Methods and Compositions for the Treatment of Immune Disorders

Abstract

The present invention provides a method of treating an immune-related disorder in a subject, comprising administering to the subject an effective amount of an inhibitor of plexin-A4 activity, which results in reducing the plexin-A4 activity in the subject, and thereby treating the immune-related disorder. Inhibitors of plexin-A4 activity include, for example, plexin-A4 antibodies and plexin-A4 fusion proteins. The present invention further provides a method of treating an immune-related disorder in a subject, comprising administering to the subject an effective amount of an inhibitor of semaphorin-3A (Sema3A) activity, which results in reducing the Sema3A activity in the subject, thereby treating the immune-related disorder. Inhibitors of Sema3A activity include, for example, Sema3A antibodies and Sema3A fusion proteins.

Description

STATEMENT OF PRIORITY

[0001] The present application is a continuation application of, and claims priority to, U.S. application Ser. No. 13/511,933, filed Jun. 12, 2012, now U.S. Pat. No. 8,871,205, which is a 35 U.S.C. §371 national phase application of International Application No. PCT/US2010/057807, filed Nov. 23, 2010, which claims the benefit, under 35 U.S.C. §119(e), of U.S. Provisional Application Ser. No. 61/264,338, filed Nov. 25, 2009, the entire contents of each of which are incorporated by reference herein.

STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

[0003] A Sequence Listing in ASCII text format, submitted under 37 C.F.R. §1.821, entitled 5470-536TSCT_ST25.txt, 129,544 bytes in size, generated on Oct. 2, 2014 and filed via EFS-Web, is provided in lieu of a paper copy. This Sequence Listing is incorporated herein by reference into the specification for its disclosures.

FIELD OF THE INVENTION

[0004] The present invention relates to methods and compositions for treatment of immune related disorders and diseases.

BACKGROUND OF THE INVENTION

[0005] The functions of plexins and their ligands, semaphorins, have been extensively studied in the central nervous system (CNS). They represent two large families of molecules that can transduce signals essential for the regulation of neuronal repulsion and attraction, cell shape, motility and cell-cell interactions (Kruger et al., 2005; Tran et al., 2007). In addition to their roles in the CNS, the diverse functions of plexins and semaphorins have also been identified in cardiac development (Toyofuku et al., 2004), vascularization and angiogenesis (Gu et al., 2003a; Serini et al., 2003), and tumorigenesis (Neufeld and Kessler, 2008; Sierra et al., 2008). More recent data strongly indicate a role for these molecules in the immune system (Kikutani and Kumanogoh, 2003; Suzuki et al., 2008). For example, plexin-A1 is expressed by dendritic cells (DCs) and regulates DC interaction with T cells to affect adaptive immunity (Takegahara et al., 2006; Wong et al., 2003). Plexin-C1 is also found on DCs, although its role is less defined and it only mildly affects T cell activation (Walzer et al., 2005). A further paper showed the high expression of plexin-Dl in double-positive (DP) thymocytes and a role for this protein in the control of intrathymic migration of these cells from the cortical to medullary region (Choi et al., 2008). Therefore, plexins are involved in diverse functions in the immune system.

[0006] Plexin-A4 belongs to the plexin A-type group (Kruger et al., 2005) and serves as a guidance cue molecule in sensory and sympathetic neurons (Waimey et al., 2008; Yaron et al., 2005) and hippocampal mossy fibers (Suto et al., 2007). One study identified plexin-A4 as a negative regulator in T cell activation (Yamamoto et al., 2008). T cells lacking plexin-A4 (Plxna4.sup.-/-) exhibited hyperproliferative responses upon stimulations in vivo and in vitro. In addition, Plxna4.sup.-/- mice developed exacerbated experimental autoimmune encephalomyelitis (EAE) when immunized with myelin oligodendrocyte glycoprotein (MOG)-derived peptides. However, given the much higher expression of plexin-A4 in myeloid cells relative to lymphoid cells (Yamamoto et al., 2008), the role of plexin-A4 in cells of myeloid lineage such as macrophages and DCs needed to be elucidated.

[0007] The innate immune system constitutes the first line of defense by rapidly detecting invading pathogens and nonmicrobial danger signals through the pattern recognition receptors (PRRs). Several classes of PRRs have been identified; the best-characterized are the Toll-like receptors (TLRs) (Iwasaki and Medzhitov, 2004). TLR family members are localized either on the cell surface (TLRs 1, 2, 4, 5 and 6) or in endosomal compartments (TLRs 3, 7, 8, 9) to detect a multitude of pathogen-associated molecular patterns (PAMPs) (Akira et al., 2006; Iwasaki and Medzhitov, 2004). TLR activation leads to the direct interactions of the TLR toll-interleukin 1 receptor (TIR) domain with a cytoplasmic TIR-containing adaptive molecule such as Myd88, TRIF, TRAM or TIRAP. Activation of Myd88-dependent signaling pathway results in the activation of IRAK kinases, the ubiquitin ligase TRAF6, TAK1 kinase complex, NF-κB transcription factor, and mitogen-activated protein kinases (MAPKs) (Akira and Takeda, 2004; Akira et al., 2006; Iwasaki and Medzhitov, 2004). TRIF-dependent type I interferon (IFN) requires a cascade involving the adaptor TRAF3, the kinase TBK1, the inhibitor of κB kinase ε (IKKε), and the transcription factor interferon-regulatory factor 3 (IRF3) (Akira et al., 2006; Kawai and Akira, 2006).

[0008] The present invention addresses the shortcomings in the art by providing methods and compositions for the treatment of immune related and inflammatory disorders and diseases based on new therapeutic targets.

SUMMARY OF THE INVENTION

[0009] Provided herein is a method of treating an immune-related and/or inflammatory disorder in a subject (e.g., a subject in need thereof), comprising administering to the subject in need of treatment an effective amount of an inhibitor of plexin-A4 activity, whereby the plexin-A4 activity in said subject is reduced, thereby treating the immune-related disorder and/or inflammatory disorder. In some embodiments, the inhibitor of plexin-A4 activity is a plexin-A4 fusion protein, a plexin-A4 antibody or an active fragment thereof, or any combination thereof. In some embodiments, the plexin-A4 fusion protein comprises the extracellular domain of plexin-A4 and the Fc region of immunoglobulin G (IgG).

[0010] Also provided is a method of reducing cytokine production in a subject (e.g., a subject in need thereof), comprising administering to the subject an effective amount of an inhibitor of plexin-A4 activity. In some embodiments, the inhibitor of plexin-A4 activity is a plexin-A4 fusion protein, a plexin-A4 antibody or an active fragment thereof, or any combination thereof. In some embodiments, the plexin-A4 fusion protein comprises the extracellular domain of plexin-A4 and the Fc region of IgG.

[0011] Further provided is a method of identifying a substance having an inhibitory effect on plexin-A4 activity, comprising contacting the substance with macrophage/dendritic cells under conditions whereby plexin-A4 activity can occur and measuring the amount of plexin-A4 activity in the presence and absence of the substance, whereby a decrease in plexin-A4 activity in the presence of the substance as compared to the amount of plexin-A4 activity in the absence of the substance identifies a substance having an inhibitory effect on plexin-A4 activity.

BRIEF DESCRIPTION OF THE FIGURES

[0012] FIGS. 1A-C show that plexin-A4 is required for tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) production in macrophages. (FIG. 1A) Expression of Plxna4 mRNA in different immune cells was normalized to Actb mRNA level. T, B, NK, bone marrow (BM) plasmacytoid DCs (pDCs), and splenic myeloid DCs (mDCs) were isolated by fluorescence activated cell sorting (FACS) according to appropriate cell surface markers described in the Examples section. BM-derived macrophages (BMMs) and BM-derived dendritic cells (BMDCs) were respectively cultured from BM cells in the presence of L929 cell supernatant and granulocyte-macrophage colony-stimulating factor (GM-CSF) plus interleukin-4 (IL-4). Peritoneal and splenic macrophages were separated from other cells by adherence for 2 h at 37° C. FIG. 1B and FIG. 1C illustrate stimulation of peritoneal macrophages with Pam3Cys (5 μg/ml), poly(I:C) (10 μg/ml), ultrapure lipopolysaccharide (LPS) (1 μg/ml), R837 (10 μg/ml), or CpG-B (4 Wimp for 4 h. Messenger RNA (FIG. 1B) and protein (FIG. 1C) levels of TNF-α, IL-6 and interferon-beta (IFN-β) were measured by RT-PCR and ELISA, respectively. The results shown are representative of five independent experiments and are expressed as mean±s.d. *P<0.05 compared to WT peritoneal macrophages.

[0013] FIGS. 2A-B show that plexin-A4 is required for cytokine production in response to bacterial challenge. Peritoneal macrophages isolated from WT or Plxna4.sup.-/- mice were stimulated with Escherichia coli, Salmonella typhimurium, Listeria monocytogenes or Staphylococcus aureus at a multiplicity of infection (MOI) of 40. Messenger RNA (FIG. 2A) and protein (FIG. 2B) levels of TNF-α, IL-6 and IFN-β were measured by RT-PCR and ELISA, respectively. The results shown are representative of three independent experiments and are expressed as mean±s.d. * P<0.05 compared to WT peritoneal macrophages.

[0014] FIGS. 3A-E shows that plexin-A4 is required for LPS-induced activation of nuclease factor-kappa B (NF-κB), c-Jun-N-terminal kinase (JNK) and small GTPase Rac1. Immunoblot analysis of NF-κB and mitogen-activated protein kinase (MAPK) signaling molecules was performed with WT and Plxna4.sup.-/- peritoneal macrophages left untreated (0) or treated for 15, 30 or 60 min with 1 μg/ml ultrapure LPS (FIG. 3A). FIG. 3B and FIG. 3C show chromatin immunoprecipitation (ChIP) assays of the binding of p65 or c-Jun at the promoter regions of Tnfa and Il6 gene in WT and Plxna4.sup.-/- peritoneal macrophages left untreated or treated with 10 μg/ml poly(I:C), 1 μg/ml ultrapure LPS (B), 5 μg/ml Pam3Cys or 4 μg/ml CpG (C) for 1 h. Rho family GTPase (Rac1, Cdc42 and RhoA) pull-down analyses in WT and Plxna4.sup.-/- peritoneal macrophages is shown in FIG. 3D. Cells were left untreated (0) or treated for 15 or 30 min with 1 μg/ml ultrapure LPS. Rac1, Cdc42 and RhoA pull-down analysis were performed as described in the Examples section. Total Rac1, Cdc42 and RhoA in cell lysate were used as controls. WT peritoneal macrophages were pretreated with or without a Rac1 inhibitor, NSC23766 (200 μM) for 1 h, or left untreated, followed by stimulation with 5 μg/ml Pam3Cys, 10 μg/ml poly(I:C), 1 μg/ml ultrapure LPS, 10 μg/ml R837, or 4 μg/ml CpG for 4 h as shown in FIG. 3E. IFN-43 production in WT macrophages was not affected by Rac1 inhibition as shown in FIG. 3F. Protein levels of TNF-α and IL-6 were measured by ELISA. The results shown are representative of three independent experiments and are expressed as mean±s.d. * P<0.05 compared to WT peritoneal macrophages without NSC23766 pretreatment.

[0015] FIGS. 4A-C show that Plxna4.sup.-/- mice are protected from septic shock induced by poly(I:C) or LPS. FIG. 4A illustrates the survival of WT and Plxna4.sup.-/- mice (n=8 per group) given intraperitoneal injection of poly(I:C) (20 mg per kg body weight; left panel) or LPS (12.5 mg per kg body weight; right panel). FIG. 4B and FIG. 4C show the results of ELISA of inflammatory cytokines in peritoneal lavage (FIG. 4B) and serum (FIG. 4C) 4 h after the administration of poly(I:C), LPS, or PBS control (n=4 per group). The results shown are representative of two experiments and are expressed as mean±s.d. * P<0.05 compared to WT mice.

[0016] FIGS. 5A-D show that Plxna4.sup.-/- mice are protected from polymicrobial peritonitis induced by cecal ligation and puncture (CLP). FIG. 5A shows the survival of WT and Plxna4.sup.-/- mice (n=10 per group) after CLP procedure with two punctures (left panel) or nine punctures (right panel). Results are representative of three individual experiments. FIGS. 5B-D show the results of ELISA of inflammatory cytokines in peritoneal lavage (FIG. 5B), serum (FIG. 5C) and lung homogenate (FIG. 5D) before CLP or 4, 24, and 72 h after CLP (n=5 per group). The results are representative of at least two individual experiments. * P<0.05 compared with cytokine levels measured in peritoneal lavage, serum, or lung homogenate from WT mice.

[0017] FIGS. 6A-E shows that plexin-A4 is not required for macrophage phagocytosis and bacteria killing. WT and Plxna4.sup.-/- mice were subjected to CLP surgery (FIG. 6A). Bacterial loads in peritoneal lavage (left panel), blood (middle panel) and lung homogenate (right panel) were determined by counting the colony forming units. FIG. 6B provides FACS histograms showing phagocytosis of E. coli-GFP (MOI=100) by WT (black lines) or Plxna4.sup.-/- (tinted) peritoneal macrophages at 37° C. The two show a complete overlap. GFP fluorescence is plotted on the x axis, and cell number is plotted on they axis. FIG. 6C and FIG. 6D provide the mean fluorescence intensities (MFIs) of GFP showing phagocytosis of E. coli-GFP after various time periods (FIG. 6C) or at different MOI (FIG. 6D) by WT and Plxna4.sup.-/- peritoneal macrophages at 37° C. FIG. 6E shows the results of treatment of WT and Plxna4.sup.-/- peritoneal macrophages with E. coli (strain LF82) at a MOI of 10 (left panel) or 100 (right panel) for 1 h at 37° C., followed by the addition of 100 μg/ml gentamicin. Intracellular alive bacteria were determined by plate counting after various time periods (0, 1, 2 or 3 h).

[0018] FIGS. 7A-G show that Semaphorin 3A (Sema3A) enhances LPS-induced cytokine production in a plexin-A4-dependent manner. WT and Plxna4.sup.-/- peritoneal macrophages were stimulated with LPS (1 μg/ml) for 4 h in the absence or presence of either Sema3A-Fc or Sema6A-Fc fusion proteins (20 or 100 μg/ml) (FIG. 7A and FIG. 7B). Messenger RNA (FIG. 7A) or protein (FIG. 7B) levels of TNF-α and IL-6 were measured by RT-PCR and ELISA, respectively. The IgG Fc fragment (100 μg/ml) was used as a negative control. WT and Plxna4.sup.-/- mice (n=7˜9 per group) were pretreated with either Sema3A-Fc or IgG Fc control proteins at a dosage of 25 μg per kg body weight 1 h prior to CLP procedure (FIG. 7C). Survival rate was monitored for 5 days. τ P<0.05 compared with WT mice pretreated with Sema3A-Fc. WT mice (n=4 per group) were pretreated with either Sema3A-Fc or IgG Fc control proteins, followed by CLP surgery (FIG. 7D). Inflammatory cytokines in peritoneal lavage collected 4 h after CLP were determined by ELISA. * P<0.05 compared to peritoneal lavage from WT mice pretreated with Sema3A-Fc (FIG. 7E). FIG. 7F shows a schematic of the cellular interactions during cytokine storm. WT and Plxna4.sup.-/- mice (n=4 per group experiment) were pretreated with either Sema3A-Fc or IgG Fc control proteins at a dosage of 25 ug/kg body weight 1 hour before CLP procedure. Inflammatory cytokines in peritoneal lavage collected 4 h after CLP were determined by ELISA.*P<0.05 compared with peritoneal lavage from WT mice pretreated with Sema3A-Fc (FIG. 7G).

[0019] FIGS. 8A-B show that plexin-A4 does not modulate the presentation of OVA antigen by bone marrow-derived dendritic cells (BMDCs). BMDCs generated from WT or Plxna4.sup.-/- mice were pulsed with either 1 μg/ml OVA peptide_323-339 (FIG. 8A) or 50 μg/ml OVA whole protein (FIG. 8B), and co-cultured with carboxyfluorescein succinimidyl ester (CFSE)-labeled splenic CD4⁺ T cells isolated from naive TCR-transgenic OTII mice. Two or four days later, T cell proliferation was analyzed by the dilution of CFSE fluorescence.

[0020] FIG. 9 shows that plexin-A4 does not affect TLR-induced IFN-α production by plasmacytoid dendritic cells (pDCs). Plasmacytoid DCs were isolated from the bone marrows of WT and Plxna4.sup.-/- mice by FACS, and stimulated with 10 μg/ml R837 (TLR7) or 4 μg/ml CpG-B for 16 h. IFN-α protein levels in the supernatants were determined by ELISA.

[0021] FIGS. 10A-C show that plexin-A4 does not affect the number of peritoneal macrophages. Total peritoneal cells were harvested from WT and Plxna4.sup.-/- mice by peritoneal lavage. Cytospins were prepared and stained by hematoxylin and eosin (Panel A). The percent of peritoneal macrophages from WT and Plxna4.sup.-/- mice (Panel B) were multiplied by the total cell count to determine the absolute numbers (Panel C).

[0022] FIGS. 11A-F show that plexin-A4 does not affect immune cell composition in the spleen. Cells of myeloid or lymphoid lineage in the spleen were analyzed by FACS, including CD11b⁺CD11c⁺ myeloid DCs (FIG. 11A), B220⁺mPDCA1⁺CD11c^low pDCs (FIG. 11B), CD11b⁺Ly6G⁺ neutrophils (FIG. 11C), CD11b⁺Ly6c⁺ monocytes and CD11b⁺F4/80⁺ macrophages (FIG. 11D), CD3⁺CD4⁺ T cells and CD3⁺CD8⁺ T cells (FIG. 11E), and CD19⁺ B cells and NK1.1⁺ NK cells (FIG. 11F).

[0023] FIGS. 12A-C show amino acid sequences for three plexin-A4 proteins. The residues indicated in bold show the sequence for the extracellular domain of the protein.

[0024] FIG. 13 shows the nucleotide sequence of the plexin-A4 coding sequence.

[0025] FIG. 14 shows the nucleotide sequence of the plexin-A4 cDNA. The nucleotides in bold are the primer sites and start (ATG) and stop codons (TGA).

[0026] FIGS. 15A-C show the amino acid sequence of the plexin-A4 fusion protein. In FIG. 15A, the portion of the sequence in bold indicates the extracellular domain of plexin-A4 and the non-bolded portion indicates the Fc region of IgG1. The two residues in bold and larger font between the plexin-A4 extracellular domain and the IgG Fc comprise the linker peptide of the fusion protein. In FIG. 15B is shown the amino acid sequence of the extracellular domain of plexin-A4 and in FIG. 15C is shown the amino acid sequence of the Fc region of IgG1.

[0027] FIGS. 16A-C show the nucleotide sequence of the plexin-A4 fusion protein. In FIG. 16A, the portion of the sequence in bold indicates the plexin-A4 extracellular domain and the non-bolded portion indicates the Fc region of IgG1. The six nucleotides in bold and larger font between the plexin-A4 extracellular domain and the IgG Fc show the nucleotide sequence encoding the linker peptide. FIG. 16B shows the nucleotide sequence of the extracellular domain of plexin-A4 and FIG. 16C shows the nucleotide sequence of the Fc region of IgG1.

[0028] FIGS. 17A-C. FIG. 17A shows plexin-A4 mRNA levels in a variety of different cell types. FIG. 17B shows the amino acid sequence for IgG with the Fc region of the sequence in bold. FIG. 17C shows a protein immunoblot for three different fusion proteins: plexin-A4/IgG Fc fusion protein (lanes 1, 2, 4, and 5), a Sema6D/IgG Fc fusion protein (lane 3) and a sema6D-IgG Fc fusion protein (lane 6). Lanes 1-3 were probed with antibody to plexin-A4 and lanes 5-6 were probed with antibody to hIgG.

[0029] FIGS. 18A-E. FIG. 18A shows the amino acid sequence for the Semaphorin 3A (Sema3A) polypeptide. FIG. 18B shows the nucleotide sequence of the Sema3A coding sequence. FIG. 18C shows nucleotide sequence of the Sema3A cDNA. The nucleotides in bold are the primer sites and start (ATG) and stop codons (TGA). FIG. 18D shows the amino acid sequence of the fusion protein between full-length human Sema3A and human IgG Fc. Full-length human Sema3A is in bold and the non-bolded portion of the sequence indicates the Fc region of IgG1. The two residues in bold and larger font between the Sema3A and the IgG Fc comprise a linker peptide. FIG. 18E shows the nucleotide sequence encoding the fusion protein between full-length human Sema3A and human IgG Fc. In FIG. 18E, the portion of the sequence in bold indicates the full length human Sema3A nucleotide sequence and the non-bolded portion indicates nucleotide sequence of the Fc region of IgG1. The six nucleotides in bold and larger font between Sema3A and IgG Fc show the nucleotide sequence encoding a linker peptide.

[0030] FIGS. 19A-B show that plexin-A4 is required for TLR-induced cytokine production in bone marrow-derived macrophages (BMMs) and bone marrow-derived dendritic cells (BMDCs). BMMs (FIG. 19A) and BMDCs (FIG. 19B) were generated from the bone marrow cells of WT and Plxna4.sup.-/- mice, and stimulated with various TLR agonists for 4 h. Protein levels of TNF-α and IL-6 in the supernatant were determined by ELISA.

[0031] FIGS. 20A-C show that plexin-A4 does not affect TNF-α, CD40, or PI3K/Akt signaling pathways. Peritoneal macrophages were isolated from WT and Plxna4.sup.-/- mice by peritoneal lavage. Cells were treated either with recombinant mouse TNF-α (20 ng/ml) or anti-CD40 (5 μg/ml) after TNF-α (1 ng/ml) priming for 3 h. TNF-α priming is required to upregulate CD40 expression (Lich et al., 2007). Immunoblot of MAPK signaling molecules was performed (FIG. 20A). Cells were treated with recombinant mouse IFN-γ, and STAT1 phosphorylation at tyrosine 701 was detected by immunoblot (FIG. 20B). Cells were treated with either Pam3Cys (5 μg/ml) or LPS (1 μg/ml). Akt phosphorylated at serine 473 was detected by immunoblot (FIG. 20C).

[0032] FIG. 21 shows that plexin-A4 does not affect toll-like receptor mRNA expression in peritoneal macrophages. Messenger RNA levels of Tlr2, 3, 4, 7 and 9 in WT and Plxna4.sup.-/- peritoneal macrophages were analyzed by RT-PCR using different primer sets, as described in Example 21, Table 1, below.

[0033] FIG. 22 shows the expression of Sema3a and Sema6a mRNA in different immune cells. Messenger RNA levels of Sema3a and Sema6a in different immune subpopulations were analyzed by RT-PCR and normalized to Actb mRNA level.

DETAILED DESCRIPTION

[0034] The present invention will now be described more fully hereinafter with reference to the accompanying drawings and specification, in which preferred embodiments of the invention are shown. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0036] All publications, patent applications, patents and other references cited herein are incorporated by reference in their entireties for the teachings relevant to the sentence and/or paragraph in which the reference is presented.

[0037] As used herein, "a," "an" or "the" can mean one or more than one. For example, "a" cell can mean a single cell or a multiplicity of cells.

[0038] Also as used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

[0039] The term "about," as used herein when referring to a measurable value such as an amount of dose (e.g., an amount of a non-viral vector) and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount.

[0040] As used herein, the transitional phrase "consisting essentially of" (and grammatical variants) means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim, "and those that do not materially affect the basic and novel characteristic(s)" of the claimed invention. Thus, the term "consisting essentially of" when used in a claim of this invention is not intended to be interpreted to be equivalent to "comprising."

[0041] Plexins are large (˜200 Kd) transmembrane glycoproteins with a conserved extracellular "Sema" domain. Plexin binds to its ligand, semaphorin, through Sema-Sema domain interaction, and transduces signals essential for cell migration in various tissue types (Kruger et al., 2005; Tran et al., 2007). The cytoplasmic domains of plexins have two highly conserved regions that share homology with GTPase-activating proteins (GAPs) that are known to activate the Ras superfamily of small GTPases. The present invention is based on the unexpected discovery that plexin-A4 in macrophages is required for optimal cytokine production upon TLR stimulation and bacterial challenge. Semaphorin 3A (Sema3A) serves as a ligand for plexin-A4 and enhances LPS-induced macrophage activation and cytokine production in a plexin-A4-dependent manner. Thus, through the inhibition of plexin-A4 activity, immune-related and/or inflammatory disorders including disorders/diseases that result in exacerbated production of proinflammatory cytokines and chemokines (those where TLR signaling is implicated) can be ameliorated.

[0042] Accordingly, in one embodiment, the present invention provides a method of treating an immune-related and/or inflammatory disorder in a subject in need thereof, comprising administering to the subject an effective amount of an inhibitor of plexin-A4 activity, whereby plexin-A4 activity in said subject is reduced, thereby treating the immune-related and/or inflammatory disorder.

[0043] Accordingly, in one embodiment, the present invention provides a method of treating an immune-related and/or inflammatory disorder in a subject in need thereof, comprising administering to the subject an effective amount of an inhibitor of plexin-A4 expression, whereby plexin-A4 expression in said subject is reduced, thereby treating the immune-related and/or inflammatory disorder.

[0044] Accordingly, in one embodiment, the present invention provides a method of treating an immune-related and/or inflammatory disorder in a subject in need thereof, comprising administering to the subject an effective amount of an inhibitor of Sema3A activity, whereby Sema3A activity in said subject is reduced, thereby treating the immune-related and/or inflammatory disorder.

[0045] Accordingly, in one embodiment, the present invention provides a method of treating an immune-related and/or inflammatory disorder in a subject in need thereof, comprising administering to the subject an effective amount of an inhibitor of Sema3A expression, whereby Sema3A expression in said subject is reduced, thereby treating the immune-related and/or inflammatory disorder.

[0046] As used herein, the terms "express," "expressing," or "expression" (or grammatical variants thereof) in reference to a gene or coding sequence can refer to transcription to produce an RNA and, optionally translation to produce a polypeptide. Thus, unless the context indicates otherwise, the terms "express," "expressing," "expression" and the like can refer to events at the transcriptional, post-transcriptional, translational and/or post-translational level.

[0047] In some embodiments of the present invention, the immune related and/or inflammatory disease or disorder can be, but is not limited to, sepsis; colitis; malignancies; systemic lupus erythematosis (SLE); arthritis, including, but not limited to, rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, and spondyloarthropathies; systemic sclerosis; idiopathic inflammatory myopathies; Sjogren's syndrome; systemic vasculitis; sarcoidosis; autoimmune hemolytic anemia; autoimmune thrombocytopenia; thyroiditis; diabetes mellitus; immune-mediated renal disease; demyelinating diseases of the central and peripheral nervous systems such as multiple sclerosis, idiopathic demyelinating polyneuropathy or Guillain-Barre syndrome, and chronic inflammatory demyelinating polyneuropathy; Alzheimer's disease; myocarditis; kidney disease; obesity; cardiovascular disease; hepatobiliary diseases such as infectious, autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis; inflammatory bowel disease; gluten-sensitive enteropathy; Whipple's disease; autoimmune or immune-mediated skin diseases including bullous skin diseases, erythema multiforme and contact dermatitis or hypersensitivity; psoriasis; allergic diseases such as asthma, allergic rhinitis, atopic dermatitis, food hypersensitivity and urticaria; immunologic diseases of the lung such as asthma, allergies, COPD (chronic obstructive pulmonary disease), eosinophilic pneumonias, idiopathic pulmonary fibrosis and hypersensitivity pneumonitis; transplantation associated diseases including graft rejection and graft-versus-host-disease; inflammation of the eye including but not limited to retinitis and uveitis; and any/or combination thereof.

[0048] In some embodiments, the immune related disease or disorder can be sepsis, arthritis, hepatitis, systemic lupus erythematosus (SLE), multiple sclerosis, Guillain-Barre syndrome, Alzheimer's disease, colitis, psoriasis, contact hypersensitivity, retinitis, uveitis, malignancies, systemic lupus erythematosis, asthma, myocarditis, hepatitis, kidney diseases, diabetes, obesity, cardiovascular diseases, inflammatory bowel disease and any/or combination thereof. In other embodiments, the immune-related disease or disorder is sepsis.

[0049] A "subject" of this invention includes any subject that is susceptible to the various diseases and/or disorders described herein. Nonlimiting examples of subjects of this invention include mammals, such as humans, nonhuman primates, domesticated mammals (e.g., dogs, cats, rabbits, guinea pigs, rats), livestock and agricultural mammals (e.g., horses, bovine, pigs, goats). In other embodiments, a subject may additionally be an animal such as a bird or reptile. Thus, in some embodiments, a subject can be any domestic, commercially or clinically valuable animal. Subjects may be male or female and may be any age including neonate, infant, juvenile, adolescent, adult, and geriatric subjects. In particular embodiments, the subject is a human. A human subject of this invention can be of any age, gender, race or ethnic group (e.g., Caucasian (white), Asian, African, black, African American, African European, Hispanic, Mideastern, etc.).

[0050] A "subject in need thereof" is a subject known to have, or suspected of having, diagnosed with, or at risk of having an immune related and/or inflammatory disease or disorder. A subject of this invention can also include a subject not previously known or suspected to have an immune related and/or inflammatory disease or disorder or in need of treatment for an immune related and/or inflammatory disease or disorder. For example, a subject of this invention can be administered the compositions of this invention even if it is not known or suspected that the subject has an immune related and/or inflammatory disease or disorder (e.g., prophylactically). A subject of this invention is also a subject known or believed to be at risk of developing an immune related and/or inflammatory disease or disorder. Accordingly, a subject in need thereof also includes a subject known to need, suspected of needing, or at risk of needing reduced cytokine production, reduced plexin-A4 activity or expression, or reduced Sema3A activity or expression. A subject of this invention can also include a subject not previously known or suspected to need reduced cytokine production, reduced plexin-A4 activity or expression, or reduced Sema3A activity or expression or to need treatment to reduce cytokine production, reduce plexin-A4 activity or expression, or reduce Sema3A activity or expression. For example, a subject of this invention can be administered the compositions of this invention even if it is not known or suspected that the subject needs reduced cytokine production or needs reduced plexin-A4 activity or expression or needs reduced Sema3A activity or expression (e.g., prophylactically). A subject of this invention is also a subject known or believed to be at risk of developing a need for reduced cytokine production, or for reduced plexin-A4 activity or expression, or for reduced Sema3A activity or expression.

[0051] A "subject in need thereof" is additionally a subject known to need, or suspected of needing, diagnosed with needing, or at risk of needing increased plexin-A4 activity or expression, or increased Sema3A activity or expression. A subject of this invention can also include a subject not previously known or suspected to need increased plexin-A4 activity or expression, or increased Sema3A activity or expression, or to need treatment to increase cytokine production, or to increase plexin-A4 activity or expression, or to increase Sema3A activity or expression. For example, a subject of this invention can be administered the compositions of this invention even if it is not known or suspected that the subject needs increased plexin-A4 activity or expression or increased Sema3A activity or expression (e.g., prophylactically). A subject of this invention is also a subject known or believed to be at risk of developing a need for increased plexin-A4 activity or expression or increased Sema3A activity or expression.

[0052] Also provided herein is a method of reducing cytokine production in a subject (e.g., a subject in need thereof), comprising administering to a subject an effective amount of an inhibitor of plexin-A4 activity. In other embodiments, a method of reducing plexin-A4 activity or expression in a subject is provided, comprising administering to a subject in need thereof an effective amount of an inhibitor of plexin-A4 activity. A subject in need thereof in these embodiments is a subject in need of reduced cytokine production or reduced plexin-A4 activity or expression, including, but not limited to, a subject having an immune-related and/or inflammatory disease or disorder including, but not limited to, those described herein.

[0053] Additionally provided herein is a method of reducing cytokine production in a subject (e.g., a subject in need thereof), comprising administering to a subject an effective amount of an inhibitor of Sema3A activity. In other embodiments, a method of reducing Sema3A activity or expression in a subject is provided, comprising administering to a subject in need thereof an effective amount of an inhibitor of Sema3A activity. A subject in need thereof in these embodiments is a subject in need of reduced cytokine production or reduced Sema3A activity or expression, including, but not limited to, a subject having an immune-related and/or inflammatory disease or disorder including, but not limited to, those described herein.

[0054] In further embodiments, an inhibitor of plexin-A4 activity includes, but is not limited to, a plexin-A4 fusion protein, a plexin-A4 antibody or an active fragment thereof or any combination thereof. In some embodiments, the plexin-A4 antibody is a monoclonal antibody and/or is derived from a monoclonal antibody. In other embodiments, the plexin-A4 antibody or active fragment thereof is a polyclonal antibody and/or is derived from a polyclonal antibody. In yet other embodiments, the present invention provides a composition comprising an inhibitor of plexin-A4 activity in an admixture with a pharmaceutically acceptable carrier. Thus, in some embodiments, a plexin-A4 antibody or active fragment thereof or a plexin-A4 fusion protein is provided in a composition further comprising a pharmaceutically acceptable carrier.

[0055] In still further embodiments, an inhibitor of Sema3A activity includes, but is not limited to, a Sema3A fusion protein, a Sema3A antibody or an active fragment thereof or any combination thereof. In some embodiments, the Sema3A antibody is a monoclonal antibody and/or is derived from a monoclonal antibody. In other embodiments, the Sema3A antibody or active fragment thereof is a polyclonal antibody and/or is derived from a polyclonal antibody. In yet other embodiments, the present invention provides a composition comprising an inhibitor of Sema3A activity in an admixture with a pharmaceutically acceptable carrier. Thus, in some embodiments, a Sema3A antibody or active fragment thereof or a Sema3A fusion protein is provided in a composition further comprising a pharmaceutically acceptable carrier.

[0056] In yet further embodiments, an inhibitor of plexin-A4 expression or Sema3A expression includes, but is not limited to, siRNA, shRNA, miRNA, antisense RNA and ribozymes.

[0057] The present invention further contemplates that enhancers or activators of plexin-A4 or Sema3A activity may be useful for treating subjects having immune deficiencies. Immune deficiencies can be inherited, or acquired through infection and/or other illness, and/or produced as an inadvertent side effect of particular drug treatments. Non-limiting examples of immune deficiencies include severe combined immunodeficiency disease (SCID), chronic fatigue and immune dysfunction syndrome (CFIDS), acquired immune deficiency syndrome (AIDS), and/or immune deficiencies resulting from chemotherapeutics administered to subjects (e.g., cancer patients) undergoing chemotherapy.

[0058] Accordingly, the present invention provides a method of treating an immune deficiency in a subject, comprising administering to a subject in need thereof an effective amount of an enhancer and/or activator of plexin-A4 or Sema3A activity or expression, whereby the plexin-A4 or Sema3A activity or expression in said subject is increased, thereby treating the immune deficiency. In some embodiments, the present invention provides a method for increasing cytokine production in a subject, comprising administering to a subject in need thereof an effective amount of an enhancer and/or activator of plexin-A4 activity or expression, or an effective amount of an enhancer and/or activator of Sema3A activity or expression. In other embodiments, a method is provided for increasing plexin-A4 activity or expression in a subject, comprising administering to a subject in need thereof an effective amount of an enhancer and/or activator of plexin-A4 activity or expression. In still other embodiments, a method is provided for increasing Sema3A activity or expression in a subject, comprising administering to a subject in need thereof an effective amount of an enhancer and/or activator of Sema3A activity or expression. The increase in cytokine production and/or in plexin-A4 activity and/or expression and/or in Sema3A activity and/or expression is any increase as compared to the level of cytokine production, and/or plexin-A4 activity and/or expression and/or in Sema3A activity and/or expression in the absence of the enhancer and/or activator.

[0059] As used herein an activator and/or enhancer of plexin-A4 activity and/or expression is a substance, or combination of substances that increase plexin-A4 activity and/or expression over the level of the plexin-A4 activity and/or expression in the absence of the substance. As used herein an activator and/or enhancer of Sema3A activity and/or expression is a substance, or combination of substances that increase Sema3A activity and/or expression over the level of the Sema3A activity and/or expression in the absence of the substance.

[0060] In some particular embodiments, an enhancer and/or activator of plexin-A4 activity and/or expression includes, but is not limited to, a plexin-A4 antibody or an active fragment thereof, or any combination thereof. In other particular embodiments, an enhancer and/or activator of Sema3A activity and/or expression includes, but is not limited to, a Sema3A antibody or an active fragment thereof, or any combination thereof.

[0061] As used herein, the term "antibody" includes intact immunoglobin molecules as well as active fragments thereof, such as Fab, F(ab')2, and Fc, which are capable of binding the epitopic determinant of an antigen (i.e., antigenic determinant). Antibodies that bind the polypeptides of this invention are prepared using intact polypeptides and/or fragments containing small peptides of interest as the immunizing antigen. The polypeptide or fragment used to immunize an animal can be derived from enzymatic cleavage, recombinant expression, isolation from biological materials, synthesis, etc., and can be conjugated to a carrier protein, if desired. Commonly used carriers that are chemically coupled to peptides and proteins for the production of antibody include, but are not limited to, bovine serum albumin, thyroglobulin and keyhole limpet hemocyanin. The coupled peptide or protein is then used to immunize a host animal (e.g., a mouse, rat, goat, sheep, human or rabbit). The polypeptide or peptide antigens can also be administered with an adjuvant, as described herein and as otherwise known in the art.

[0062] The terms "antibody" and "antibodies" as used herein refer to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE. The antibody can be monoclonal or polyclonal and can be of any species of origin, including, for example, mouse, rat, rabbit, horse, goat, sheep or human, and/or can be a chimeric or humanized antibody. See, e.g., Walker et al., Molec. Immunol. 26:403-11 (1989). The antibodies can be recombinant monoclonal antibodies produced according to the methods disclosed in U.S. Pat. No. 4,474,893 or U.S. Pat. No. 4,816,567. The antibodies can also be chemically constructed according to the method disclosed in U.S. Pat. No. 4,676,980. The antibody can further be a single chain antibody or bispecific antibody.

[0063] Techniques for the production of chimeric antibodies or humanized antibodies by splicing mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity can be used (Morrison et al. 1984. Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger et al. 1984. Nature 312:604-608; Takeda et al. 1985. Nature 314:452-454). Alternatively, techniques described for the production of single chain antibodies can be adapted, using methods known in the art, to produce single chain antibodies specific for the polypeptides and/or fragments and/or epitopes of this invention. Antibodies with related specificity, but of distinct idiotypic composition, can be generated by chain shuffling from random combinatorial immunoglobin libraries (Burton 1991. Proc. Natl. Acad. Sci. 88:11120-3).

[0064] Active antibody fragments included within the scope of the present invention include, for example, Fab, F(ab')2, and Fc fragments, and the corresponding fragments obtained from antibodies other than IgG. Such fragments can be produced by known techniques. For example, F(ab')2 fragments can be produced by pepsin digestion of the antibody molecule, and Fab fragments can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries can be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse et al., (1989) Science 254:1275-1281).

[0065] Monoclonal antibodies can be produced in a hybridoma cell line according to the technique of Kohler and Milstein (Nature 265:495-97 (1975)). For example, a solution containing the appropriate antigen can be injected into a mouse and, after a sufficient time, the mouse sacrificed and spleen cells obtained. The spleen cells are then immortalized by fusing them with myeloma cells or with lymphoma cells, typically in the presence of polyethylene glycol, to produce hybridoma cells. The hybridoma cells are then grown in a suitable medium and the supernatant screened for monoclonal antibodies having the desired specificity. Monoclonal Fab fragments can be produced in bacterial cell such as E. coli by recombinant techniques known to those skilled in the art. See, e.g., W. Huse, (1989) Science 246:1275-81.

[0066] Antibodies can also be obtained by phage display techniques known in the art or by immunizing a heterologous host with a cell containing an epitope or immunogen of interest.

[0067] In some embodiments of the present invention, the plexin-A4 fusion protein comprises, consists essentially of, or consists of an extracellular domain of plexin-A4 and an Fc region of IgG. The extracellular domain of plexin-A4 and the Fc region of IgG can be derived from a plexin-A4 or IgG from any species (e.g., mouse, human). In particular embodiments, the extracellular domain of plexin-A4 and the IgG Fc region are derived from human.

[0068] A plexin-A4 polypeptide of this invention includes, but is not limited to, the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3. It is further contemplated that plexin-A4 polypeptides useful for the present invention include those known in the art, including the non-limiting examples of the plexin-A4 polypeptides having NCBI Accession Nos. NM_--020911, NM_--181775, and NM_--001105543.

[0069] As discussed above, in some embodiments of the invention, the plexin-A4 fusion protein comprises the extracellular domain of a plexin-A4 polypeptide. Thus, in some embodiments, the extracellular domain of a plexin-A4 polypeptide includes, but is not limited to, the amino acid sequence of SEQ ID NO:7.

[0070] In some embodiments, the plexin-A4 fusion protein further comprises a region of an immunoglobulin, for example, IgG, IgM, IgA, IgD, and IgE. In further embodiments, the plexin-A4 fusion protein comprises a region of IgG. The amino acid sequence of a representative IgG is SEQ ID NO:12 (FIG. 17B). In other embodiments of the invention, the plexin-A4 fusion protein comprises the Fc region of an immunoglobulin. In yet other embodiments of the invention, the plexin-A4 fusion protein comprises the Fc region of IgG. Thus, in some embodiments of the present invention, the plexin-A4 fusion protein comprises IgG Fc having the amino acid sequence of SEQ ID NO:8 (FIG. 15C).

[0071] Accordingly, in some embodiments of the present invention a plexin-A4 fusion protein is provided comprising the amino acid sequences of SEQ ID NO:7 (FIG. 15B) and SEQ ID NO:8 (FIG. 15C).

[0072] In further embodiments of the present invention, the fusion protein comprises an extracellular domain of plexin-A4 and the Fc region of IgG, wherein the plexin-A4 extracellular domain and the IgG Fc region are linked by a peptide or peptide fragment. In some embodiments, the peptide or peptide fragment linker is a length from about 1 amino acid residue to about 20 amino acid residues. Thus, the peptide linker can be a length of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length, Thus, in further embodiments, the length of the peptide linker can be from about 1 to about 5 amino acid residues, from about 1 to about 10 amino acid residues, from about 1 to about 15 amino acid residues, from about 2 to about 5 amino acid residues, from about 2 to about 10 amino acid residues, from about 2 to about 15 amino acid residues, from about 2 to about 20 amino acid residues, from about 5 to about 10 amino acid residues, from about 5 to about 15 amino acid residues, from about 5 to about 20 amino acid residues, and the like. In some embodiments, the peptide linker comprises, consists essentially of, and/or consists of 2 amino acids in length. The peptide linker can be any amino acid residue or combination of amino acid residues in any order and/or in any multiplicity of the same amino acid residues. In some embodiments, the peptide linker of the present invention comprises glycine and/or cysteine. In particular embodiments, the peptide linker comprises a glycine residue and a cysteine residue (i.e., G-C; gly-cys).

[0073] Accordingly, the present invention further provides embodiments wherein the plexin-A4 fusion protein comprises the amino acid sequence of SEQ ID NO:6 (FIG. 15A).

[0074] In some embodiments of the present invention, the Sema3A fusion protein comprises, consists essentially of, or consists of full length Sema3A and an Fc region of IgG. The Sema3A and the Fc region of IgG can be derived from a Sema3A or IgG from any species (e.g., mouse, human). In particular embodiments, the Sema3A and the IgG Fc region are derived from human.

[0075] A Sema3A polypeptide of this invention includes, but is not limited to, the amino acid sequence of SEQ ID NO:13 (FIG. 18A). It is further contemplated that Sema3A polypeptides useful for the present invention include those known in the art, including the non-limiting example of a Sema3A polypeptide having NCBI Accession No. NM_--006080.

[0076] As discussed above, in some embodiments of the invention, the Sema3A fusion protein comprises the full length polypeptide. In other embodiments, the Sema3A fusion protein comprises fragments of the full length polypeptide. Thus, in some embodiments, the Sema3A fusion protein includes, but is not limited to, fragments of the amino acid sequence of SEQ ID NO:13 (FIG. 18A).

[0077] In some embodiments, the fusion protein further comprises a region of an immunoglobulin, for example, IgG, IgM, IgA, IgD, and IgE. In further embodiments, the fusion protein comprises a region of IgG. The amino acid sequence of a representative IgG is SEQ ID NO:12 (FIG. 17B). In other embodiments of the invention, the fusion protein comprises the Fc region of an immunoglobulin. In yet other embodiments of the invention, the fusion protein comprises the Fc region of IgG. Thus, in some embodiments of the present invention, the fusion protein comprises IgG Fc region having the amino acid sequence of SEQ ID NO:8 (FIG. 15).

[0078] Accordingly, in some embodiments of the present invention a Sema3A fusion protein is provided comprising the amino acid sequences of SEQ ID NO:13 and SEQ ID NO:8.

[0079] In further embodiments of the present invention, the Sema3A fusion protein comprises a full length Sema3A and the Fc region of IgG, wherein the Sema3A and the IgG Fc region are linked by a peptide or peptide fragment. In some embodiments, the peptide or peptide fragment linker is a length from about 1 amino acid residue to about 20 amino acid residues. Thus, the peptide linker can be a length of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length, Thus, in further embodiments, the length of the peptide linker can be from about 1 to about 5 amino acid residues, from about 1 to about 10 amino acid residues, from about 1 to about 15 amino acid residues, from about 2 to about 5 amino acid residues, from about 2 to about 10 amino acid residues, from about 2 to about 15 amino acid residues, from about 2 to about 20 amino acid residues, from about 5 to about 10 amino acid residues, from about 5 to about 15 amino acid residues, from about 5 to about 20 amino acid residues, and the like. In some embodiments, the peptide linker comprises, consists essentially of and/or consists of 2 amino acids in length. The peptide linker can be any amino acid residue or combination of amino acid residues in any order and/or in any multiplicity of the same amino acid residues. In some embodiments, the peptide linker of the present invention comprises glycine and/or cysteine. In particular embodiments, the peptide linker comprises a glycine residue and a cysteine residue (i.e., G-C; gly-cys).

[0080] Accordingly, the present invention further provides embodiments wherein the plexin-A4 fusion protein comprises the amino acid sequence of SEQ ID NO:16 (FIG. 18D).

[0081] As used herein, the term "polypeptide" encompasses both peptides and proteins, unless indicated otherwise.

[0082] The terms "polypeptide," "protein," and "peptide" refer to a chain of covalently linked amino acids. In general, the term "peptide" can refer to shorter chains of amino acids (e.g., 2-50 amino acids); however, all three terms overlap with respect to the length of the amino acid chain. Polypeptides, proteins, and peptides may comprise naturally occurring amino acids, non-naturally occurring amino acids, or a combination of both. The polypeptides, proteins, and peptides may be isolated from sources (e.g., cells or tissues) in which they naturally occur, produced recombinantly in cells in vivo or in vitro or in a test tube in vitro, and/or synthesized chemically. Such techniques are known to those skilled in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd Ed. (Cold Spring Harbor, N.Y., 1989); Ausubel et al. Current Protocols in Molecular Biology (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York).

[0083] The term "fragment," as applied to a polypeptide, will be understood to mean an amino acid sequence of reduced length relative to a reference polypeptide or amino acid sequence and comprising, consisting essentially of, and/or consisting of an amino acid sequence of contiguous amino acids identical, or substantially identical, to the reference polypeptide or amino acid sequence. Such a polypeptide fragment according to the invention may be, where appropriate, included in a larger polypeptide of which it is a constituent. In some embodiments, such fragments can comprise, consist essentially of, and/or consist of peptides having a length of at least about 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, or more consecutive amino acids of a polypeptide or amino acid sequence according to the invention.

[0084] A fragment of a polypeptide or protein of this invention can be produced by methods well known and routine in the art. Fragments of this invention can be produced, for example, by enzymatic or other cleavage of naturally occurring peptides or polypeptides or by synthetic protocols that are well known. Such fragments can be tested for one or more of the biological activities of this invention (e.g., plexin-A4 binding to Sema3A) according to the methods described herein, which are routine methods for testing activities of polypeptides, and/or according to any art-known and routine methods for identifying such activities. Such production and testing to identify biologically active fragments of the polypeptides described herein would be well within the scope of one of ordinary skill in the art and would be routine.

[0085] Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific fashion. Ribozymes have specific catalytic domains that possess endonuclease activity (Kim et al. (1987); Gerlach et al. (1987); Forster et al. (1987)). For example, a large number of ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate (Michel et al. (1990); Reinhold-Hurek et al. (1992)). This specificity has been attributed to the requirement that the substrate bind via specific base-pairing interactions to the internal guide sequence ("IGS") of the ribozyme prior to chemical reaction.

[0086] Ribozyme catalysis has primarily been observed as part of sequence-specific cleavage/ligation reactions involving nucleic acids (Joyce (1989)). For example, U.S. Pat. No. 5,354,855 reports that certain ribozymes can act as endonucleases with a sequence specificity greater than that of known ribonucleases and approaching that of the DNA restriction enzymes. Thus, sequence-specific ribozyme-mediated inhibition of nucleic acid expression may be particularly suited to therapeutic applications (Scanlon et al. (1991); Sarver et al. (1990); Sioud, M. et al. (1992)).

[0087] MicroRNAs (miRNA) are RNA molecules, generally 21-23 nucleotides long, that can down-regulate gene expression by hybridizing to miRNA. Over-expression or diminution of a particular miRNA can be used to treat a dysfunction and has been shown to be effective in a number of disease states and animal models of disease (Couzin 2008). Mature miRNAs are produced from a primary transcript (pri-miRNA) that is processed into a short stem-loop structure (a pre-miRNA) that then forms the final miRNA product.

[0088] The term "antisense oligonucleotide" (including "antisense RNA") as used herein, refers to a nucleic acid that is complementary to and specifically hybridizes to a specified DNA or RNA sequence such as a plexin-A4 or Sema3A DNA or RNA sequence. Antisense oligonucleotides and nucleic acids that encode the same can be made in accordance with conventional techniques. See, e.g., U.S. Pat. No. 5,023,243 to Tullis; U.S. Pat. No. 5,149,797 to Pederson et al.

[0089] Those skilled in the art will appreciate that it is not necessary that the antisense oligonucleotide be fully complementary to the target sequence as long as the degree of sequence similarity is sufficient for the antisense nucleotide sequence to specifically hybridize to its target (as defined above) and reduces production of the protein product (e.g., by at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more).

[0090] To determine the specificity of hybridization, hybridization of such oligonucleotides to target sequences can be carried out under conditions of reduced stringency, medium stringency or even high stringency conditions, as described herein. Exemplary conditions for reduced, medium and high stringency hybridization are as follows: (e.g., conditions represented by a wash stringency of 35-40% Formamide with 5×Denhardt's solution, 0.5% SDS and 1×SSPE at 37° C.; conditions represented by a wash stringency of 40-45% Formamide with 5×Denhardt's solution, 0.5% SDS, and 1×SSPE at 42° C.; and conditions represented by a wash stringency of 50% Formamide with 5×Denhardt's solution, 0.5% SDS and 1×SSPE at 42° C., respectively). See, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual (2d Ed. 1989) (Cold Spring Harbor Laboratory).

[0091] Alternatively stated, in particular embodiments, the antisense oligonucleotide has at least about 60%, 70%, 80%, 90%, 95%, 97%, 98% or higher sequence similarity with the complement of the target sequence and reduce production of the protein product (as defined above). In some embodiments, the antisense sequence contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more mismatches as compared with the target sequence.

[0092] Methods of determining percent identity of nucleic acid sequences are described in more detail elsewhere herein.

[0093] The length of the antisense oligonucleotide is not critical as long as it specifically hybridizes to the intended target and reduces production of the protein product and can be determined in accordance with routine procedures. In general, the antisense oligonucleotide is at least about eight, ten or twelve or fifteen nucleotides in length and/or less than about 20, 30, 40, 50, 60, 70, 80, 100 or 150 nucleotides in length.

[0094] An antisense oligonucleotide can be constructed using chemical synthesis and enzymatic ligation reactions by procedures known in the art. For example, an antisense oligonucleotide can be chemically synthesized using naturally occurring nucleotides or various modified nucleotides designed to increase the biological stability of the molecules and/or to increase the physical stability of the duplex formed between the antisense and sense nucleotide sequences, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.

[0095] Examples of modified nucleotides which can be used to generate the antisense oligonucleotide include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomet-hyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopenten-yladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.

[0096] The antisense oligonucleotides can further include nucleotide sequences wherein at least one, or all, of the internucleotide bridging phosphate residues are modified phosphates, such as methyl phosphonates, methyl phosphonothioates, phosphoromorpholidates, phosphoropiperazidates and phosphoramidates. For example, every other one of the internucleotide bridging phosphate residues can be modified as described.

[0097] As another non-limiting example, one or all of the nucleotides in the oligonucleotide can contain a 2' loweralkyl moiety (e.g., C₁-C₄, linear or branched, saturated or unsaturated alkyl, such as methyl, ethyl, ethenyl, propyl, 1-propenyl, 2-propenyl, and isopropyl). For example, every other one of the nucleotides can be modified as described. See also, Furdon et al. (1989) Nucleic Acids Res. 17, 9193-9204; Agrawal et al. (1990) Proc. Natl. Acad. Sci. USA 87, 1401-1405; Baker et al. (1990) Nucleic Acids Res. 18, 3537-3543; Sproat et al. (1989) Nucleic Acids Res. 17, 3373-3386; Walder and Walder (1988) Proc. Natl. Acad. Sci. USA 85, 5011-5015.

[0098] The antisense oligonucleotide can be chemically modified (e.g., at the 3' and/or 5' end) to be covalently conjugated to another molecule. To illustrate, the antisense oligonucleotide can be conjugated to a molecule that facilitates delivery to a cell of interest, enhances absorption by the nasal mucosa (e.g., by conjugation to a lipophilic moiety such as a fatty acid), provides a detectable marker, increases the bioavailability of the oligonucleotide, increases the stability of the oligonucleotide, improves the formulation or pharmacokinetic characteristics, and the like. Examples of conjugated molecules include but are not limited to cholesterol, lipids, polyamines, polyamides, polyesters, intercalators, reporter molecules, biotin, dyes, polyethylene glycol, human serum albumin, an enzyme, an antibody or antibody fragment, or a ligand for a cellular receptor.

[0099] Other modifications to nucleic acids to improve the stability, nuclease-resistance, bioavailability, formulation characteristics and/or pharmacokinetic properties are known in the art.

[0100] RNA interference (RNAi) is another useful approach for reducing production of a protein product (e.g., shRNA or siRNA). RNAi is a mechanism of post-transcriptional gene silencing in which double-stranded RNA (dsRNA) corresponding to a target sequence of interest (e.g., plexin-A4 and/or Sema3A) is introduced into a cell or an organism, resulting in degradation of the corresponding mRNA. The mechanism by which RNAi achieves gene silencing has been reviewed in Sharp et al. (2001) Genes Dev 15: 485-490; and Hammond et al. (2001) Nature Rev Gen 2:110-119). The RNAi effect persists for multiple cell divisions before gene expression is regained. RNAi is therefore a powerful method for making targeted knockouts or "knockdowns" at the RNA level. RNAi has proven successful in human cells, including human embryonic kidney and HeLa cells (see, e.g., Elbashir et al., Nature (2001) 411:494-8).

[0101] The RNAi molecule (including an siRNA molecule) can be a short hairpin RNA (shRNA; see Paddison et al. (2002) PNAS USA 99:1443-1448), which is believed to be processed in the cell by the action of the RNase III like enzyme Dicer into 20-25 mer siRNA molecules. The shRNAs generally have a stem-loop structure in which two inverted repeat sequences are separated by a short spacer sequence that loops out. There have been reports of shRNAs with loops ranging from 3 to 23 nucleotides in length. The loop sequence is generally not critical. Exemplary loop sequences include the following motifs: AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU, CCACACC and UUCAAGAGA.

[0102] The RNAi can further comprise a circular molecule comprising sense and antisense regions with two loop regions on either side to form a "dumbbell" shaped structure upon dsRNA formation between the sense and antisense regions. This molecule can be processed in vitro or in vivo to release the dsRNA portion, e.g., a siRNA.

[0103] International patent publication WO 01/77350 describes a vector for bi-directional transcription to generate both sense and antisense transcripts of a heterologous sequence in a eukaryotic cell. This technique can be employed to produce RNAi for use according to the invention.

[0104] Shinagawa et al. (2003) Genes & Dev. 17:1340 reported a method of expressing long dsRNAs from a CMV promoter (a pol II promoter), which method is also applicable to tissue specific pol II promoters. Likewise, the approach of Xia et al. (2002) Nature Biotech. 20:1006, avoids poly(A) tailing and can be used in connection with tissue-specific promoters.

[0105] Methods of generating RNAi include chemical synthesis, in vitro transcription, digestion of long dsRNA by Dicer (in vitro or in vivo), expression in vivo from a delivery vector, and expression in vivo from a PCR-derived RNAi expression cassette (see, e.g., TechNotes 10(3) "Five Ways to Produce siRNAs," from Ambion, Inc., Austin Tex.).

[0106] Guidelines for designing siRNA molecules are available (see e.g., literature from Ambion, Inc., Austin Tex.). In particular embodiments, the siRNA sequence has about 30-50% G/C content. Further, long stretches of greater than four T or A residues are generally avoided if RNA polymerase III is used to transcribe the RNA. Online siRNA target finders are available, e.g., from Ambion, Inc., through the Whitehead Institute of Biomedical Research or from Dharmacon Research, Inc.

[0107] The antisense region of the RNAi molecule can be completely complementary to the target sequence (e.g., plexin-A4 and/or Sema3A), but need not be as long as it specifically hybridizes to the target sequence and reduces production of the protein product (e.g., by at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more). In some embodiments, hybridization of such oligonucleotides to target sequences can be carried out under conditions of reduced stringency, medium stringency or even high stringency conditions, as defined herein.

[0108] In other embodiments, the antisense region of the RNAi has at least about 60%, 70%, 80%, 90%, 95%, 97%, 98% or higher sequence identity with the complement of the target sequence and reduces production of the protein product (e.g., by at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more). In some embodiments, the antisense region contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mismatches as compared with the target sequence. Mismatches are generally tolerated better at the ends of the dsRNA than in the center portion.

[0109] In particular embodiments, the RNAi is formed by intermolecular complexing between two separate sense and antisense molecules. The RNAi comprises a ds region formed by the intermolecular basepairing between the two separate strands. In other embodiments, the RNAi comprises a ds region formed by intramolecular basepairing within a single nucleic acid molecule comprising both sense and antisense regions, typically as an inverted repeat (e.g., a shRNA or other stem loop structure, or a circular RNAi molecule). The RNAi can further comprise a spacer region between the sense and antisense regions.

[0110] The RNAi molecule can contain modified sugars, nucleotides, backbone linkages and other modifications as described above for antisense oligonucleotides.

[0111] Generally, RNAi molecules are highly selective. If desired, those skilled in the art can readily eliminate candidate RNAi that are likely to interfere with expression of nucleic acids other than the target by searching relevant databases to identify RNAi sequences that do not have substantial sequence homology with other known sequences, for example, using BLAST.

[0112] Kits for the production of RNAi are commercially available, e.g., from New England Biolabs, Inc. and Ambion, Inc.

[0113] A further aspect of the present invention is an isolated nucleic acid encoding a plexin-A4 polypeptide, an extracellular domain of a plexin-A4 polypeptide, an immunoglobulin Fc fragment, and/or a plexin-A4 fusion protein (e.g., a plexin-A4/Fc IgG fusion protein). Examples include (a) nucleic acids as disclosed herein, such as isolated nucleic acids having the nucleotide sequence as set forth in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11 (FIGS. 13, 14, 16A-C); (b) nucleic acids that hybridize to isolated nucleic acids of (a) above or the complement thereof (e.g., under stringent conditions), and/or have substantial sequence identity to nucleic acids of (a) above (e.g., are about 75, 80, 85, 90 95 or 99% identical to nucleic acids of (a) above), and encode a plexin-A4 polypeptide, an extracellular domain of a plexin-A4 polypeptide, an immunoglobulin Fc fragment, or a plexin-A4 fusion protein; and (c) nucleic acids that differ from the nucleic acids of (a) or (b) above due to the degeneracy of the genetic code, and code for a plexin-A4, an extracellular domain of plexin-A4, an immunoglobulin Fc fragment, or a plexin-A4 fusion protein.

[0114] Thus, in particular embodiments, the present invention provides an isolated plexin-A4 polypeptide, a plexin-A4 fusion protein, a nucleic acid comprising a nucleotide sequence encoding a plexin-A4 polypeptide or a plexin-A4 fusion protein of this invention, a vector comprising said nucleic acid and a cell containing said vector. The plexin-A4 polypeptide and fusion protein, the nucleic acid, the vector and/or the cell can be present singly and/or in any combination in a composition comprising a pharmaceutically acceptable carrier.

[0115] Accordingly, in some embodiments, a plexin-A4 coding sequence includes, but is not limited to, the nucleotide sequence of SEQ ID NO:4. In other embodiments, a plexin-A4 cDNA includes, but is not limited to, the nucleotide sequence of SEQ ID NO:5. The genomic sequence of the plexin-A4 gene is also contemplated to be a part of the present invention.

[0116] The present invention further provides a nucleic acid comprising an isolated nucleotide sequence encoding the extracellular domain of a plexin-4A polypeptide. Thus, in some embodiments, the extracellular domain of a plexin-4A polypeptide is encoded by the nucleotide sequence of SEQ ID NO:10. The present invention additionally provides a nucleic acid comprising a nucleotide sequence encoding the Fc region of IgG. In some embodiments, the IgG Fc region is encoded by the nucleotide sequence of SEQ ID NO:11. In still further embodiments, the present invention provides a nucleic acid comprising, consisting essentially of, and/or consisting of a nucleotide sequence encoding a plexin-A4/Fc IgG fusion protein. In additional embodiments of the invention, the nucleic acid comprising a nucleotide sequence encoding a plexin-A4/Fc IgG fusion protein comprises the nucleotide sequences of SEQ ID NO:10 and SEQ ID NO:11.

[0117] The present invention further provides a nucleic acid comprising a nucleotide sequence encoding a plexin-A4/Fc IgG fusion protein, wherein the plexin-A4/IgG Fc fusion protein comprises an extracellular domain of plexin-A4 fused to the Fc region of an IgG via a peptide linker. Thus, in some embodiments of the invention, the plexin-A4/IgG Fc fusion protein is encoded by the nucleotide sequence of SEQ ID NO:9.

[0118] An additional aspect of the present invention is an isolated nucleic acid encoding a Sema3A polypeptide, an immunoglobulin Fc fragment, and/or a Sema3A fusion protein (e.g., a Sema3A/Fc IgG fusion protein). Examples include (a) nucleic acids as disclosed herein, such as isolated nucleic acids having the nucleotide sequence as set forth in SEQ ID NO:11 (FIG. 16C), SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO: 17 (FIGS. 18B,C and E); (b) nucleic acids that hybridize to isolated nucleic acids of (a) above or the complement thereof (e.g., under stringent conditions), and/or have substantial sequence identity to nucleic acids of (a) above (e.g., are about 75, 80, 85, 90 95 or 99% identical to nucleic acids of (a) above), and encode a Sema3A polypeptide, an immunoglobulin Fc fragment, or a Sema3A fusion protein; and (c) nucleic acids that differ from the nucleic acids of (a) or (b) above due to the degeneracy of the genetic code, and code for a Sema3A polypeptide, an immunoglobulin Fc fragment, or a Sema3A fusion protein.

[0119] Thus, in particular embodiments, the present invention provides an isolated Sema3A polypeptide, a Sema3A fusion protein, a nucleic acid comprising a nucleotide sequence encoding a Sema3A polypeptide or a Sema3A fusion protein of this invention, a vector comprising said nucleic acid and a cell containing said vector. The Sema3A polypeptide and fusion protein, the nucleic acid, the vector and/or the cell can be present singly and/or in any combination in a composition comprising a pharmaceutically acceptable carrier.

[0120] Accordingly, in some embodiments, a Sema3A coding sequence includes, but is not limited to, the nucleotide sequence of SEQ ID NO:14 (FIG. 18B). In other embodiments, a Sema3A cDNA includes, but is not limited to, the nucleotide sequence of SEQ ID NO:15 (FIG. 18C). The genomic sequence of the Sema3A gene is also contemplated to be a part of the present invention.

[0121] As discussed above, the present invention additionally provides a nucleic acid comprising a nucleotide sequence encoding the Fc region of IgG. In some embodiments, the IgG Fc region is encoded by the nucleotide sequence of SEQ ID NO:11. In still further embodiments, the present invention provides a nucleic acid comprising, consisting essentially of, and/or consisting of a nucleotide sequence encoding a Sema3A/Fc IgG fusion protein. In additional embodiments of the invention, the nucleic acid comprising a nucleotide sequence encoding a Sema3A/Fc IgG fusion protein comprises the nucleotide sequences of SEQ ID NO:14 and SEQ ID NO:11.

[0122] The present invention further provides a nucleic acid comprising a nucleotide sequence encoding a Sema3A/Fc IgG fusion protein, wherein the Sema3A/IgG Fc fusion protein comprises Sema3A fused to the Fc region of an IgG via a peptide linker. Thus, in some embodiments of the invention, the Sema3A/IgG Fc fusion protein is encoded by the nucleotide sequence of SEQ ID NO:17.

[0123] As used herein, "nucleic acid," "nucleic acid molecule," "nucleotide sequence" and "polynucleotide" encompass both RNA and DNA, including cDNA, genomic DNA, mRNA, synthetic (e.g., chemically synthesized) DNA and chimeras of RNA and DNA [e.g., DNA-RNA hybrid sequences (including both naturally occurring and non-naturally occurring nucleotides)], but are typically either single or double stranded DNA or RNA sequences.

[0124] The term polynucleotide, nucleotide sequence, or nucleic acid refers to a chain of nucleotides without regard to length of the chain. The nucleic acid can be double-stranded or single-stranded (i.e., including the complementary nucleic acid). Where single-stranded, the nucleic acid can be a sense strand or an antisense strand. The nucleic acid can be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such oligonucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases. The present invention further provides a nucleic acid that is the complement (which can be either a full complement or a partial complement) of a nucleic acid or nucleotide sequence of this invention.

[0125] The term "nucleic acid fragment" will be understood to mean a nucleotide sequence of reduced length relative to a reference nucleic acid or nucleotide sequence and comprising, consisting essentially of and/or consisting of a nucleotide sequence of contiguous nucleotides identical or almost identical (e.g., about 75%, 80%, 82%, 85%, 88%, 90%, 92%, 95%, 98%, 99% identical) to the reference nucleic acid or nucleotide sequence. Such a nucleic acid fragment according to the invention may be, where appropriate, included in a larger polynucleotide of which it is a constituent. In some embodiments, such fragments can comprise, consist essentially of and/or consist of, oligonucleotides having a length of at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25. 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000 or more, consecutive nucleotides of a nucleic acid or nucleotide sequence according to the invention.

[0126] The terms "complementary" or "complementarity," as used herein, refer to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing. For example, the sequence "A-G-T" binds to the complementary sequence "T-C-A." Complementarity between two single-stranded molecules may be "partial," in which only some of the nucleotides bind, or it may be complete when total complementarity exists between the single stranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. Similarly, a sequence "homologous" to a target nucleic acid can mean that the sequence is of exact, or nearly exact, identity to the nucleic acid, such that it would specifically hybridize to a nucleic acid exactly complementary to the target nucleic acid. As used herein, stringent hybridization conditions are those conditions that provide selective hybridization for a selected target(s). By "selective hybridization" as used herein is meant that a nucleic acid can hybridize to a target nucleic acid under sufficient stringency conditions without significant hybridization to a nucleic acid likely to be present in the reaction but not of interest. Conditions can be selected to allow for detection of, for example, one or more related nucleic acids having, e.g., one, two, or three mismatches, as in, for example, related strains of the same organism, or they can be selected to provide that hybridization only occurs with one selected target, e.g., such that it differentiates between two strains, depending upon the purpose of the hybridization, amplification and detection. Such conditions are well known to persons of ordinary skill in the art.

[0127] Hybridization is typically performed under stringent hybridization conditions. Stringent hybridization conditions are described in, e.g., Sambrook, et. al, Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Lab. Press, December 1989. However, hybridization conditions can be modified and selected to suit the particular reaction design, including the T_ms of the selected primers and the temperature at which the amplification reaction is to be performed. One of skill in the art can suitably design the appropriate reaction conditions for the selected type of amplification and for the selected combination of targets and primers.

[0128] Thus, stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms. Post-hybridization washes determine stringency conditions. Higher stringency conditions utilize buffers with lower ionic strength and/or a higher reaction temperature, and tend to require a more perfect match between probe/primer and a target sequence in order to form a stable duplex. If the stringency is too high, however, hybridization may not occur at all. In contrast, lower stringency conditions utilize buffers with higher ionic strength and/or a lower reaction temperature, and permit the formation of stable duplexes with more mismatched bases between a probe/primer and a target sequence.

[0129] Non-limiting examples of high stringency hybridization conditions include: (1) prehybridization with a solution containing 5× standard saline phosphate EDTA (SSPE), 0.5% NaDodSO₄ (SDS) at 55° C., and incubating probe with target nucleic acid molecules in the same solution at the same temperature, followed by washing with a solution containing 2×.SSPE, and 0.1% SDS at 55° C. or room temperature; (2) a series of washes starting with 6×SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 60° C. for 30 min; (3) alternatively, the last two washes of (2) can be carried out at a higher temperature of 65° C.; and/or (4) a single wash at 0.1×SSC, 65° C.

[0130] Non-limiting examples of moderate stringency hybridization conditions include: (1) a series of washes starting with 6×SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min; (2) a wash with a solution of about 50 mM KCl at about 46° C.; (3) a wash with a solution of about 100 mM KCl at about 46° C.; and/or (4) a wash with a solution of 2×SSC, 65° C.

[0131] Further exemplary conditions for reduced, medium and high stringency hybridization are as follows: (e.g., conditions represented by a wash stringency of 35-40% Formamide with 5×Denhardt's solution, 0.5% SDS and 1×SSPE at 37° C.; conditions represented by a wash stringency of 40-45% Formamide with 5×Denhardt's solution, 0.5% SDS, and 1×SSPE at 42° C.; and conditions represented by a wash stringency of 50% Formamide with 5×Denhardt's solution, 0.5% SDS and 1×SSPE at 42° C., respectively). See, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual (2d Ed. 1989) (Cold Spring Harbor Laboratory).

[0132] The term "isolated" can refer to a nucleic acid, nucleotide sequence, polypeptide, peptide or fragment that is substantially free of cellular material, viral material, and/or culture medium (e.g., when produced by recombinant DNA techniques), or chemical precursors or other chemicals (e.g., when chemically synthesized). Moreover, an "isolated fragment" is a fragment of a nucleic acid, nucleotide sequence or polypeptide that is not naturally occurring as a fragment and would not be found as such in the natural state. "Isolated" does not mean that the preparation is technically pure (homogeneous), but it is sufficiently pure to provide the polypeptide or nucleic acid in a form in which it can be used for the intended purpose.

[0133] An "isolated nucleic acid" is a nucleotide sequence (e.g., DNA or RNA) that is not immediately contiguous with nucleotide sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived. Thus, in one embodiment, an isolated nucleic acid includes some or all of the 5' non-coding (e.g., promoter) sequences that are immediately contiguous to a coding sequence. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment), independent of other sequences. It also includes a recombinant DNA that is part of a hybrid nucleic acid encoding an additional polypeptide or peptide sequence.

[0134] Different nucleic acids or proteins having homology are referred to herein as "homologues." The term homologue includes homologous sequences from the same and other species and orthologous sequences from the same and other species. "Homology" refers to the level of similarity between two or more nucleic acid and/or amino acid sequences in terms of percent of positional identity (i.e., sequence similarity or identity). Homology also refers to the concept of similar functional properties among different nucleic acids or proteins.

[0135] As is well known in the art, nucleic acid sequences can have changes in one or more nucleotide bases that results in substitution of one or more amino acids, but which do not affect the functional properties of the polypeptide encoded by the nucleotide sequence. It is therefore understood that the invention encompasses more than the specific exemplary nucleotide or amino acid sequences and includes functional equivalents thereof.

[0136] For example, alterations in the nucleotide sequence of a gene which result in the production of a chemically equivalent amino acid at a given site, but do not affect the functional properties of the encoded protein, are well known in the art. Such alterations include "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties. In particular, such changes can be guided by known similarities between amino acids in physical features such as charge density, hydrophobicity/hydrophilicity, size and configuration, so that amino acids are substituted with other amino acids having essentially the same functional properties. For example: Ala may be replaced with Val or Ser; Val may be replaced with Ala, Leu, Met, or Ile, preferably Ala or Leu; Leu may be replaced with Ala, Val or Ile, preferably Val or Ile; Gly may be replaced with Pro or Cys, preferably Pro; Pro may be replaced with Gly, Cys, Ser, or Met, preferably Gly, Cys, or Ser; Cys may be replaced with Gly, Pro, Ser, or Met, preferably Pro or Met; Met may be replaced with Pro or Cys, preferably Cys; His may be replaced with Phe or Gln, preferably Phe; Phe may be replaced with His, Tyr, or Trp, preferably His or Tyr; Tyr may be replaced with His, Phe or Trp, preferably Phe or Trp; Trp may be replaced with Phe or Tyr, preferably Tyr; Asn may be replaced with Gln or Ser, preferably Gln; Gln may be replaced with His, Lys, Glu, Asn, or Ser, preferably Asn or Ser; Ser may be replaced with Gln, Thr, Pro, Cys or Ala; Thr may be replaced with Gln or Ser, preferably Ser; Lys may be replaced with Gln or Arg; Arg may be replaced with Lys, Asp or Glu, preferably Lys or Asp; Asp may be replaced with Lys, Arg, or Glu, preferably Arg or Glu; and Glu may be replaced with Arg or Asp, preferably Asp. Once made, changes can be routinely screened to determine their effects on function. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products.

[0137] Alternatively, alterations in the coding sequence of a gene may involve "nonconservative" changes (e.g., replacement of glycine with tryptophan). Analogous minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological activity may be found using computer programs well known in the art, such as for example, LASERGENE® software.

[0138] In particular embodiments, a resultant variant polypeptide has at least about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more amino acid sequence similarity or identity with the amino acid sequence of a naturally occurring protein or reference amino acid sequence (e.g., plexin-A4, Sema3A or IgG-Fc).

[0139] Thus, the invention further provides homologues, as well as methods of obtaining homologues, of the polypeptides and/or fragments of this invention from other organisms included in this invention. As used herein, an amino acid sequence or protein is defined as a homologue of a polypeptide or fragment of the present invention if it shares significant homology or identity to a polypeptide, peptide and/or fragment of the present invention. Significant homology or identity means at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, and/or 100% homology or identity with another amino acid sequence. Specifically, by using the nucleic acids that encode the proteins, peptides and fragments of this invention, as a probe or primer, and techniques such as PCR amplification and colony/plaque hybridization, one skilled in the art can identify homologues of the polypeptides, peptides and/or fragments of this invention in other organisms on the basis of information available in the art.

[0140] The term "percent identity," as known in the art, describes a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences as determined by the match between strings of such sequences. "Identity" and "similarity" can be readily calculated by known methods including, but not limited to, those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991).

[0141] Accordingly, the present invention further provides nucleotide sequences having significant sequence similarity or identity to the nucleotide sequences of the present invention. Significant sequence similarity or identity means at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, and/or 100% similarity or identity with another nucleotide sequence.

[0142] Exemplary methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations can be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences may be performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Exemplary default parameters for pairwise alignments using the Clustal method can be selected: KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

[0143] The term "sequence analysis software" refers to any computer algorithm or software program that is useful for the analysis of nucleotide and/or amino acid sequences. Sequence analysis software is commercially available or can be independently developed. Typical sequence analysis software will include but is not limited to the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis.), BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215:403-410 (1990), and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison, Wis. 53715 USA). Within the context of this application, it will be understood that where sequence analysis software is used for analysis, the results of the analysis will be based on the "default values" of the program referenced, unless otherwise specified. As used herein "default values" will mean any set of values or parameters, which originally load with the software when first initialized.

[0144] A percentage amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the "longer" sequence in the aligned region. The "longer" sequence is the one having the most actual residues in the aligned region (gaps introduced by WU-Blast-2 to maximize the alignment score are ignored).

[0145] The alignment can include the introduction of gaps in the sequences to be aligned. In addition, for sequences which contain either more or fewer amino acids than the polypeptides specifically disclosed herein, it is understood that in one embodiment, the percentage of sequence identity will be determined based on the number of identical amino acids in relation to the total number of amino acids. Thus, for example, in one embodiment, sequence identity of sequences shorter than a sequence specifically disclosed herein, will be determined using the number of amino acids in the shorter sequence. In percent identity calculations relative weight is not assigned to various manifestations of sequence variation, such as insertions, deletions, substitutions, etc.

[0146] In other embodiments, only identities are scored positively (+1) and all forms of sequence variation including gaps are assigned a value of "0," which obviates the need for a weighted scale or parameters as described below for sequence similarity calculations. Percent sequence identity can be calculated, for example, by dividing the number of matching identical residues by the total number of residues of the "shorter" sequence in the aligned region and multiplying by 100. The "longer" sequence is the one having the most actual residues in the aligned region.

[0147] The nucleotide sequences and/or nucleic acid fragments of the instant invention may be used to isolate cDNAs and genes encoding homologous proteins from the same or different organisms. Isolation of homologous genes using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to, methods of nucleic acid hybridization, and methods of DNA and RNA amplification as exemplified by various uses of nucleic acid amplification technologies (e.g., polymerase chain reaction, ligase chain I reaction, etc.).

[0148] For example, genes encoding other plexin-A4 and/or Sema3A polypeptides, either as cDNAs or genomic DNAs, could be isolated directly by using all or a substantial portion of the nucleic acid sequences or fragments of the present invention as DNA hybridization probes to screen libraries from any desired organism employing methodology well known to those skilled in the art. Specific oligonucleotide probes based upon the instant nucleic acid sequences can be designed and synthesized by methods known in the art (See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd Ed. (Cold Spring Harbor, N.Y., 1989); Ausubel et al. Current Protocols in Molecular Biology (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York). Moreover, the entire sequence(s) can be used directly to synthesize DNA probes by methods known to the skilled artisan such as random primer DNA labeling, nick translation, end-labeling techniques, or RNA probes using available in vitro transcription systems. In addition, specific primers can be designed and used to amplify a part or all of the instant sequences. The resulting amplification products can be labeled directly during amplification reactions or labeled after amplification reactions, and used as probes to isolate full length cDNA or genomic fragments under conditions of appropriate stringency.

[0149] In addition, two short segments of the instant nucleic acid fragments may be used in polymerase chain reaction protocols to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA. The polymerase chain reaction may also be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the instant nucleic acid fragments, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3' end of the mRNA precursor encoding the plexin-A4 genes or the Sema3A genes. Alternatively, the second primer sequence may be based upon sequences derived from the cloning vector. For example, the skilled artisan can follow the RACE protocol (Frohman et al. (1988) Proc. Natl. Acad. Sci. USA 85:8998-9002) to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3' or 5' end. Primers oriented in the 3' and 5' directions can be designed from the instant sequences. Using commercially available 3' RACE or 5' RACE systems (BRL), specific 3' or 5' cDNA fragments can be isolated (Ohara et al. (1989) Proc. Natl. Acad. Sci. USA 86:5673-5677; Loh et al. (1989) Science 243:217-220). Products generated by the 3' and 5' RACE procedures can be combined to generate full-length cDNAs (Frohman and Martin (1989) Techniques 1:165).

[0150] The term "therapeutically effective amount" or "effective amount," as used herein, refers to that amount of a composition of this invention that imparts a modulating effect, which, for example, can be a beneficial effect, to a subject afflicted with a disorder, disease or cosmetic condition, including improvement in the disease or disorder of the subject (e.g., in one or more symptoms), delay or reduction in the progression of the disease or disorder, prevention or delay of the onset of the disease or disorder, and/or change in clinical parameters of the disease or disorder, as would be well known in the art. The effective amount will vary with the age, general condition of the subject, the severity of the disease, disorder or condition being treated, the particular agent or composition administered, the duration of the treatment, the nature of any concurrent treatment, the pharmaceutically acceptable carrier used, and like factors within the knowledge and expertise of those skilled in the art. As appropriate, an "effective amount" in any individual case can be determined by one of ordinary skill in the art by reference to the pertinent texts and literature and/or by using routine experimentation. (See, for example, Remington, The Science and Practice of Pharmacy (20th ed. 2000)). For example, a therapeutically effective amount or effective amount can refer to the amount of a composition, compound, or agent that improves a disease or disorder in a subject by at least 5%, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%.

[0151] Although individual needs may vary, the determination of optimal ranges for effective amounts of a composition of this invention is within the skill of the art. Human doses can also readily be extrapolated from animal studies (Katocs et al., Chapter 27 In: Remington's Pharmaceutical Sciences, 18^th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990). Generally, the dosage required to provide an effective amount of a composition of this invention, which can be adjusted by one skilled in the art, will vary depending on several factors, including the age, health, physical condition, weight, type and extent of the disease or disorder of the recipient, frequency of treatment, the nature of concurrent therapy, if required, and the nature and scope of the desired effect(s) (Nies et al., Chapter 3 In: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9^th Ed., Hardman et al., eds., McGraw-Hill, New York, N.Y., 1996). In some embodiments, a daily dosage of fusion protein can be about 1 μg to 100 milligrams per kilogram of body weight. In other embodiments, the dosage can be 0.5 μg to 50 milligrams per kilogram of body weight, and preferably 1 to 10 milligrams per kilogram per day given in divided doses 1 to 6 times a day or in sustained release form is effective to obtain desired results.

[0152] The pharmaceutical compositions according to the present invention may be administered as a single dose or in multiple doses. The pharmaceutical compositions of the present invention may be administered either as individual therapeutic agents or in combination with other therapeutic agents, which when combined may be administered sequentially or simultaneously.

[0153] For antibodies, the dosage administered to a patient is typically 0.1 mg/kg to 100 mg/kg of the patient's body weight. In some aspects of the invention, the dosage administered to a patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight. In other aspects of the invention, the dosage can be 1 mg/kg to 10 mg/kg of the patient's body weight. Generally, human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible. Further, the dosage and frequency of administration of antibodies of the invention may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation.

[0154] By the terms "treat," "treating" or "treatment of" (or grammatically equivalent terms) it is meant that the severity of the subject's disease or disorder is reduced or at least partially improved or ameliorated and/or that some alleviation, mitigation or decrease in at least one clinical symptom is achieved and/or there is a delay in the progression of the disease or disorder, as would be well known in the art. Thus, in some embodiments, the terms "treat," "treating" or "treatment of" refer only to therapeutic regimens. In other embodiments, the terms "treat," "treating" or "treatment of" (or grammatically equivalent terms) refer to both prophylactic and therapeutic regimens. As an example, a patient exhibiting bacteremia may be treated with the compositions of the present invention prior to the onset of sepsis in order to prevent the onset of said disease.

[0155] The terms "prevent," "preventing" and "prevention" (and grammatical variations thereof) refer to avoidance, prevention and/or delay of the onset of a disease, disorder and/or a clinical symptom(s) in a subject and/or a reduction in the severity of the onset of the disease, disorder and/or clinical symptom(s) relative to what would occur in the absence of the methods of the invention. The prevention can be complete, e.g., the total absence of the disease, disorder and/or clinical symptom(s). The prevention can also be partial, such that the occurrence of the disease, disorder and/or clinical symptom(s) in the subject and/or the severity of onset is delayed and/or is less than what would occur in the absence of the method of the present invention.

[0156] An "effective amount," as used herein, refers to an amount that imparts a desired effect, which is optionally a therapeutic or prophylactic effect.

[0157] A "treatment effective" amount as used herein is an amount that is sufficient to provide some improvement or benefit to the subject. Alternatively stated, a "treatment effective" amount is an amount that will provide some alleviation, mitigation, decrease or stabilization in at least one clinical symptom in the subject. Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.

[0158] A "prevention effective" amount as used herein is an amount that is sufficient to prevent and/or delay the onset of a disease, disorder and/or clinical symptoms in a subject and/or to reduce and/or delay the severity of the onset of a disease, disorder and/or clinical symptoms in a subject relative to what would occur in the absence of the methods of the invention. Those skilled in the art will appreciate that the level of prevention need not be complete, as long as some benefit is provided to the subject.

[0159] Pharmaceutical compositions comprising the plexin-A4/Fc IgG fusion proteins, plexin-A4 antibodies and/or fragments thereof of this invention, and a pharmaceutically acceptable carrier are also provided. Additionally provided herein are pharmaceutical compositions comprising the Sema3A/Fc IgG fusion proteins, Sema3A antibodies and/or fragments thereof of this invention, and a pharmaceutically acceptable carrier. The compositions described herein can be formulated for administration in a pharmaceutical carrier in accordance with known techniques. See, e.g., Remington, The Science And Practice of Pharmacy (latest edition). In the manufacture of a pharmaceutical composition according to embodiments of the present invention, the composition of this invention is typically admixed with, inter alia, a pharmaceutically acceptable carrier. By "pharmaceutically acceptable carrier" is meant a carrier that is compatible with other ingredients in the pharmaceutical composition and that is not harmful or deleterious to the subject. The carrier may be a solid or a liquid, or both, and is preferably formulated with the composition of this invention as a unit-dose formulation, for example, a tablet, which may contain from about 0.01 or 0.5% to about 95% or 99% by weight of the composition. The pharmaceutical compositions are prepared by any of the well-known techniques of pharmacy including, but not limited to, admixing the components, optionally including one or more accessory ingredients. In certain embodiments, the pharmaceutically acceptable carrier is sterile and would be deemed suitable for administration into human subjects according to regulatory guidelines for pharmaceutical compositions comprising the carrier.

[0160] Furthermore, a "pharmaceutically acceptable" component such as a salt, carrier, excipient or diluent of a composition according to the present invention is a component that (i) is compatible with the other ingredients of the composition in that it can be combined with the compositions of the present invention without rendering the composition unsuitable for its intended purpose, and (ii) is suitable for use with subjects as provided herein without undue adverse side effects (such as toxicity, irritation, and allergic response). Side effects are "undue" when their risk outweighs the benefit provided by the composition. Non-limiting examples of pharmaceutically acceptable components include any of the standard pharmaceutical carriers such as phosphate buffered saline solutions, water, emulsions such as oil/water emulsion, microemulsions and various types of wetting agents.

[0161] Exemplary modes of administration of the proteins, peptides, fragments, nucleic acids and/or vectors of this invention can include oral, rectal, transmucosal, topical, intranasal, inhalation (e.g., via an aerosol), buccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, in utero (or in ovo), parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular [including administration to skeletal, diaphragm and/or cardiac muscle], intraperitoneal, intradermal, intrapleural, intracerebral, intracranial, and intraarticular), topical (e.g., to both skin and mucosal surfaces, including airway surfaces, and transdermal administration, and the like, as well as direct tissue or organ injection (e.g., to liver, skeletal muscle, cardiac muscle, diaphragm muscle or brain). The most suitable route in any given case will depend on the nature and severity of the condition being treated and on the nature of the particular protein, peptide, fragment, nucleic acid and/or vector that is being used.

[0162] The compositions of the present invention may be administered to a subject in need of treatment prior to, during or after onset of the disease or disorder. For instance, a patient exhibiting bacteremia may be treated with a composition of the present invention prior to onset of sepsis to prevent progression to sepsis. Thus, the compositions of the present invention can be used to treat ongoing immune-related and/or inflammatory diseases or disorders or to prevent diseases or delay the development of immune-related and/or inflammatory diseases or disorders.

[0163] In some embodiments, an effective dose or effective amount can comprise one or more (e.g., two or three or four or more) doses of the composition of this invention at any time interval (e.g., hourly, daily, weekly, monthly, yearly, as needed, etc.) so as to achieve and/or maintain the desired therapeutic benefit.

[0164] Several methods known in the art may be used to produce a polynucleotide and/or vector according to this invention. A "vector" is any nucleic acid molecule for the cloning and/or amplification of nucleic acid as well as for the transfer of nucleic acid into a subject (e.g., a cell of the subject). A vector may be a replicon to which another nucleotide sequence may be attached to allow for replication of the attached nucleotide sequence. A "replicon" can be any genetic element (e.g., plasmid, phage, cosmid, chromosome, viral genome) that functions as an autonomous unit of nucleic acid replication in vivo, i.e., capable of replication under its own control. The term "vector" includes both viral and nonviral nucleic acid molecules for introducing a nucleic acid into a cell in vitro, ex vivo, and/or in vivo.

[0165] A large number of vectors known in the art may be used to manipulate nucleic acids, incorporate response elements and promoters into genes, etc. Such vectors include, for example, plasmids or modified viruses including, for example bacteriophages such as lambda derivatives, or plasmids such as pBR322 or pUC plasmid derivatives, or the Bluescript vector. For example, the insertion of the nucleic acid fragments corresponding to response elements and promoters into a suitable vector can be accomplished by ligating the appropriate nucleic acid fragments into a chosen vector that has complementary cohesive termini. Alternatively, the ends of the nucleic acid molecules may be enzymatically modified or any site may be produced by ligating nucleotide sequences (linkers) to the nucleic acid termini. Such vectors may be engineered to contain sequences encoding selectable markers that provide for the selection of cells that contain the vector and/or have incorporated the nucleic acid of the vector into the cellular genome. Such markers allow identification and/or selection of host cells that incorporate and express the proteins encoded by the marker.

[0166] Vectors have been used in a wide variety of gene delivery applications in cells, as well as in living animal subjects. Viral vectors that can be used include, but are not limited to, retrovirus, lentivirus, adeno-associated virus, poxvirus, alphavirus, baculovirus, vaccinia virus, herpes virus, Epstein-Barr virus, and adenovirus vectors, as well as any combination thereof. Nonlimiting examples of non-viral vectors include plasmids, liposomes, electrically charged lipids (cytofectins), nucleic acid-protein complexes, and biopolymers, as well as any combination thereof. In addition to a nucleic acid of interest, a vector may also comprise one or more regulatory regions (e.g., promoters, enhancers, termination sequences, etc.), and/or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (delivery to specific tissues, duration of expression, etc.).

[0167] In embodiments in which a nucleic acid of this invention is delivered in a viral vector (e.g., a virus particle), the dosage of virus particles to be administered to a subject will depend upon the mode of administration, the disease or disorder to be treated, the individual subject's condition, the particular virus vector, and the nucleic acid to be delivered, and can be determined in a routine manner. Exemplary doses are virus titers of at least about 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10³, 10¹⁴, 10¹⁵ transducing units or more, preferably about 10⁸-10¹³ transducing units, yet more preferably 10¹² transducing units.

[0168] The present invention further provides a method of identifying a substance having an inhibitory effect on plexin-A4 activity, comprising contacting the substance with macrophage/dendritic cells under conditions whereby plexin-A4 activity can occur and measuring the amount of plexin-A4 activity in the presence and absence of the substance, whereby a decrease in plexin-A4 activity in the presence of the substance as compared to the amount of plexin-A4 activity in the absence of the substance identifies a substance having an inhibitory effect on plexin-A4 activity.

[0169] In addition, the present invention provides a method of identifying a substance having an inhibitory effect on plexin-A4 expression, comprising contacting the substance with macrophage/dendritic cells under conditions whereby plexin-A4 expression can occur and measuring the amount of plexin-A4 expression in the presence and absence of the substance, whereby a decrease in plexin-A4 expression in the presence of the substance as compared to the amount of plexin-A4 expression in the absence of the substance identifies a substance having an inhibitory effect on plexin-A4 expression.

[0170] Plexin-A4 activity and expression can be measured by many means as known in the art. For example, plexin-A4 expression can be measured at the RNA level by conventional protocols including, but not limited to, Northern blots, real-time PCR, reverse transcribed PCR, RNase protection protocols, and/or in situ hybridization. Plexin-A4 protein can be measured by protocols including, but not limited to, immunodetection such as ELISA, flow cytometric analysis, binding to Sema3A, and/or a combination of microscopic and immunolabelling and immunoblot methods. The activity of plexin-A4 protein can be measured by its ability optimize TLR activation by TLR agonists in the absence or presence of plexin-A4 fusion protein. TLR activation can be measured by events such as the secretion of proinflammatory cytokines (non-limiting examples of which are IL-6 and TNF) and chemokines, and by the activation of Rac, NF-kB and MAP kinases.

[0171] The present invention further provides a method of identifying a substance having an inhibitory effect on Sema3A activity, comprising contacting the substance with macrophage/dendritic cells under conditions whereby SemaA3 activity can occur and measuring the amount of Sema3A activity in the presence and absence of the substance, whereby a decrease in Sema3A activity in the presence of the substance as compared to the amount of Sema3A activity in the absence of the substance identifies a substance having an inhibitory effect on Sema3A activity.

[0172] In addition, the present invention provides a method of identifying a substance having an inhibitory effect on Sema3A expression, comprising contacting the substance with macrophage/dendritic cells under conditions whereby Sema3A expression can occur and measuring the amount of Sema3A expression in the presence and absence of the substance, whereby a decrease in Sema3A expression in the presence of the substance as compared to the amount of Sema3A expression in the absence of the substance identifies a substance having an inhibitory effect on Sema3A expression.

[0173] Sema3A activity and expression can be measured by many means as known in the art. For example, Sema3A expression can be measured at the RNA level by conventional protocols including, but not limited to, Northern blots, real-time PCR, reverse transcribed PCR, RNase protection protocols, and/or in situ hybridization. Sema3A protein can be measured by protocols including, but not limited to, immunodetection such as ELISA, flow cytometric analysis, binding to plexin-A4, and/or a combination of microscopic and immunolabelling and immunoblot methods. The activity of Sema3A protein can be measured by its ability optimize TLR activation by TLR agonists in the absence or presence of Sema3A fusion protein. TLR activation can be measured by events such as the secretion of proinflammatory cytokines (non-limiting examples of which are IL-6 and TNF) and chemokines, and by the activation of Rac, NF-kB and MAP kinases.

[0174] The present invention further provides a method of identifying a substance having an enhancing effect on plexin-A4 activity or expression, said method comprising contacting the substance with macrophage/dendritic cells under conditions whereby plexin A4 activity or expression can occur and measuring the amount of plexin-A4 activity or expression in the presence and absence of the substance, whereby an increase in plexin-A4 activity or expression in the presence of the substance as compared to the amount of plexin-A4 activity or expression in the absence of the substance identifies a substance having an enhancing effect on plexin-A4 activity or expression. Methods for detecting plexin-A4 activity or expression are known in the art and discussed above.

[0175] The present invention additionally provides a method of identifying a substance having an enhancing effect on Sema3A activity or expression, said method comprising contacting the substance with macrophage/dendritic cells under conditions whereby Sema3A activity or expression can occur and measuring the amount of Sema3A activity or expression in the presence and absence of the substance, whereby an increase in Sema3A activity or expression in the presence of the substance as compared to the amount of Sema3A activity or expression in the absence of the substance identifies a substance having an enhancing effect on Sema3A activity or expression. Methods for detecting Sema3A activity or expression are known in the art and discussed above.

[0176] The following examples are included to demonstrate various embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of the invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLES

Example 1

Mice

[0177] C57BL/6 mice were purchased from the Jackson Laboratory. Plxna4.sup.-/- mice were provided by M. Tessier-Lavigne (Stanford University) (Yaron et al., 2005) and were backcrossed for nine generations onto the C57BL/6 background. OT-II TCR-transgenic mice were obtained from M. Croft (La Jolla Institute of Allergy and Immunology). Mice were treated in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. The Institutional Animal Care and Use Committee (IACUC) of the University of North Carolina at Chapel Hill approved all experimental procedures.

[0178] Reagents.

[0179] Pam3Cys, poly(I:C), Ultrapure LPS, imiquimod (R837) and mouse CpG-B (ODN 1826) were from InvivoGen. LPS (E. coli 0111:B4) used for the in vivo injections was purchased from Sigma. Recombinant human Sema3A-Fc (1250-S3), human Sema6A-Fc (1146-S6) and human IgG₁ Fc (110-HG) were from R&D. The Rac1 inhibitor NSC23766 (Gao et al., 2004) was from Calbiochem. In immunoblotting experiments, the primary antibodies specific for NF-κB p65 (3034), NF-κB p65 phosphorylated at Ser536 (93H1; 3033), IκBα phosphorylated at Ser32 (14D4; 2859), phospho-ERK1/2 (Thr202/Thr204) (197G2; 4377), phospho-JNK1/2 (Thr183/Tyr185) (81E11; 4668) and phospho-p38 (Thr180/Tyr182) (9211) were from Cell Signaling Technology. Anti-β-actin (C-11; sc-1615 HRP) was from Santa Cruz Biotechnology. Antibody specific for mouse plexin-A4 has been previously described (Suto et al., 2007) and was kindly provided by F. Suto (Tohoku University). In ChIP analysis, the antibodies specific for NF-κB p65 (A) X (sc-109) and c-Jun phosphorylated at Ser63 (9261) were from Santa Cruz Biotechnology (Santa Cruz, Calif.) and Cell Signaling Technology (Danvers, Mass.), respectively. In assays for GTP-bound Rac1, Cdc42 and RhoA, glutathione-SEPHAROSE® (crosslinked polysaccharide polymer) beads conjugated with either GST-Pak1 PBD (for GTP-bound Rac1 and Cdc42) or GST-rhotekin RBD (for GTP-bound RhoA) were kindly provided by K. Burridge (University of North Carolina at Chapel Hill). The primary antibodies specific for Rac1 (C-14; sc-217), Cdc42 (B-8; sc-8401) and RhoA (26C4; sc-418) were from Santa Cruz Biotechnology.

Example 2

Peritoneal Macrophage Isolation and In Vitro Stimulation

[0180] Total peritoneal cells were harvested from the peritoneal cavities of naive WT or Plxna4⁴'' mice with two 10 ml cold sterile PBS wash. Lavage was pooled for mice in the same group. RBCs were lysed in ammonium chloride buffer (150 mM NH₄CL, 10 mM NaHCO3, 1 mM EDTA-tetrasodium). The remaining cells were thoroughly washed with PBS and counted in a hemocytometer. Cytospins were prepared and stained with Diff-Quik solutions, and the number of peritoneal macrophages was determined. Cells were resuspended at a concentration of 10⁶ macrophages/ml in RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg streptomycin. Macrophages were plated in 96-well cell culture plate (2×10⁵/well) and incubated for 2 h at 37° C. Non-adherent cells were removed, and adherent cells were washed with complete RPMI 1640 medium, followed by stimulation with various reagents. In some experiments, cells were pretreated with 200 μM NSC23766 for 1 h followed by the stimulation with TLR agonists.

Example 3

Bacterial Infection In Vitro

[0181] L. monocytogenes and S. typhimurium were from the ATCC (ATCC 43251 and ATCC14028, respectively). E. coli strain LF82 has been previously described (Carvalho et al., 2008) and was provided by R. Balfour Sartor (University of North Carolina at Chapel Hill). S. aureus strain RN6390 has been previously described (Labandeira-Rey et al., 2007) and was provided by M. Gabriela Bowden (Texas A&M University). All bacterial strains were grown to the late exponential phase. The number of viable bacteria used in each experiment was calibrated by colony counting. After three washes with sterile cold PBS, bacteria were added to peritoneal macrophages at a multiplicity of infection (MOI) of 40. After incubation for 1 h at 37° C., cells were treated with 100 μg/ml gentamicin to kill extracellular bacteria and were further incubated for various time periods (S. typhimurium for 1 h; L. monocytogenes, E. coli and S. aureus for 3 h).

Example 4

ELISA

[0182] Cytokines generated by in vitro cultured peritoneal macrophages were quantified using the ELISA Set for mouse TNF-α or IL-6 (BD Biosciences) and mouse IFN-β ELISA Kit (PBL InterferonSource). For the in vivo experiments, concentrations of TNF-α, IL-1β, IL-6, IL-12p70, CCL2 and CCL3 were measured in cell-free peritoneal lavage fluid, serum, and lung homogenate using a Customized Mouse Cytokine 6-plex Panel (BioLegend) and a Luminex Bio-Plex 200 system (Bio-Rad Laboratories).

Example 5

Immunoblotting

[0183] Electrophoresis of proteins was performed by using the NuPAGE system (Invitrogen) according to the manufacturer's protocol. Briefly, peritoneal macrophage stimulated with LPS or left unstimulated were collected and lysed with buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% TRITON® X-100 (a non-ionic surfactant), 0.1% SDS, and protease inhibitor cocktail). Proteins were separated on a NuPAGE® SDS PAGE gel and were transferred onto nitrocellulose membranes (Bio-Rad Laboratories). The membranes were blocked with 10% milk proteins in 1×PBS and 0.1% Tween-20 and then probed with the primary antibodies. Appropriate HRP-conjugated secondary antibodies were used and proteins were detected using an Enhanced Chemiluminescent (ECL) Western Blotting Substrate reagent (peroxidase substrate) from Thermo Scientific.

Example 6

Chromatin Immunoprecipitation (ChIP) Assay

[0184] ChIP assays were performed as described previously (Wen et al., 2008). Briefly, resident peritoneal macrophages were stimulated with various TLR agonists for 1 hour. DNA-protein structure was then crosslinked by 1% formaldehyde for 10 min at 37° C. Cells were collected and lysed in 400 μl SDS lysis buffer. The resulting lysates were sonicated to obtain DNA fragments ranging from 200-1,000 bp using a Sonics VCX 750 (Sonics & Materials, Inc.) 4× for periods of 15 s each. After centrifugation, the supernatant containing chromatin was diluted and an aliquot (2% volume) was saved to quantitate the input DNA in each sample. The remaining chromatin fractions were pre-cleared with salmon sperm DNA/protein A agarose beads (Invitrogen) followed by immunoprecipitation overnight with primary antibodies at 4° C. with gentle rotation. Cross-linking was reversed for 4 h at 65° C. and was followed by proteinase K digestion. DNA was purified by standard phenol/chloroform and ethanol precipitation, and subjected to real-time PCR. Primers for mouse Tnfa promoter κB binding site: forward, 5'-TGAAAGGAGAAGGCTTGTGA (SEQ ID NO:18); reverse, 5'-TAATGGGATGAGTATGGGGCA (SEQ ID NO:19). Primers for mouse Tnfa promoter c-Jun binding site: forward, 5'-CCCAACTTTCCAAACCCTCT (SEQ ID NO:20); reverse, 5'-ACCATGATCTCATGTGGAGGA (SEQ ID NO:21). Primers for mouse Il6 promoter: forward, 5'-GCCTCAAGGATGACTTAAGCA (SEQ ID NO:22); reverse, 5'-AGATTGCACAATGTGACGTCG (SEQ ID NO:23).

Example 7

Detection of GTP-Bound Rac1, Cdc42 and RhoA

[0185] The assays for GTP-bound Rac1, Cdc42 and RhoA were performed as described (Noren et al., 2000). Briefly, peritoneal macrophages were stimulated with 1 μg/ml LPS for various time periods. Cells were lysed and GTP-bound Rac1 and Cdc42 were affinity precipitated using Rac1/Cdc42-binding domain of PAK (PBD) fused with GST. GTP-bound RhoA was affinity precipitated using RhoA-binding domain of rhotekin (RBD) fused with GST. Bound proteins were resolved on a NuPAGE® SDS PAGE gel and were transferred onto nitrocellulose membranes. Membranes were immunoblotted with the appropriate primary antibodies against Rac1, Cdc42 or RhoA.

Example 8

Experimental Sepsis Induced by CLP

[0186] CLP surgery was performed on mice as previously described with minor modifications (Wen et al., 2008). In brief, mice were anesthetized with an intraperitoneal injection of 8 mg of 2,2,2-tribromoethanol (Avertin) (Sigma). Under sterile surgical conditions, a 1-cm midline incision was made to the ventral surface of the abdomen and the cecum was exposed. The cecum was partially ligated at its base with a 3.0 silk suture and punctured two or nine times with 21-gauge needle. The cecum was returned to the peritoneal cavity and the abdominal incision was closed using surgical staples. Mice were rehydrated with 1 ml saline subcutaneously and placed on a heating pad until they recovered from anesthetic. Sham-operated mice were subjected to a similar laparotomy without ligation and puncture. For survival studies, mice subjected to surgery were monitored for 5 days. For analysis of septic inflammatory responses, CLP mice were anesthetized and bled at 4, 24 and 72 h after surgery. Peritoneal lavage was performed with 1 ml of cold sterile PBS wash. Serum and cell-free peritoneal lavage fluid were collected for cytokine protein analyses. To determine bacterial loads after CLP surgery, peritoneal lavage fluid, EDTA-treated blood and lung homogenate from 24 h post-CLP were placed on ice and serially diluted in sterile PBS. A 10 μl aliquot of each dilution was spread on LB agar plates without antibiotics and incubated at 37° C. overnight. Colonies were counted and expressed as CFU/ml. In some experiments, recombinant human Sema3A-Fc or human IgG₁ Fc control protein were injected intraperitoneally at a dosage of 25 μg/kg 1 h prior to CLP procedure.

Example 9

Statistics

[0187] Statistical analysis was carried out with Prism 4 for Macintosh. In survival studies a log-rank test was used to test for significance. For all other studies results were presented as the mean±s.d., and unpaired Student's t-test (one tailed) was applied to evaluate significance. P values less than 0.05 were considered statistically significant.

Example 10

Antigen Presenting Assay

[0188] Bone marrow-derived dendritic cells (BMDCs) were generated from WT and Plxna4.sup.-/- mice as described previously (Eun et al., 2006). BMDCs were incubated with either 1 μg/ml OVA peptide_323-339(ISQAVHAAHAEINEAGR (SEQ ID NO:60); New England Peptide) or 50 μg/ml OVA whole protein (Worthington Biochemical Corp.) at 37° C. overnight. Soluble antigen was removed from DC culture by washing cells two times with medium. CD4⁺ T cells isolated from spleens of OT II mice with Mouse CD4 Subset Column (R&D) were labeled with 2 μM carboxyfluorescein succinimidyl ester (CFSE) and then added to the DC culture. Two or four days later, cells were collected, and T cell proliferation was analyzed by the dilution of CFSE fluorescence detected on a CyAn ADP flow cytometer (DAKO).

Example 11

Sorting and Stimulation of Plasmacytoid DCs

[0189] Total bone marrow cells were harvested from the femurs and tibiae of mice. Single cell suspensions were prepared, and RBCs were lysed. Plasmacytoid DCs were sorted to more than 98% purity by FACS by labeling cells with 3 colors: anti-mPDCA1-PE, antiCD11c-APC and anti-B220-pacific blue (all from BD Biosciences). Sorted pDCs (B220⁺CD11c.sup.intmPDCA1⁺) were stimulated with either TLR7 ligand Imiquimod (R837) or TLR9 ligand B-type CpG oligodeoxynucleotide (ODN1826) in 96-well plates for 16 h. IFN-α protein levels in cell-free supernatant were determined using a mouse IFN-α ELISA kit (PBL InterferonSource).

Example 12

Flow Cytometry Analysis

[0190] Spleens were collected from naive WT and Plxna4.sup.-/- mice and dispersed in 0.5% collagenase A (Roche). After lysing RBCs with ammonium chloride buffer, total cell numbers were determined using a hemocytometer. Fc binding was blocked via a 10 min incubation with purified rat anti-mouse CD16/CD32 (FcγIII/II receptor). The cells were stained with the following monoclonal antibodies to identify different mouse cell types: anti-CD11b-APC, anti-F4/80-PE-Cy5, anti-Ly6C-PE, anti-Ly6G-FITC, anti-B220-Pacific Blue, anti-CD11c-PE-Cy7, anti-mPDCA1-PE, anti-NK1.1-FITC, anti-CD3-PE, anti-CD4-PerCP, anti-CD8-Pacific Blue, anti-CD19-PE-Cy7. All antibodies were purchased from eBioscience, except for anti-F4/80 (Serotec). The cells were fixed in 1% paraformaldehyde and kept in the dark at 4° C. until analysis with a CyAn ADP flow cytometer (DAKO).

Example 13

Plexin-A4 is Expressed by Myeloid and Monocytic Cells

[0191] Plexin-A4 is highly expressed in the nervous system and functions as an axon guidance factor during neuronal development (Suto et al., 2007; Yaron et al., 2005). To investigate the role of plexin-A4 in the immune system, the Plxna4 mRNA expression level was analyzed in different immune subpopulations. Cells of the lymphoid lineage (T, B and NK cells) showed no detectable expression, while cells of the myeloid lineage including macrophages and conventional DCs expressed relatively high levels of the gene with the exception of plasmacytoid DCs (pDCs), which expressed a low level of the gene (FIG. 1A). The highest level of Plxna4 mRNA was observed in peritoneal macrophages. Plexin-A4 protein was detected on the surface of wild type (WT), but not of Plxna4.sup.-/- peritoneal macrophages as determined by immunofluorescence staining and flow cytometry analysis.

[0192] Due to the selective expression of plexin-A4 in myeloid cells, the role of plexin-A4 was investigated in functions that are well studied in cells of this lineage. Previous work has shown that plexin-A1 on DCs mediates the activation of T cells (Takegahara et al., 2006; Wong et al., 2003). In contrast, plexin-A4 is not involved in DC antigen presentation, since OVA_323-339-specific OTII CD4⁺ T cell proliferation was comparable when stimulated with either WT or Plxna4.sup.-/- bone marrow-derived DCs (BMDCs) that have been loaded with either OVA_323-339 peptide or whole OVA protein (FIG. 8). The role of plexin-A4 in type I IFN production by pDCs was also evaluated. WT and Plxna4.sup.-/- pDCs isolated from the bone marrow generated comparable amounts of IFN-α in response to stimulation with TLR7 (R837) or TLR9 (B-type CpG oligodeoxynucleotide, CpG-B) agonists. This indicates that plexin-A4 does not play a role in type 1 IFN production by pDCs during TLR stimulation (FIG. 9).

Example 14

Plexin-A4 is Required for TNF-α and IL-6 Production in Macrophages

[0193] The role of plexin-A4 in cytokine production in response to TLR activation was examined next. Plxna4.sup.-/- and WT mice showed no difference in the percent or absolute number of peritoneal macrophages as determined by the analysis of a cytospin sample (FIGS. 10A-10C). A variety of TLR agonists were used to stimulate naive peritoneal macrophages, including Pam3Cys (TLR2), polyinosinic-polycytidylic acid (poly(I:C), TLR3), lipopolysaccharide (LPS, TLR4), R837 and CpG-B. During TLR stimulation with these diverse agonists, Plxna4.sup.-/- peritoneal macrophages generated significantly lower amount of TNF-α and IL-6 mRNA and protein compared to similarly treated WT controls (FIGS. 1B-C). In contrast, there was no difference in IFN-β production between WT and Plxna4.sup.-/- peritoneal macrophages upon TLR activation. In addition to peritoneal macrophages, Plxna4.sup.-/- bone marrow-derived macrophages (BMMs) (FIG. 19A) and BMDCs (FIG. 19B) exhibited defective TNF-α and IL-6 production upon TLR stimulation when compared to their WT counterparts. These findings show that plexin-A4 is required for TLR-initiated proinflammatory cytokines but not type 1 IFN production.

[0194] The role of macrophage plexin-A4 in response to bacterial challenge was further tested. Both Gram-positive (Listeria monocytogenes, Staphylococcus aureus) and -negative (Escherichia coli, Salmonella typhimurium) bacteria were used to stimulate peritoneal macrophages. In all cases, bacteria-challenged Plxna4.sup.-/- macrophages produced significantly reduced levels of TNF-α and IL-6 mRNA and protein, but a similar level of IFN-β when compared to WT macrophages (FIGS. 2A and 2B). These data suggest that plexin-A4 is important in the bacteria-induced, inflammatory cytokine response.

Example 15

Plexin-A4 Mediates the Activation of NF-κB and JNK but not ERK or p38

[0195] NF-κB and MAPK signaling pathways control the production of inflammatory cytokines during macrophage activation in response to TLR agonists and microbial pathogens (Vallabhapurapu and Karin, 2009). The defective production of TNF-α and IL-6 by Plxna4.sup.-/- macrophages prompted us to investigate the function of plexin-A4 in these signaling pathways. After LPS stimulation, naive WT peritoneal macrophages exhibited enhanced phosphorylation of p65, JNK, p38 and ERK1/2 MAPKs as expected. However, Plxna4.sup.-/- macrophages showed a dramatic defect in the phosphorylation of p65 and JNK, but not ERK1/2 and p38, suggesting a specific regulatory function of plexin-A4 in the activation of NF-κB and JNK kinase (FIG. 3A). Phosphorylation and activation of p65 requires phosphorylation of the inhibitor of κB (IκB). In accordance with defective p65 phosphorylation during LPS stimulation, Plxna4.sup.-/- macrophages showed a defect in IκB phosphorylation compared with WT macrophages (FIG. 3A). In contrast, Plxna4.sup.-/- macrophages were not defective in response to TNF-α, anti-CD40 and IFN-γ treatment. The phosphorylation of MAPKs upon TNF-α or anti-CD40 treatment (FIG. 20A) and of STAT1 (signal transducer activators of transcription 1) upon IFN-γ treatment (FIG. 20B) was not altered in Plxna4.sup.-/- macrophages. This indicates that plexin-A4 specifically mediates TLR activation. Additionally, Plxna4.sup.-/- peritoneal macrophages showed no defect in Akt phosphorylation in response to TLR activation by Pam3Cys and LPS stimulation (FIG. 20C). These findings indicate that plexin-A4 deficiency leads to a TLR-specific defect in NF-κB and JNK activation. Furthermore, this defect is not due to decreased TLR expression, since the mRNA levels of Tlr2, 3, 4, 7 or 9 were similar between WT and Plxna4.sup.-/- peritoneal macrophages (FIG. 21).

[0196] To determine if impaired NF-κB and JNK activation in Plxna4.sup.-/- macrophages causes defective binding of the downstream transcription factors, p65 and c-Jun, at the promoter regions of Tnfa and Il6, chromatin immunoprecipitation (ChIP) assays were performed by using specific anti-p65 and anti-phospho-c-Jun antibodies. Appropriate primer sets were designed that flanked the well-defined binding sites of κB and c-Jun at the promoter regions of Tnfa and Il6 (Galvez et al., 2009; Leng et al., 2009). One hour after stimulation with various TLR agonists, WT macrophages showed enhanced binding of p65 and c-Jun at both Tnfa and Il6 promoters, which correlated with the increased expression of these inflammatory genes (FIGS. 3B and 3C). Conversely, Plxna4.sup.-/- macrophages showed significantly decreased p65 and c-Jun binding at these promoters compared with similarly treated WT controls. This data suggests that plexin-A4 mediates TLR-induced TNF-α and IL-6 production by inducing the activities of NF-κB and JNK to cause the epigenetic activation of these genes.

Example 16

Plexin-a4 Causes the Activation of the Small GTPase, Rac1, but not RhoA or Cdc42

[0197] Several members of the plexin family, such as plexin-B1 (Perrot et al., 2002; Swiercz et al., 2002) and plexin-A1 (Turner et al., 2004), mediate their downstream effects through the activation of Rho family of small GTPases such as RhoA, Rac1 and Cdc42. More importantly, Rac1 has been shown to serve an essential role upstream of NF-κB activation and cytokine production in response to either TLR2 (Arbibe et al., 2000) or TLR4 (Sanlioglu et al., 2001) agonists. To determine if plexin-A4 regulates the activation of small GTPases during TLR activation, a small GTPase pull-down assay was used in which the binding domain of the downstream effector of a particular GTPase was used to pull down the GTP-bound and hence activated form of that GTPase. LPS-stimulated WT macrophages displayed an increase in the GTP-bound form of Rac1 and Cdc42 when compared to unstimulated controls (FIG. 3D). LPS-stimulated Plxna4.sup.-/- macrophages exhibited a dramatic reduction in the quantity of GTP-bound Rac1 when compared to WT cells, while GTP-bound Cdc42 and RhoA were indistinguishable. Thus, plexin-A4 affects the activation of Rac1, which is known to promote NF-κB activation and cytokine production in TLR-stimulated macrophages (Arbibe et al., 2000; Sanlioglu et al., 2001). In support of the importance of Rac1 in TLR-stimulated gene expression, a specific Rac1 inhibitor NSC23766 (Gao et al., 2004) was used and found to significantly attenuate TNF-α and IL-6 production in WT peritoneal macrophages stimulated by various TLR agonists (FIG. 3E). However, NSC23766 showed no inhibitory effect on cytokine production by TLR-activated Plxna4.sup.-/- macrophages. This is compatible with the conclusion that Rac1 activation is significantly attenuated in Plxna4 macrophages such that a Rac1 inhibitor has no further suppressive effect on these cells. IFN-β production in WT macrophages was not affected by Rac1 inhibition (FIG. 3F). This is consistent with the earlier observation that plexin-A4 does not affect TLR-induced type I IFN production.

Example 17

Plxna4.sup.-/- Mice Exhibit Reduced Cytokine Storm and are Protected from Lethal Challenge with LPS and Poly(I:C)

[0198] Next, the physiological relevance of these findings was evaluated. The number of immune cells in the Plxna4.sup.-/- mice was first determined. No apparent defect was detected for all immune cell types tested, including myeloid DCs (mDCs), pDCs, neutrophils, monocytes, macrophages, CD4⁺ or CD8⁺ T cells, B cells, NK cells (FIG. 11). Thus, any physiological difference observed between WT and Plxna4.sup.-/- mice is not due to a change in immune subpopulations.

[0199] Since Plxna4.sup.-/- macrophages showed decreased TNF-α and IL-6 production during TLR stimulation in vitro, the in vivo role of plexin-A4 was examined next. Mice were injected intraperitoneally with either LPS (12.5 mg per kg body weight) or poly(I:C) (20 mg per kg body weight), and the peritoneal lavage fluid was analyzed for inflammatory cytokines by the Multi-plex ELISA assay. Four hours after the injection of LPS or poly(I:C), both localized (FIG. 4B) and systemic (FIG. 4C) levels of inflammatory cytokines, including TNF-α, IL-1β, IL-6, IL-12p70, CCL2 and CCL3, were attenuated in Plxna4.sup.-/- mice compared to WT mice.

[0200] The production of a plethora of inflammatory cytokines is frequently detrimental to the animals and referred to as the cytokine storm (FIG. 7F). Thus, morbidity among animals injected with TLR agonists was examined. While WT mice succumbed to high doses of TLR agonists, Plxna4.sup.-/- mice were significantly protected from LPS or poly(I:C)-induced lethality (FIG. 4A). These findings indicate that plexin-A4 enhances the in vivo inflammatory responses induced by TLR agonists.

Example 18

Plxna4.sup.-/- Mice are Resistant to Septic Inflammation Induced by Cecal Ligation and Puncture (CLP)

[0201] To further evaluate the role of plexin-A4 during in vivo bacterial infection, a well-defined CLP-induced polymicrobial peritonitis model was utilized, which closely replicates the nature and course of clinical sepsis (Hubbard et al., 2005). Acute sepsis results in a high mortality rate, which is primarily due to the overzealous production of proinflammatory cytokines. Given that the number of cecal punctures may determine the severity of sepsis, both moderate sepsis (two punctures) and severe sepsis (nine puncture) models were employed. WT mice with two or nine punctures respectively showed 80% and 100% mortality rate as early as 3 days after CLP (FIG. 5A). However, Plxna4.sup.-/- mice were significantly protected from CLP-induced lethality in both moderate sepsis (p=0.0285) and severe sepsis (p=0.0012). Hence the absence of plexin-A4 provided a clear survival benefit. Inflammatory cytokine levels were also examined in the peritoneal lavage, serum, and lung homogenate before and after CLP-induced peritonitis. At 4 h and 24 h after CLP, Plxna4.sup.-/- mice produced significantly reduced levels of inflammatory cytokines such as TNF-α, IL-1β, IL-6, IL-12p70, CCL2 and CCL3 in the peritoneal lavage fluid (FIG. 5B), serum (FIG. 5C) and lung homogenate (FIG. 5D) compared to WT mice. Therefore, plexin-A4 enhances the septic inflammatory response and promotes a cytokine storm in an experimental peritonitis model.

Example 19

Plexin-A4 Shows No Role in Macrophage Phagocytosis and Bacteria Killing

[0202] Another essential factor that contributes to host survival during acute sepsis is efficient phagocytosis and killing of invading bacteria (Matsukawa et al., 2000). The dramatic survival benefit in Plxna4⁴'' mice prompted an investigation of the function of plexin-A4 in macrophage phagocytosis and bacteria killing. Twenty-four hours after CLP surgery, there were similar levels of bacterial loads in the peritoneal lavage, blood and lung homogenate from WT and Plxna4.sup.-/- mice, indicating that plexin-A4 is not required for bacterial clearance (FIG. 6A). WT and Plxna4.sup.-/- peritoneal macrophages did not differ in the in vitro phagocytosis of GFP-expressing E. coli following different points of incubation (30, 60 or 120 min) (FIG. 6B and FIG. 6C) or infection with different MOIs (10 or 100) (FIG. 6D). Furthermore, there was no difference in the bacterial killing capacity of WT and Plxna4.sup.-/- peritoneal macrophages, because similar numbers of E. coli were recovered from macrophages of both genotypes after the extracellular bacteria were killed by antibiotics and the E. coli-loaded macrophages were incubated for different time periods (FIG. 6E). Taken together, these data suggest that plexin-A4 does not play a role in bacterial phagocytosis and killing in macrophages.

Example 20

Sema3A Enhances LPS-Induced Cytokine Production in a Plexin-A4-Dependent Manner

[0203] Previous studies in the CNS have identified Sema3A and Sema6A as the ligands for plexin-A4 (Suto et al., 2007; Yaron et al., 2005). Sema3a and Sema6a mRNA expression levels were first analyzed in immune subpopulations (FIG. 22). The highest level of Sema3a mRNA was observed in cells of the lymphoid lineage such as T, B and NK cells. Myeloid and monocytic cells also express Sema3a, but at a lower level. Sema6a mRNA is expressed at a relatively low level in all immune subpopulations that we analyzed. The level of Sema6a was nearly 100× lower than that of Sema3a when normalized to the Actb signal.

[0204] To determine if soluble Sema3A or Sema6A also functions in the immune system as a ligand for plexin-A4, WT or Plxna4.sup.-/- peritoneal macrophages were stimulated with increasing concentrations of Sema3A or Sema6A in the absence or presence of LPS. These ligands were expressed as chimeric proteins consisting of the human IgG1 Fc fragment fused to either Sema3A or Sema6A. The IgG1 Fc fragment alone was used as a negative control. Neither Sema3A nor Sema6A alone induced TNF-α and IL-6 production in macrophages. In the presence of a suboptimal concentration of LPS (50 ng/ml), Sema3A, but not Sema6A or IgG Fc control, enhanced LPS-induced TNF-α and IL-6 production in WT peritoneal macrophages in a dose-dependent manner (FIG. 7A and FIG. 7B). However, this enhancement in LPS-responsiveness was completely abolished in Plxna4.sup.-/- macrophages (FIG. 7A and FIG. 7B). The absence of a functional effect of Sema6A agrees with its low expression in different immune populations shown earlier.

[0205] The data presented suggest that LPS via TLR and Sema3A via plexin-A4 might synergistically induce macrophage activation. To assess a converging point for TLR and plexin-A4, we assayed for GTP-bound Rac1. WT macrophages stimulated by LPS or Sema3A resulted in Rac1 activation, while the two stimuli together enhanced this activation. However, Rac1 activation was not observed upon stimulation by either LPS or Sema3A in Plxna4.sup.-/- macrophages, suggesting that plexin-A4 is required for signals that emanate from the binding of TLR agonists and Sema3A (FIG. 7C). Similarly, neither Sema3A nor LPS activated Rac1 in Myd88.sup.-/- macrophages. This finding suggests that MyD88 lies downstream of the Sema3A/plexin-A4 axis. These findings further suggest that Sema3A and LPS both require intact MyD88 signaling to synergistically promote Rac1 activation.

[0206] We next assessed if Sema3A engagement of plexin-A4 could synergistically exacerbate CLP-induced septic inflammation. A single peritoneal injection of Sema3A or control IgG Fc at a dosage of 25 μg/kg body weight was administrated to WT and Plxna4 mice 1 h prior to CLP procedure. Sema3A, but not IgG control, significantly exacerbated CLP-induced lethality in WT mice (FIG. 7D). In contrast, Sema3A did not affect the survival rate of Plxna4.sup.-/- mice in the same experiment. This indicates that the observed biologic effect of Sema3A is dependent on plexin-A4. To further determine if decreased survival observed in Sema3A-pretreated WT mice was associated with dysregulated cytokine production, cytokine production in the peritoneal lavage was measured 4 h after CLP. Indeed, Sema3A-pretreated mice had significantly higher levels of inflammatory cytokines than IgG Fc-treated controls (FIG. 7E).

[0207] The above experiments were performed in cell culture with highly enriched macrophage preparations, thus it was important to assess the physiologic relevance of these findings. To address this, we assessed if Sema3A engagement of plexin-A4 could synergistically exacerbate CLP-induced septic inflammation. A single peritoneal injection of Sema3A or control IgG Fc at a dosage of 25 μg/kg body weight was administrated to WT and Plxna4.sup.-/- mice 1 h prior to CLP procedure. We measured cytokine production in the peritoneal lavage 4 h after CLP. Sema3A-pretreated mice had significantly higher levels of inflammatory cytokines than IgG Fc-treated controls in WT mice, but not in Plxna4.sup.-/- mice (FIG. 8G). Collectively, these in vitro and in vivo results suggest that plexin-A4 binding by Sema3A synergizes with TLR engagement by its agonist(s) to amplify innate inflammatory responses. Strategies to reduce Sema3A engagement of plexin-A4 should be beneficial in controlling an adverse inflammatory response associated with endotoxin shock or sepsis.

Example 21

Preparation of a Fusion Protein Between Human Plexin-A4 Extracellular Fragment and Human IgG1 Fc

[0208] Total RNA was isolated from human myeloid leukemia cell line KG1 cells, which expresses a relatively high PLXNA4 mRNA level (FIG. 17A). Reverse transcription was performed to yield complementary DNA. Human plexin-A4 extracellular fragment (hPLXNA4EF) (bold in FIG. 12A) was cloned using a primer set that contained HindIII (forward primer) digestion site or BamHI (reverse primer) digestion site: forward, 5'-AAGCTTGCCACCCATGAAAGCCATGCCCTGGAACT (SEQ ID NO:24); reverse, 5'-GGATCCGTCCGGGGCAATGTACACCATC (SEQ ID NO:25). Using these enzyme digestion sites, human plexin-A4 extracellular fragment was subcloned into pCDNA3 vector containing human IgG1 Fc fragment (hIgG Fc) (bold in FIG. 17B). Then human plexin-A4 extracellular fragment is upstream of human IgG1 Fc with a linker of two amino acids between them. Human PLXNA4 EF-hIgG Fc fragment was further subcloned into pCEP4 vector to generate fusion protein between human plexin-A4 extracellular fragment and human IgG1 Fc (FIG. 15A; FIG. 17C).

Example 22

Primers

Amplifying Primers:

[0209] Full-Length hPLXNA4:

TABLE-US-00001 Forward (Hind3): (SEQ ID NO: 24) AAGCTTGCCACCCATGAAAGCCATGCCCTGGAACT Reverse (BamHI): (SEQ ID NO: 26) GGATCCAAGGACGGTTCTCAGCTGTCTA.

hPLXNA4 Extracellular Fragment:

TABLE-US-00002 Forward (Hind3): (SEQ ID NO: 24) AAGCTTGCCACCCATGAAAGCCATGCCCTGGAACT Reverse (BamHI): (SEQ ID NO: 25) GGATCCGTCCGGGGCAATGTACACCATC

Sequencing Primers:

TABLE-US-00003

[0210] (SEQ ID NO: 27) 1. GATTGCCTGTGGGAGCCTGTA (SEQ ID NO: 28) 2. TATGTAGAGGTGCCCATTGGCT (SEQ ID NO: 29) 3. TATGAGACGGTGCAGGTGGT (SEQ ID NO: 30) 4. TTCGCCAGCACCAGCTTT (SEQ ID NO: 31) 5. TGACCCAGACTTCGCATGT (SEQ ID NO: 32) 6. AACCTGAATGCCGGAAGCAA (SEQ ID NO: 33) 7. GTGTTTGAGGCCTTTGGTCC (SEQ ID NO: 34) 8. GGATTCCGTTCCTGGACTATA (SEQ ID NO: 35) 9. TTGAGCGAGGACAAGCTCAT (SEQ ID NO: 36) 10. TGACCTGGAGAGTGGAGTCAA Forward-Primer 19 SEQ ID NO: 37 GCCTCTGCCATGAAAGCCA Reverse-Primer 23 SEQ ID NO: 38 AAGGACGGTTCTCAGCTGTCTAA

TABLE-US-00004 TABLE 1 Sequences of RT-PCR primers Mouse genes Forward Reverse Sema3a GCCTGCAGAAGAAGGATTCA TCAGGTTGGGGTGGTTAATG (SEQ ID NOs: 39, 40) Sema6a AATGGCCAGATGCCCT CCGAGTAGAGTTTTCCATTGCA TATG (SEQ ID NOs: 41, 42) Tlr2 CTCCTGAAGCTGTTGCGTTAC TACTTTACCCAGCTCGCTC ACTAC (SEQ ID NOs: 43, 44) Tlr3 TCTTCTTTACGAAA TTGCCAATTGTCTGGAAACACC GTTGGACTTGTC (SEQ ID NOs: 45, 46) Tlr4 ATGGCATGGCTTACACCACC GAGGCCAATTTTGTCTCCACA (SEQ ID NOs: 47, 48) Tlr7 CTGGAGTTCAGAGGCAAC GTTATCACCGGCTCTCCATAGAA CATT (SEQ ID NOs: 49, 50) Tlr9 AGCTGAACATGAACGGCATCT TGAGCGTGTACTTGTTGAGCG (SEQ ID NOs: 51, 52) Ifnb1 ATGAGTGGTGGTT TGACCTTTCAAATGCAGTAGA GCAGGC TTCA (SEQ ID NOs: 53, 54)

Example 23

Preparation of a Fusion Protein Between Human Semaphorin 3A and Human IgG1 Fc

[0211] An expression vector pCR-Blunt-TOPO containing full-length human SEMA3A was purchased from Mammalian Gene Collection (MGC). SEMA3A sequence was amplified by a primer set that contained HindIII (forward primer) digestion site or BamHI (reverse primer) digestion site: forward, 5'-AAGCTTGCCACCCATGGGCTGGTTAACTAGGATTGT (SEQ ID NO:55); reverse, 5'-GGATCCGACACTCCTGGGTGCCCTCTCA (SEQ ID NO:56). Using these enzyme digestion sites, human SEMA3A was subcloned into pCDNA3 vector containing human IgG1 Fc fragment (hIgG Fc) (bold portion in FIG. 17B). Then human SEMA3A is upstream of human IgG1 Fc with a linker of two amino acids between them. Human SEMA3A-hIgG Fc fragment was further subcloned into pCEP4 vector to generate fusion protein between human SEMA3A and human IgG1 Fc (FIG. 16A).

Sequencing Primers:

TABLE-US-00005

[0212] (SEQ ID NO: 57) 1 TGGGAAGAGTCCATATGACCCT (SEQ ID NO: 58) 2 ATTTTCAAGGGATCAGCCGT (SEQ ID NO: 59) 3 CTATATATTGGTTCAACGGC

Example 24

Preparation and Testing of Monoclonal Antibodies to Plexin-A4

[0213] Monoclonal antibodies were prepared against plexin-A4 using standard protocols (See, e.g., Monoclonal Antibodies. Shepherd and Dean, eds. New York: Oxford University Press, 2000; Monoclonal Antibodies: Preparation and Use of Monoclonal Antibodies and Engineered Antibody Derivatives. Heddy Zola, ed. Oxford: BIOS Scientific Publishers Limited, 2000). Western blot analysis was used to assay sixteen of the monoclonal antibodies for the ability to bind to plexin-A4. Briefly, expression vector encoding full-length human PLXNA4 was transfected into 293T cell line by using FuGENE® 6 Transfection Reagent (Roche). Sixteen hours later, cell were collected and lysed with buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 2 mM EDTA, % Triton X-100, 0.1% SDS, and protease inhibitor cocktail). Proteins were separated on a NuPAGE gel (Invitrogen) and were transferred onto nitrocellulose membranes (Bio-Rad Laboratories). The membranes were blocked with 10% milk proteins in 1×PBS and 0.1% Tween-20 and then probed with each of sixteen anti-human plexin-A4 antibodies. HRP-conjugated anti-rat secondary antibodies were used and overexpressed human plexin-A4 were detected using an Enhanced Chemiluminescent (ECL) reagent (Thermo Scientific).

Example 25

Functional Testing of Monoclonal Anti-Plexin-A4 Antibodies in Macrophages

[0214] We have identified plexin-A4 as an essential cell surface molecule involved in macrophage activation and cytokine production. Therefore, we plan to test the efficacy of monoclonal anti-plexin-A4 antibody in inhibiting macrophage cytokine production such as TNF and IL-6. Briefly, resident peritoneal macrophages are isolated and stimulated with a series of TLR agonists in the absence or presence of each of sixteen anti-plexin-A4 antibodies. The supernatants are collected and TNF and IL-6 will be measured by ELISA. Furthermore, the efficacy of anti-plexin-A4 in the treatment of experimental sepsis is tested by using polymicrobial peritonitis animal model, which has been described above.

Example 26

[0215] Prior to the present study, none of the plexin molecules had been shown to regulate TLR signaling. The present invention shows the novel intersection of the plexin-A4 and TLR pathways through the intracellular activation of Rac1, JNK and NF-κB (FIG. 7F). This intersection results in epigenetic changes at the promoters of proinflammatory genes, followed by a pan-cytokine inflammatory response in response to TLR stimulation and bacterial challenge. It cumulates in a cytokine storm that precipitates endotoxin shock induced by TLR agonists and sepsis induced by bacterial infection. The data presented herein show that the plexin-A4 ligand, Sema3A, together with endotoxin can synergistically induce cytokine production in a plexin-A4-dependent manner. Collectively, these data reveal a pivotal role of plexin-A4 in TLR-induced macrophage cytokine production through activation of Rac-1, NF-κB and JNK.

[0216] Whereas it is generally believed that TLR engagement by TLR agonists is able to fully activate immune cells, the present study identifies another requirement during this process involving the engagement of membrane plexin-A4 by Sema3A. Without wishing to be bound by any particular theory of the invention, it appears that TLR and plexin-A4 constitute components of a "two-signal" model for TLR activation. Signal 1 is initiated upon the binding of TLRs with their corresponding ligands and transduced sequentially through MyD88, IRAK, TRAF6, TAK1 kinase complex, IKK and MAPK. Meanwhile, signal 2 is initiated by plexin-A4 binding to its soluble ligand, Sema3A, which triggers Rac1, leading to the activation of NF-κB and JNK. In the absence of TLR stimulation, plexin-A4 activation by Sema3A failed to induce cytokine production, indicating that plexin-A4-mediated signal 2 is not sufficient for the induction of cytokine production. In the absence of plexin-A4, TLR signaling is significantly attenuated but not abolished, indicating that TLR signaling is amplified by plexin-A4 activation but is not initiated by this activation. The data presented herein show that in macrophages lacking MyD88, which is the adaptor for all TLRs except for TLR3, Rac1 activation is attenuated in response to Sema3A stimulation. This indicates that Sema3A/plexin-A4 signaling also requires MyD88. Whether this is achieved via direct binding of plexin-A4 and MyD88 or an indirect route remains to be elucidated. In contrast to its role in the promotion of proinflammatory cytokines, plexin-A4 is not required for TLR-induced TRIF-dependent type 1 IFN production, suggesting that plexin-A4 does not affect TRIF signaling.

[0217] The findings of the present invention show that while plexin-A4 is most highly expressed by monocytic and myeloid cell lineages and significantly reduced on lymphoid cells, Sema3A is highly expressed by lymphoid cells with reduced expression by myeloid/monocytic cell types. Thus it is possible that during an early inflammatory response, the smaller amount of Sema3A produced by myeloid and monocytic cells together with TLR ligands is sufficient to activate the higher density of plexin-A4 on macrophage membrane. Later when T cells are called in, the higher amounts of Sema3A expressed by activated T cells then serve as a feed-back-loop to downregulate T cells, which express less plexin-A4 receptors.

[0218] The biologic function of plexin-A4 in the activation of a pan-cytokine response has broad clinical implications. New anti-inflammatory therapeutic methods are needed in the treatment of immune-related and/or inflammatory diseases. In addition, pathogen-induced sepsis represents a major burden to the US health care system and has become increasingly significant over the past decades (Martin et al., 2003). The hallmark of sepsis in the acute phase is an exacerbated production of proinflammatory cytokines and chemokines, leading to the cytokine storm. While these inflammatory mediators are essential in providing an immediate host defense, their overzealous production can be deleterious to the host if left uncontrolled. The present invention suggests a role for plexin-A4 in mediating the production of proinflammatory cytokines, which affects host survival in models of sepsis. Therefore, the interaction of plexin-A4 and Sema3A presents a new therapeutic target for anti-inflammatory and anti-sepsis treatment.

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Sequence CWU 1

1

6011894PRTHomo sapiens 1Met Lys Ala Met Pro Trp Asn Trp Thr Cys Leu Leu Ser His Leu Leu 1 5 10 15 Met Val Gly Met Gly Ser Ser Thr Leu Leu Thr Arg Gln Pro Ala Pro 20 25 30 Leu Ser Gln Lys Gln Arg Ser Phe Val Thr Phe Arg Gly Glu Pro Ala 35 40 45 Glu Gly Phe Asn His Leu Val Val Asp Glu Arg Thr Gly His Ile Tyr 50 55 60 Leu Gly Ala Val Asn Arg Ile Tyr Lys Leu Ser Ser Asp Leu Lys Val 65 70 75 80 Leu Val Thr His Glu Thr Gly Pro Asp Glu Asp Asn Pro Lys Cys Tyr 85 90 95 Pro Pro Arg Ile Val Gln Thr Cys Asn Glu Pro Leu Thr Thr Thr Asn 100 105 110 Asn Val Asn Lys Met Leu Leu Ile Asp Tyr Lys Glu Asn Arg Leu Ile 115 120 125 Ala Cys Gly Ser Leu Tyr Gln Gly Ile Cys Lys Leu Leu Arg Leu Glu 130 135 140 Asp Leu Phe Lys Leu Gly Glu Pro Tyr His Lys Lys Glu His Tyr Leu 145 150 155 160 Ser Gly Val Asn Glu Ser Gly Ser Val Phe Gly Val Ile Val Ser Tyr 165 170 175 Ser Asn Leu Asp Asp Lys Leu Phe Ile Ala Thr Ala Val Asp Gly Lys 180 185 190 Pro Glu Tyr Phe Pro Thr Ile Ser Ser Arg Lys Leu Thr Lys Asn Ser 195 200 205 Glu Ala Asp Gly Met Phe Ala Tyr Val Phe His Asp Glu Phe Val Ala 210 215 220 Ser Met Ile Lys Ile Pro Ser Asp Thr Phe Thr Ile Ile Pro Asp Phe 225 230 235 240 Asp Ile Tyr Tyr Val Tyr Gly Phe Ser Ser Gly Asn Phe Val Tyr Phe 245 250 255 Leu Thr Leu Gln Pro Glu Met Val Ser Pro Pro Gly Ser Thr Thr Lys 260 265 270 Glu Gln Val Tyr Thr Ser Lys Leu Val Arg Leu Cys Lys Glu Asp Thr 275 280 285 Ala Phe Asn Ser Tyr Val Glu Val Pro Ile Gly Cys Glu Arg Ser Gly 290 295 300 Val Glu Tyr Arg Leu Leu Gln Ala Ala Tyr Leu Ser Lys Ala Gly Ala 305 310 315 320 Val Leu Gly Arg Thr Leu Gly Val His Pro Asp Asp Asp Leu Leu Phe 325 330 335 Thr Val Phe Ser Lys Gly Gln Lys Arg Lys Met Lys Ser Leu Asp Glu 340 345 350 Ser Ala Leu Cys Ile Phe Ile Leu Lys Gln Ile Asn Asp Arg Ile Lys 355 360 365 Glu Arg Leu Gln Ser Cys Tyr Arg Gly Glu Gly Thr Leu Asp Leu Ala 370 375 380 Trp Leu Lys Val Lys Asp Ile Pro Cys Ser Ser Ala Leu Leu Thr Ile 385 390 395 400 Asp Asp Asn Phe Cys Gly Leu Asp Met Asn Ala Pro Leu Gly Val Ser 405 410 415 Asp Met Val Arg Gly Ile Pro Val Phe Thr Glu Asp Arg Asp Arg Met 420 425 430 Thr Ser Val Ile Ala Tyr Val Tyr Lys Asn His Ser Leu Ala Phe Val 435 440 445 Gly Thr Lys Ser Gly Lys Leu Lys Lys Ile Arg Val Asp Gly Pro Arg 450 455 460 Gly Asn Ala Leu Gln Tyr Glu Thr Val Gln Val Val Asp Pro Gly Pro 465 470 475 480 Val Leu Arg Asp Met Ala Phe Ser Lys Asp His Glu Gln Leu Tyr Ile 485 490 495 Met Ser Glu Arg Gln Leu Thr Arg Val Pro Val Glu Ser Cys Gly Gln 500 505 510 Tyr Gln Ser Cys Gly Glu Cys Leu Gly Ser Gly Asp Pro His Cys Gly 515 520 525 Trp Cys Val Leu His Asn Thr Cys Thr Arg Lys Glu Arg Cys Glu Arg 530 535 540 Ser Lys Glu Pro Arg Arg Phe Ala Ser Glu Met Lys Gln Cys Val Arg 545 550 555 560 Leu Thr Val His Pro Asn Asn Ile Ser Val Ser Gln Tyr Asn Val Leu 565 570 575 Leu Val Leu Glu Thr Tyr Asn Val Pro Glu Leu Ser Ala Gly Val Asn 580 585 590 Cys Thr Phe Glu Asp Leu Ser Glu Met Asp Gly Leu Val Val Gly Asn 595 600 605 Gln Ile Gln Cys Tyr Ser Pro Ala Ala Lys Glu Val Pro Arg Ile Ile 610 615 620 Thr Glu Asn Gly Asp His His Val Val Gln Leu Gln Leu Lys Ser Lys 625 630 635 640 Glu Thr Gly Met Thr Phe Ala Ser Thr Ser Phe Val Phe Tyr Asn Cys 645 650 655 Ser Val His Asn Ser Cys Leu Ser Cys Val Glu Ser Pro Tyr Arg Cys 660 665 670 His Trp Cys Lys Tyr Arg His Val Cys Thr His Asp Pro Lys Thr Cys 675 680 685 Ser Phe Gln Glu Gly Arg Val Lys Leu Pro Glu Asp Cys Pro Gln Leu 690 695 700 Leu Arg Val Asp Lys Ile Leu Val Pro Val Glu Val Ile Lys Pro Ile 705 710 715 720 Thr Leu Lys Ala Lys Asn Leu Pro Gln Pro Gln Ser Gly Gln Arg Gly 725 730 735 Tyr Glu Cys Ile Leu Asn Ile Gln Gly Ser Glu Gln Arg Val Pro Ala 740 745 750 Leu Arg Phe Asn Ser Ser Ser Val Gln Cys Gln Asn Thr Ser Tyr Ser 755 760 765 Tyr Glu Gly Met Glu Ile Asn Asn Leu Pro Val Glu Leu Thr Val Val 770 775 780 Trp Asn Gly His Phe Asn Ile Asp Asn Pro Ala Gln Asn Lys Val His 785 790 795 800 Leu Tyr Lys Cys Gly Ala Met Arg Glu Ser Cys Gly Leu Cys Leu Lys 805 810 815 Ala Asp Pro Asp Phe Ala Cys Gly Trp Cys Gln Gly Pro Gly Gln Cys 820 825 830 Thr Leu Arg Gln His Cys Pro Ala Gln Glu Ser Gln Trp Leu Glu Leu 835 840 845 Ser Gly Ala Lys Ser Lys Cys Thr Asn Pro Arg Ile Thr Glu Ile Ile 850 855 860 Pro Val Thr Gly Pro Arg Glu Gly Gly Thr Lys Val Thr Ile Arg Gly 865 870 875 880 Glu Asn Leu Gly Leu Glu Phe Arg Asp Ile Ala Ser His Val Lys Val 885 890 895 Ala Gly Val Glu Cys Ser Pro Leu Val Asp Gly Tyr Ile Pro Ala Glu 900 905 910 Gln Ile Val Cys Glu Met Gly Glu Ala Lys Pro Ser Gln His Ala Gly 915 920 925 Phe Val Glu Ile Cys Val Ala Val Cys Arg Pro Glu Phe Met Ala Arg 930 935 940 Ser Ser Gln Leu Tyr Tyr Phe Met Thr Leu Thr Leu Ser Asp Leu Lys 945 950 955 960 Pro Ser Arg Gly Pro Met Ser Gly Gly Thr Gln Val Thr Ile Thr Gly 965 970 975 Thr Asn Leu Asn Ala Gly Ser Asn Val Val Val Met Phe Gly Lys Gln 980 985 990 Pro Cys Leu Phe His Arg Arg Ser Pro Ser Tyr Ile Val Cys Asn Thr 995 1000 1005 Thr Ser Ser Asp Glu Val Leu Glu Met Lys Val Ser Val Gln Val 1010 1015 1020 Asp Arg Ala Lys Ile His Gln Asp Leu Val Phe Gln Tyr Val Glu 1025 1030 1035 Asp Pro Thr Ile Val Arg Ile Glu Pro Glu Trp Ser Ile Val Ser 1040 1045 1050 Gly Asn Thr Pro Ile Ala Val Trp Gly Thr His Leu Asp Leu Ile 1055 1060 1065 Gln Asn Pro Gln Ile Arg Ala Lys His Gly Gly Lys Glu His Ile 1070 1075 1080 Asn Ile Cys Glu Val Leu Asn Ala Thr Glu Met Thr Cys Gln Ala 1085 1090 1095 Pro Ala Leu Ala Leu Gly Pro Asp His Gln Ser Asp Leu Thr Glu 1100 1105 1110 Arg Pro Glu Glu Phe Gly Phe Ile Leu Asp Asn Val Gln Ser Leu 1115 1120 1125 Leu Ile Leu Asn Lys Thr Asn Phe Thr Tyr Tyr Pro Asn Pro Val 1130 1135 1140 Phe Glu Ala Phe Gly Pro Ser Gly Ile Leu Glu Leu Lys Pro Gly 1145 1150 1155 Thr Pro Ile Ile Leu Lys Gly Lys Asn Leu Ile Pro Pro Val Ala 1160 1165 1170 Gly Gly Asn Val Lys Leu Asn Tyr Thr Val Leu Val Gly Glu Lys 1175 1180 1185 Pro Cys Thr Val Thr Val Ser Asp Val Gln Leu Leu Cys Glu Ser 1190 1195 1200 Pro Asn Leu Ile Gly Arg His Lys Val Met Ala Arg Val Gly Gly 1205 1210 1215 Met Glu Tyr Ser Pro Gly Met Val Tyr Ile Ala Pro Asp Ser Pro 1220 1225 1230 Leu Ser Leu Pro Ala Ile Val Ser Ile Ala Val Ala Gly Gly Leu 1235 1240 1245 Leu Ile Ile Phe Ile Val Ala Val Leu Ile Ala Tyr Lys Arg Lys 1250 1255 1260 Ser Arg Glu Ser Asp Leu Thr Leu Lys Arg Leu Gln Met Gln Met 1265 1270 1275 Asp Asn Leu Glu Ser Arg Val Ala Leu Glu Cys Lys Glu Ala Phe 1280 1285 1290 Ala Glu Leu Gln Thr Asp Ile His Glu Leu Thr Ser Asp Leu Asp 1295 1300 1305 Gly Ala Gly Ile Pro Phe Leu Asp Tyr Arg Thr Tyr Thr Met Arg 1310 1315 1320 Val Leu Phe Pro Gly Ile Glu Asp His Pro Val Leu Arg Asp Leu 1325 1330 1335 Glu Val Pro Gly Tyr Arg Gln Glu Arg Val Glu Lys Gly Leu Lys 1340 1345 1350 Leu Phe Ala Gln Leu Ile Asn Asn Lys Val Phe Leu Leu Ser Phe 1355 1360 1365 Ile Arg Thr Leu Glu Ser Gln Arg Ser Phe Ser Met Arg Asp Arg 1370 1375 1380 Gly Asn Val Ala Ser Leu Ile Met Thr Val Leu Gln Ser Lys Leu 1385 1390 1395 Glu Tyr Ala Thr Asp Val Leu Lys Gln Leu Leu Ala Asp Leu Ile 1400 1405 1410 Asp Lys Asn Leu Glu Ser Lys Asn His Pro Lys Leu Leu Leu Arg 1415 1420 1425 Arg Thr Glu Ser Val Ala Glu Lys Met Leu Thr Asn Trp Phe Thr 1430 1435 1440 Phe Leu Leu Tyr Lys Phe Leu Lys Glu Cys Ala Gly Glu Pro Leu 1445 1450 1455 Phe Ser Leu Phe Cys Ala Ile Lys Gln Gln Met Glu Lys Gly Pro 1460 1465 1470 Ile Asp Ala Ile Thr Gly Glu Ala Arg Tyr Ser Leu Ser Glu Asp 1475 1480 1485 Lys Leu Ile Arg Gln Gln Ile Asp Tyr Lys Thr Leu Val Leu Ser 1490 1495 1500 Cys Val Ser Pro Asp Asn Ala Asn Ser Pro Glu Val Pro Val Lys 1505 1510 1515 Ile Leu Asn Cys Asp Thr Ile Thr Gln Val Lys Glu Lys Ile Leu 1520 1525 1530 Asp Ala Ile Phe Lys Asn Val Pro Cys Ser His Arg Pro Lys Ala 1535 1540 1545 Ala Asp Met Asp Leu Glu Trp Arg Gln Gly Ser Gly Ala Arg Met 1550 1555 1560 Ile Leu Gln Asp Glu Asp Ile Thr Thr Lys Ile Glu Asn Asp Trp 1565 1570 1575 Lys Arg Leu Asn Thr Leu Ala His Tyr Gln Val Pro Asp Gly Ser 1580 1585 1590 Val Val Ala Leu Val Ser Lys Gln Val Thr Ala Tyr Asn Ala Val 1595 1600 1605 Asn Asn Ser Thr Val Ser Arg Thr Ser Ala Ser Lys Tyr Glu Asn 1610 1615 1620 Met Ile Arg Tyr Thr Gly Ser Pro Asp Ser Leu Arg Ser Arg Thr 1625 1630 1635 Pro Met Ile Thr Pro Asp Leu Glu Ser Gly Val Lys Met Trp His 1640 1645 1650 Leu Val Lys Asn His Glu His Gly Asp Gln Lys Glu Gly Asp Arg 1655 1660 1665 Gly Ser Lys Met Val Ser Glu Ile Tyr Leu Thr Arg Leu Leu Ala 1670 1675 1680 Thr Lys Gly Thr Leu Gln Lys Phe Val Asp Asp Leu Phe Glu Thr 1685 1690 1695 Ile Phe Ser Thr Ala His Arg Gly Ser Ala Leu Pro Leu Ala Ile 1700 1705 1710 Lys Tyr Met Phe Asp Phe Leu Asp Glu Gln Ala Asp Lys His Gly 1715 1720 1725 Ile His Asp Pro His Val Arg His Thr Trp Lys Ser Asn Cys Leu 1730 1735 1740 Pro Leu Arg Phe Trp Val Asn Met Ile Lys Asn Pro Gln Phe Val 1745 1750 1755 Phe Asp Ile His Lys Asn Ser Ile Thr Asp Ala Cys Leu Ser Val 1760 1765 1770 Val Ala Gln Thr Phe Met Asp Ser Cys Ser Thr Ser Glu His Arg 1775 1780 1785 Leu Gly Lys Asp Ser Pro Ser Asn Lys Leu Leu Tyr Ala Lys Asp 1790 1795 1800 Ile Pro Ser Tyr Lys Asn Trp Val Glu Arg Tyr Tyr Ser Asp Ile 1805 1810 1815 Gly Lys Met Pro Ala Ile Ser Asp Gln Asp Met Asn Ala Tyr Leu 1820 1825 1830 Ala Glu Gln Ser Arg Met His Met Asn Glu Phe Asn Thr Met Ser 1835 1840 1845 Ala Leu Ser Glu Ile Phe Ser Tyr Val Gly Lys Tyr Ser Glu Glu 1850 1855 1860 Ile Leu Gly Pro Leu Asp His Asp Asp Gln Cys Gly Lys Gln Lys 1865 1870 1875 Leu Ala Tyr Lys Leu Glu Gln Val Ile Thr Leu Met Ser Leu Asp 1880 1885 1890 Ser 2522PRTHomo sapiens 2Met Lys Ala Met Pro Trp Asn Trp Thr Cys Leu Leu Ser His Leu Leu 1 5 10 15 Met Val Gly Met Gly Ser Ser Thr Leu Leu Thr Arg Gln Pro Ala Pro 20 25 30 Leu Ser Gln Lys Gln Arg Ser Phe Val Thr Phe Arg Gly Glu Pro Ala 35 40 45 Glu Gly Phe Asn His Leu Val Val Asp Glu Arg Thr Gly His Ile Tyr 50 55 60 Leu Gly Ala Val Asn Arg Ile Tyr Lys Leu Ser Ser Asp Leu Lys Val 65 70 75 80 Leu Val Thr His Glu Thr Gly Pro Asp Glu Asp Asn Pro Lys Cys Tyr 85 90 95 Pro Pro Arg Ile Val Gln Thr Cys Asn Glu Pro Leu Thr Thr Thr Asn 100 105 110 Asn Val Asn Lys Met Leu Leu Ile Asp Tyr Lys Glu Asn Arg Leu Ile 115 120 125 Ala Cys Gly Ser Leu Tyr Gln Gly Ile Cys Lys Leu Leu Arg Leu Glu 130 135 140 Asp Leu Phe Lys Leu Gly Glu Pro Tyr His Lys Lys Glu His Tyr Leu 145 150 155 160 Ser Gly Val Asn Glu Ser Gly Ser Val Phe Gly Val Ile Val Ser Tyr 165 170 175 Ser Asn Leu Asp Asp Lys Leu Phe Ile Ala Thr Ala Val Asp Gly Lys 180 185 190 Pro Glu Tyr Phe Pro Thr Ile Ser Ser Arg Lys Leu Thr Lys Asn Ser 195 200 205 Glu Ala Asp Gly Met Phe Ala Tyr Val Phe His Asp Glu Phe Val Ala 210 215 220 Ser Met Ile Lys Ile Pro Ser Asp Thr Phe Thr Ile Ile Pro Asp Phe 225 230 235 240 Asp Ile Tyr Tyr Val Tyr Gly Phe Ser Ser Gly Asn Phe Val Tyr Phe 245 250 255 Leu Thr Leu Gln Pro Glu Met Val Ser Pro Pro Gly Ser Thr Thr Lys 260 265 270 Glu Gln Val Tyr Thr Ser Lys Leu Val Arg Leu Cys Lys Glu Asp Thr 275 280 285 Ala Phe Asn Ser Tyr Val Glu Val Pro Ile Gly Cys Glu Arg Ser Gly 290 295 300 Val Glu Tyr Arg Leu Leu Gln Ala Ala Tyr Leu Ser Lys Ala Gly Ala 305 310 315 320 Val Leu Gly Arg Thr Leu Gly Val His Pro Asp Asp Asp Leu Leu Phe 325 330 335 Thr Val Phe Ser Lys Gly Gln Lys Arg Lys Met Lys Ser Leu Asp Glu 340 345 350 Ser Ala Leu Cys Ile Phe Ile Leu Lys Gln Ile Asn Asp Arg Ile Lys 355

360 365 Glu Arg Leu Gln Ser Cys Tyr Arg Gly Glu Gly Thr Leu Asp Leu Ala 370 375 380 Trp Leu Lys Val Lys Asp Ile Pro Cys Ser Ser Ala Leu Leu Thr Ile 385 390 395 400 Asp Asp Asn Phe Cys Gly Leu Asp Met Asn Ala Pro Leu Gly Val Ser 405 410 415 Asp Met Val Arg Gly Ile Pro Val Phe Thr Glu Asp Arg Asp Arg Met 420 425 430 Thr Ser Val Ile Ala Tyr Val Tyr Lys Asn His Ser Leu Ala Phe Val 435 440 445 Gly Thr Lys Ser Gly Lys Leu Lys Lys Ser Phe Gly Thr Gly Pro Gln 450 455 460 Gly Gly Ile Thr Gln Glu Trp Ile Gly Val Glu Gly Asp Pro Pro Gly 465 470 475 480 Ala Asn Ile Ala Ser Gln Glu Gln Met Leu Cys Val Tyr Leu Gln Cys 485 490 495 Ser Ser His Lys Ala Ile Ser Asp Gln Arg Val Gln Pro Leu Leu Cys 500 505 510 Cys Phe Leu Asn Val Pro Gly Asn Ser Ser 515 520 3492PRTHomo sapiens 3Met Lys Ala Met Pro Trp Asn Trp Thr Cys Leu Leu Ser His Leu Leu 1 5 10 15 Met Val Gly Met Gly Ser Ser Thr Leu Leu Thr Arg Gln Pro Ala Pro 20 25 30 Leu Ser Gln Lys Gln Arg Ser Phe Val Thr Phe Arg Gly Glu Pro Ala 35 40 45 Glu Gly Phe Asn His Leu Val Val Asp Glu Arg Thr Gly His Ile Tyr 50 55 60 Leu Gly Ala Val Asn Arg Ile Tyr Lys Leu Ser Ser Asp Leu Lys Val 65 70 75 80 Leu Val Thr His Glu Thr Gly Pro Asp Glu Asp Asn Pro Lys Cys Tyr 85 90 95 Pro Pro Arg Ile Val Gln Thr Cys Asn Glu Pro Leu Thr Thr Thr Asn 100 105 110 Asn Val Asn Lys Met Leu Leu Ile Asp Tyr Lys Glu Asn Arg Leu Ile 115 120 125 Ala Cys Gly Ser Leu Tyr Gln Gly Ile Cys Lys Leu Leu Arg Leu Glu 130 135 140 Asp Leu Phe Lys Leu Gly Glu Pro Tyr His Lys Lys Glu His Tyr Leu 145 150 155 160 Ser Gly Val Asn Glu Ser Gly Ser Val Phe Gly Val Ile Val Ser Tyr 165 170 175 Ser Asn Leu Asp Asp Lys Leu Phe Ile Ala Thr Ala Val Asp Gly Lys 180 185 190 Pro Glu Tyr Phe Pro Thr Ile Ser Ser Arg Lys Leu Thr Lys Asn Ser 195 200 205 Glu Ala Asp Gly Met Phe Ala Tyr Val Phe His Asp Glu Phe Val Ala 210 215 220 Ser Met Ile Lys Ile Pro Ser Asp Thr Phe Thr Ile Ile Pro Asp Phe 225 230 235 240 Asp Ile Tyr Tyr Val Tyr Gly Phe Ser Ser Gly Asn Phe Val Tyr Phe 245 250 255 Leu Thr Leu Gln Pro Glu Met Val Ser Pro Pro Gly Ser Thr Thr Lys 260 265 270 Glu Gln Val Tyr Thr Ser Lys Leu Val Arg Leu Cys Lys Glu Asp Thr 275 280 285 Ala Phe Asn Ser Tyr Val Glu Val Pro Ile Gly Cys Glu Arg Ser Gly 290 295 300 Val Glu Tyr Arg Leu Leu Gln Ala Ala Tyr Leu Ser Lys Ala Gly Ala 305 310 315 320 Val Leu Gly Arg Thr Leu Gly Val His Pro Asp Asp Asp Leu Leu Phe 325 330 335 Thr Val Phe Ser Lys Gly Gln Lys Arg Lys Met Lys Ser Leu Asp Glu 340 345 350 Ser Ala Leu Cys Ile Phe Ile Leu Lys Gln Ile Asn Asp Arg Ile Lys 355 360 365 Glu Arg Leu Gln Ser Cys Tyr Arg Gly Glu Gly Thr Leu Asp Leu Ala 370 375 380 Trp Leu Lys Val Lys Asp Ile Pro Cys Ser Ser Ala Leu Leu Thr Ile 385 390 395 400 Asp Asp Asn Phe Cys Gly Leu Asp Met Asn Ala Pro Leu Gly Val Ser 405 410 415 Asp Met Val Arg Gly Ile Pro Val Phe Thr Glu Asp Arg Asp Arg Met 420 425 430 Thr Ser Val Ile Ala Tyr Val Tyr Lys Asn His Ser Leu Ala Phe Val 435 440 445 Gly Thr Lys Ser Gly Lys Leu Lys Lys Met Pro Gly Thr Ser Leu Cys 450 455 460 Pro Thr Leu Glu Leu Gln Thr Gly Pro Arg Ser His Arg Ala Thr Val 465 470 475 480 Thr Leu Glu Leu Leu Phe Ser Ser Cys Ser Ser Asn 485 490 45685DNAHomo sapiens 4atgaaagcca tgccctggaa ctggacctgc cttctctccc acctcctcat ggtgggcatg 60ggctcctcca ctttgctcac ccggcagcca gccccgctgt cccagaagca gcggtcattt 120gtcacattcc gaggagagcc cgccgagggt ttcaatcacc tggtggtgga tgagaggaca 180ggacacattt acttgggggc cgtcaatcgg atttacaagc tctccagcga cctgaaggtc 240ttggtgacgc atgagacagg gccggacgag gacaacccca agtgttaccc accccgcatc 300gtccagacct gcaatgagcc cctgaccacc accaacaatg tcaacaagat gctcctcata 360gactacaagg agaacaggct gattgcctgt gggagcctgt accaaggcat ctgcaagctg 420ctgaggctgg aggacctctt caagctgggg gagccttatc ataagaagga gcactatctg 480tcaggtgtca acgagagcgg ctcagtcttt ggagtgatcg tctcctacag caacctggat 540gacaagctgt tcattgccac ggcagtggat gggaagcccg agtattttcc caccatctcc 600agccggaaac tgaccaagaa ctctgaggcg gatggcatgt tcgcgtacgt cttccatgat 660gagttcgtgg cctcgatgat taagatccct tcggacacct tcaccatcat ccctgacttt 720gatatctact atgtctatgg ttttagcagt ggcaactttg tctacttttt gaccctccaa 780cctgagatgg tgtctccacc aggctccacc accaaggagc aggtgtatac atccaagctc 840gtgaggcttt gcaaggagga cacagccttc aactcctatg tagaggtgcc cattggctgt 900gagcgcagtg gggtggagta ccgcctgctg caggctgcct acctgtccaa agcgggggcc 960gtgcttggca ggacccttgg agtccatcca gatgatgacc tgctcttcac cgtcttctcc 1020aagggccaga agcggaaaat gaaatccctg gatgagtcgg ccctgtgcat cttcatcttg 1080aagcagataa atgaccgcat taaggagcgg ctgcagtctt gttaccgggg cgagggcacg 1140ctggacctgg cctggctcaa ggtgaaggac atcccctgca gcagtgcgct cttaaccatt 1200gacgataact tctgtggcct ggacatgaat gctcccctgg gagtgtccga catggtgcgt 1260ggaattcccg tcttcacgga ggacagggac cgcatgacgt ctgtcatcgc atatgtctac 1320aagaaccact ctctggcctt tgtgggcacc aaaagtggca agctgaagaa gatccgggtg 1380gatggaccca ggggcaacgc cctccagtat gagacggtgc aggtggtgga ccccggccca 1440gtcctccggg atatggcctt ctccaaggac cacgagcaac tctacatcat gtcagagagg 1500cagctcacca gagtccctgt ggagtcctgt ggtcagtatc agagctgcgg cgagtgcctt 1560ggctcaggcg acccccactg tggctggtgt gtgctgcaca acacttgcac ccggaaggag 1620cggtgtgagc ggtccaagga gccccgcagg tttgcctcgg agatgaagca gtgtgtccgg 1680ctgacggtcc atcccaacaa tatctccgtc tctcagtaca acgtgctgct ggtcctggag 1740acgtacaatg tcccggagct gtcagctggc gtcaactgca cctttgagga cctgtcagag 1800atggatgggc tggtcgtggg caatcagatc cagtgctact cccctgcagc caaggaggtg 1860ccccggatca tcacagagaa tggggaccac catgtcgtac agcttcagct caaatcaaag 1920gagaccggca tgaccttcgc cagcaccagc tttgtcttct acaattgcag cgtccacaat 1980tcgtgcctgt cctgcgtgga gagtccatac cgctgccact ggtgtaaata ccggcatgtc 2040tgcacccatg accccaagac ctgctccttc caggaaggcc gagtgaagct gcccgaggac 2100tgcccccagc tgctgcgagt ggacaagatc ctggtgcccg tggaggtgat caagcctatc 2160acgctgaagg ccaagaacct cccccagccc cagtctgggc agcgtggcta cgaatgcatc 2220ctcaacattc agggcagcga gcagcgagtg cccgccctgc gcttcaacag ctccagcgta 2280cagtgccaga acacctctta ttcctatgaa gggatggaga tcaacaacct gcccgtggag 2340ttgacagtcg tgtggaatgg gcacttcaac attgacaacc cagctcagaa taaagttcac 2400ctctacaagt gtggagccat gcgtgagagc tgcgggctgt gcctcaaggc tgacccagac 2460ttcgcatgtg gctggtgcca gggcccaggc cagtgcaccc tgcgccagca ctgccctgcc 2520caggagagcc agtggctgga gctgtctggt gccaaaagca agtgcacaaa cccccgcatc 2580acagagataa tcccggtgac aggcccccgg gaagggggca ccaaggtcac tatccgaggg 2640gagaacctgg gcctggaatt tcgcgacatc gcctcccatg tcaaggttgc tggcgtggag 2700tgcagccctt tagtggatgg ttacatccct gcagaacaga tcgtgtgtga gatgggggag 2760gccaagccca gccagcatgc aggcttcgtg gagatctgcg tggctgtgtg tcggcctgaa 2820ttcatggccc ggtcctcaca gctctattac ttcatgacac tgactctctc agatctgaag 2880cccagccggg ggcccatgtc cggagggacc caagtgacca tcacaggcac caacctgaat 2940gccggaagca acgtggtggt gatgtttgga aagcagccct gtctcttcca caggcgatct 3000ccatcctaca ttgtctgcaa caccacatcc tcagatgagg tgctagagat gaaggtgtcg 3060gtgcaggtgg acagggccaa gatccaccag gacctggtct ttcagtatgt ggaagacccc 3120accatcgtgc ggattgagcc agaatggagc attgtcagtg gaaacacacc catcgccgta 3180tgggggaccc acctggacct catacagaac ccccagatcc gtgccaagca tggagggaag 3240gagcacatca atatctgtga ggttctgaac gctactgaga tgacctgtca ggcgcccgcc 3300ctcgctctgg gtcctgacca ccagtcagac ctgaccgaga ggcccgagga gtttggcttc 3360atcctggaca acgtccagtc cctgctcatc ctcaacaaga ccaacttcac ctactatccc 3420aacccggtgt ttgaggcctt tggtccctca ggaatcctgg agctcaagcc tggcacgccc 3480atcatcctaa agggcaagaa cctgatcccg cctgtggctg ggggcaacgt gaagctgaac 3540tacactgtgc tggttgggga gaagccgtgc accgtgaccg tgtcagatgt ccagctgctc 3600tgcgagtccc ccaacctcat cggcaggcac aaagtgatgg cccgtgtcgg tggcatggag 3660tactccccgg ggatggtgta cattgccccg gacagcccgc tcagcctgcc cgccatcgtc 3720agcatcgcag tggctggcgg cctcctcatc attttcatcg tggccgtgct cattgcctat 3780aaacgcaagt cccgcgaaag tgacctcacg ctgaagcggc tgcagatgca gatggacaac 3840ctggagtccc gtgtggccct ggagtgcaag gaagcctttg ccgagctgca gacggacatc 3900catgagctga ccagtgacct ggatggagcc gggattccgt tcctggacta tagaacttac 3960accatgcggg tgctgttccc aggaattgaa gaccaccctg tcctccggga ccttgaggtc 4020ccgggctacc ggcaggagcg tgtggagaaa ggcctgaagc tcttcgccca gctcatcaac 4080aacaaggtgt tcctgctgtc cttcatccgc acgcttgagt cccagcgtag cttctccatg 4140cgcgaccgtg gcaacgtggc ctcactcatc atgaccgtgc tgcagagcaa gctggagtac 4200gccactgatg tgctgaagca gctgctggcc gacctcattg acaagaacct ggagagcaag 4260aaccacccta agctgctgct caggaggact gagtcagtgg ctgagaagat gctgaccaat 4320tggtttactt tcctcctcta caagttcctc aaggagtgtg ctggggagcc cctcttctcc 4380ctgttctgtg ccatcaagca gcagatggag aagggcccca ttgacgccat cacgggcgag 4440gcccgctact ccttgagcga ggacaagctc atccgccagc agattgacta caaaaccctg 4500gtcctgagct gtgtcagccc agacaatgcc aacagccccg aggtcccagt aaagatcctc 4560aactgtgaca ccatcactca ggtcaaggag aagattctgg atgccatctt caagaatgtg 4620ccttgctccc accggcccaa agctgcagat atggatctgg agtggcgaca aggaagtggg 4680gcaaggatga tcttgcagga tgaagacatc accaccaaga ttgagaatga ttggaagcga 4740ctgaacacac tggcccacta ccaggtgcca gatggttccg tggtggcatt agtgtccaag 4800caggtgacag cctataacgc agtgaacaac tccaccgtct ccaggacctc agcaagtaaa 4860tatgaaaaca tgatccggta cacgggcagc cccgacagcc tccgctcacg gacacctatg 4920atcactcctg acctggagag tggagtcaag atgtggcacc tagtgaagaa ccacgagcac 4980ggagaccaga aggaggggga ccgggggagc aagatggtgt ctgaaatcta cctgacccga 5040ctcctggcca ctaagggcac actgcagaag tttgtggatg acctctttga gaccatcttc 5100agcacggcac accgtggctc tgccctgccc ctggccatca agtacatgtt tgacttcctg 5160gatgagcagg ctgataaaca tggcattcat gacccgcacg tccgccatac ctggaagagc 5220aattgcctgc ccctgaggtt ttgggtcaac atgatcaaga acccgcagtt tgtgtttgac 5280atccataaga acagcatcac agacgcctgc ctctctgtgg tggctcagac cttcatggac 5340tcttgctcca cgtcagagca ccggctgggc aaggactcgc cctccaacaa gctgctgtat 5400gccaaggaca tccccagcta caagaattgg gtggagaggt attactcaga catagggaag 5460atgccagcca tcagcgacca agacatgaac gcatacctgg ctgagcagtc ccggatgcac 5520atgaatgagt tcaacaccat gagtgcactc tcagagatct tctcctatgt gggcaaatac 5580agcgaggaga tccttggacc tctggaccac gatgaccagt gtgggaagca gaaactggcc 5640tacaaactag aacaagtcat aaccctcatg agcttagaca gctga 5685513061DNAHomo sapiens 5tgcgttcggg gctggggctg gaggaggcag ccacacgcgc gcacacgcac acgttcagag 60gagggcgaga ggcagcggca taggctccat ctgcagtgtc aatgcggcgc tcccgctgaa 120ggagggaaac gcggcgcgtc cagtagggga gactgcattg ctgagtcctg gccctctgag 180gggacgactg tgcctgagtg ctgctgtgcc actgggaccc gcctctgcca tgaaagccat 240gccctggaac tggacctgcc ttctctccca cctcctcatg gtgggcatgg gctcctccac 300tttgctcacc cggcagccag ccccgctgtc ccagaagcag cggtcatttg tcacattccg 360aggagagccc gccgagggtt tcaatcacct ggtggtggat gagaggacag gacacattta 420cttgggggcc gtcaatcgga tttacaagct ctccagcgac ctgaaggtct tggtgacgca 480tgagacaggg ccggacgagg acaaccccaa gtgttaccca ccccgcatcg tccagacctg 540caatgagccc ctgaccacca ccaacaatgt caacaagatg ctcctcatag actacaagga 600gaacaggctg attgcctgtg ggagcctgta ccaaggcatc tgcaagctgc tgaggctgga 660ggacctcttc aagctggggg agccttatca taagaaggag cactatctgt caggtgtcaa 720cgagagcggc tcagtctttg gagtgatcgt ctcctacagc aacctggatg acaagctgtt 780cattgccacg gcagtggatg ggaagcccga gtattttccc accatctcca gccggaaact 840gaccaagaac tctgaggcgg atggcatgtt cgcgtacgtc ttccatgatg agttcgtggc 900ctcgatgatt aagatccctt cggacacctt caccatcatc cctgactttg atatctacta 960tgtctatggt tttagcagtg gcaactttgt ctactttttg accctccaac ctgagatggt 1020gtctccacca ggctccacca ccaaggagca ggtgtataca tccaagctcg tgaggctttg 1080caaggaggac acagccttca actcctatgt agaggtgccc attggctgtg agcgcagtgg 1140ggtggagtac cgcctgctgc aggctgccta cctgtccaaa gcgggggccg tgcttggcag 1200gacccttgga gtccatccag atgatgacct gctcttcacc gtcttctcca agggccagaa 1260gcggaaaatg aaatccctgg atgagtcggc cctgtgcatc ttcatcttga agcagataaa 1320tgaccgcatt aaggagcggc tgcagtcttg ttaccggggc gagggcacgc tggacctggc 1380ctggctcaag gtgaaggaca tcccctgcag cagtgcgctc ttaaccattg acgataactt 1440ctgtggcctg gacatgaatg ctcccctggg agtgtccgac atggtgcgtg gaattcccgt 1500cttcacggag gacagggacc gcatgacgtc tgtcatcgca tatgtctaca agaaccactc 1560tctggccttt gtgggcacca aaagtggcaa gctgaagaag atccgggtgg atggacccag 1620gggcaacgcc ctccagtatg agacggtgca ggtggtggac cccggcccag tcctccggga 1680tatggccttc tccaaggacc acgagcaact ctacatcatg tcagagaggc agctcaccag 1740agtccctgtg gagtcctgtg gtcagtatca gagctgcggc gagtgccttg gctcaggcga 1800cccccactgt ggctggtgtg tgctgcacaa cacttgcacc cggaaggagc ggtgtgagcg 1860gtccaaggag ccccgcaggt ttgcctcgga gatgaagcag tgtgtccggc tgacggtcca 1920tcccaacaat atctccgtct ctcagtacaa cgtgctgctg gtcctggaga cgtacaatgt 1980cccggagctg tcagctggcg tcaactgcac ctttgaggac ctgtcagaga tggatgggct 2040ggtcgtgggc aatcagatcc agtgctactc ccctgcagcc aaggaggtgc cccggatcat 2100cacagagaat ggggaccacc atgtcgtaca gcttcagctc aaatcaaagg agaccggcat 2160gaccttcgcc agcaccagct ttgtcttcta caattgcagc gtccacaatt cgtgcctgtc 2220ctgcgtggag agtccatacc gctgccactg gtgtaaatac cggcatgtct gcacccatga 2280ccccaagacc tgctccttcc aggaaggccg agtgaagctg cccgaggact gcccccagct 2340gctgcgagtg gacaagatcc tggtgcccgt ggaggtgatc aagcctatca cgctgaaggc 2400caagaacctc ccccagcccc agtctgggca gcgtggctac gaatgcatcc tcaacattca 2460gggcagcgag cagcgagtgc ccgccctgcg cttcaacagc tccagcgtac agtgccagaa 2520cacctcttat tcctatgaag ggatggagat caacaacctg cccgtggagt tgacagtcgt 2580gtggaatggg cacttcaaca ttgacaaccc agctcagaat aaagttcacc tctacaagtg 2640tggagccatg cgtgagagct gcgggctgtg cctcaaggct gacccagact tcgcatgtgg 2700ctggtgccag ggcccaggcc agtgcaccct gcgccagcac tgccctgccc aggagagcca 2760gtggctggag ctgtctggtg ccaaaagcaa gtgcacaaac ccccgcatca cagagataat 2820cccggtgaca ggcccccggg aagggggcac caaggtcact atccgagggg agaacctggg 2880cctggaattt cgcgacatcg cctcccatgt caaggttgct ggcgtggagt gcagcccttt 2940agtggatggt tacatccctg cagaacagat cgtgtgtgag atgggggagg ccaagcccag 3000ccagcatgca ggcttcgtgg agatctgcgt ggctgtgtgt cggcctgaat tcatggcccg 3060gtcctcacag ctctattact tcatgacact gactctctca gatctgaagc ccagccgggg 3120gcccatgtcc ggagggaccc aagtgaccat cacaggcacc aacctgaatg ccggaagcaa 3180cgtggtggtg atgtttggaa agcagccctg tctcttccac aggcgatctc catcctacat 3240tgtctgcaac accacatcct cagatgaggt gctagagatg aaggtgtcgg tgcaggtgga 3300cagggccaag atccaccagg acctggtctt tcagtatgtg gaagacccca ccatcgtgcg 3360gattgagcca gaatggagca ttgtcagtgg aaacacaccc atcgccgtat gggggaccca 3420cctggacctc atacagaacc cccagatccg tgccaagcat ggagggaagg agcacatcaa 3480tatctgtgag gttctgaacg ctactgagat gacctgtcag gcgcccgccc tcgctctggg 3540tcctgaccac cagtcagacc tgaccgagag gcccgaggag tttggcttca tcctggacaa 3600cgtccagtcc ctgctcatcc tcaacaagac caacttcacc tactatccca acccggtgtt 3660tgaggccttt ggtccctcag gaatcctgga gctcaagcct ggcacgccca tcatcctaaa 3720gggcaagaac ctgatcccgc ctgtggctgg gggcaacgtg aagctgaact acactgtgct 3780ggttggggag aagccgtgca ccgtgaccgt gtcagatgtc cagctgctct gcgagtcccc 3840caacctcatc ggcaggcaca aagtgatggc ccgtgtcggt ggcatggagt actccccggg 3900gatggtgtac attgccccgg acagcccgct cagcctgccc gccatcgtca gcatcgcagt 3960ggctggcggc ctcctcatca ttttcatcgt ggccgtgctc attgcctata aacgcaagtc 4020ccgcgaaagt gacctcacgc tgaagcggct gcagatgcag atggacaacc tggagtcccg 4080tgtggccctg gagtgcaagg aagcctttgc cgagctgcag acggacatcc atgagctgac 4140cagtgacctg gatggagccg ggattccgtt cctggactat agaacttaca ccatgcgggt 4200gctgttccca ggaattgaag accaccctgt cctccgggac cttgaggtcc cgggctaccg 4260gcaggagcgt gtggagaaag gcctgaagct cttcgcccag ctcatcaaca acaaggtgtt 4320cctgctgtcc ttcatccgca cgcttgagtc ccagcgtagc ttctccatgc gcgaccgtgg 4380caacgtggcc tcactcatca tgaccgtgct gcagagcaag ctggagtacg ccactgatgt 4440gctgaagcag ctgctggccg acctcattga caagaacctg gagagcaaga accaccctaa 4500gctgctgctc aggaggactg agtcagtggc tgagaagatg ctgaccaatt ggtttacttt 4560cctcctctac aagttcctca aggagtgtgc tggggagccc ctcttctccc tgttctgtgc 4620catcaagcag cagatggaga agggccccat tgacgccatc acgggcgagg cccgctactc 4680cttgagcgag gacaagctca tccgccagca gattgactac aaaaccctgg tcctgagctg 4740tgtcagccca gacaatgcca acagccccga ggtcccagta aagatcctca actgtgacac 4800catcactcag gtcaaggaga agattctgga tgccatcttc aagaatgtgc cttgctccca 4860ccggcccaaa gctgcagata tggatctgga gtggcgacaa ggaagtgggg caaggatgat 4920cttgcaggat gaagacatca ccaccaagat tgagaatgat tggaagcgac tgaacacact 4980ggcccactac caggtgccag atggttccgt ggtggcatta gtgtccaagc aggtgacagc 5040ctataacgca

gtgaacaact ccaccgtctc caggacctca gcaagtaaat atgaaaacat 5100gatccggtac acgggcagcc ccgacagcct ccgctcacgg acacctatga tcactcctga 5160cctggagagt ggagtcaaga tgtggcacct agtgaagaac cacgagcacg gagaccagaa 5220ggagggggac cgggggagca agatggtgtc tgaaatctac ctgacccgac tcctggccac 5280taagggcaca ctgcagaagt ttgtggatga cctctttgag accatcttca gcacggcaca 5340ccgtggctct gccctgcccc tggccatcaa gtacatgttt gacttcctgg atgagcaggc 5400tgataaacat ggcattcatg acccgcacgt ccgccatacc tggaagagca attgcctgcc 5460cctgaggttt tgggtcaaca tgatcaagaa cccgcagttt gtgtttgaca tccataagaa 5520cagcatcaca gacgcctgcc tctctgtggt ggctcagacc ttcatggact cttgctccac 5580gtcagagcac cggctgggca aggactcgcc ctccaacaag ctgctgtatg ccaaggacat 5640ccccagctac aagaattggg tggagaggta ttactcagac atagggaaga tgccagccat 5700cagcgaccaa gacatgaacg catacctggc tgagcagtcc cggatgcaca tgaatgagtt 5760caacaccatg agtgcactct cagagatctt ctcctatgtg ggcaaataca gcgaggagat 5820ccttggacct ctggaccacg atgaccagtg tgggaagcag aaactggcct acaaactaga 5880acaagtcata accctcatga gcttagacag ctgagaaccg tccttccagg gccgccctgg 5940agggggacac accaagccgt gcctcagtct agattatcat ctttaccaag tgcaagttcc 6000gactggcatc agcagcatcc cctgagcagc gctgtttctc tctctttctc tctgcctctt 6060tccgtttctc cctccttcct ggatctcttc tcttccagtt gctctgccaa cacgattgga 6120ccaagccact gaccctcagt tagtccaaga atggccaggc ccatggcaag ggagctgacc 6180agaagatgtc agagaggcct ctgtctccca ggtgctcctg accctgtgca tgtcagcagc 6240agggtgcaaa taacgaatga ggagccaggg acaggggaca tttctgtgct gctacttcac 6300cttccacttt ggcagcccct gctttggtct gagccttggc ctagggaaga ggcaaggaag 6360gacttcagta ttatctttac tgggaagaca tcacctggct ctcccttccc acagttccat 6420ctccagtggt tcagccagtg gtctgatcgc tttgcagctg tgagaagaaa ggctacacct 6480cctgcatgtg gctggagcag ggcatgtgtg ggcagctggg aggtgctcct tgaggctcct 6540tctcccccac tgggctggtg tccagaggct tcctgtcctt ttccaggtct ccagagggac 6600ctgcctgccc tgcctgctcc cccgccagta gaaagccagg caggagaaag aatagcaatt 6660acattccacc atggagatgc tcctgacctt ttcatctgaa tcctagtagc agaaatgtaa 6720cacaggggga gaaaaggaaa gagagttgca tctaccctgg aagcagaatt tgttttccat 6780ttaccctcaa attcaaatga gtcacaatca tagtcatagg tctagtccac taccagagcc 6840ctgagtgctg tcaagagaaa gcatctatct ccaccctcct ttgtcaacct tcatcaaggg 6900tcaacgtgaa atgcagagtg catctaggag attctacctc cagccatctc catggctcca 6960tccccatcat ccttcctgag aactccatag acggctgggg ccaacagcct agtccctgtt 7020ccctctgcag aatccggtgc cattgctatg cagatgactt tgtcactggg ctgtccagac 7080ctctttggga atgatttcat caacatctca gctgtctctc atcattctcc ttcctcatct 7140cttcagcagt catccttgaa agaaacagac ttaagcaaag cctcacggag acagcccaaa 7200atgccagcca acctcagcct ccagcttgtc agatctggga gggacaaaga gtcgagctga 7260tgggcctggc tggaattaag aagagggaca tacaaatgac cttggccttg gcatccatct 7320ccccatctgt tcttacatct acagatgcac gattttagcc aggcaggcaa atgtgtgcct 7380agaaattgat actaggtaag cagaggctat ggggagagat ggtctaatgg agggttctag 7440gaacctttca tcctaaggag accttaggtg ctgtctggtg cagtctccca tcctaagcag 7500gagtctctgt tggcacctct gctctggagt tgttcaccac tatgggagac aaggagaaac 7560atcttaggtg aggttgagga gaaggattca cagtcttgcc ttcactcccc aaacatcaga 7620catcattcct tgtcacccac tcagaatgag ccccccttgg ggaagaaacc acaccatttc 7680cagcaaagtc catggagcat ccggtacttt taagaacact tgcccctttg gatatgaata 7740tgtgcacatg tgtgtgagca catgtatgtg tgtgtgtgtg tctgccccag gtgtaggcgg 7800aaagctcaaa aggatttctt gtcctttgta ggaggatttt tgaagtgttc cccttctctt 7860tccccttgct catccattca tcctgcagct tcaggacatt tcaacactta cttgctttct 7920atgctgagag ctggtgggtg gaaggagagg gcgcttgtcc ataggaaatc agggtggtcg 7980cctgccgagg cctggacctt ggaacagggc atcatgtgac atcgcagagg acagatggtg 8040gaaaagacat gagcaaccta atgggaagag gaaaatggga aacaatgcat tggaagagga 8100agaaaaaaaa taaataacca aaggttttgg caagtgcagt accaggtgga gaagcttgac 8160ttttctatcc ttgatcattt tattccctcc caagaagtca gtcacaggac ctggaaggcc 8220agaaagggta catgtgggag acggtctgag gaagtacctc ggtcactaca atatttttgc 8280acatataaag ggttggggag gaaagagaca caaacgtatt taacacagat ttgctggatg 8340gaagctgcgt gtgtgaacgt gtgtatgagt gagtgcattt tgattttttt tttttttttt 8400tgcacagtta agagaaaaaa tcaaacaagc agaaaaaaaa aagaaaaaag acttatcacg 8460gttctgctga agcttttatt ttttactgga tgatgattat tgttattgtt actttggcgg 8520tacaggactt tattttattc catgtttttg ttataagaaa aatttcaaac acctcagaga 8580aatagaaagg ttaggaagaa agaggagaca aggacagaca aattttctgg ctgtccccat 8640ttctcctggg ggaggggttt ggggctggtt tgactttaat tggtgggtgg gttgtttctg 8700ccgctctgtt tgctgcagtc cccgtggcct gcttggggac tgagaaattt gagccaggta 8760tccagagcca cagcccatct tgcttataaa aattatcttc tgctgtttgt tttccatttc 8820ttccgtttgg attcttggtg cacgtgtgat atggtattta aaagcaaaga caagcaacat 8880tgtcaaaaag ctgtccttgc cccccatccc ccacccaaat cttttttcca aactccccca 8940gggatcttcc ttaccccact ggcagagcaa acatccaggg gctgtccatg tggcttgcgg 9000gctcccagag aaaggaattg ggccaacttt gtcctgtggg atggaggccc cttcacggcc 9060tccctcgagg caaagttaat ttgtagggtc accattatgt tgagtcatga gcagacagaa 9120ggagagaaaa ggccatcttc cttaccttcc cctccaactt atcccgtacc ctcccaggga 9180aaatggtacc agactgagcc atcaaaatca ctgacaaagt ttaggtggga attttttttg 9240catgttggag agagaagggc ttaaggtagc agggaagaag ggggctttgt ggggtcctaa 9300attttaagga ataagtagag gaagacaaga aacagagtgg taggctggtc atttctcctg 9360gccacaagtc cccccagatg cagcttttac ccattctttg tccttcccca taaggagaga 9420ccctgacatt tcttggtagc tgcaaatagt gccactaagt gaaggtggcc atcatgccag 9480ttacttcctc aggaaaatat tttcttgcct tcttctttca gtatggtttt aaatttggga 9540acagtggata acccaagtgt cccacaggcc aaggtacatt ccaatggcag catgatccct 9600gcacccaaag ccagccccta aagcctaccc cttgtgcacc cgcagcctgg taagtgagct 9660tggctgcttg tgaggagcta caagtgaaag agaagttatt ttaaataaat cccaaagttt 9720gaggcagact gtccaggact gttcccagga agaagcagga gttacccaca ggaaaagtct 9780ctgacctggt cccctcaggc ccagctacct gcgcccacca gcagtgaagg ttgatgtact 9840ggcccagcat ctccacctcc cccatgcaac caggtccctg gtaccgtgtc tcccgttgca 9900tgtctggctt ctgcctgtgc tcctcctgcc acgagcatcc tccctgtccc tcctcattcc 9960accgtgtctc tcctgcacac atagcctctg tcccagggcg atttatccac ttgagtacag 10020gagctgctca gacctctcag cccagccctc tgtgactgcc ccagccccat cctaccccac 10080ccaaagctgc cttcctggct gtaggagctc cctcgtctag ccaaggccct atgggtcccc 10140atccgaggat ccacaagcaa tgacttccca aatgacctcc actgcaagaa gaatccttac 10200cactgtttcc agagccgtga acgatgctgt gatgggccca ggtctcagca ccaccctctg 10260tgacctaaaa agaaaagctc aatttccatc tgtcttcttt cccaggacca aggggacaca 10320gtaatgtgaa gtcaaatact taaccgagca aagggccagt attgttatca gtcaaggaca 10380aacctcccac ctcacagaca gccaagcagt gagggaaaga cagacagaca taggtaggaa 10440ggtgctctgc aggcacaagg cccagagaag cccctctccg ggaacttccc ctgctccttc 10500caggaacagt gagcccagtg agcagcccca gccagctctt caaggccttc aaggggtctt 10560tccatgactg agtcacctcc aggagctcac ctgaccccca gagaagacct accccaggca 10620gctccgtgcc ctggcttctc cccatgcccc aaatcccccc ccgccatccc tcctggtcct 10680cgtctacatc aagggcctct tcccctcttc ctgccagctc tcaggacagg tgactgggag 10740gccttgaacc ctcagcctct tcctttaaaa aaaacaaaac aaaacaaaac tgtgggccat 10800ttatttggga ttttggagtt gtttggtttt tgtttgtata tcttaatagt tcgaaagtaa 10860gaagggagcc ctgctatgga tgttaagtcc aaattactcg gttagtggga gcaaaaccta 10920tgacttccaa ggggatgagg agaggttcag aggacaggag gagcctcccc cattgaaaaa 10980aaaaaatggg tcaggacatt ccctggatga ggacaatgct aggggtggca tctcacatgg 11040ctgctgctat tcctggtgct tccccacact tttgacagat ggagtccttc tcctaccgcc 11100tcctgccacc tcaccctaca ggcattctct atgtaggaaa caagagcctt attttataga 11160gtggggagct gagacacagc ctcaggtaac actgacacag ctcccgaatg aggctgggac 11220actctgcaaa cctctcctca tggtgctaag ggtggcatgc tcttgacagg aaacctaaat 11280gaccactcct ctcatttgga aagtaatcca ctgcagtaaa agtttcagac atgcaagaga 11340gagttttttt ttttttacta caaatttttg ctcccccata aaattatttt attagaggga 11400gtatccaagt tttaaaagta tatagaattt tttggttgta agagaaatac atactcatta 11460ggatcccgat taaattcctt gagtagactg gtgcctacca gaaagcaaag caaagttaaa 11520caaaacgaaa caaaatcctt catatacaaa aagaactttc tgtttgtatt ggcagaggta 11580gtgaggtgat tcaggtaggc tgaaaatcct gggttgcggg agcctcactt tattccattc 11640ccacccgctt tgatgtctat gcttggctct ctgggctgcc cctggtactg ccgaatccta 11700cacatctctt atcagctttc ctcaaacttt aaggaggctc tgtgagggat gggtcatggg 11760aagacccaag ctttccctcc gccaggattg caaaagcaag tagacttggt ctatgcagct 11820cttcttccag caatttcttt atttggaatt agaacttcct ttgttagtat ctttgatctt 11880ttgactcaag cacattttgg aagggctccc ttacaaaagt agaatttaaa acagaggata 11940cagttaaaga gcaacccaaa ggacgcttaa gaaaccgaga ccacttcacc aaacaggact 12000aaggaacact ttcgtgcaca gaagtcagcc gcaatccagg cacaggacga agatgggata 12060cacgtgctca tctgtctgtc ctcctttcct ctccctcccc gacgttctag ttagcttgtt 12120gacttgttaa accttctgtt cttaaaatga aaagctagct tacctcaaag aatcttgttt 12180ccattcggaa accaacgatt ttgtgtttta gaatggacag ccctcccctc accactccct 12240accttggcct ggtgtccttg agacatacgg tctttgctta gtcgtgtgtt ggctgctttg 12300agcaggaaca aggcctccag gccctgaggt gggaaggaag gattggatgc cactgccctc 12360ctccccactt tagcatgtag gggccagccc atctcttcca gcagggtcct gctgagttac 12420catagcaacc agcaactcca gggtaccaca acagacaatg gctcagcgag ccgacgtgtg 12480gggatgatgc aggggttttg gcccagccag aggacccaga gttgagcttc aaatgctaga 12540gaaggggaga aacaggatgg aagggtggtt taaggaaccg gcaggggtct ttgagtcaca 12600tagagaagcc gttgaaggag gtagggcagg ttatctctgt tccagtcacc cccttccagc 12660cccatcccac ttctgtttca aactaaagct cccacctcga acattgaccc tttgttagaa 12720caaagcaaag catatcttta gacaacagtg ttaaaatgag cctcaaatgt atgtggatga 12780gatctctaag aagagggtct tctggttttg atttttaaag aagagtatcc tagtaaaata 12840ttaaaaaaaa attaaaaagt ttttaaaaag gaaacctgtg ctatttaaat tggagcccag 12900ttgtaacttg gtaaaggcaa gcttctgtac ctttgttata attaattgta tacctgtgta 12960tgtaaatata aggcattcct attttgcagt tcagaacaaa aaaaacttat ttgtaatata 13020gaataaagtt tattaaaaaa taataaaaat gcagtttggg a 1306161466PRTArtificialPlexin-A4 fusion protein sequence 6Met Lys Ala Met Pro Trp Asn Trp Thr Cys Leu Leu Ser His Leu Leu 1 5 10 15 Met Val Gly Met Gly Ser Ser Thr Leu Leu Thr Arg Gln Pro Ala Pro 20 25 30 Leu Ser Gln Lys Gln Arg Ser Phe Val Thr Phe Arg Gly Glu Pro Ala 35 40 45 Glu Gly Phe Asn His Leu Val Val Asp Glu Arg Thr Gly His Ile Tyr 50 55 60 Leu Gly Ala Val Asn Arg Ile Tyr Lys Leu Ser Ser Asp Leu Lys Val 65 70 75 80 Leu Val Thr His Glu Thr Gly Pro Asp Glu Asp Asn Pro Lys Cys Tyr 85 90 95 Pro Pro Arg Ile Val Gln Thr Cys Asn Glu Pro Leu Thr Thr Thr Asn 100 105 110 Asn Val Asn Lys Met Leu Leu Ile Asp Tyr Lys Glu Asn Arg Leu Ile 115 120 125 Ala Cys Gly Ser Leu Tyr Gln Gly Ile Cys Lys Leu Leu Arg Leu Glu 130 135 140 Asp Leu Phe Lys Leu Gly Glu Pro Tyr His Lys Lys Glu His Tyr Leu 145 150 155 160 Ser Gly Val Asn Glu Ser Gly Ser Val Phe Gly Val Ile Val Ser Tyr 165 170 175 Ser Asn Leu Asp Asp Lys Leu Phe Ile Ala Thr Ala Val Asp Gly Lys 180 185 190 Pro Glu Tyr Phe Pro Thr Ile Ser Ser Arg Lys Leu Thr Lys Asn Ser 195 200 205 Glu Ala Asp Gly Met Phe Ala Tyr Val Phe His Asp Glu Phe Val Ala 210 215 220 Ser Met Ile Lys Ile Pro Ser Asp Thr Phe Thr Ile Ile Pro Asp Phe 225 230 235 240 Asp Ile Tyr Tyr Val Tyr Gly Phe Ser Ser Gly Asn Phe Val Tyr Phe 245 250 255 Leu Thr Leu Gln Pro Glu Met Val Ser Pro Pro Gly Ser Thr Thr Lys 260 265 270 Glu Gln Val Tyr Thr Ser Lys Leu Val Arg Leu Cys Lys Glu Asp Thr 275 280 285 Ala Phe Asn Ser Tyr Val Glu Val Pro Ile Gly Cys Glu Arg Ser Gly 290 295 300 Val Glu Tyr Arg Leu Leu Gln Ala Ala Tyr Leu Ser Lys Ala Gly Ala 305 310 315 320 Val Leu Gly Arg Thr Leu Gly Val His Pro Asp Asp Asp Leu Leu Phe 325 330 335 Thr Val Phe Ser Lys Gly Gln Lys Arg Lys Met Lys Ser Leu Asp Glu 340 345 350 Ser Ala Leu Cys Ile Phe Ile Leu Lys Gln Ile Asn Asp Arg Ile Lys 355 360 365 Glu Arg Leu Gln Ser Cys Tyr Arg Gly Glu Gly Thr Leu Asp Leu Ala 370 375 380 Trp Leu Lys Val Lys Asp Ile Pro Cys Ser Ser Ala Leu Leu Thr Ile 385 390 395 400 Asp Asp Asn Phe Cys Gly Leu Asp Met Asn Ala Pro Leu Gly Val Ser 405 410 415 Asp Met Val Arg Gly Ile Pro Val Phe Thr Glu Asp Arg Asp Arg Met 420 425 430 Thr Ser Val Ile Ala Tyr Val Tyr Lys Asn His Ser Leu Ala Phe Val 435 440 445 Gly Thr Lys Ser Gly Lys Leu Lys Lys Ile Arg Val Asp Gly Pro Arg 450 455 460 Gly Asn Ala Leu Gln Tyr Glu Thr Val Gln Val Val Asp Pro Gly Pro 465 470 475 480 Val Leu Arg Asp Met Ala Phe Ser Lys Asp His Glu Gln Leu Tyr Ile 485 490 495 Met Ser Glu Arg Gln Leu Thr Arg Val Pro Val Glu Ser Cys Gly Gln 500 505 510 Tyr Gln Ser Cys Gly Glu Cys Leu Gly Ser Gly Asp Pro His Cys Gly 515 520 525 Trp Cys Val Leu His Asn Thr Cys Thr Arg Lys Glu Arg Cys Glu Arg 530 535 540 Ser Lys Glu Pro Arg Arg Phe Ala Ser Glu Met Lys Gln Cys Val Arg 545 550 555 560 Leu Thr Val His Pro Asn Asn Ile Ser Val Ser Gln Tyr Asn Val Leu 565 570 575 Leu Val Leu Glu Thr Tyr Asn Val Pro Glu Leu Ser Ala Gly Val Asn 580 585 590 Cys Thr Phe Glu Asp Leu Ser Glu Met Asp Gly Leu Val Val Gly Asn 595 600 605 Gln Ile Gln Cys Tyr Ser Pro Ala Ala Lys Glu Val Pro Arg Ile Ile 610 615 620 Thr Glu Asn Gly Asp His His Val Val Gln Leu Gln Leu Lys Ser Lys 625 630 635 640 Glu Thr Gly Met Thr Phe Ala Ser Thr Ser Phe Val Phe Tyr Asn Cys 645 650 655 Ser Val His Asn Ser Cys Leu Ser Cys Val Glu Ser Pro Tyr Arg Cys 660 665 670 His Trp Cys Lys Tyr Arg His Val Cys Thr His Asp Pro Lys Thr Cys 675 680 685 Ser Phe Gln Glu Gly Arg Val Lys Leu Pro Glu Asp Cys Pro Gln Leu 690 695 700 Leu Arg Val Asp Lys Ile Leu Val Pro Val Glu Val Ile Lys Pro Ile 705 710 715 720 Thr Leu Lys Ala Lys Asn Leu Pro Gln Pro Gln Ser Gly Gln Arg Gly 725 730 735 Tyr Glu Cys Ile Leu Asn Ile Gln Gly Ser Glu Gln Arg Val Pro Ala 740 745 750 Leu Arg Phe Asn Ser Ser Ser Val Gln Cys Gln Asn Thr Ser Tyr Ser 755 760 765 Tyr Glu Gly Met Glu Ile Asn Asn Leu Pro Val Glu Leu Thr Val Val 770 775 780 Trp Asn Gly His Phe Asn Ile Asp Asn Pro Ala Gln Asn Lys Val His 785 790 795 800 Leu Tyr Lys Cys Gly Ala Met Arg Glu Ser Cys Gly Leu Cys Leu Lys 805 810 815 Ala Asp Pro Asp Phe Ala Cys Gly Trp Cys Gln Gly Pro Gly Gln Cys 820 825 830 Thr Leu Arg Gln His Cys Pro Ala Gln Glu Ser Gln Trp Leu Glu Leu 835 840 845 Ser Gly Ala Lys Ser Lys Cys Thr Asn Pro Arg Ile Thr Glu Ile Ile 850 855 860 Pro Val Thr Gly Pro Arg Glu Gly Gly Thr Lys Val Thr Ile Arg Gly 865 870 875 880 Glu Asn Leu Gly Leu Glu Phe Arg Asp Ile Ala Ser His Val Lys Val 885 890 895 Ala Gly Val Glu Cys Ser Pro Leu Val Asp Gly Tyr Ile Pro Ala Glu 900 905 910 Gln Ile Val Cys Glu Met Gly Glu Ala Lys Pro Ser Gln His Ala Gly 915 920 925 Phe Val Glu Ile Cys Val Ala Val Cys Arg Pro Glu Phe Met Ala Arg 930 935 940 Ser Ser Gln Leu Tyr Tyr Phe Met Thr Leu Thr Leu Ser Asp Leu Lys 945 950 955 960 Pro Ser Arg Gly Pro Met Ser Gly Gly Thr Gln Val Thr Ile Thr Gly 965 970 975 Thr Asn Leu Asn Ala Gly Ser Asn Val Val Val Met Phe Gly Lys Gln 980 985 990 Pro Cys Leu Phe His Arg Arg Ser Pro Ser Tyr Ile Val Cys Asn Thr 995 1000 1005 Thr Ser Ser Asp Glu Val Leu Glu Met Lys Val Ser Val Gln Val 1010 1015 1020 Asp Arg Ala Lys Ile His Gln Asp Leu Val Phe Gln Tyr Val Glu 1025 1030 1035 Asp Pro Thr Ile Val Arg Ile Glu Pro Glu Trp Ser Ile Val Ser 1040 1045 1050 Gly Asn Thr Pro Ile Ala Val Trp Gly Thr His Leu Asp Leu Ile 1055 1060

1065 Gln Asn Pro Gln Ile Arg Ala Lys His Gly Gly Lys Glu His Ile 1070 1075 1080 Asn Ile Cys Glu Val Leu Asn Ala Thr Glu Met Thr Cys Gln Ala 1085 1090 1095 Pro Ala Leu Ala Leu Gly Pro Asp His Gln Ser Asp Leu Thr Glu 1100 1105 1110 Arg Pro Glu Glu Phe Gly Phe Ile Leu Asp Asn Val Gln Ser Leu 1115 1120 1125 Leu Ile Leu Asn Lys Thr Asn Phe Thr Tyr Tyr Pro Asn Pro Val 1130 1135 1140 Phe Glu Ala Phe Gly Pro Ser Gly Ile Leu Glu Leu Lys Pro Gly 1145 1150 1155 Thr Pro Ile Ile Leu Lys Gly Lys Asn Leu Ile Pro Pro Val Ala 1160 1165 1170 Gly Gly Asn Val Lys Leu Asn Tyr Thr Val Leu Val Gly Glu Lys 1175 1180 1185 Pro Cys Thr Val Thr Val Ser Asp Val Gln Leu Leu Cys Glu Ser 1190 1195 1200 Pro Asn Leu Ile Gly Arg His Lys Val Met Ala Arg Val Gly Gly 1205 1210 1215 Met Glu Tyr Ser Pro Gly Met Val Tyr Ile Ala Pro Asp Gly Cys 1220 1225 1230 Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 1235 1240 1245 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 1250 1255 1260 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 1265 1270 1275 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 1280 1285 1290 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 1295 1300 1305 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 1310 1315 1320 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 1325 1330 1335 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 1340 1345 1350 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 1355 1360 1365 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 1370 1375 1380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 1385 1390 1395 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 1400 1405 1410 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 1415 1420 1425 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 1430 1435 1440 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 1445 1450 1455 Ser Leu Ser Leu Ser Pro Gly Lys 1460 1465 71231PRTHomo sapiens 7Met Lys Ala Met Pro Trp Asn Trp Thr Cys Leu Leu Ser His Leu Leu 1 5 10 15 Met Val Gly Met Gly Ser Ser Thr Leu Leu Thr Arg Gln Pro Ala Pro 20 25 30 Leu Ser Gln Lys Gln Arg Ser Phe Val Thr Phe Arg Gly Glu Pro Ala 35 40 45 Glu Gly Phe Asn His Leu Val Val Asp Glu Arg Thr Gly His Ile Tyr 50 55 60 Leu Gly Ala Val Asn Arg Ile Tyr Lys Leu Ser Ser Asp Leu Lys Val 65 70 75 80 Leu Val Thr His Glu Thr Gly Pro Asp Glu Asp Asn Pro Lys Cys Tyr 85 90 95 Pro Pro Arg Ile Val Gln Thr Cys Asn Glu Pro Leu Thr Thr Thr Asn 100 105 110 Asn Val Asn Lys Met Leu Leu Ile Asp Tyr Lys Glu Asn Arg Leu Ile 115 120 125 Ala Cys Gly Ser Leu Tyr Gln Gly Ile Cys Lys Leu Leu Arg Leu Glu 130 135 140 Asp Leu Phe Lys Leu Gly Glu Pro Tyr His Lys Lys Glu His Tyr Leu 145 150 155 160 Ser Gly Val Asn Glu Ser Gly Ser Val Phe Gly Val Ile Val Ser Tyr 165 170 175 Ser Asn Leu Asp Asp Lys Leu Phe Ile Ala Thr Ala Val Asp Gly Lys 180 185 190 Pro Glu Tyr Phe Pro Thr Ile Ser Ser Arg Lys Leu Thr Lys Asn Ser 195 200 205 Glu Ala Asp Gly Met Phe Ala Tyr Val Phe His Asp Glu Phe Val Ala 210 215 220 Ser Met Ile Lys Ile Pro Ser Asp Thr Phe Thr Ile Ile Pro Asp Phe 225 230 235 240 Asp Ile Tyr Tyr Val Tyr Gly Phe Ser Ser Gly Asn Phe Val Tyr Phe 245 250 255 Leu Thr Leu Gln Pro Glu Met Val Ser Pro Pro Gly Ser Thr Thr Lys 260 265 270 Glu Gln Val Tyr Thr Ser Lys Leu Val Arg Leu Cys Lys Glu Asp Thr 275 280 285 Ala Phe Asn Ser Tyr Val Glu Val Pro Ile Gly Cys Glu Arg Ser Gly 290 295 300 Val Glu Tyr Arg Leu Leu Gln Ala Ala Tyr Leu Ser Lys Ala Gly Ala 305 310 315 320 Val Leu Gly Arg Thr Leu Gly Val His Pro Asp Asp Asp Leu Leu Phe 325 330 335 Thr Val Phe Ser Lys Gly Gln Lys Arg Lys Met Lys Ser Leu Asp Glu 340 345 350 Ser Ala Leu Cys Ile Phe Ile Leu Lys Gln Ile Asn Asp Arg Ile Lys 355 360 365 Glu Arg Leu Gln Ser Cys Tyr Arg Gly Glu Gly Thr Leu Asp Leu Ala 370 375 380 Trp Leu Lys Val Lys Asp Ile Pro Cys Ser Ser Ala Leu Leu Thr Ile 385 390 395 400 Asp Asp Asn Phe Cys Gly Leu Asp Met Asn Ala Pro Leu Gly Val Ser 405 410 415 Asp Met Val Arg Gly Ile Pro Val Phe Thr Glu Asp Arg Asp Arg Met 420 425 430 Thr Ser Val Ile Ala Tyr Val Tyr Lys Asn His Ser Leu Ala Phe Val 435 440 445 Gly Thr Lys Ser Gly Lys Leu Lys Lys Ile Arg Val Asp Gly Pro Arg 450 455 460 Gly Asn Ala Leu Gln Tyr Glu Thr Val Gln Val Val Asp Pro Gly Pro 465 470 475 480 Val Leu Arg Asp Met Ala Phe Ser Lys Asp His Glu Gln Leu Tyr Ile 485 490 495 Met Ser Glu Arg Gln Leu Thr Arg Val Pro Val Glu Ser Cys Gly Gln 500 505 510 Tyr Gln Ser Cys Gly Glu Cys Leu Gly Ser Gly Asp Pro His Cys Gly 515 520 525 Trp Cys Val Leu His Asn Thr Cys Thr Arg Lys Glu Arg Cys Glu Arg 530 535 540 Ser Lys Glu Pro Arg Arg Phe Ala Ser Glu Met Lys Gln Cys Val Arg 545 550 555 560 Leu Thr Val His Pro Asn Asn Ile Ser Val Ser Gln Tyr Asn Val Leu 565 570 575 Leu Val Leu Glu Thr Tyr Asn Val Pro Glu Leu Ser Ala Gly Val Asn 580 585 590 Cys Thr Phe Glu Asp Leu Ser Glu Met Asp Gly Leu Val Val Gly Asn 595 600 605 Gln Ile Gln Cys Tyr Ser Pro Ala Ala Lys Glu Val Pro Arg Ile Ile 610 615 620 Thr Glu Asn Gly Asp His His Val Val Gln Leu Gln Leu Lys Ser Lys 625 630 635 640 Glu Thr Gly Met Thr Phe Ala Ser Thr Ser Phe Val Phe Tyr Asn Cys 645 650 655 Ser Val His Asn Ser Cys Leu Ser Cys Val Glu Ser Pro Tyr Arg Cys 660 665 670 His Trp Cys Lys Tyr Arg His Val Cys Thr His Asp Pro Lys Thr Cys 675 680 685 Ser Phe Gln Glu Gly Arg Val Lys Leu Pro Glu Asp Cys Pro Gln Leu 690 695 700 Leu Arg Val Asp Lys Ile Leu Val Pro Val Glu Val Ile Lys Pro Ile 705 710 715 720 Thr Leu Lys Ala Lys Asn Leu Pro Gln Pro Gln Ser Gly Gln Arg Gly 725 730 735 Tyr Glu Cys Ile Leu Asn Ile Gln Gly Ser Glu Gln Arg Val Pro Ala 740 745 750 Leu Arg Phe Asn Ser Ser Ser Val Gln Cys Gln Asn Thr Ser Tyr Ser 755 760 765 Tyr Glu Gly Met Glu Ile Asn Asn Leu Pro Val Glu Leu Thr Val Val 770 775 780 Trp Asn Gly His Phe Asn Ile Asp Asn Pro Ala Gln Asn Lys Val His 785 790 795 800 Leu Tyr Lys Cys Gly Ala Met Arg Glu Ser Cys Gly Leu Cys Leu Lys 805 810 815 Ala Asp Pro Asp Phe Ala Cys Gly Trp Cys Gln Gly Pro Gly Gln Cys 820 825 830 Thr Leu Arg Gln His Cys Pro Ala Gln Glu Ser Gln Trp Leu Glu Leu 835 840 845 Ser Gly Ala Lys Ser Lys Cys Thr Asn Pro Arg Ile Thr Glu Ile Ile 850 855 860 Pro Val Thr Gly Pro Arg Glu Gly Gly Thr Lys Val Thr Ile Arg Gly 865 870 875 880 Glu Asn Leu Gly Leu Glu Phe Arg Asp Ile Ala Ser His Val Lys Val 885 890 895 Ala Gly Val Glu Cys Ser Pro Leu Val Asp Gly Tyr Ile Pro Ala Glu 900 905 910 Gln Ile Val Cys Glu Met Gly Glu Ala Lys Pro Ser Gln His Ala Gly 915 920 925 Phe Val Glu Ile Cys Val Ala Val Cys Arg Pro Glu Phe Met Ala Arg 930 935 940 Ser Ser Gln Leu Tyr Tyr Phe Met Thr Leu Thr Leu Ser Asp Leu Lys 945 950 955 960 Pro Ser Arg Gly Pro Met Ser Gly Gly Thr Gln Val Thr Ile Thr Gly 965 970 975 Thr Asn Leu Asn Ala Gly Ser Asn Val Val Val Met Phe Gly Lys Gln 980 985 990 Pro Cys Leu Phe His Arg Arg Ser Pro Ser Tyr Ile Val Cys Asn Thr 995 1000 1005 Thr Ser Ser Asp Glu Val Leu Glu Met Lys Val Ser Val Gln Val 1010 1015 1020 Asp Arg Ala Lys Ile His Gln Asp Leu Val Phe Gln Tyr Val Glu 1025 1030 1035 Asp Pro Thr Ile Val Arg Ile Glu Pro Glu Trp Ser Ile Val Ser 1040 1045 1050 Gly Asn Thr Pro Ile Ala Val Trp Gly Thr His Leu Asp Leu Ile 1055 1060 1065 Gln Asn Pro Gln Ile Arg Ala Lys His Gly Gly Lys Glu His Ile 1070 1075 1080 Asn Ile Cys Glu Val Leu Asn Ala Thr Glu Met Thr Cys Gln Ala 1085 1090 1095 Pro Ala Leu Ala Leu Gly Pro Asp His Gln Ser Asp Leu Thr Glu 1100 1105 1110 Arg Pro Glu Glu Phe Gly Phe Ile Leu Asp Asn Val Gln Ser Leu 1115 1120 1125 Leu Ile Leu Asn Lys Thr Asn Phe Thr Tyr Tyr Pro Asn Pro Val 1130 1135 1140 Phe Glu Ala Phe Gly Pro Ser Gly Ile Leu Glu Leu Lys Pro Gly 1145 1150 1155 Thr Pro Ile Ile Leu Lys Gly Lys Asn Leu Ile Pro Pro Val Ala 1160 1165 1170 Gly Gly Asn Val Lys Leu Asn Tyr Thr Val Leu Val Gly Glu Lys 1175 1180 1185 Pro Cys Thr Val Thr Val Ser Asp Val Gln Leu Leu Cys Glu Ser 1190 1195 1200 Pro Asn Leu Ile Gly Arg His Lys Val Met Ala Arg Val Gly Gly 1205 1210 1215 Met Glu Tyr Ser Pro Gly Met Val Tyr Ile Ala Pro Asp 1220 1225 1230 8233PRTArtificialIgG Fc portion of plexin-A4 fusion protein 8Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 10 15 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 20 25 30 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 35 40 45 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 50 55 60 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 65 70 75 80 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 85 90 95 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 100 105 110 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 115 120 125 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 130 135 140 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 145 150 155 160 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 165 170 175 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 180 185 190 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 195 200 205 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 210 215 220 Lys Ser Leu Ser Leu Ser Pro Gly Lys 225 230 94398DNAArtificialCoding sequence for plexin-A4 fusion protein 9atgaaagcca tgccctggaa ctggacctgc cttctctccc acctcctcat ggtgggcatg 60ggctcctcca ctttgctcac ccggcagcca gccccgctgt cccagaagca gcggtcattt 120gtcacattcc gaggagagcc cgccgagggt ttcaatcacc tggtggtgga tgagaggaca 180ggacacattt acttgggggc cgtcaatcgg atttacaagc tctccagcga cctgaaggtc 240ttggtgacgc atgagacagg gccggacgag gacaacccca agtgttaccc accccgcatc 300gtccagacct gcaatgagcc cctgaccacc accaacaatg tcaacaagat gctcctcata 360gactacaagg agaacaggct gattgcctgt gggagcctgt accaaggcat ctgcaagctg 420ctgaggctgg aggacctctt caagctgggg gagccttatc ataagaagga gcactatctg 480tcaggtgtca acgagagcgg ctcagtcttt ggagtgatcg tctcctacag caacctggat 540gacaagctgt tcattgccac ggcagtggat gggaagcccg agtattttcc caccatctcc 600agccggaaac tgaccaagaa ctctgaggcg gatggcatgt tcgcgtacgt cttccatgat 660gagttcgtgg cctcgatgat taagatccct tcggacacct tcaccatcat ccctgacttt 720gatatctact atgtctatgg ttttagcagt ggcaactttg tctacttttt gaccctccaa 780cctgagatgg tgtctccacc aggctccacc accaaggagc aggtgtatac atccaagctc 840gtgaggcttt gcaaggagga cacagccttc aactcctatg tagaggtgcc cattggctgt 900gagcgcagtg gggtggagta ccgcctgctg caggctgcct acctgtccaa agcgggggcc 960gtgcttggca ggacccttgg agtccatcca gatgatgacc tgctcttcac cgtcttctcc 1020aagggccaga agcggaaaat gaaatccctg gatgagtcgg ccctgtgcat cttcatcttg 1080aagcagataa atgaccgcat taaggagcgg ctgcagtctt gttaccgggg cgagggcacg 1140ctggacctgg cctggctcaa ggtgaaggac atcccctgca gcagtgcgct cttaaccatt 1200gacgataact tctgtggcct ggacatgaat gctcccctgg gagtgtccga catggtgcgt 1260ggaattcccg tcttcacgga ggacagggac cgcatgacgt ctgtcatcgc atatgtctac 1320aagaaccact ctctggcctt tgtgggcacc aaaagtggca agctgaagaa gatccgggtg 1380gatggaccca ggggcaacgc cctccagtat gagacggtgc aggtggtgga ccccggccca 1440gtcctccggg atatggcctt ctccaaggac cacgagcaac tctacatcat gtcagagagg 1500cagctcacca gagtccctgt ggagtcctgt ggtcagtatc agagctgcgg cgagtgcctt 1560ggctcaggcg acccccactg tggctggtgt gtgctgcaca acacttgcac ccggaaggag 1620cggtgtgagc ggtccaagga gccccgcagg tttgcctcgg agatgaagca gtgtgtccgg 1680ctgacggtcc atcccaacaa tatctccgtc tctcagtaca acgtgctgct ggtcctggag 1740acgtacaatg tcccggagct gtcagctggc gtcaactgca cctttgagga cctgtcagag 1800atggatgggc tggtcgtggg caatcagatc cagtgctact cccctgcagc caaggaggtg 1860ccccggatca tcacagagaa tggggaccac catgtcgtac agcttcagct caaatcaaag 1920gagaccggca tgaccttcgc cagcaccagc tttgtcttct acaattgcag cgtccacaat 1980tcgtgcctgt cctgcgtgga gagtccatac cgctgccact ggtgtaaata ccggcatgtc 2040tgcacccatg accccaagac ctgctccttc caggaaggcc gagtgaagct gcccgaggac 2100tgcccccagc tgctgcgagt ggacaagatc ctggtgcccg tggaggtgat caagcctatc 2160acgctgaagg ccaagaacct cccccagccc cagtctgggc agcgtggcta cgaatgcatc 2220ctcaacattc agggcagcga gcagcgagtg cccgccctgc gcttcaacag ctccagcgta 2280cagtgccaga acacctctta ttcctatgaa gggatggaga tcaacaacct gcccgtggag 2340ttgacagtcg tgtggaatgg gcacttcaac attgacaacc cagctcagaa taaagttcac 2400ctctacaagt gtggagccat gcgtgagagc tgcgggctgt gcctcaaggc tgacccagac 2460ttcgcatgtg gctggtgcca gggcccaggc cagtgcaccc tgcgccagca ctgccctgcc 2520caggagagcc agtggctgga gctgtctggt

gccaaaagca agtgcacaaa cccccgcatc 2580acagagataa tcccggtgac aggcccccgg gaagggggca ccaaggtcac tatccgaggg 2640gagaacctgg gcctggaatt tcgcgacatc gcctcccatg tcaaggttgc tggcgtggag 2700tgcagccctt tagtggatgg ttacatccct gcagaacaga tcgtgtgtga gatgggggag 2760gccaagccca gccagcatgc aggcttcgtg gagatctgcg tggctgtgtg tcggcctgaa 2820ttcatggccc ggtcctcaca gctctattac ttcatgacac tgactctctc agatctgaag 2880cccagccggg ggcccatgtc cggagggacc caagtgacca tcacaggcac caacctgaat 2940gccggaagca acgtggtggt gatgtttgga aagcagccct gtctcttcca caggcgatct 3000ccatcctaca ttgtctgcaa caccacatcc tcagatgagg tgctagagat gaaggtgtcg 3060gtgcaggtgg acagggccaa gatccaccag gacctggtct ttcagtatgt ggaagacccc 3120accatcgtgc ggattgagcc agaatggagc attgtcagtg gaaacacacc catcgccgta 3180tgggggaccc acctggacct catacagaac ccccagatcc gtgccaagca tggagggaag 3240gagcacatca atatctgtga ggttctgaac gctactgaga tgacctgtca ggcgcccgcc 3300ctcgctctgg gtcctgacca ccagtcagac ctgaccgaga ggcccgagga gtttggcttc 3360atcctggaca acgtccagtc cctgctcatc ctcaacaaga ccaacttcac ctactatccc 3420aacccggtgt ttgaggcctt tggtccctca ggaatcctgg agctcaagcc tggcacgccc 3480atcatcctaa agggcaagaa cctgatcccg cctgtggctg ggggcaacgt gaagctgaac 3540tacactgtgc tggttgggga gaagccgtgc accgtgaccg tgtcagatgt ccagctgctc 3600tgcgagtccc ccaacctcat cggcaggcac aaagtgatgg cccgtgtcgg tggcatggag 3660tactccccgg ggatggtgta cattgccccg gacggatccg agcccaaatc ttgtgacaaa 3720actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttcctc 3780ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 3840gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 3900gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 3960gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 4020gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 4080ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag 4140gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 4200agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 4260tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 4320ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 4380ctgtctccgg gtaaatga 4398103693DNAHomo sapiens 10atgaaagcca tgccctggaa ctggacctgc cttctctccc acctcctcat ggtgggcatg 60ggctcctcca ctttgctcac ccggcagcca gccccgctgt cccagaagca gcggtcattt 120gtcacattcc gaggagagcc cgccgagggt ttcaatcacc tggtggtgga tgagaggaca 180ggacacattt acttgggggc cgtcaatcgg atttacaagc tctccagcga cctgaaggtc 240ttggtgacgc atgagacagg gccggacgag gacaacccca agtgttaccc accccgcatc 300gtccagacct gcaatgagcc cctgaccacc accaacaatg tcaacaagat gctcctcata 360gactacaagg agaacaggct gattgcctgt gggagcctgt accaaggcat ctgcaagctg 420ctgaggctgg aggacctctt caagctgggg gagccttatc ataagaagga gcactatctg 480tcaggtgtca acgagagcgg ctcagtcttt ggagtgatcg tctcctacag caacctggat 540gacaagctgt tcattgccac ggcagtggat gggaagcccg agtattttcc caccatctcc 600agccggaaac tgaccaagaa ctctgaggcg gatggcatgt tcgcgtacgt cttccatgat 660gagttcgtgg cctcgatgat taagatccct tcggacacct tcaccatcat ccctgacttt 720gatatctact atgtctatgg ttttagcagt ggcaactttg tctacttttt gaccctccaa 780cctgagatgg tgtctccacc aggctccacc accaaggagc aggtgtatac atccaagctc 840gtgaggcttt gcaaggagga cacagccttc aactcctatg tagaggtgcc cattggctgt 900gagcgcagtg gggtggagta ccgcctgctg caggctgcct acctgtccaa agcgggggcc 960gtgcttggca ggacccttgg agtccatcca gatgatgacc tgctcttcac cgtcttctcc 1020aagggccaga agcggaaaat gaaatccctg gatgagtcgg ccctgtgcat cttcatcttg 1080aagcagataa atgaccgcat taaggagcgg ctgcagtctt gttaccgggg cgagggcacg 1140ctggacctgg cctggctcaa ggtgaaggac atcccctgca gcagtgcgct cttaaccatt 1200gacgataact tctgtggcct ggacatgaat gctcccctgg gagtgtccga catggtgcgt 1260ggaattcccg tcttcacgga ggacagggac cgcatgacgt ctgtcatcgc atatgtctac 1320aagaaccact ctctggcctt tgtgggcacc aaaagtggca agctgaagaa gatccgggtg 1380gatggaccca ggggcaacgc cctccagtat gagacggtgc aggtggtgga ccccggccca 1440gtcctccggg atatggcctt ctccaaggac cacgagcaac tctacatcat gtcagagagg 1500cagctcacca gagtccctgt ggagtcctgt ggtcagtatc agagctgcgg cgagtgcctt 1560ggctcaggcg acccccactg tggctggtgt gtgctgcaca acacttgcac ccggaaggag 1620cggtgtgagc ggtccaagga gccccgcagg tttgcctcgg agatgaagca gtgtgtccgg 1680ctgacggtcc atcccaacaa tatctccgtc tctcagtaca acgtgctgct ggtcctggag 1740acgtacaatg tcccggagct gtcagctggc gtcaactgca cctttgagga cctgtcagag 1800atggatgggc tggtcgtggg caatcagatc cagtgctact cccctgcagc caaggaggtg 1860ccccggatca tcacagagaa tggggaccac catgtcgtac agcttcagct caaatcaaag 1920gagaccggca tgaccttcgc cagcaccagc tttgtcttct acaattgcag cgtccacaat 1980tcgtgcctgt cctgcgtgga gagtccatac cgctgccact ggtgtaaata ccggcatgtc 2040tgcacccatg accccaagac ctgctccttc caggaaggcc gagtgaagct gcccgaggac 2100tgcccccagc tgctgcgagt ggacaagatc ctggtgcccg tggaggtgat caagcctatc 2160acgctgaagg ccaagaacct cccccagccc cagtctgggc agcgtggcta cgaatgcatc 2220ctcaacattc agggcagcga gcagcgagtg cccgccctgc gcttcaacag ctccagcgta 2280cagtgccaga acacctctta ttcctatgaa gggatggaga tcaacaacct gcccgtggag 2340ttgacagtcg tgtggaatgg gcacttcaac attgacaacc cagctcagaa taaagttcac 2400ctctacaagt gtggagccat gcgtgagagc tgcgggctgt gcctcaaggc tgacccagac 2460ttcgcatgtg gctggtgcca gggcccaggc cagtgcaccc tgcgccagca ctgccctgcc 2520caggagagcc agtggctgga gctgtctggt gccaaaagca agtgcacaaa cccccgcatc 2580acagagataa tcccggtgac aggcccccgg gaagggggca ccaaggtcac tatccgaggg 2640gagaacctgg gcctggaatt tcgcgacatc gcctcccatg tcaaggttgc tggcgtggag 2700tgcagccctt tagtggatgg ttacatccct gcagaacaga tcgtgtgtga gatgggggag 2760gccaagccca gccagcatgc aggcttcgtg gagatctgcg tggctgtgtg tcggcctgaa 2820ttcatggccc ggtcctcaca gctctattac ttcatgacac tgactctctc agatctgaag 2880cccagccggg ggcccatgtc cggagggacc caagtgacca tcacaggcac caacctgaat 2940gccggaagca acgtggtggt gatgtttgga aagcagccct gtctcttcca caggcgatct 3000ccatcctaca ttgtctgcaa caccacatcc tcagatgagg tgctagagat gaaggtgtcg 3060gtgcaggtgg acagggccaa gatccaccag gacctggtct ttcagtatgt ggaagacccc 3120accatcgtgc ggattgagcc agaatggagc attgtcagtg gaaacacacc catcgccgta 3180tgggggaccc acctggacct catacagaac ccccagatcc gtgccaagca tggagggaag 3240gagcacatca atatctgtga ggttctgaac gctactgaga tgacctgtca ggcgcccgcc 3300ctcgctctgg gtcctgacca ccagtcagac ctgaccgaga ggcccgagga gtttggcttc 3360atcctggaca acgtccagtc cctgctcatc ctcaacaaga ccaacttcac ctactatccc 3420aacccggtgt ttgaggcctt tggtccctca ggaatcctgg agctcaagcc tggcacgccc 3480atcatcctaa agggcaagaa cctgatcccg cctgtggctg ggggcaacgt gaagctgaac 3540tacactgtgc tggttgggga gaagccgtgc accgtgaccg tgtcagatgt ccagctgctc 3600tgcgagtccc ccaacctcat cggcaggcac aaagtgatgg cccgtgtcgg tggcatggag 3660tactccccgg ggatggtgta cattgccccg gac 369311699DNAArtificialCoding sequence for IgG Fc portion of plexin-A4 fusion protein 11gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg 60gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 120acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 180aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 240tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 300ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 360atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 420gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 480gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 540cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 600aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 660tacacgcaga agagcctctc cctgtctccg ggtaaatga 69912475PRTHomo sapiens 12Met Glu Phe Gly Leu Ser Cys Val Phe Leu Val Ala Ile Phe Lys Gly 1 5 10 15 Val His Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Gly Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Ala Phe Thr Leu 35 40 45 Ser Arg His Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Tyr Val Ser Gly Ile Ser Asn Ser Glu Asn Ser Thr Tyr Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Tyr Lys Asn 85 90 95 Thr Leu Tyr Leu Gln Leu Gly Ser Leu Arg Ala Glu Asp Lys Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Ala Arg Cys Arg Gly Asp Thr Cys Leu Asn Phe 115 120 125 Tyr Tyr Gly Leu Asp Val Trp Gly Gln Gly Thr Thr Val Ile Val Ser 130 135 140 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 145 150 155 160 Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 165 170 175 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 180 185 190 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 195 200 205 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln 210 215 220 Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 225 230 235 240 Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 245 250 255 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 260 265 270 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 275 280 285 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 290 295 300 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 305 310 315 320 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 325 330 335 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 340 345 350 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 355 360 365 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 370 375 380 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 385 390 395 400 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 405 410 415 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 420 425 430 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 435 440 445 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 450 455 460 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 465 470 475 13771PRTHomo sapiens 13Met Gly Trp Leu Thr Arg Ile Val Cys Leu Phe Trp Gly Val Leu Leu 1 5 10 15 Thr Ala Arg Ala Asn Tyr Gln Asn Gly Lys Asn Asn Val Pro Arg Leu 20 25 30 Lys Leu Ser Tyr Lys Glu Met Leu Glu Ser Asn Asn Val Ile Thr Phe 35 40 45 Asn Gly Leu Ala Asn Ser Ser Ser Tyr His Thr Phe Leu Leu Asp Glu 50 55 60 Glu Arg Ser Arg Leu Tyr Val Gly Ala Lys Asp His Ile Phe Ser Phe 65 70 75 80 Asp Leu Val Asn Ile Lys Asp Phe Gln Lys Ile Val Trp Pro Val Ser 85 90 95 Tyr Thr Arg Arg Asp Glu Cys Lys Trp Ala Gly Lys Asp Ile Leu Lys 100 105 110 Glu Cys Ala Asn Phe Ile Lys Val Leu Lys Ala Tyr Asn Gln Thr His 115 120 125 Leu Tyr Ala Cys Gly Thr Gly Ala Phe His Pro Ile Cys Thr Tyr Ile 130 135 140 Glu Ile Gly His His Pro Glu Asp Asn Ile Phe Lys Leu Glu Asn Ser 145 150 155 160 His Phe Glu Asn Gly Arg Gly Lys Ser Pro Tyr Asp Pro Lys Leu Leu 165 170 175 Thr Ala Ser Leu Leu Ile Asp Gly Glu Leu Tyr Ser Gly Thr Ala Ala 180 185 190 Asp Phe Met Gly Arg Asp Phe Ala Ile Phe Arg Thr Leu Gly His His 195 200 205 His Pro Ile Arg Thr Glu Gln His Asp Ser Arg Trp Leu Asn Asp Pro 210 215 220 Lys Phe Ile Ser Ala His Leu Ile Ser Glu Ser Asp Asn Pro Glu Asp 225 230 235 240 Asp Lys Val Tyr Phe Phe Phe Arg Glu Asn Ala Ile Asp Gly Glu His 245 250 255 Ser Gly Lys Ala Thr His Ala Arg Ile Gly Gln Ile Cys Lys Asn Asp 260 265 270 Phe Gly Gly His Arg Ser Leu Val Asn Lys Trp Thr Thr Phe Leu Lys 275 280 285 Ala Arg Leu Ile Cys Ser Val Pro Gly Pro Asn Gly Ile Asp Thr His 290 295 300 Phe Asp Glu Leu Gln Asp Val Phe Leu Met Asn Phe Lys Asp Pro Lys 305 310 315 320 Asn Pro Val Val Tyr Gly Val Phe Thr Thr Ser Ser Asn Ile Phe Lys 325 330 335 Gly Ser Ala Val Cys Met Tyr Ser Met Ser Asp Val Arg Arg Val Phe 340 345 350 Leu Gly Pro Tyr Ala His Arg Asp Gly Pro Asn Tyr Gln Trp Val Pro 355 360 365 Tyr Gln Gly Arg Val Pro Tyr Pro Arg Pro Gly Thr Cys Pro Ser Lys 370 375 380 Thr Phe Gly Gly Phe Asp Ser Thr Lys Asp Leu Pro Asp Asp Val Ile 385 390 395 400 Thr Phe Ala Arg Ser His Pro Ala Met Tyr Asn Pro Val Phe Pro Met 405 410 415 Asn Asn Arg Pro Ile Val Ile Lys Thr Asp Val Asn Tyr Gln Phe Thr 420 425 430 Gln Ile Val Val Asp Arg Val Asp Ala Glu Asp Gly Gln Tyr Asp Val 435 440 445 Met Phe Ile Gly Thr Asp Val Gly Thr Val Leu Lys Val Val Ser Ile 450 455 460 Pro Lys Glu Thr Trp Tyr Asp Leu Glu Glu Val Leu Leu Glu Glu Met 465 470 475 480 Thr Val Phe Arg Glu Pro Thr Ala Ile Ser Ala Met Glu Leu Ser Thr 485 490 495 Lys Gln Gln Gln Leu Tyr Ile Gly Ser Thr Ala Gly Val Ala Gln Leu 500 505 510 Pro Leu His Arg Cys Asp Ile Tyr Gly Lys Ala Cys Ala Glu Cys Cys 515 520 525 Leu Ala Arg Asp Pro Tyr Cys Ala Trp Asp Gly Ser Ala Cys Ser Arg 530 535 540 Tyr Phe Pro Thr Ala Lys Arg Arg Thr Arg Arg Gln Asp Ile Arg Asn 545 550 555 560 Gly Asp Pro Leu Thr His Cys Ser Asp Leu His His Asp Asn His His 565 570 575 Gly His Ser Pro Glu Glu Arg Ile Ile Tyr Gly Val Glu Asn Ser Ser 580 585 590 Thr Phe Leu Glu Cys Ser Pro Lys Ser Gln Arg Ala Leu Val Tyr Trp 595 600 605 Gln Phe Gln Arg Arg Asn Glu Glu Arg Lys Glu Glu Ile Arg Val Asp 610 615 620 Asp His Ile Ile Arg Thr Asp Gln Gly Leu Leu Leu Arg Ser Leu Gln 625 630 635 640 Gln Lys Asp Ser Gly Asn Tyr Leu Cys His Ala Val Glu His Gly Phe 645 650 655 Ile Gln Thr Leu Leu Lys Val Thr Leu Glu Val Ile Asp Thr Glu His 660 665 670 Leu Glu Glu Leu Leu His Lys Asp Asp Asp Gly Asp Gly Ser Lys Thr 675 680 685 Lys Glu Met Ser Asn Ser Met Thr Pro Ser Gln Lys Val Trp Tyr Arg 690 695 700 Asp Phe Met Gln Leu Ile Asn His Pro Asn Leu Asn Thr Met Asp Glu 705 710 715 720 Phe Cys Glu Gln Val Trp Lys Arg Asp Arg Lys Gln Arg Arg Gln Arg 725 730 735 Pro Gly His Thr Pro Gly Asn Ser Asn Lys Trp Lys His Leu Gln Glu 740 745 750 Asn Lys Lys Gly Arg Asn Arg Arg Thr His Glu Phe Glu Arg Ala Pro 755 760 765 Arg Ser Val 770 142316DNAHomo sapiens 14atgggctggt taactaggat tgtctgtctt ttctggggag tattacttac agcaagagca 60aactatcaga atgggaagaa caatgtgcca aggctgaaat tatcctacaa agaaatgttg 120gaatccaaca atgtgatcac tttcaatggc ttggccaaca gctccagtta tcataccttc 180cttttggatg aggaacggag taggctgtat gttggagcaa aggatcacat attttcattc 240gacctggtta atatcaagga ttttcaaaag attgtgtggc cagtatctta caccagaaga 300gatgaatgca agtgggctgg aaaagacatc ctgaaagaat gtgctaattt catcaaggta 360cttaaggcat ataatcagac tcacttgtac gcctgtggaa cgggggcttt tcatccaatt 420tgcacctaca ttgaaattgg

acatcatcct gaggacaata tttttaagct ggagaactca 480cattttgaaa acggccgtgg gaagagtcca tatgacccta agctgctgac agcatccctt 540ttaatagatg gagaattata ctctggaact gcagctgatt ttatggggcg agactttgct 600atcttccgaa ctcttgggca ccaccaccca atcaggacag agcagcatga ttccaggtgg 660ctcaatgatc caaagttcat tagtgcccac ctcatctcag agagtgacaa tcctgaagat 720gacaaagtat actttttctt ccgtgaaaat gcaatagatg gagaacactc tggaaaagct 780actcacgcta gaataggtca gatatgcaag aatgactttg gagggcacag aagtctggtg 840aataaatgga caacattcct caaagctcgt ctgatttgct cagtgccagg tccaaatggc 900attgacactc attttgatga actgcaggat gtattcctaa tgaactttaa agatcctaaa 960aatccagttg tatatggagt gtttacgact tccagtaaca ttttcaaggg atcagccgtg 1020tgtatgtata gcatgagtga tgtgagaagg gtgttccttg gtccatatgc ccacagggat 1080ggacccaact atcaatgggt gccttatcaa ggaagagtcc cctatccacg gccaggaact 1140tgtcccagca aaacatttgg tggttttgac tctacaaagg accttcctga tgatgttata 1200acctttgcaa gaagtcatcc agccatgtac aatccagtgt ttcctatgaa caatcgccca 1260atagtgatca aaacggatgt aaattatcaa tttacacaaa ttgtcgtaga ccgagtggat 1320gcagaagatg gacagtatga tgttatgttt atcggaacag atgttgggac cgttcttaaa 1380gtagtttcaa ttcctaagga gacttggtat gatttagaag aggttctgct ggaagaaatg 1440acagtttttc gggaaccgac tgctatttca gcaatggagc tttccactaa gcagcaacaa 1500ctatatattg gttcaacggc tggggttgcc cagctccctt tacaccggtg tgatatttac 1560gggaaagcgt gtgctgagtg ttgcctcgcc cgagaccctt actgtgcttg ggatggttct 1620gcatgttctc gctattttcc cactgcaaag agacgcacaa gacgacaaga tataagaaat 1680ggagacccac tgactcactg ttcagactta caccatgata atcaccatgg ccacagccct 1740gaagagagaa tcatctatgg tgtagagaat agtagcacat ttttggaatg cagtccgaag 1800tcgcagagag cgctggtcta ttggcaattc cagaggcgaa atgaagagcg aaaagaagag 1860atcagagtgg atgatcatat catcaggaca gatcaaggcc ttctgctacg tagtctacaa 1920cagaaggatt caggcaatta cctctgccat gcggtggaac atgggttcat acaaactctt 1980cttaaggtaa ccctggaagt cattgacaca gagcatttgg aagaacttct tcataaagat 2040gatgatggag atggctctaa gaccaaagaa atgtccaata gcatgacacc tagccagaag 2100gtctggtaca gagacttcat gcagctcatc aaccacccca atctcaacac aatggatgag 2160ttctgtgaac aagtttggaa aagggaccga aaacaacgtc ggcaaaggcc aggacatacc 2220ccagggaaca gtaacaaatg gaagcactta caagaaaata agaaaggtag aaacaggagg 2280acccacgaat ttgagagggc acccaggagt gtctga 2316155672DNAHomo sapiens 15aagcaccact gcagcagacc ttgttaattt tttttttttt tctttccaca caacagttgt 60gcctcattat ccggtgcctg gctcggaatt tttttttttt tttttctttt tggagggttt 120gaagtttctg tgcttcagtg actgttacag aagaagaggt gttagtgttg ccatgaggtc 180ttgattgtct gcatttatga atgaaactga cctaaatcac ctgttacctc cagtttccag 240attgtttgaa cttctctggc cgcacaatac aggaaggaag actaaagcag caaagggacc 300tacagcgtct gcagcatggg ctggttaact aggattgtct gtcttttctg gggagtatta 360cttacagcaa gagcaaacta tcagaatggg aagaacaatg tgccaaggct gaaattatcc 420tacaaagaaa tgttggaatc caacaatgtg atcactttca atggcttggc caacagctcc 480agttatcata ccttcctttt ggatgaggaa cggagtaggc tgtatgttgg agcaaaggat 540cacatatttt cattcgacct ggttaatatc aaggattttc aaaagattgt gtggccagta 600tcttacacca gaagagatga atgcaagtgg gctggaaaag acatcctgaa agaatgtgct 660aatttcatca aggtacttaa ggcatataat cagactcact tgtacgcctg tggaacgggg 720gcttttcatc caatttgcac ctacattgaa attggacatc atcctgagga caatattttt 780aagctggaga actcacattt tgaaaacggc cgtgggaaga gtccatatga ccctaagctg 840ctgacagcat cccttttaat agatggagaa ttatactctg gaactgcagc tgattttatg 900gggcgagact ttgctatctt ccgaactctt gggcaccacc acccaatcag gacagagcag 960catgattcca ggtggctcaa tgatccaaag ttcattagtg cccacctcat ctcagagagt 1020gacaatcctg aagatgacaa agtatacttt ttcttccgtg aaaatgcaat agatggagaa 1080cactctggaa aagctactca cgctagaata ggtcagatat gcaagaatga ctttggaggg 1140cacagaagtc tggtgaataa atggacaaca ttcctcaaag ctcgtctgat ttgctcagtg 1200ccaggtccaa atggcattga cactcatttt gatgaactgc aggatgtatt cctaatgaac 1260tttaaagatc ctaaaaatcc agttgtatat ggagtgttta cgacttccag taacattttc 1320aagggatcag ccgtgtgtat gtatagcatg agtgatgtga gaagggtgtt ccttggtcca 1380tatgcccaca gggatggacc caactatcaa tgggtgcctt atcaaggaag agtcccctat 1440ccacggccag gaacttgtcc cagcaaaaca tttggtggtt ttgactctac aaaggacctt 1500cctgatgatg ttataacctt tgcaagaagt catccagcca tgtacaatcc agtgtttcct 1560atgaacaatc gcccaatagt gatcaaaacg gatgtaaatt atcaatttac acaaattgtc 1620gtagaccgag tggatgcaga agatggacag tatgatgtta tgtttatcgg aacagatgtt 1680gggaccgttc ttaaagtagt ttcaattcct aaggagactt ggtatgattt agaagaggtt 1740ctgctggaag aaatgacagt ttttcgggaa ccgactgcta tttcagcaat ggagctttcc 1800actaagcagc aacaactata tattggttca acggctgggg ttgcccagct ccctttacac 1860cggtgtgata tttacgggaa agcgtgtgct gagtgttgcc tcgcccgaga cccttactgt 1920gcttgggatg gttctgcatg ttctcgctat tttcccactg caaagagacg cacaagacga 1980caagatataa gaaatggaga cccactgact cactgttcag acttacacca tgataatcac 2040catggccaca gccctgaaga gagaatcatc tatggtgtag agaatagtag cacatttttg 2100gaatgcagtc cgaagtcgca gagagcgctg gtctattggc aattccagag gcgaaatgaa 2160gagcgaaaag aagagatcag agtggatgat catatcatca ggacagatca aggccttctg 2220ctacgtagtc tacaacagaa ggattcaggc aattacctct gccatgcggt ggaacatggg 2280ttcatacaaa ctcttcttaa ggtaaccctg gaagtcattg acacagagca tttggaagaa 2340cttcttcata aagatgatga tggagatggc tctaagacca aagaaatgtc caatagcatg 2400acacctagcc agaaggtctg gtacagagac ttcatgcagc tcatcaacca ccccaatctc 2460aacacaatgg atgagttctg tgaacaagtt tggaaaaggg accgaaaaca acgtcggcaa 2520aggccaggac ataccccagg gaacagtaac aaatggaagc acttacaaga aaataagaaa 2580ggtagaaaca ggaggaccca cgaatttgag agggcaccca ggagtgtctg agctgcatta 2640cctctagaaa cctcaaacaa gtagaaactt gcctagacaa taactggaaa aacaaatgca 2700atatacatga acttttttca tggcattatg tggatgttta caatggtggg aaattcagct 2760gagttccacc aattataaat taaatccatg agtaactttc ctaataggct ttttttccta 2820ataccaccac ctaacagaga acacaggtga atgcagatgt tcactttagc agacttaatg 2880tttcctatga gatttcactg tacaggtttg tctttcttct ttgcctgaga aataaaaatg 2940tcatttgcca tattgccatc taaaggagaa aaactgcatc agcaaagcca ttgtattgaa 3000ctaaaagttt aaaatgaact gcatggattt actaagctga tgaatattcc aaaacgtggt 3060tggattcaag gatatatttt gtctaccggc cctcatgttt gtatgtactt gaggagtaaa 3120atgagtaaaa tgatactgaa tgaaatgttc tgtggaaata ttaaaaaaaa aaaaaaacat 3180aagccatcca tcatccagaa gaaaaatgga atacactgat ctactactga tgtcttcttt 3240cagctttgat ctaaagatgt attttattaa aactataatt taaatgtacc atgaaaaata 3300tgcagtaaaa attagttgtt ttctaagcta gagtaggatt tgtcttacaa ttattgtgct 3360atgtagtttt tgttttaaaa attccaatgg tgtgctgctt tctttggaca ttttattttc 3420aattctataa gagggataga tgacattgtt ctagaaacac atatacatca ttaagagtga 3480atctctaaaa ccaggatata aattatgctt tatttctctg agaaaatcaa acaaatggaa 3540gctgttcaca cctccccttc tttaagcatt atctaaatta atttttactt gcataatgtt 3600cttagaaaaa aaaacagaac atttaagcag gaaaaaagga agaaacaagt tgatttttaa 3660gtgcatttta ctataatgaa tcaatgaagg gaaaaggaac tgcatatttc atgaaaataa 3720taagcattgt cttaatatac tgttaataga aaatgtgtct taattccgtg cttgaatccc 3780tgcatgatat ttgagactaa gatctctctt atgattctac caagaattat atctgtgtca 3840cttaattttt ttaaaagaga gagatcaata actattcaga gcaacatgtt aaaggcaaag 3900tttccaatca tttacatctg tatcaggtgc ctcttacctt tccttattta agacaattat 3960ttgtacaaga aacacatgac tcttttcata tcaatgggag ggacttttct acaaagtatt 4020ttccaggatg caacccacat ttaaacaatg taaaattctt tgtttcctgc aacaacttac 4080aaaataaggt aaaagactaa aattcaagat ttgcttcctt cattgtccta agacgattcg 4140ttgagaatca ctgactttga gatatttaaa actttcagca ttatactgtg gtttcttttg 4200cactgcactc acctattcag gactcctccc ccaggttcct catcatgcac aaaaatgcaa 4260agaaaacatc ttattagtaa ttaatgaagc aacattgaaa ttctaactct agctgtcttt 4320ggattctaat taactcagca tcaatttctc acctcagact acagtgaatt tttatttcct 4380atcagctgaa atatttcaca gatggaagct catgtttcag ttttaatgac tgccttgaat 4440aaacaagttg ttgccacttg tttcaaacaa aagcctaaaa ataatctaca ttcaatttta 4500ggctccattg actaatatgg tgttgctttt ggaagtactg tatatcctca catggaagcc 4560aaattgttaa attatttgaa ggacacacca ctgtacagaa agtagtgttt caaatataaa 4620tcgaagaaca aagagtgctc caaaaaatag gtcattcttt tattttcata aagtatctaa 4680actgtactaa cattcagtgt tgtgtttcat tctaaatttg cagctgaaat aaatttattt 4740gcgatagcag aaatatctta ttattcatcc tcagaaataa aggatttgaa gggatagaga 4800ttatatgata aatttataga agactttcag aatttgaatg cattttgttt agtgttatga 4860aatgacaata gaaaaaagtc tcgacttcaa ttaaaagtta cacaaacaaa caaatctaca 4920ggcatgtctt tatataccat caggtctaag ttttcaaaga aaattgtaga tataacttgc 4980agataactca ttacagtcat aatctctgcc catgtgtatt gagagggggc agtttgcacg 5040aaaaagaatt attggcccat ttaataattc agctttaaat agactttgtc atatgcatga 5100atcatcagag atgaaactgt ttgagagact catgtgacct tacgaaaatt acaacagcag 5160tcttaaagta tgaaaaagat gcatcacagc agagacatta tggcccagtt gatatcaaat 5220gtaaaatgta aatgcatgta aatgcacact tcattttatg tattatttag taatttgcag 5280tggtatgtgt ttaatatttt tgctacctac acattaggca aaaaaaagat gtaaataatt 5340tgggagaaaa agaggaagaa cagtgtaaaa taaaactttc tataagtact ccatttcaat 5400gtgttcaaca tcatcctaaa aggcaagatt ttcccacgca ggtgacaagg tggtttatgt 5460actatttaag ggcggaaggt gcgtgcccgt tcaataagca tgttttttgc caggtaggaa 5520atatgttcca tatctttact tatcattgca tttcagatgg gaactagaaa aactggagag 5580aaaaatgtaa tgaaactgct gctgtaaatt attcctttta gcatgtattc acttgctaaa 5640tacacatttc ttcaaaataa aaaaaaaaaa aa 5672161006PRTArtificialSema3A/Fc IgG fusion protein sequence 16Met Gly Trp Leu Thr Arg Ile Val Cys Leu Phe Trp Gly Val Leu Leu 1 5 10 15 Thr Ala Arg Ala Asn Tyr Gln Asn Gly Lys Asn Asn Val Pro Arg Leu 20 25 30 Lys Leu Ser Tyr Lys Glu Met Leu Glu Ser Asn Asn Val Ile Thr Phe 35 40 45 Asn Gly Leu Ala Asn Ser Ser Ser Tyr His Thr Phe Leu Leu Asp Glu 50 55 60 Glu Arg Ser Arg Leu Tyr Val Gly Ala Lys Asp His Ile Phe Ser Phe 65 70 75 80 Asp Leu Val Asn Ile Lys Asp Phe Gln Lys Ile Val Trp Pro Val Ser 85 90 95 Tyr Thr Arg Arg Asp Glu Cys Lys Trp Ala Gly Lys Asp Ile Leu Lys 100 105 110 Glu Cys Ala Asn Phe Ile Lys Val Leu Lys Ala Tyr Asn Gln Thr His 115 120 125 Leu Tyr Ala Cys Gly Thr Gly Ala Phe His Pro Ile Cys Thr Tyr Ile 130 135 140 Glu Ile Gly His His Pro Glu Asp Asn Ile Phe Lys Leu Glu Asn Ser 145 150 155 160 His Phe Glu Asn Gly Arg Gly Lys Ser Pro Tyr Asp Pro Lys Leu Leu 165 170 175 Thr Ala Ser Leu Leu Ile Asp Gly Glu Leu Tyr Ser Gly Thr Ala Ala 180 185 190 Asp Phe Met Gly Arg Asp Phe Ala Ile Phe Arg Thr Leu Gly His His 195 200 205 His Pro Ile Arg Thr Glu Gln His Asp Ser Arg Trp Leu Asn Asp Pro 210 215 220 Lys Phe Ile Ser Ala His Leu Ile Ser Glu Ser Asp Asn Pro Glu Asp 225 230 235 240 Asp Lys Val Tyr Phe Phe Phe Arg Glu Asn Ala Ile Asp Gly Glu His 245 250 255 Ser Gly Lys Ala Thr His Ala Arg Ile Gly Gln Ile Cys Lys Asn Asp 260 265 270 Phe Gly Gly His Arg Ser Leu Val Asn Lys Trp Thr Thr Phe Leu Lys 275 280 285 Ala Arg Leu Ile Cys Ser Val Pro Gly Pro Asn Gly Ile Asp Thr His 290 295 300 Phe Asp Glu Leu Gln Asp Val Phe Leu Met Asn Phe Lys Asp Pro Lys 305 310 315 320 Asn Pro Val Val Tyr Gly Val Phe Thr Thr Ser Ser Asn Ile Phe Lys 325 330 335 Gly Ser Ala Val Cys Met Tyr Ser Met Ser Asp Val Arg Arg Val Phe 340 345 350 Leu Gly Pro Tyr Ala His Arg Asp Gly Pro Asn Tyr Gln Trp Val Pro 355 360 365 Tyr Gln Gly Arg Val Pro Tyr Pro Arg Pro Gly Thr Cys Pro Ser Lys 370 375 380 Thr Phe Gly Gly Phe Asp Ser Thr Lys Asp Leu Pro Asp Asp Val Ile 385 390 395 400 Thr Phe Ala Arg Ser His Pro Ala Met Tyr Asn Pro Val Phe Pro Met 405 410 415 Asn Asn Arg Pro Ile Val Ile Lys Thr Asp Val Asn Tyr Gln Phe Thr 420 425 430 Gln Ile Val Val Asp Arg Val Asp Ala Glu Asp Gly Gln Tyr Asp Val 435 440 445 Met Phe Ile Gly Thr Asp Val Gly Thr Val Leu Lys Val Val Ser Ile 450 455 460 Pro Lys Glu Thr Trp Tyr Asp Leu Glu Glu Val Leu Leu Glu Glu Met 465 470 475 480 Thr Val Phe Arg Glu Pro Thr Ala Ile Ser Ala Met Glu Leu Ser Thr 485 490 495 Lys Gln Gln Gln Leu Tyr Ile Gly Ser Thr Ala Gly Val Ala Gln Leu 500 505 510 Pro Leu His Arg Cys Asp Ile Tyr Gly Lys Ala Cys Ala Glu Cys Cys 515 520 525 Leu Ala Arg Asp Pro Tyr Cys Ala Trp Asp Gly Ser Ala Cys Ser Arg 530 535 540 Tyr Phe Pro Thr Ala Lys Arg Arg Thr Arg Arg Gln Asp Ile Arg Asn 545 550 555 560 Gly Asp Pro Leu Thr His Cys Ser Asp Leu His His Asp Asn His His 565 570 575 Gly His Ser Pro Glu Glu Arg Ile Ile Tyr Gly Val Glu Asn Ser Ser 580 585 590 Thr Phe Leu Glu Cys Ser Pro Lys Ser Gln Arg Ala Leu Val Tyr Trp 595 600 605 Gln Phe Gln Arg Arg Asn Glu Glu Arg Lys Glu Glu Ile Arg Val Asp 610 615 620 Asp His Ile Ile Arg Thr Asp Gln Gly Leu Leu Leu Arg Ser Leu Gln 625 630 635 640 Gln Lys Asp Ser Gly Asn Tyr Leu Cys His Ala Val Glu His Gly Phe 645 650 655 Ile Gln Thr Leu Leu Lys Val Thr Leu Glu Val Ile Asp Thr Glu His 660 665 670 Leu Glu Glu Leu Leu His Lys Asp Asp Asp Gly Asp Gly Ser Lys Thr 675 680 685 Lys Glu Met Ser Asn Ser Met Thr Pro Ser Gln Lys Val Trp Tyr Arg 690 695 700 Asp Phe Met Gln Leu Ile Asn His Pro Asn Leu Asn Thr Met Asp Glu 705 710 715 720 Phe Cys Glu Gln Val Trp Lys Arg Asp Arg Lys Gln Arg Arg Gln Arg 725 730 735 Pro Gly His Thr Pro Gly Asn Ser Asn Lys Trp Lys His Leu Gln Glu 740 745 750 Asn Lys Lys Gly Arg Asn Arg Arg Thr His Glu Phe Glu Arg Ala Pro 755 760 765 Arg Ser Val Gly Cys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 770 775 780 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 785 790 795 800 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 805 810 815 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 820 825 830 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 835 840 845 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 850 855 860 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 865 870 875 880 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 885 890 895 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 900 905 910 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 915 920 925 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 930 935 940 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 945 950 955 960 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 965 970 975 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 980 985 990 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 995 1000 1005 173018DNAArtificialCoding sequence for Sema3A/Fc IgG fusion protein 17atgggctggt taactaggat tgtctgtctt ttctggggag tattacttac agcaagagca 60aactatcaga atgggaagaa caatgtgcca aggctgaaat tatcctacaa agaaatgttg 120gaatccaaca atgtgatcac tttcaatggc ttggccaaca gctccagtta tcataccttc 180cttttggatg aggaacggag taggctgtat gttggagcaa aggatcacat attttcattc 240gacctggtta atatcaagga ttttcaaaag attgtgtggc cagtatctta caccagaaga 300gatgaatgca agtgggctgg aaaagacatc ctgaaagaat gtgctaattt catcaaggta 360cttaaggcat ataatcagac tcacttgtac gcctgtggaa cgggggcttt tcatccaatt 420tgcacctaca ttgaaattgg acatcatcct gaggacaata tttttaagct ggagaactca 480cattttgaaa acggccgtgg gaagagtcca tatgacccta agctgctgac agcatccctt 540ttaatagatg gagaattata ctctggaact gcagctgatt ttatggggcg agactttgct 600atcttccgaa ctcttgggca ccaccaccca atcaggacag agcagcatga ttccaggtgg 660ctcaatgatc caaagttcat tagtgcccac ctcatctcag agagtgacaa tcctgaagat 720gacaaagtat actttttctt ccgtgaaaat

gcaatagatg gagaacactc tggaaaagct 780actcacgcta gaataggtca gatatgcaag aatgactttg gagggcacag aagtctggtg 840aataaatgga caacattcct caaagctcgt ctgatttgct cagtgccagg tccaaatggc 900attgacactc attttgatga actgcaggat gtattcctaa tgaactttaa agatcctaaa 960aatccagttg tatatggagt gtttacgact tccagtaaca ttttcaaggg atcagccgtg 1020tgtatgtata gcatgagtga tgtgagaagg gtgttccttg gtccatatgc ccacagggat 1080ggacccaact atcaatgggt gccttatcaa ggaagagtcc cctatccacg gccaggaact 1140tgtcccagca aaacatttgg tggttttgac tctacaaagg accttcctga tgatgttata 1200acctttgcaa gaagtcatcc agccatgtac aatccagtgt ttcctatgaa caatcgccca 1260atagtgatca aaacggatgt aaattatcaa tttacacaaa ttgtcgtaga ccgagtggat 1320gcagaagatg gacagtatga tgttatgttt atcggaacag atgttgggac cgttcttaaa 1380gtagtttcaa ttcctaagga gacttggtat gatttagaag aggttctgct ggaagaaatg 1440acagtttttc gggaaccgac tgctatttca gcaatggagc tttccactaa gcagcaacaa 1500ctatatattg gttcaacggc tggggttgcc cagctccctt tacaccggtg tgatatttac 1560gggaaagcgt gtgctgagtg ttgcctcgcc cgagaccctt actgtgcttg ggatggttct 1620gcatgttctc gctattttcc cactgcaaag agacgcacaa gacgacaaga tataagaaat 1680ggagacccac tgactcactg ttcagactta caccatgata atcaccatgg ccacagccct 1740gaagagagaa tcatctatgg tgtagagaat agtagcacat ttttggaatg cagtccgaag 1800tcgcagagag cgctggtcta ttggcaattc cagaggcgaa atgaagagcg aaaagaagag 1860atcagagtgg atgatcatat catcaggaca gatcaaggcc ttctgctacg tagtctacaa 1920cagaaggatt caggcaatta cctctgccat gcggtggaac atgggttcat acaaactctt 1980cttaaggtaa ccctggaagt cattgacaca gagcatttgg aagaacttct tcataaagat 2040gatgatggag atggctctaa gaccaaagaa atgtccaata gcatgacacc tagccagaag 2100gtctggtaca gagacttcat gcagctcatc aaccacccca atctcaacac aatggatgag 2160ttctgtgaac aagtttggaa aagggaccga aaacaacgtc ggcaaaggcc aggacatacc 2220ccagggaaca gtaacaaatg gaagcactta caagaaaata agaaaggtag aaacaggagg 2280acccacgaat ttgagagggc acccaggagt gtcggatccg agcccaaatc ttgtgacaaa 2340actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttcctc 2400ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 2460gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 2520gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 2580gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 2640gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 2700ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag 2760gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 2820agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 2880tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 2940ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 3000ctgtctccgg gtaaatga 30181820DNAArtificialPCT primer 18tgaaaggaga aggcttgtga 201921DNAArtificialPCR primer 19taatgggatg agtatggggc a 212020DNAArtificialPCR primer 20cccaactttc caaaccctct 202121DNAArtificialPCR primer 21accatgatct catgtggagg a 212221DNAArtificialPCR primer 22gcctcaagga tgacttaagc a 212321DNAArtificialPCR primer 23agattgcaca atgtgacgtc g 212435DNAArtificialCloning primer 24aagcttgcca cccatgaaag ccatgccctg gaact 352528DNAArtificialCloning primer 25ggatccgtcc ggggcaatgt acaccatc 282628DNAArtificialCloning primer 26ggatccaagg acggttctca gctgtcta 282721DNAArtificialSequencing primer 27gattgcctgt gggagcctgt a 212822DNAArtificialSequencing primer 28tatgtagagg tgcccattgg ct 222920DNAArtificialSequencing primer 29tatgagacgg tgcaggtggt 203018DNAArtificialSequencing primer 30ttcgccagca ccagcttt 183119DNAArtificialSequencing primer 31tgacccagac ttcgcatgt 193220DNAArtificialSequencing primer 32aacctgaatg ccggaagcaa 203320DNAArtificialSequencing primer 33gtgtttgagg cctttggtcc 203421DNAArtificialSequencing primer 34ggattccgtt cctggactat a 213520DNAArtificialSequencing primer 35ttgagcgagg acaagctcat 203621DNAArtificialSequencing primer 36tgacctggag agtggagtca a 213719DNAArtificialSequencing primer 37gcctctgcca tgaaagcca 193823DNAArtificialSequencing primer 38aaggacggtt ctcagctgtc taa 233920DNAArtificialRT-PCR primer 39gcctgcagaa gaaggattca 204020DNAArtificialRT-PCR primer 40tcaggttggg gtggttaatg 204120DNAArtificialRT-PCR primer 41aatggccaga tgcccttatg 204222DNAArtificialRT-PCR primer 42ccgagtagag ttttccattg ca 224321DNAArtificialRT-PCR primer 43ctcctgaagc tgttgcgtta c 214424DNAArtificialRT-PCR primer 44tactttaccc agctcgctca ctac 244526DNAArtificialRT-PCR primer 45tcttctttac gaaagttgga cttgtc 264622DNAArtificialRT-PCR primer 46ttgccaattg tctggaaaca cc 224720DNAArtificialRT-PCR primer 47atggcatggc ttacaccacc 204821DNAArtificialRT-PCR primer 48gaggccaatt ttgtctccac a 214922DNAArtificialRT-PCR primer 49ctggagttca gaggcaacca tt 225023DNAArtificialRT-PCR primer 50gttatcaccg gctctccata gaa 235121DNAArtificialRT-PCR primer 51agctgaacat gaacggcatc t 215221DNAArtificialRT-PCR primer 52tgagcgtgta cttgttgagc g 215319DNAArtificialRT-PCR primer 53atgagtggtg gttgcaggc 195425DNAArtificialRT-PCR primer 54tgacctttca aatgcagtag attca 255536DNAArtificialPCR primer 55aagcttgcca cccatgggct ggttaactag gattgt 365628DNAArtificialPCR primer 56ggatccgaca ctcctgggtg ccctctca 285722DNAArtificialSequencing primer 57tgggaagagt ccatatgacc ct 225820DNAArtificialSequencing primer 58attttcaagg gatcagccgt 205920DNAArtificialSequencing primer 59ctatatattg gttcaacggc 206017PRTArtificialOVA peptide sequence 60Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile Asn Glu Ala Gly 1 5 10 15 Arg

Claims

1. A method of treating an immune-related and/or inflammatory disorder in a subject, comprising administering to the subject an effective amount of an inhibitor of plexin-A4 activity, whereby the plexin-A4 activity in said subject is reduced, thereby treating the immune-related disorder and/or inflammatory disorder.

2. A method of reducing cytokine production in a subject, comprising administering to the subject an effective amount of an inhibitor of plexin-A4 activity, thereby reducing the cytokine production the subject.

3. The method of claim 1, wherein the inhibitor of plexin-A4 activity is selected from the group consisting of a plexin-A4 fusion protein, a plexin-A4 antibody or an active fragment thereof, or any combination thereof.

4. The method of claim 3, wherein the plexin-A4 antibody or active fragment thereof is a monoclonal antibody or is derived from a monoclonal antibody.

5. The method of claim 3, wherein the plexin-A4 fusion protein comprises the extracellular domain of plexin-A4 and the Fc region of IgG.

6. The method of claim 5, wherein the extracellular domain of plexin-A4 and the IgG1 Fc region is human.

7. The method of claim 5, wherein the extracellular domain of plexin-A4 comprises the amino acid sequence of SEQ ID NO:7 and the IgG Fc region comprises the amino acid sequence of SEQ ID NO:8.

8. The method of claim 3, wherein the plexin-A4 fusion protein comprises the amino acid sequence of SEQ ID NO:6.

9. The method of claim 1, wherein the immune-related and/or inflammatory disorder is selected from the group consisting of sepsis, arthritis, hepatitis, systemic lupus erythematosus (SLE), multiple sclerosis, Guillain-Barre syndrome, Alzheimer's disease, colitis, psoriasis, contact hypersensitivity, retinitis, uveitis, malignancies, systemic lupus erythematosis, asthma, myocarditis, hepatitis, kidney diseases, diabetes, obesity, cardiovascular diseases, and inflammatory bowel disease, or any combination thereof.

10. The method of claim 3, wherein the inhibitor is administered subcutaneously, intramuscularly, intraperitoneally, topically, intravenously, and any combination thereof.

11. (canceled)

12. A method of treating an immune-related and/or inflammatory disorder in a subject, comprising administering to the subject an effective amount of an inhibitor of semaphorin-3A (Sema3A) activity, whereby the Sema3A activity in said subject is reduced, thereby treating the immune-related disorder and/or inflammatory disorder.

13. A method of reducing cytokine production in a subject, comprising administering to the subject an effective amount of an inhibitor of Sema3A activity, thereby reducing the cytokine production the subject.

14. The method of claim 12, wherein the inhibitor of Sema3A activity is selected from the group consisting of a Sema3A fusion protein, a Sema3A antibody or an active fragment thereof, or any combination thereof.

15. The method of claim 14, wherein the plexin-A4 antibody or active fragment thereof is a monoclonal antibody or is derived from a monoclonal antibody.

16. The method of claim 14, wherein the Sema3A fusion protein comprises the amino acid sequence encoding Sema3A and the Fe region of IgG.

17. The method of claim 16, wherein the amino acid sequence encoding Sema3A and the IgG1 Fc region is human.

18. The method of claim 16, wherein the amino acid sequence encoding Sema3A comprises the amino acid sequence of SEQ ID NO:13 and the IgG Fe region comprises the amino acid sequence of SEQ ID NO:8.

19. The method of claim 14, wherein the Sema3A fusion protein comprises the amino acid sequence of SEQ ID NO:16.

20. The method of claim 12, wherein the immune-related and/or inflammatory disorder is selected from the group consisting of sepsis, arthritis, hepatitis, systemic lupus erythematosus (SLE), multiple sclerosis, Guillain-Barre syndrome, Alzheimer's disease, colitis, psoriasis, contact hypersensitivity, retinitis, uveitis, malignancies, systemic lupus erythematosis, asthma, myocarditis, hepatitis, kidney diseases, diabetes, obesity, cardiovascular diseases, and inflammatory bowel disease, or any combination thereof.

21. The method of claim 14, wherein the inhibitor is administered subcutaneously, intramuscularly, intraperitoneally, topically, intravenously, and any combination thereof.

22. (canceled)

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