.com. sequences and designing Zinc finger binding domains may be Additional enzymes that cleave DNA are known (e.g., S1 found at http://www.zincfingertools.org and http://bindr. Nuclease; mung bean nuclease; pancreatic DNase I; micro gdcb.iastate.edu/ZiFiT/, respectively (Mandell et al. (2006) coccal nuclease; yeast HO endonuclease). See also Linn et al. Nuc. Acid Res. 34:W516-W523: Sander et al. (2007) Nuc. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, Acid Res. 35:W599-W605). 1993. One or more of these enzymes (or functional fragments 0037. A zinc finger binding domain may be designed to thereof) may be used as a source of cleavage domains. recognize a DNA sequence ranging from about 3 nucleotides 0043. A cleavage domain also may be derived from an to about 21 nucleotides in length, or from about 8 to about 19 enzyme or portion thereof, as described above, that requires nucleotides in length. In general, the Zinc finger binding dimerization for cleavage activity. Two Zinc finger nucleases domains of the Zinc finger nucleases disclosed herein com may be required for cleavage, as each nuclease comprises a prise at least three Zinc finger recognition regions (i.e., Zinc monomer of the active enzyme dimer. Alternatively, a single fingers). In one embodiment, the Zinc finger binding domain Zinc finger nuclease may comprise both monomers to create may comprise four Zinc finger recognition regions. In another an active enzyme dimer. As used herein, an “active enzyme embodiment, the Zinc finger binding domain may comprise dimer' is an enzyme dimer capable of cleaving a nucleic acid five Zinc finger recognition regions. In still another embodi molecule. The two cleavage monomers may be derived from ment, the Zinc finger binding domain may comprise six Zinc the same endonuclease (or functional fragments thereof), or finger recognition regions. A Zinc finger binding domain may each monomer may be derived from a different endonuclease be designed to bind to any suitable target DNA sequence. See (or functional fragments thereof). US 2011/001 6543 A1 Jan. 20, 2011

0044) When two cleavage monomers are used to forman 534, 537, and 538 of Fok I are all targets for influencing active enzyme dimer, the recognition sites for the two Zinc dimerization of the FokI cleavage half-domains. Exemplary finger nucleases are preferably disposed such that binding of engineered cleavage monomers of FokI that form obligate the two Zinc finger nucleases to their respective recognition heterodimers include a pair in which a first cleavage mono sites places the cleavage monomers in a spatial orientation to mer includes mutations at amino acid residue positions 490 each other that allows the cleavage monomers to form an and 538 of Fok I and a second cleavage monomer that active enzyme dimer, e.g., by dimerizing. As a result, the near includes mutations at amino-acid residue positions 486 and edges of the recognition sites may be separated by about 5 to 499. about 18 nucleotides. For instance, the near edges may be 0048 Thus, in one embodiment, a mutation at amino acid separated by about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 position 490 replaces Glu (E) with Lys (K); a mutation at or 18 nucleotides. It will however be understood that any amino acid residue 538 replaces Iso (I) with Lys (K); a muta integral number of nucleotides or nucleotide pairs may inter tion at amino acid residue 486 replaces Gln (Q) with Glu (E): Vene between two recognition sites (e.g., from about 2 to and a mutation at position 499 replaces Iso (I) with Lys (K). about 50 nucleotide pairs or more). The near edges of the Specifically, the engineered cleavage monomers may be pre recognition sites of the Zinc finger nucleases, such as for pared by mutating positions 490 from E to Kand 538 from Ito example those described in detail herein, may be separated by Kin one cleavage monomer to produce an engineered cleav 6 nucleotides. In general, the site of cleavage lies between the age monomer designated “E490K:I538K and by mutating recognition sites. positions 486 from Q to E and 499 from Ito L in another 0045 Restriction endonucleases (restriction enzymes) are cleavage monomer to produce an engineered cleavage mono present in many species and are capable of sequence-specific mer designated “Q486E:I499L. The above described engi binding to DNA (at a recognition site), and cleaving DNA at neered cleavage monomers are obligate heterodimer mutants or near the site of binding. Certain restriction enzymes (e.g., in which aberrant cleavage is minimized or abolished. Engi Type IIS) cleave DNA at sites removed from the recognition neered cleavage monomers may be prepared using a Suitable site and have separable binding and cleavage domains. For method, for example, by site-directed mutagenesis of wild example, the Type IIS enzyme Fok I catalyzes double type cleavage monomers (Fok I) as described in U.S. Patent stranded cleavage of DNA, at 9 nucleotides from its recogni Publication No. 20050064474 (see Example 5). tion site on one strand and 13 nucleotides from its recognition 0049. The zinc finger nuclease described above may be site on the other. See, for example, U.S. Pat. Nos. 5,356,802: engineered to introduce a double stranded break at the tar 5,436,150 and 5.487,994; as well as Li et al. (1992) Proc. geted site of integration. The double stranded break may beat Natl. Acad. Sci. USA 89:4275-4279; Li et al. (1993) Proc. the targeted site of integration, or it may be up to 1, 2, 3, 4, 5, Natl. Acad. Sci. USA90:2764-2768; Kimetal. (1994a) Proc. 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, or 1000 nucleotides Natl. Acad. Sci. USA 91:883-887: Kim et al. (1994b) J. Biol. away from the site of integration. In some embodiments, the Chem. 269:31, 978-31,982. Thus, a zinc finger nuclease may double stranded break may be up to 1, 2, 3, 4, 5, 10, 15, or 20 comprise the cleavage domain from at least one Type IIS nucleotides away from the site of integration. In other restriction enzyme and one or more Zinc finger binding embodiments, the double stranded break may be up to 10, 15, domains, which may or may not be engineered. Exemplary 20, 25, 30, 35, 40, 45, or 50 nucleotides away from the site of Type IIS restriction enzymes are described for example in integration. In yet other embodiments, the double stranded International Publication WO 07/014,275, the disclosure of break may be up to 50, 100, or 1000 nucleotides away from which is incorporated by reference herein in its entirety. Addi the site of integration. tional restriction enzymes also contain separable binding and cleavage domains, and these also are contemplated by the (b) Optional Donor Polynucleotide present disclosure. See, for example, Roberts et al. (2003) 0050. The method for editing chromosomal sequences Nucleic Acids Res. 31:418-420. encoding inflammation-related proteins may further com 0046. An exemplary Type IIS restriction enzyme, whose prise introducing at least one donor polynucleotide compris cleavage domain is separable from the binding domain, is Fok ing a sequence encoding an inflammation-related protein into I. This particular enzyme is active as a dimmer (Bitinaite et al. the embryo or cell. A donor polynucleotide comprises at least (1998) Proc. Natl. Acad. Sci. USA 95: 10, 570-10, 575). three components: the sequence coding the inflammation Accordingly, for the purposes of the present disclosure, the related protein, an upstream sequence, and a downstream portion of the FokI enzyme used in a Zinc finger nuclease is sequence. The sequence encoding the protein is flanked by considered a cleavage monomer. Thus, for targeted double the upstream and downstream sequence, wherein the Stranded cleavage using a FokI cleavage domain, two Zinc upstream and downstream sequences share sequence similar finger nucleases, each comprising a FokI cleavage monomer, ity with either side of the site of integration in the chromo may be used to reconstitute an active enzyme dimer. Alterna SO. tively, a single polypeptide molecule containing a Zinc finger 0051 Typically, the donor polynucleotide will be DNA. binding domain and two FokI cleavage monomers may also The donor polynucleotide may be a DNA plasmid, a bacterial be used. artificial chromosome (BAC), a yeast artificial chromosome 0047. In certain embodiments, the cleavage domain may (YAC), a viral vector, a linear piece of DNA, a PCR fragment, comprise one or more engineered cleavage monomers that a naked nucleic acid, or a nucleic acid complexed with a minimize or prevent homodimerization, as described, for delivery vehicle Such as a liposome or poloxamer. An exem example, in U.S. Patent Publication Nos. 20050064.474, plary donor polynucleotide comprising the sequence encod 2006018.8987, and 20080131962, each of which is incorpo ing the inflammation-related protein may be a BAC. rated by reference herein in its entirety. By way of non 0.052 The sequence of the donor polynucleotide that limiting example, amino acid residues at positions 446, 447. encodes the inflammation-related protein may include coding 479, 483, 484, 486, 487, 490, 491, 496, 498,499, 500, 531, (i.e., exon) sequence, as well as intron sequences and US 2011/001 6543 A1 Jan. 20, 2011

upstream regulatory sequences (such as, e.g., a promoter). otide into the chromosome. Thus, endogenous chromosomal Depending upon the identity and the source of the sequence sequence may be converted to the sequence of the donor encoding the inflammation-related protein, the size of the polynucleotide. sequence encoding the inflammation-related protein will vary. For example, the sequence encoding the inflammation (c) Optional Exchange Polynucleotide related protein may range in size from about 1 kb to about 0058. The method for editing chromosomal sequences 5,000 kb. encoding an inflammation-related protein may further com 0053. The donor polynucleotide also comprises upstream prise introducing into the embryo or cell at least one exchange and downstream sequence flanking the sequence encoding polynucleotide comprising a sequence that is substantially the inflammation-related protein. The upstream and down identical to the chromosomal sequence at the site of cleavage stream sequences in the donor polynucleotide are selected to and which further comprises at least one specific nucleotide promote recombination between the chromosomal sequence change. of interest and the donor polynucleotide. The upstream 0059) Typically, the exchange polynucleotide will be sequence, as used herein, refers to a nucleic acid sequence DNA. The exchange polynucleotide may be a DNA plasmid, that shares sequence similarity with the chromosomal a bacterial artificial chromosome (BAC), a yeast artificial sequence upstream of the targeted site of integration. Simi chromosome (YAC), a viral vector, a linear piece of DNA, a larly, the downstream sequence refers to a nucleic acid PCR fragment, a naked nucleic acid, or a nucleic acid com sequence that shares sequence similarity with the chromo plexed with a delivery vehicle such as a liposome or polox Somal sequence downstream of the targeted site of integra amer. An exemplary exchange polynucleotide may be a DNA tion. The upstream and downstream sequences in the donor plasmid. polynucleotide may share about 75%, 80%, 85%, 90%. 95%, 0060. The sequence in the exchange polynucleotide is or 100% sequence identity with the targeted chromosomal Substantially identical to a portion of the chromosomal sequence. In other embodiments, the upstream and down sequence at the site of cleavage. In general, the sequence of stream sequences in the donor polynucleotide may share the exchange polynucleotide will share enough sequence identity with the chromosomal sequence Such that the two about 95%,96%.97%.98%, 99%, or 100% sequence identity sequences may be exchanged by homologous recombination. with the targeted chromosomal sequence. In an exemplary For example, the sequence in the exchange polynucleotide embodiment, the upstream and downstream sequences in the may have at least about 80, 81, 82, 83, 84,85, 86, 87, 88, 89, donor polynucleotide may share about 99% or 100% 90,91, 92,93, 94, 95, 96, 97,98, or 99% sequence identity sequence identity with the targeted chromosomal sequence. with a portion of the chromosomal sequence. 0054 An upstream or downstream sequence may com 0061 Importantly, the sequence in the exchange poly prise from about 50 bp to about 2500 bp. In one embodiment, nucleotide comprises at least one specific nucleotide change an upstream or downstream sequence may comprise about with respect to the sequence of the corresponding chromo 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, Somal sequence. For example, one nucleotide in a specific 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, codon may be changed to another nucleotide such that the 2200, 2300, 2400, or 2500 bp. An exemplary upstream or codon codes for a different amino acid. In one embodiment, downstream sequence may comprise about 200 bp to about the sequence in the exchange polynucleotide may comprise 2000 bp, about 600 bp to about 1000 bp, or more particularly one specific nucleotide change Such that the encoded protein about 700 bp to about 1000 bp. comprises one amino acid change. In other embodiments, the 0055. In some embodiments, the donor polynucleotide sequence in the exchange polynucleotide may comprise two, may further comprise a marker. Such a marker may make it three, four, or more specific nucleotide changes such that the easy to Screen for targeted integrations. Non-limiting encoded protein comprises one, two, three, four, or more amino acid changes. In still other embodiments, the sequence examples of Suitable markers include restriction sites, fluo in the exchange polynucleotide may comprise a three nucle rescent proteins, or selectable markers. otide deletion or insertion such that the reading frame of the 0056. One of skill in the art would be able to construct a coding reading is not altered (and a functional protein is donor polynucleotide as described herein using well-known produced). The expressed protein, however, would comprise standard recombinant techniques (see, for example, Sam a single amino acid deletion or insertion. brook et al., 2001 and Ausubel et al., 1996). 0062. The length of the sequence in the exchange poly 0057. In the method detailed above for integrating a nucleotide that is substantially identical to a portion of the sequence encoding the inflammation-related protein, a chromosomal sequence at the site of cleavage can and will double stranded break introduced into the chromosomal vary. In general, the sequence in the exchange polynucleotide sequence by the Zinc finger nuclease is repaired, via homolo may range from about 50 bp to about 10,000 bp in length. In gous recombination with the donor polynucleotide, Such that various embodiments, the sequence in the exchange poly the sequence encoding the inflammation-related protein is nucleotide may be about 100, 200, 400, 600,800, 1000, 1200, integrated into the chromosome. The presence of a double 1400, 1600, 1800, 2000, 2200, 2400, 2600, 2800, 3000, 3200, Stranded break facilitates integration of the sequence encod 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, or 5000 bp ing the inflammation-related protein. A donor polynucleotide in length. In other embodiments, the sequence in the may be physically integrated or, alternatively, the donor poly exchange polynucleotide may be about 5500, 6000, 6500, nucleotide may be used as a template for repair of the break, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, or 10,000 resulting in the introduction of the sequence encoding the bp in length. inflammation-related protein as well as all or part of the 0063. One of skill in the art would be able to construct an upstream and downstream sequences of the donor polynucle exchange polynucleotide as described herein using well US 2011/001 6543 A1 Jan. 20, 2011 known standard recombinant techniques (see, for example, nuclease, as well as the optional donor (or exchange) poly Sambrook et al., 2001 and Ausubel et al., 1996). nucleotides, may be introduced sequentially. 0064. In the method detailed above for modifying a chro mosomal sequence, a double stranded break introduced into (e) Culturing the Embryo or Cell the chromosomal sequence by the Zinc finger nuclease is 0069. The method of inducing genomic editing with a zinc repaired, via homologous recombination with the exchange finger nuclease further comprises culturing the embryo or cell polynucleotide, Such that the sequence in the exchange poly comprising the introduced nucleic acid(s) to allow expression nucleotide may be exchanged with a portion of the chromo of the Zinc finger nuclease. An embryo may be cultured in somal sequence. The presence of the double stranded break vitro (e.g., in cell culture). Typically, the embryo is cultured at facilitates homologous recombination and repair of the break. an appropriate temperature and in appropriate media with the The exchange polynucleotide may be physically integrated necessary O/CO ratio to allow the expression of the zinc or, alternatively, the exchange polynucleotide may be used as finger nuclease. Suitable non-limiting examples of media a template for repair of the break, resulting in the exchange of include M2, M16, KSOM, BMOC, and HTF media. A skilled the sequence information in the exchange polynucleotide artisan will appreciate that culture conditions can and will with the sequence information in that portion of the chromo vary depending on the species of embryo. Routine optimiza Somal sequence. Thus, a portion of the endogenous chromo tion may be used, in all cases, to determine the best culture Somal sequence may be converted to the sequence of the conditions for a particular species of embryo. In some cases, exchange polynucleotide. The changed nucleotide(s) may be a cell line may be derived from an in vitro-cultured embryo at or near the site of cleavage. Alternatively, the changed (e.g., an embryonic stem cell line). nucleotide(s) may be anywhere in the exchanged sequences. 0070 Alternatively, an embryo may be cultured in vivo by As a consequence of the exchange, however, the chromo transferring the embryo into the uterus of a female host. Somal sequence is modified. Generally speaking the female host is from the same or simi lar species as the embryo. Preferably, the female host is (d) Delivery of Nucleic Acids pseudo-pregnant. Methods of preparing pseudo-pregnant female hosts are known in the art. Additionally, methods of 0065. To mediate Zinc finger nuclease genomic editing, at transferring an embryo into a female host are known. Cultur least one nucleic acid molecule encoding a Zinc finger ing an embryo in vivo permits the embryo to develop and may nuclease and, optionally, at least one exchange polynucle result in a live birth of an animal derived from the embryo. otide or at least one donor polynucleotide are delivered to the Such an animal would comprise the edited chromosomal embryo or the cell of interest. Typically, the embryo is a sequence encoding the inflammation-related protein in every fertilized one-cell stage embryo of the species of interest. cell of the body. 0066 Suitable methods of introducing the nucleic acids to 0071 Similarly, cells comprising the introduced nucleic the embryo or cell include microinjection, electroporation, acids may be cultured using standard procedures to allow Sonoporation, biolistics, calcium phosphate-mediated trans expression of the Zinc finger nuclease. Standard cell culture fection, cationic transfection, liposome transfection, den techniques are described, for example, in Santiago et al. drimer transfection, heat shock transfection, nucleofection (2008) PNAS 105:5809-5814; Moehle et al. (2007) PNAS transfection, magnetofection, lipofection, impalefection, 104:3055-3060: Urnov et al. (2005)Nature 435:646-651; and optical transfection, proprietary agent-enhanced uptake of Lombardo et al (2007) Nat. Biotechnology 25:1298-1306. nucleic acids, and delivery via liposomes, immunoliposomes, Those of skill in the art appreciate that methods for culturing Virosomes, or artificial virions. In one embodiment, the cells are known in the art and can and will vary depending on nucleic acids may be introduced into an embryo by microin the cell type. Routine optimization may be used, in all cases, jection. The nucleic acids may be microinjected into the to determine the best techniques for a particular cell type. nucleus or the cytoplasm of the embryo. In another embodi 0072. Upon expression of the zinc finger nuclease, the ment, the nucleic acids may be introduced into a cell by chromosomal sequence may be edited. In cases in which the nucleofection. embryo or cell comprises an expressed Zinc finger nuclease 0067. In embodiments in which both a nucleic acid encod but no donor (or exchange) polynucleotide, the Zinc finger ing a Zinc finger nuclease and a donor (or exchange) poly nuclease recognizes, binds, and cleaves the target sequence in nucleotide are introduced into an embryo or cell, the ratio of the chromosomal sequence of interest. The double-stranded donor (or exchange) polynucleotide to nucleic acid encoding break introduced by the Zinc finger nuclease is repaired by an a Zinc finger nuclease may range from about 1:10 to about error-prone non-homologous end-joining DNA repair pro 10:1. In various embodiments, the ratio of donor (or cess. Consequently, a deletion, insertion or nonsense muta exchange) polynucleotide to nucleic acid encoding a Zinc tion may be introduced in the chromosomal sequence Such finger nuclease may be about 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, that the sequence is inactivated. 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In 0073. In cases in which the embryo or cell comprises an one embodiment, the ratio may be about 1:1. expressed Zinc finger nuclease as well as a donor (or 0068. In embodiments in which more than one nucleic exchange) polynucleotide, the Zinc finger nuclease recog acid encoding a Zinc finger nuclease and, optionally, more nizes, binds, and cleaves the target sequence in the chromo than one donor (or exchange) polynucleotide are introduced some. The double-stranded break introduced by the zinc fin into an embryo or cell, the nucleic acids may be introduced ger nuclease is repaired, via homologous recombination with simultaneously or sequentially. For example, nucleic acids the donor (or exchange) polynucleotide. Such that the encoding the Zinc finger nucleases, each specific for a distinct sequence in the donor polynucleotide is integrated into the recognition sequence, as well as the optional donor (or chromosomal sequence (or a portion of the chromosomal exchange) polynucleotides, may be introduced at the same sequence is converted to the sequence in the exchange poly time. Alternatively, each nucleic acid encoding a Zinc finger nucleotide). As a consequence, a sequence may be integrated US 2011/001 6543 A1 Jan. 20, 2011

into the chromosomal sequence (or a portion of the chromo modified rat, such that the information gained therefrom may Somal sequence may be modified). be used to predict the effect of the agent in a human. In 0074 The genetically modified animals disclosed herein general, the method comprises contacting a genetically modi may be crossbred to create animals comprising more than one fied animal comprising at least one edited chromosomal edited chromosomal sequence or to create animals that are sequence encoding an inflammation-related protein, and homozygous for one or more edited chromosomal sequences. comparing results of a selected parameter to results obtained For example, two animals comprising the same edited chro from contacting a control genetically modified animal with mosomal sequence may be crossbred to create an animal the same agent. Non limiting examples of disease states or homozygous for the edited chromosomal sequence. Alterna disorders that may be associated with inflammation-related tively, animals with different edited chromosomal sequences proteins include allergies, , arthritis, asthma, may be crossbred to create an animal comprising both edited atherosclerosis, amyloid diseases, acne, , infections, chromosomal sequences. ischaemic heart disease, inflammatory bowel disorders, inter 0075 For example, animal A comprising an inactivated Stitial cystitis, hypersensitivities, inflammatory bowel dis PPARA chromosomal sequence may be crossed with animal eases, reperfusion injury, transplant rejection, obesity, myo B comprising a chromosomally integrated sequence encod pathies, leukopenia, Vitamin deficiencies, pelvic ing a human PPARA to give rise to a “humanized PPARA inflammatory disease, glomeronephritis, graft versus host offspring comprising both the inactivated PPARA chromo disease (transplant rejection), preterm labor, vasculitis, viti Somal sequence and the chromosomally integrated human ligo, HIV infection and progression to AIDS. PPARA gene. Similarly, an animal comprising an inactivated 0079 Also provided are methods to assess the effect(s) of IL-4 chromosomal sequence may be crossed with an animal an agent in an isolated cell comprising at least one edited comprising chromosomally integrated sequence encoding chromosomal sequence encoding an inflammation-related the human IL-4 protein to generate “humanized IL-4 off protein, as well as methods of using lysates of Such cells (or spring. Moreover, a humanized PPARA animal may be cells derived from a genetically modified animal disclosed crossed with a humanized IL-4 animal to create a humanized herein) to assess the effect(s) of an agent. For example, the PPARA/IL-4 animal. Those of skill in the art will appreciate role of a particular inflammation-related protein in the that many combinations are possible. metabolism of a particular agent may be determined using 0076. In other embodiments, an animal comprising an Such methods. Similarly, Substrate specificity and pharmaco edited chromosomal sequence disclosed herein may be cross kinetic parameter may be readily determined using Such bred to combine the edited chromosomal sequence with other methods. genetic backgrounds. By way of non-limiting example, other 0080 Yet another aspect encompasses a method for genetic backgrounds may include wild type genetic back assessing the therapeutic efficacy of a potential gene therapy grounds, genetic backgrounds with deletion mutations, strategy. That is, a chromosomal sequence encoding an genetic backgrounds with another targeted integration, and inflammation-related protein may be modified such that the genetic backgrounds with non-targeted integrations. inflammation is reduced or eliminated. In particular, the method comprises editing a chromosomal sequence encoding (IV) Applications an inflammation-related protein such that an altered protein product is produced. The genetically modified animal may 0077. A further aspect of the present disclosure encom further be exposed to a test conditions such as exposure to a passes a method for using the genetically modified animals. test compound, and cellular, and/or molecular responses mea In one embodiment, the genetically modified animals may be Sured and compared to those of a wild-type animal exposed to used to study the effects of mutations on the progression of the same test conditions. Consequently, the therapeutic inflammation using measures commonly used in the study of potential of the inflammation-related gene therapy regime inflammation. Alternatively, the animals of the invention may may be assessed. be used to study the effects of the mutations on the progres I0081. Still yet another aspect encompasses a method of sion of a disease state or disorder associated with inflamma generating a cell line or cell lysate using a genetically modi tion-related proteins using measures commonly used in the fied animal comprising an edited chromosomal sequence study of said disease state or disorder. Non-limiting examples encoding an inflammation-related protein. An additional of measures that may be used include spontaneous behaviors other aspect encompasses a method of producing purified of the genetically modified animal, performance during biological components using a genetically modified cell or behavioral testing, physiological anomalies, differential animal comprising an edited chromosomal sequence encod responses to a compound, abnormalities in tissues or cells, ing an inflammation-related protein. Non-limiting examples and biochemical or molecular differences between geneti of biological components include antibodies, , sig cally modified animals and wild type animals. nal proteins, enzymes, receptoragonists and receptor antago 0078. In another embodiment, the genetically modified nists. animals and cells may be used for assessing the effect(s) of an agent on inflammation. Alternatively, the animals and cells of DEFINITIONS the invention may be used for assessing the effect(s) of an agent on the progression of a disease state or disorder asso 0082 Unless defined otherwise, all technical and scien ciated with inflammation-related proteins. Suitable agents tific terms used herein have the meaning commonly under include without limit pharmaceutically active ingredients, stood by a person skilled in the art to which this invention drugs, food additives, pesticides, herbicides, toxins, indus belongs. The following references provide one of skill with a trial chemicals, household chemicals and other environmen general definition of many of the terms used in this invention: tal chemicals, viral vectors encoding therapeutic properties, Singleton et al., Dictionary of Microbiology and Molecular stem cell-based therapeutic agents. For example, the effect(s) Biology (2nd ed. 1994); The Cambridge Dictionary of Sci of an agent may be measured in a "humanized genetically ence and Technology (Walker ed., 1988); The Glossary of US 2011/001 6543 A1 Jan. 20, 2011

Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag two polynucleotides or polypeptide sequences, respectively. (1991); and Hale & Marham, The Harper Collins Dictionary Two or more sequences (polynucleotide oramino acid) can be of Biology (1991). As used herein, the following terms have compared by determining their percent identity. The percent the meanings ascribed to them unless specified otherwise. identity of two sequences, whether nucleic acid oramino acid 0083. A “gene.” as used herein, refers to a DNA region sequences, is the number of exact matches between two (including exons and introns) encoding a gene product, as aligned sequences divided by the length of the shorter well as all DNA regions, which regulate the production of the sequences and multiplied by 100. An approximate alignment gene product, whether or not such regulatory sequences are for nucleic acid sequences is provided by the local homology adjacent to coding and/or transcribed sequences. Accord algorithm of Smith and Waterman, Advances in Applied ingly, a gene includes, but is not necessarily limited to, pro Mathematics 2:482-489 (1981). This algorithm can be moter sequences, terminators, translational regulatory applied to amino acid sequences by using the scoring matrix sequences such as ribosome binding sites and internal ribo developed by Dayhoff, Atlas of Protein Sequences and Struc Some entry sites, enhancers, silencers, insulators, boundary ture, M. O. Dayhoff ed., 5 suppl. 3:353-358, National Bio elements, replication origins, matrix attachment sites, and medical Research Foundation, Washington, D.C., USA, and locus control regions. normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763 I0084. The terms “nucleic acid” and “polynucleotide” refer (1986). An exemplary implementation of this algorithm to to a deoxyribonucleotide or ribonucleotide polymer, in linear determine percent identity of a sequence is provided by the or circular conformation, and in either single- or double Genetics Computer Group (Madison, Wis.) in the “BestFit' stranded form. For the purposes of the present disclosure, utility application. Other Suitable programs for calculating these terms are not to be construed as limiting with respect to the percent identity or similarity between sequences are gen the length of a polymer. The terms can encompass known erally known in the art, for example, another alignment pro analogs of natural nucleotides, as well as nucleotides that are gram is BLAST, used with default parameters. For example, modified in the base, Sugar and/or phosphate moieties (e.g., BLASTN and BLASTP can be used using the following phosphorothioate backbones). In general, an analog of a par default parameters: genetic code-standard; filter none; ticular nucleotide has the same base-pairing specificity; i.e., strand–both: cutoff=60; expect=10; Matrix=BLOSUM62: an analog of A will base-pair with T. Descriptions=50 sequences; sort by HIGH SCORE: I0085. The terms “polypeptide' and “protein” are used Databases non-redundant, GenBank--EMBL-DDBJ interchangeably to refer to a polymer of amino acid residues. PDB+GenBank CDS translations-FSwiss protein--Spup I0086. The term “recombination” refers to a process of date+PIR. Details of these programs can be found on the exchange of genetic information between two polynucle GenBank website. With respect to sequences described otides. For the purposes of this disclosure, "homologous herein, the range of desired degrees of sequence identity is recombination” refers to the specialized form of such approximately 80% to 100% and any integer value therebe exchange that takes place, for example, during repair of tween. Typically the percent identities between sequences are double-strand breaks in cells. This process requires sequence at least 70-75%, preferably 80-82%, more preferably similarity between the two polynucleotides, uses a “donor” or 85-90%, even more preferably 92%, still more preferably exchange molecule to template repair of a “target molecule 95%, and most preferably 98% sequence identity. (i.e., the one that experienced the double-strand break), and is I0089 Alternatively, the degree of sequence similarity variously known as “non-crossover gene conversion' or between polynucleotides can be determined by hybridization “short tract gene conversion.” because it leads to the transfer of polynucleotides under conditions that allow formation of of genetic information from the donor to the target. Without stable duplexes between regions that share a degree of being bound by any particular theory, such transfer can sequence identity, followed by digestion with single involve mismatch correction of heteroduplex DNA that forms Stranded-specific nuclease(s), and size determination of the between the broken target and the donor, and/or “synthesis digested fragments. Two nucleic acid, or two polypeptide dependent strand annealing.” in which the donor is used to sequences are Substantially similar to each other when the resynthesize genetic information that will become part of the sequences exhibit at least about 70%-75%, preferably 80%- target, and/or related processes. Such specialized homolo 82%, more-preferably 85%-90%, even more preferably 92%, gous recombination often results in an alteration of the still more preferably 95%, and most preferably 98% sequence sequence of the target molecule Such that part or all of the identity over a defined length of the molecules, as determined sequence of the donor polynucleotide is incorporated into the using the methods above. As used herein, Substantially simi target polynucleotide. lar also refers to sequences showing complete identity to a 0087 As used herein, the terms “target site' or “target specified DNA or polypeptide sequence. DNA sequences that sequence” refer to a nucleic acid sequence that defines a are substantially similar can be identified in a Southern portion of a chromosomal sequence to be edited and to which hybridization experiment under, for example, stringent con a Zinc finger nuclease is engineered to recognize and bind, ditions, as defined for that particular system. Defining appro provided sufficient conditions for binding exist. priate hybridization conditions is within the skill of the art. 0088 Techniques for determining nucleic acid and amino See, e.g., Sambrook et al., supra; Nucleic Acid Hybridization: acid sequence identity are known in the art. Typically, Such A Practical Approach, editors B. D. Hames and S. J. Higgins, techniques include determining the nucleotide sequence of (1985) Oxford; Washington, D.C.; IRL Press). the mRNA for a gene and/or determining the amino acid 0090 Selective hybridization of two nucleic acid frag sequence encoded thereby, and comparing these sequences to ments can be determined as follows. The degree of sequence a second nucleotide or amino acid sequence. Genomic identity between two nucleic acid molecules affects the effi sequences can also be determined and compared in this fash ciency and strength of hybridization events between Such ion. In general, identity refers to an exact nucleotide-to molecules. A partially identical nucleic acid sequence will at nucleotide or amino acid-to-amino acid correspondence of least partially inhibit the hybridization of a completely iden US 2011/001 6543 A1 Jan. 20, 2011 20 tical sequence to a target molecule. Inhibition of hybridiza EXAMPLES tion of the completely identical sequence can be assessed 0093. The following examples are included to illustrate using hybridization assays that are well known in the art (e.g., the invention. Southern (DNA) blot, Northern (RNA) blot, solution hybrid ization, or the like, see Sambrook, et al., Molecular Cloning: Example 1 A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.). Such assays can be conducted using varying Genome Editing of CCR2 in a Model Organism degrees of selectivity, for example, using conditions varying 0094 Zinc finger nuclease (ZFN)-mediated genome edit from low to high Stringency. If conditions of low stringency ing may be used to study the effects of a “knockout' mutation are employed, the absence of non-specific binding can be in an inflammation-related chromosomal sequence, Such as a assessed using a secondary probe that lacks even a partial chromosomal sequence encoding the CCR2 protein, in a degree of sequence identity (for example, a probehaving less genetically modified model animal and cells derived from the than about 30% sequence identity with the target molecule), animal. Such a model animal may be a rat. In general, ZFNS Such that, in the absence of non-specific binding events, the that bind to the rat chromosomal sequence encoding the secondary probe will not hybridize to the target. inflammation-related protein CCR2 may be used to introduce a non-sense mutation into the coding region of the CCR2 0091. When utilizing a hybridization-based detection sys gene. Such that an active CCR2 protein may not be produced. tem, a nucleic acid probe is chosen that is complementary to (0095 Capped, polyadenylated mRNA encoding the ZFN a reference nucleic acid sequence, and then by selection of may be produced using known molecular biology techniques, appropriate conditions the probe and the reference sequence including but not limited to a technique Substantially similar selectively hybridize, or bind, to each other to form a duplex to the technique described in Science (2009) 325:433, which molecule. A nucleic acid molecule that is capable of hybrid is incorporated by reference herein in its entirety. The mRNA izing selectively to a reference sequence under moderately may be transfected into rat embryos. The rat embryos may be stringent hybridization conditions typically hybridizes under at the single cell stage when microinjected. Control embryos conditions that allow detection of a target nucleic acid may be injected with 0.1 mM EDTA. The frequency of ZFN sequence of at least about 10-14 nucleotides in length having induced double strand chromosomal breaks may be deter at least approximately 70% sequence identity with the mined using the Cel-1 nuclease assay. This assay detects sequence of the selected nucleic acid probe. Stringent hybrid alleles of the target locus that deviate from wild type (WT) as ization conditions typically allow detection of target nucleic a result of non-homologous end joining (NHEJ)-mediated acid sequences of at least about 10-14 nucleotides in length imperfect repair of ZFN-induced DNA double strand breaks. having a sequence identity of greater than about 90-95% with PCR amplification of the targeted region from a pool of the sequence of the selected nucleic acid probe. Hybridization ZFN-treated cells may generate a mixture of WT and mutant conditions useful for probe/reference sequence hybridiza amplicons. Melting and reannealing of this mixture results in tion, where the probe and reference sequence have a specific mismatches forming between heteroduplexes of the WT and degree of sequence identity, can be determined as is known in mutant alleles. A DNA “bubble' formed at the site of mis the art (see, for example, Nucleic Acid Hybridization: A match is cleaved by the surveyor nuclease Cel-1, and the Practical Approach, editors B. D. Hames and S. J. Higgins, cleavage products can be resolved by gel electrophoresis. The (1985) Oxford; Washington, D.C.: IRL Press). Conditions for relative intensity of the cleavage products compared with the hybridization are well-known to those of skill in the art. parental band is a measure of the level of Cel-1 cleavage of the 0092. Hybridization stringency refers to the degree to heteroduplex. This, in turn, reflects the frequency of ZFN which hybridization conditions disfavor the formation of mediated cleavage of the endogenous target locus that has hybrids containing mismatched nucleotides, with higher subsequently undergone imperfect repair by NHEJ. stringency correlated with a lower tolerance for mismatched 0096. The development of the embryos following micro hybrids. Factors that affect the stringency of hybridization are injection, and the development of inflammation-related well-known to those of skill in the art and include, but are not symptoms and disorders caused by the CCR2 “knockout' limited to, temperature, pH, ionic strength, and concentration may be assessed in the genetically modified rat. For CCR2, of organic solvents such as, for example, formamide and inflammation-related symptoms and disorders may include dimethylsulfoxide. As is known to those of skill in the art, development of and an altered inflam hybridization stringency is increased by higher temperatures, matory response against tumors. The results may be com lower ionic strength and lower solvent concentrations. With pared to the control rat injected with 0.1 mM EDTA, where respect to stringency conditions for hybridization, it is well the chromosomal region encoding the CCR2 protein is not known in the art that numerous equivalent conditions can be altered. In addition, molecular analysis of inflammation-re employed to establish a particular stringency by varying, for lated pathways may be performed in cells derived from the example, the following factors: the length and nature of the genetically modified animal comprising a CCR2 "knockout'. sequences, base composition of the various sequences, con centrations of salts and other hybridization solution compo Example 2 nents, the presence or absence of blocking agents in the hybridization solutions (e.g., dextran Sulfate, and polyethyl Generation of a Humanized Rat Expressing a Mutant ene glycol), hybridization reaction temperature and time Form of Human Perforin-1 parameters, as well as, varying wash conditions. A particular 0097 Missense mutations in perforin-1, a critical effector set of hybridization conditions may be selected following of lymphocyte cytotoxicity, lead to a spectrum of diseases, standard methods in the art (see, for example, Sambrook, et from familial hemophagocytic lymphohistiocytosis to an al., Molecular Cloning: A Laboratory Manual, Second Edi increased risk of tumorigenesis. One Such mutation is the tion, (1989) Cold Spring Harbor, N.Y.). V50M missense mutation where the valine amino acid at US 2011/001 6543 A1 Jan. 20, 2011

position 50 in perforin-1 is replaced with methionine. ZFN either incubated in vitro, or transferred to pseudopregnant mediated genome editing may be used to generate a human female rats to be carried to parturition. The resulting ized rat wherein the rat PRF1 gene is replaced with a mutant embryos/fetus, or the toe/tail clip of live born animals were form of the human PRF1 gene comprising the V50M muta harvested for DNA extraction and analysis. DNA was isolated tion. Such a humanized rat may be used to study the devel using standard procedures. The targeted region of the Ragl opment of the diseases associated with the mutant human locus was PCR amplified using appropriate primers. The perforin-1 protein. In addition, the humanized rat may be amplified DNA was subcloned into a suitable vector and used to assess the efficacy of potential therapeutic agents sequenced using standard methods. FIG. 1 presents DNA targeted at the inflammatory pathway comprising perforin-1. sequences of edited Rag1 loci in two animals (SEQID NOS: 0098. The genetically modified rat may be generated using 5 and 6). One animal had a 808 bp deletion in exon 2, and a the methods described in Example 1 above. However, to second animal had a 29bp deletion in the target sequence of generate the humanized rat, the ZFN mRNA may be co exon 2. These deletions disrupt the reading frame of the Rag1 injected with the human chromosomal sequence encoding the coding region. mutant perforin-1 protein into the rat embryo. The rat chro mosomal sequence may then be replaced by the mutant Example 6 human sequence by homologous recombination, and a humanized rat expressing a mutant form of the perforin-1 Identification of ZFNs that Edit the Rag2 Locus protein may be produced. 0102 ZFNs that target and cleave the Rag2 gene were identified essentially as described above. The rat Rag2 gene Example 3 (XM 00107.9235) was scanned for putative zinc finger bind ing sites. ZFNs were assembled and tested essentially as Editing the Pten Locus described in Example 1. This assay revealed that the ZFN pair 0099 ZFNs that target and cleave the Pten locus in rats targeted to bind 5'-acGTGGTATATaGCCGAGgaaaaagtgt-3' were designed and tested for activity essentially as described (SEQ ID NO: 7; contact sites in uppercase) and 5'-atAC above in Example 1. An active pair of ZFNs was identified. CACGTCAATGGAAtggccatatct-'3' (SEQID NO:8) cleaved The DNA binding sites were 5'-CCCCAGTTTGTGGTCt within the Rag2 locus. gcca-3' (SEQID NO:1) and 5'-gcTAAAGGTGAAGATCTA 3' (SEQID NO:2). Capped, polyadenylated mRNA encoding Example 7 the active pair may be microinjected into rat embryos and the Editing the Rag2 Locus resultant embryos may be analyzed as described in Example 0103 Rat embryos were microinjected with mRNA 1. Accordingly, the Pten locus may be edited to contain a encoding the active pair of Rag2 ZFNs essentially as deletion or an insertion Such that the coding region is dis described in Example 2. The injected embryos were incu rupted and no functional gene product is made. bated and DNA was extracted from the resultant animals. The Example 4 targeted region of the Rag2 locus was PCR amplified using appropriate primers. The amplified DNA was subcloned into Identification of ZFNs that Edit the Rag1 Locus a Suitable vector and sequenced using standard methods. FIG. 2 presents DNA sequences of edited Rag2 loci in two animals. 0100. The Rag1 gene was chosen for Zinc finger nuclease One animal had a 13 bp deletion in the target sequence in exon (ZFN) mediated genome editing. ZFNs were designed, 3, and a second animal had a 2 bp deletion in the target assembled, and validated using strategies and procedures sequence of exon3. These deletions disrupt the reading frame described in the examples above. ZFN design made use of an of the Rag2 coding region. archive of pre-validated 1-finger and 2-finger modules. The rat Rag1 gene region (XM 00107.9242) was scanned for Example 8 putative Zinc finger binding sites to which existing modules could be fused to generate a pair of 4-, 5-, or 6-finger proteins Identification of ZFNS that Edit the FoxN 1 Locus that would bind a 12-18 bp sequence on one Strand and a 0104 ZFNs that target and cleave the FoxN1 gene were 12-18 bp sequence on the other strand, with about 5-6 bp identified essentially as described above in Example 1. The between the two binding sites. Capped, polyadenylated rat FoxN1 gene (XM 220632) was scanned for putative zinc mRNA encoding each pair of ZFNs was produced and trans finger binding sites. ZFNs were assembled and tested essen fected into rat cells. Control cells were injected with mRNA tially as described in Example 1. This assay revealed two encoding GFP. Active ZFN pairs were identified by detecting pairs of active ZFNs that cleaved within the FoxN1 locus: a ZFN-induced double strand chromosomal breaks using the first pair targeted to bind 5'-ttAAGGGCCATGAAGATgag Cel- 1 nuclease assay. This assay revealed that the ZFN pair gatgctac-3' (SEQ ID NO: 9; contact sites in uppercase) and targeted to bind 5'-ttCCTTGGGCAGTAGACctgactgtgag-3 5'-caGCAAGACCGGAAGCCttccagticagt-'3' (SEQ ID NO: (SEQ ID NO:3: contact sites in upper case) and 5'-gtGAC 10); and a second pair targeted to bind 5'-ttGTCGATTTTG CGTGGAGTGGCAcccccacacac-3 (SEQID NO: 4) cleaved GAAGGattgaggg.ccc-3' (SEQ ID NO: 11) and 5'-atGCAG within the Ragl gene. GAAGAGCTGCAgaagtggaaga-'3' (SEQID NO: 12) Example 5 Example 9 Editing the Ragl Locus Identification of ZFNS that Edit the DNAPK Locus 0101 Capped, polyadenylated mRNA encoding the active 0105 ZFNs that target and cleave the DNAPK gene were pair of ZFNs was microinjected into fertilized rat embryos as identified essentially as described above in Example 1. The described in the examples above. The injected embryos were rat DNAPK gene (NM 001108327) was scanned for puta US 2011/001 6543 A1 Jan. 20, 2011 22 tive zinc finger binding sites. ZFNs were assembled and tested essentially as described in Example 1. This assay - Continued revealed that the ZFN pair targeted to bind5'-taCACAAGTC CtTCTCCAggagctagaa-3' (SEQ ID NO: 13; contact sites in SEQ ID uppercase) and 5'-acAAAGCTTATGAAGGTcttagtgaaaa-3' Name Sequence of Zinc Finger Helices NO : (SEQ ID NO: 14) cleaved within the DNAPK locus. 0106 The table below presents the amino acid sequences RAG2 QSGNLAR RSDSLSV OSADRTK RSDTLST DRKTRIN 18 of helices of the active ZFNs. FOXN1TSGNLTR OSGNLAR DRSHLTR RLDNRTA 19

FOXN1DRSDLSR OSGNLAR RSDTLSE OROHRTT ONATRIK

SEQ ID FOXN1RSDHLSA OSGHLSR DSESLNA TSSNLSR DRSSRKR 21 Name Sequence of Zinc Finger Helices NO : FOXN1QSGSLTR OSSDLRR ORTHLTO QSGHLOR OSGDLTR 22 RAG1 DRSNLSR OSGSLTR ERGTLAR RSDHLTT HKTSLKD 15 DNAPKOSGDLTR SSSDRKK DSSDRKK RSDNLST DNSNRIN 23 RAG1 ONATRIK RSDALSR OSGHLSR RSADLTE DRANLSR 16 DNAPKTSGHLSR OSGNLAR QSSDLSR OSGNRTT 24 RAG2 RSDNLSR DSSTRKK NSGNLDK OSGAL.AR RSDALAR 17

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 26

<21 Os SEQ ID NO 1 &211s LENGTH: 2O &212s. TYPE: DNA &213s ORGANISM: Rattus rattus

<4 OOs SEQUENCE: 1 ccc.cagtttgttggtctgcca

<21 Os SEQ ID NO 2 &211s LENGTH: 18 &212s. TYPE: DNA &213s ORGANISM: Rattus rattus

<4 OOs SEQUENCE: 2

gctaaaggtg aagat ct a 18

<21 Os SEQ ID NO 3 &211s LENGTH: 28 &212s. TYPE: DNA &213s ORGANISM: Rattus rattus

<4 OOs SEQUENCE: 3

titcCttgggc agtag acct g actgtgag 28

<21 Os SEQ ID NO 4 &211s LENGTH: 28 &212s. TYPE: DNA &213s ORGANISM: Rattus rattus

<4 OOs SEQUENCE: 4

gtgaccgtgg agtggcaccC cc acacac 28

<21 Os SEQ ID NO 5 &211s LENGTH: 1.OOO &212s. TYPE: DNA &213s ORGANISM: Rattus rattus

<4 OOs SEQUENCE: 5

US 2011/001 6543 A1 Jan. 20, 2011 24

- Continued <4 OOs, SEQUENCE: 9 ttaagggc.ca talagatgag gatgctac 28

<210s, SEQ ID NO 10 &211s LENGTH: 28 &212s. TYPE: DNA &213s ORGANISM: Rattus rattus

<4 OOs, SEQUENCE: 10

Cagcaaga cc ggaagcct tc cagt cagt 28

<210s, SEQ ID NO 11 &211s LENGTH: 28 &212s. TYPE: DNA &213s ORGANISM: Rattus rattus

<4 OOs, SEQUENCE: 11 ttgtcgattt tdaaggatt gagggcc.c 28

<210s, SEQ ID NO 12 &211s LENGTH: 28 &212s. TYPE: DNA &213s ORGANISM: Rattus rattus

<4 OOs, SEQUENCE: 12 atgcaggaag agctgcagaa gtggalaga 28

<210s, SEQ ID NO 13 &211s LENGTH: 28 &212s. TYPE: DNA &213s ORGANISM: Rattus rattus

<4 OOs, SEQUENCE: 13 tacacaagtic citt citccagg agctagaa 28

<210s, SEQ ID NO 14 &211s LENGTH: 28 &212s. TYPE: DNA &213s ORGANISM: Rattus rattus

<4 OOs, SEQUENCE: 14 acaaagctta talaggtott agtgaaaa 28

<210s, SEQ ID NO 15 &211s LENGTH: 35 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: COMPLETELY ARTIFICIAL

<4 OOs, SEQUENCE: 15 Asp Arg Ser Asn Lieu. Ser Arg Glin Ser Gly Ser Lieu. Thir Arg Glu Arg 1. 5 15 Gly. Thir Lieu Ala Arg Arg Ser Asp His Lieu. Thir Thr His Llys Thir Ser 25 Lieu Lys Asp 35

<210s, SEQ ID NO 16 &211s LENGTH: 35 US 2011/001 6543 A1 Jan. 20, 2011 25

- Continued

212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: COMPLETELY ARTIFICIAL

<4 OOs, SEQUENCE: 16 Glin Asn Ala Thr Arg Ile Lys Arg Ser Asp Ala Lieu. Ser Arg Glin Ser 1. 5 1O 15 Gly His Lieu. Ser Arg Arg Ser Ala Asp Lieu. Thr Glu Asp Arg Ala Asn 2O 25 3O

Lieu. Ser Arg 35

<210s, SEQ ID NO 17 &211s LENGTH: 35 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: COMPLETELY ARTIFICIAL

<4 OOs, SEQUENCE: 17 Arg Ser Asp Asn Lieu. Ser Arg Asp Ser Ser Thr Arg Llys Lys Asn. Ser 1. 5 1O 15 Gly Asn Lieu. Asp Llys Glin Ser Gly Ala Lieu Ala Arg Arg Ser Asp Ala 2O 25 3O Lieu. Ala Arg 35

<210s, SEQ ID NO 18 &211s LENGTH: 35 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: COMPLETELY ARTIFICIAL

<4 OOs, SEQUENCE: 18 Glin Ser Gly Asn Lieu Ala Arg Arg Ser Asp Ser Lieu. Ser Val Glin Ser 1. 5 1O 15 Ala Asp Arg Thr Lys Arg Ser Asp Thr Lieu. Ser Thr Asp Arg Llys Thr 2O 25 3O Arg Ile Asn 35

<210s, SEQ ID NO 19 &211s LENGTH: 35 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: COMPLETELY ARTIFICIAL

<4 OOs, SEQUENCE: 19 Thir Ser Gly Asn Lieu. Thir Arg Glin Ser Gly Asn Lieu Ala Arg Lieu Lys 1. 5 1O 15 Glin Asn Lieu. Asp Ala Asp Arg Ser His Lieu. Thir Arg Arg Lieu. Asp Asn 2O 25 3O Arg Thr Ala 35

<210s, SEQ ID NO 2 O &211s LENGTH: 35 US 2011/001 6543 A1 Jan. 20, 2011 26

- Continued

212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: COMPLETELY ARTIFICIAL

<4 OOs, SEQUENCE: 2O Asp Arg Ser Asp Lieu. Ser Arg Glin Ser Gly Asn Lieu Ala Arg Arg Ser 1. 5 1O 15 Asp Thir Lieu. Ser Glu Glin Arg Gln His Arg Thr Thr Glin Asn Ala Thr 2O 25 3O Arg Ile Llys 35

<210s, SEQ ID NO 21 &211s LENGTH: 35 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: COMPLETELY ARTIFICIAL

<4 OOs, SEQUENCE: 21 Arg Ser Asp His Lieu. Ser Ala Glin Ser Gly His Lieu. Ser Arg Asp Ser 1. 5 1O 15

Glu Ser Lieu. Asn Ala Thir Ser Ser Asn Lieu. Ser Arg Asp Arg Ser Ser 2O 25 3O Arg Lys Arg 35

<210s, SEQ ID NO 22 &211s LENGTH: 35 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: COMPLETELY ARTIFICIAL

<4 OOs, SEQUENCE: 22 Gln Ser Gly Ser Lieu. Thr Arg Glin Ser Ser Asp Lieu. Arg Arg Glin Arg 1. 5 1O 15 Thr His Lieu. Thr Glin Glin Ser Gly His Lieu. Glin Arg Glin Ser Gly Asp 2O 25 3O Lieu. Thir Arg 35

<210s, SEQ ID NO 23 &211s LENGTH: 35 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: COMPLETELY ARTIFICIAL

<4 OOs, SEQUENCE: 23 Glin Ser Gly Asp Lieu. Thir Arg Ser Ser Ser Asp Arg Llys Lys Asp Ser 1. 5 1O 15 Ser Asp Arg Llys Lys Arg Ser Asp Asn Lieu. Ser Thr Asp Asn. Ser Asn 2O 25 3O Arg Ile Asn 35

<210s, SEQ ID NO 24 &211s LENGTH: 35 US 2011/001 6543 A1 Jan. 20, 2011 27

- Continued

212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: COMPLETELY ARTIFICIAL

<4 OOs, SEQUENCE: 24 Thir Ser Gly. His Lieu. Ser Arg Glin Ser Gly Asn Lieu Ala Arg His Lieu. 1. 5 1O 15 Gly Asn Lieu Lys Thr Glin Ser Ser Asp Lieu. Ser Arg Glin Ser Gly Asn 2O 25 3O Arg Thr Thr 35

<210s, SEQ ID NO 25 &211s LENGTH: 3 OO &212s. TYPE: DNA &213s ORGANISM: Rattus rattus

<4 OOs, SEQUENCE: 25 tat catccac ggagggaaaa cqccaaacaa tdagctitt.cc gataagattt atat catgtc 6 O tgtc.gcttgc aagaataa.ca aaaaagttac titt cogttgt acagagaaag act tagtagg 12 O agatgtc.cct gaa.gc.cagat atggc catt C cattgacgtg gtatatago C gaggaaaaag 18O tgttggtgtt citctittggag gacggtcata catgc ctitct acccaaagaa ccacagaaaa 24 O atggaatagt gtagctgatt gcc taccc.ca tdttitt Cttg gtagattittgaatttgggtg 3OO

<210s, SEQ ID NO 26 &211s LENGTH: 3 OO &212s. TYPE: DNA &213s ORGANISM: Rattus rattus

<4 OOs, SEQUENCE: 26 tat catccac ggagggaaaa cqccaaacaa tdagctitt.cc gataagattt atat catgtc 6 O tgtc.gcttgc aagaataa.ca aaaaagttac titt cogttgt acagagaaag act tagtagg 12 O agatgtc.cct gaa.gc.cagat atggc catt C cattgacgtg gtatatago C gaggaaaaag 18O tgttggtgtt citctittggag gacggtcata catgc ctitct acccaaagaa ccacagaaaa 24 O atggaatagt gtagctgatt gcc taccc.ca tttitt Cttg gtagattittgaatttgggtg 3OO

What is claimed is: 7. The genetically modified animal of claim 1, wherein the 1. A genetically modified animal comprising at least one inflammation-related protein is chosen from the proteins edited chromosomal sequence encoding an inflammation related protein. listed in Table A, and combinations thereof. 2. The genetically modified animal of claim 1, wherein the 8. The genetically modified animal of claim 1, wherein the edited chromosomal sequence is inactivated, modified, or inflammation-related protein is chosen from MCP1, CCR5, comprises an integrated sequence. FCGR2B, FCER1g, IFN-y, IL-4, perforin-1, COX1, COX2, 3. The genetically modified animal of claim 1, wherein the TBX21, SH2BPSM1, FGFR2, SLC22A1, PPAR-O, PTEN, edited chromosomal sequence is inactivated Such that a func IL-1C., IL-1B, IL-6, IL-10, IL-12C., IL-12B, IL-13, IL-17A, tional inflammation-related protein is not produced. IL-17B, IL-17C, IL-17D, IL-17F, IL-23, CX3CR1, CX3CL1, 4. The genetically modified animal of claim 3, wherein the RAG1, PTPN22, TNFC., NOD2, CTLA4, and combinations inactivated chromosomal sequence comprises no exog thereof. enously introduced sequence. 5. The genetically modified animal of claim 1, wherein the 9. The genetically modified animal of claim 1, further edited chromosomal sequence is modified Such that the comprising a conditional knock-out system for conditional inflammation-related protein is over-produced. expression of the inflammation-related protein. 6. The genetically modified animal of claim 3, further 10. The genetically modified animal of claim 1, wherein comprising at least one chromosomally integrated sequence the edited chromosomal sequence comprises an integrated encoding a functional inflammation-related protein. reporter sequence. US 2011/001 6543 A1 Jan. 20, 2011 28

11. The genetically modified animal of claim 1, wherein 28. The genetically modified cell of claim 19, wherein the the animal is heterozygous or homozygous for the at least one cell is heterozygous or homozygous for the at least one edited edited chromosomal sequence. chromosomal sequence. 12. The genetically modified animal of claim 1, wherein 29. The genetically modified cell of claim 19, wherein the the animal is an embryo, a juvenile, or an adult. cell is of bovine, canine, equine, feline, human, Ovine, por 13. The genetically modified animal of claim 1, wherein cine, non-human primate, or rodent origin. the animal is chosen from bovine, canine, equine, feline, 30. The genetically modified cell of claim 23, wherein the ovine, porcine, non-human primate, and rodent. cell is of rat origin and the chromosomally integrated 14. The genetically modified animal of claim 6, wherein sequence encoding an inflammation-related protein is the animal is rat and the chromosomally integrated sequence human. encoding an inflammation-related protein is human. 15. A non-human embryo, the embryo comprising at least 31. A method for assessing the effect of a genetically modi one RNA molecule encoding a Zinc finger nuclease that rec fied inflammation-related protein on the progression or symp ognizes a chromosomal sequence encoding an inflammation toms of an inflammation-related disease state in an animal, related protein, and, optionally, at least one donor polynucle the method comprising comparing a wild type animal to a otide comprising a sequence encoding an inflammation genetically modified animal comprising at least one edited related protein. chromosomal sequence encoding an inflammation-related 16. The non-human embryo of claim 15, wherein the protein, and measuring a phenotype associated with the dis inflammation-related protein is chosen from MCP1, CCR5, ease State. FCGR2B, FCER1g, IFN-y, IL-4, perforin-1, COX1, COX2, 32. The method of claim 31, wherein the at least one edited TBX21, SH2BPSM1, FGFR2, SLC22A1, PPAR-O, PTEN, chromosomal sequence is inactivated Such that a functional IL-1C., IL-1B, IL-6, IL-10, IL-12C., IL-12B, IL-13, IL-17A, inflammation-related protein is not produced. IL-17B, IL-17C, IL-17D, IL-17F, IL-23, CX3CR1, CX3CL1, 33. The method of claim 31, wherein the at least one edited RAG1, PTPN22, TNFC., NOD2, CTLA4, and combinations chromosomal sequence is inactivated Such that the inflamma thereof. tion-related protein is over-produced. 17. The non-human embryo of claim 15, wherein the 34. The method of claim 31, wherein the at least one edited embryo is chosen from bovine, canine, equine, feline, Ovine, chromosomal sequence is inactivated Such that the inflamma porcine, non-human primate, and rodent. tion-related protein is not produced or is not functional, and 18. The non-human embryo of claim 15, wherein the wherein the animal further comprises at least one chromo embryo is rat and the donor polynucleotide comprising a Somally integrated sequence encoding a functional inflam sequence encoding an inflammation-related protein is mation-related protein. human. 35. The method of claim 31, wherein the inflammation 19. A genetically modified cell, the cell comprising at least related protein is chosen from the proteins listed in Table A, one edited chromosomal sequence encoding an inflamma and combinations thereof. tion-related protein. 36. The method of claim 31, wherein the inflammation 20. The genetically modified cell of claim 19, wherein the related protein is chosen from MCP1, CCR5, FCGR2B, edited chromosomal sequence is inactivated, modified, or FCER1g, IFN-y, IL-4, perforin-1, COX1, COX2, TBX21, comprises an integrated sequence. SH2BPSM1, FGFR2, SLC22A1, PPAR-O, PTEN, IL-1C., 21. The genetically modified cell of claim 19, wherein the IL-1B, IL-6, IL-10, IL-12C., IL-12B, IL-1B, IL-17A, IL-17B, edited chromosomal sequence is inactivated Such that a func IL-17C, IL-17D, IL-17F, IL-23, CX3CR1, CX3CL1, RAG1, tional inflammation-related protein is not produced. PTPN22, TNFC, NOD2, CTLA4, and combinations thereof. 22. The genetically modified cell of claim 19, wherein the 37. The method of claim 31, wherein the disease state is edited chromosomal sequence is modified Such that the chosen from allergies, autoimmunity, arthritis, asthma, ath inflammation-related protein is over-produced. erosclerosis, amyloid diseases, acne, cancer, infections, 23. The genetically modified cell of claim 21, further com ischaemic heart disease, inflammatory bowel disorders, inter prising at least one chromosomally integrated sequence Stitial cystitis, hypersensitivities, inflammatory bowel dis encoding a functional inflammation-related protein. eases, reperfusion injury, transplant rejection, obesity, myo 24. The genetically modified cell of claim 19, wherein the pathies, leukopenia, Vitamin deficiencies, pelvic inflammation-related protein is chosen from the proteins inflammatory disease, glomeronephritis, graft versus host listed in Table A, and combinations thereof. disease (transplant rejection), preterm labor, vasculitis, viti 25. The genetically modified cell of claim 19, wherein the ligo, and HIV infection and progression to AIDS. inflammation-related protein is chosen from MCP1, CCR5, 38. A method for assessing the effect of an agent on pro FCGR2B, FCER1g, IFN-y, IL-4, perforin-1, COX1, COX2, gression or symptoms of inflammation, the method compris TBX21, SH2BPSM1, FGFR2, SLC22A1, PPAR-O, PTEN, ing: IL-1C., IL-1B, IL-6, IL-10, IL-12C., IL-12B, IL-13, IL-17A, IL-17B, IL-17C, IL-17D, IL-17F, IL-23, CX3CR1, CX3CL1, a) contacting a genetically modified animal comprising at RAG1, PTPN22, TNFC., NOD2, CTLA4, and combinations least one edited chromosomal sequence encoding an thereof. inflammation-related protein with the agent; 26. The genetically modified cell of claim 19, further com b) measuring an inflammation-related phenotype, and prising a conditional knock-out System for conditional c) comparing results of the inflammation-related pheno expression of the inflammation-related protein. type in (b) to results obtained from a control genetically 27. The genetically modified cell of claim 19, wherein the modified animal comprising said edited chromosomal edited chromosomal sequence comprises an integrated sequence encoding an inflammation-related protein not reporter sequence. contacted with the agent. US 2011/001 6543 A1 Jan. 20, 2011 29

39. The method of claim 38, wherein the agent is a phar 42. The method of claim 38, wherein the inflammation maceutically active ingredient, a drug, a toxin, or a chemical. related protein is chosen from the proteins listed in Table A, and combinations thereof. 40. The method of claim38, wherein the at least one edited 43. The method of claim 38, wherein the inflammation chromosomal sequence is inactivated Such that the inflamma related protein is chosen from MCP1, CCR5, FCGR2B, tion-related protein is not produced or is not functional. FCER1g, IFN-y, IL-4, perforin-1, COX1, COX2, TBX21, 41. The method of claim38, wherein the at least one edited SH2BPSM1, FGFR2, SLC22A1, PPAR-O, PTEN, IL-1C., chromosomal sequence is inactivated Such that the inflamma IL-1 B, IL-6, IL-10, IL-12C., IL-12|B, IL-13, IL-17A, IL-17B, tion-related protein is not produced or is not functional, and IL-17C, IL-17D, IL-17F, IL-23, CX3CR1, CX3CL1, RAG1, wherein the animal further comprises at least one chromo PTPN22, TNFC, NOD2, CTLA4, and combinations thereof. Somally integrated sequence encoding a functional inflam mation-related protein. c c c c c