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WO2006051333A2 - Proteines contenant des motifs repetes riches en leucine (lrr) - Google Patents

Proteines contenant des motifs repetes riches en leucine (lrr) Download PDF

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Publication number
WO2006051333A2
WO2006051333A2 PCT/GB2005/004390 GB2005004390W WO2006051333A2 WO 2006051333 A2 WO2006051333 A2 WO 2006051333A2 GB 2005004390 W GB2005004390 W GB 2005004390W WO 2006051333 A2 WO2006051333 A2 WO 2006051333A2
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seq
polypeptide
nucleic acid
disease
insp
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WO2006051333A3 (fr
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David Michalovich
Simon John White
Melanie Yorke
Kinsey Maundrell
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Ares Trading SA
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Ares Trading SA
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Priority to AU2005303536A priority Critical patent/AU2005303536A1/en
Priority to CA002586486A priority patent/CA2586486A1/fr
Priority to EP05803576A priority patent/EP1814903A2/fr
Priority to JP2007540722A priority patent/JP2008519593A/ja
Publication of WO2006051333A2 publication Critical patent/WO2006051333A2/fr
Publication of WO2006051333A3 publication Critical patent/WO2006051333A3/fr
Priority to IL182960A priority patent/IL182960A0/en
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/06—Antiglaucoma agents or miotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • This invention relates to proteins, termed INSP 168, INSP168-SV1, INSP 149 and INSP 169, herein identified as leucine-rich repeat (LRR) motif containing proteins, and to the use of these proteins and nucleic acid sequences from the encoding gene in the diagnosis, prevention and treatment of disease.
  • LRR leucine-rich repeat
  • bioinformatics tools increase in potency and in accuracy, these tools are rapidly replacing the conventional techniques of biochemical characterisation. Indeed, the advanced bioinformatics tools used in identifying the present invention are now capable of outputting results in which a high degree of confidence can be placed.
  • the ability of cells to make and secrete extracellular proteins is central to many biological processes. Enzymes, growth factors, extracellular matrix proteins and signalling molecules are all secreted by cells. This is through fusion of a secretory vesicle with the plasma membrane. In most cases, but not all, proteins are directed to the endoplasmic reticulum and into secretory vesicles by a signal peptide. Signal peptides are cis-acting sequences that affect the transport of polypeptide chains from the cytoplasm to a membrane bound compartment such as a secretory vesicle. Polypeptides that are targeted to the secretory vesicles are either secreted into the extracellular matrix or are retained in the plasma membrane.
  • the polypeptides that are retained in the plasma membrane will have one or more transmembrane domains.
  • Examples of signal peptide containing proteins that play a central role in the functioning of a cell are cytokines, hormones, extracellular matrix proteins, adhesion molecules, receptors, proteases, and growth and differentiation factors.
  • the photoreceptor-associated leucine-rich repeat (LRR) protein (abbreviated to PAL) is a membrane glycoprotein that is specifically expressed in the photoreceptor cells of the retina (Gomi et al, J. Neuroscience, 2000, 20(9):3206-3213).
  • PAL protein contains an LRR motif, an Ig C2-like domain and a f ⁇ bronectin type Ill-like domain, all within its extracellular region.
  • the LRR domain of PAL contains five contiguous LRRs. This combination of the three types of domain described above was identified as a new class of transmembrane protein, although some previously known proteins contain two of these three domains (e.g. Trk and NCAM) (Gomi et al, J. Neuroscience, 2000, 20(9):3206-3213).
  • PAL mRNA The abundance of PAL mRNA was observed to increase over the time course of development of the rat retina.
  • Northern blotting experiments revealed that the PAL mRNA was specific to the photoreceptor cells within the retina.
  • Western blotting and immunoprecipitation experiments with a PAL-specific polyclonal antibody showed that PAL forms a strong homodimer structure that is resistant to SDS and high temperature (Gomi et al, J. Neuroscience, 2000, 20(9):3206-3213).
  • a human homolog of PAL was also identified and was mapped to chromosome 10q23.2-23.3 by fluorescence in situ hybridisation (FISH).
  • FISH fluorescence in situ hybridisation
  • PAL may act as a receptor for a certain trophic factor or for an adhesion molecule participating in morphogenesis.
  • the human PAL protein was therefore considered to be a potential candidate disease gene for inherited retinal disorders (Gomi et al, J. Neuroscience, 2000, 20(9):3206-3213).
  • Other known retina-specific genes include rhodopsin, transducin, cGMP-gated ion channels, peripherin/rds and rom-1.
  • mutations in rhodopsin or peripherin/rds contribute to autosomal dominant retinitis pigmentosa.
  • Fibronectin Type III (FnIII) protein domains are formed by 80-100 amino acids included in several multimodular proteins, mostly associated to extracellular matrix such as tenascins (Joester A and Faissner A, Matrix Biol. 2001, 20:13-22), or Titins (Skeie GO, Cell MoI Life Sci. 2000, 57:1570-6) or to receptor proteins such as insulin receptor protein family (Marino-Buslje C et al, FEBS Lett. 1998, 441 :331-6).
  • FnIII modules share low sequence homology. Conversely, the sequence homology for the same FnIII module across multiple species is notably higher, suggesting that sequence variability is functionally significant.
  • Prolines are of particular importance since prevent aggregation in multi-modular proteins containing this domain (Craig D et al, Structure 2004, 12:21-30; Craig D et al, Proc Natl Acad Sci U S A. 2001, 98(10):5590-5; Cota E et al, J MoI Biol. 2001, 305:1185-94; Cota E et al, J MoI Biol. 2000, 302:713-25; Steward A etal, J MoI Biol. 2002, 318:935-40).
  • the paradigm of this protein module is human Fibronectin, a 2386-amino acid glycoprotein of the extracellular matrix containing several protein modules, usually categorized in three types: FnI, FnII, and FnIII (or Fl, F2, or F3; Potts JR and
  • Fibronectin circulates in a soluble form in the plasma and is also found in an insoluble, multimeric form within the extracellular matrix at appropriate sites. The formation and the degradation of these insoluble fibrils is a dynamic, cell-dependent process that is mediated by a series of events involving the actin cytoskeleton and integrin receptors. Fibronectin fibrils can bind the surfaces of mammalian and bacterial cells and various molecules including collagen, fibrin, heparin, DNA, and actin. Fibronectin is involved in cell adhesion/contractility/motility, opsonization, wound healing, and formation of fibrotic aggregates.
  • isolated fibronectin domains of extracellular matrix proteins can modulate various biological and physiological responses, such as the neuronal regeneration, hippocampal learning and synaptic plasticity (Meiners S and Mercado ML, MoI Neurobiol. 2003, 27:177-96; Strekalova T et al, MoI Cell Neurosci. 2002, 21:173-87), osteoblast adhesion, proliferation and differentiation (Kim TI et al, Biotechnol Lett. 2003, 25:2007-11), tissue degradation, inflammation and tumor progression (Labat-Robert J, Ageing Res Rev. 2004, 3:233-47).
  • Fragments or splicing variants of FnIII domain-containing proteins may become target for antibodies and other proteins blocking them having important therapeutic or diagnostic applications, such as cancer (Ebbinghaus C et al, Curr Pliarm Des. 2004, 10:1537-49), inflammatory arthritis (Barilla ML and Carsons SE, Semin Arthritis Rheum. 2000, 29:252-65), or organ transplantation (Coito AJ et al, Dev Immunol. 2000;7:239-48).
  • the LRR motif is a relatively short motif of around 22-28 residues, and is found in a variety of cytoplasmic, membrane and extracellular proteins. Proteins containing LRRs are associated with a very wide range of biological functions, although all are thought to be involved in protein-protein interaction or cell adhesion.
  • the LRR motif is a repetitive motif made up of several copies of the sequence LxxLxxLxLxxNxLxxL xxxxFxx. LRRs are often flanked by cysteine-rich repeat regions, an N-terminal LRR motif or a leucine- rich repeat C-terminal domain (LRRCT).
  • the immunoglobulin (Ig) domain is a well characterised domain present in hundreds of proteins of varying functions.
  • the basic Ig domain structure is a tetramer of two light chains and two heavy chains linked by disulphide bonds.
  • Immunoglobulin domain-containing cell surface recognition molecules have been shown to play a role in diverse physiological functions, many of which can play a role in disease processes. Alteration of their activity is a means to alter the disease phenotype and as such identification of novel immunoglobulin domain-containing cell surface recognition molecules is highly relevant as they may play a role in many diseases, particularly inflammatory disease, oncology, and cardiovascular disease.
  • Immunoglobulin domain-containing cell surface recognition molecules are involved in a range of biological processes, including: embryogenesis, maintenance of tissue integrity, leukocyte extravasation/inflammation, oncogenesis, angiogenesis, bone resorption, neurological dysfunction, thrombogenesis, and invasion/adherence of bacterial pathogens to the host cell.
  • Immunoglobulin domain containing cell surface recognition molecules are involved in virtually every aspect of biology from embryogenesis to apoptosis. They are essential to the structural integrity and homeostatic functioning of most tissues. It is therefore not surprising that defects in immunoglobulin domain containing cell surface recognition molecules cause disease and that many diseases involve modulation of immunoglobulin domain containing cell surface recognition molecule function.
  • Immunoglobulin domain-containing cell surface recognition molecules are thus known to play a role in diverse physiological functions, many of which can play a role in disease processes. Alteration of their activity is a means to alter the disease phenotype and as such identification of novel immunoglobulin domain-containing cell surface recognition molecules is highly relevant as they may play a role in many diseases, particularly immunology, inflammatory disease, oncology, cardiovascular disease, central nervous system disorders and infection.
  • cell surface recognition molecules including those containing LRRs, Ig domains or fibronectin type 3 domains, have been shown to play a role in diverse physiological functions, many of which can play a role in disease processes. Alteration of their activity is a means to alter the disease phenotype and as such identification of novel adhesion molecules is highly relevant as they may play a role in many diseases, particularly inflammatory disease, oncology, cardiovascular disease and bacterial infection.
  • identification of further retina-specific cell surface recognition molecules including paralogs of the human PAL protein (which will contain LRRs, Ig domains and fibronectin type 3 domains) is of great importance in the ongoing investigation of retinal developments and retinal pathologies. Their identification will allow the development of new methods for the treatment and diagnosis of retinal diseases and disorders. Accordingly, there remains a need for the identification of such proteins to enable new drugs to be developed for the treatment and prevention of human disease.
  • the invention is based on the discovery that the INSP168, INSP168-SV1, INSP149 and INSP 169 proteins are splice variants of a leucine-rich repeat (LRR) motif containing sequence with similarity to PAL (SwissProt Ace. Code PALP_HUMAN) and to a no go receptor homolog (SwissProt Ace. Code Q6X814).
  • LRR leucine-rich repeat
  • the invention is based on the finding that polypeptides of the present invention are PAL-like and/or nogo-receptor like molecules.
  • polypeptide which polypeptide:
  • (ii) is a fragment thereof which functions as a biologically active polypeptide and/or has an antigenic determinant in common with the polypeptides of (i); or
  • a polypeptide according to part (i) of the first aspect of the invention may comprise the amino acid sequence as recited in SEQ ID NO:4 (mature INSP149), SEQ ID NO:8 (mature INSP168-SV1) or SEQ ID NO:10 (mature INSP169).
  • a polypeptide according to part (i) of the first aspect of the invention may comprise the amino acid sequence as recited in SEQ ID NO:30 (full length INSP 168), SEQ ID NO:32 (full length INSP 149), SEQ ID NO:34 (foil length INSP 168-SV 1), SEQ ID NO:36 (foil length INSP169) and SEQ ID NO:67 (INSP169 cloned extracellular region).
  • a polypeptide according to part (i) of the first aspect of the invention may comprise the extracellular portion of the amino acid sequence as recited in SEQ ID NO:4 (mature INSP149) or SEQ ID NO:10 (mature INSP169).
  • a polypeptide according to part (i) of the first aspect of the invention may comprise the amino acid sequence as recited in SEQ ID NO:6 (INSP149 extracellular region) or SEQ ID NO: 12 (INSP 169 extracellular region).
  • a polypeptide according to part (i) of the first aspect of the invention may comprise the amino acid sequence as recited in SEQ ID NO:67 (INSP 169 cloned extracellular region).
  • the cloned extracellular region of INSP169 differs from the predicted extracellular region of INSP169 at amino acid 524 (see Example 7 and Figure 8).
  • the INSP 168, INSP 168-SV1 , INSP 149 and INSP 169 proteins each contain a leucine- rich repeat motif.
  • the amino acid sequence of the leucine-rich repeat motif present in the INSP168, INSP168-SV1, INSP149 and INSP169 proteins is recited in SEQ ID NO:14.
  • preferred fragments of the INSP168, INSP168-SV1, INSP149 and INSP 169 proteins are fragments that comprise or consist of the amino acid sequence as recited in SEQ ID NO: 14 (LRR motif).
  • a polypeptide according to part (i) of the first aspect of the invention may comprise the amino acid sequence as recited in SEQ ID NO:2 (mature INSP 168), SEQ ID NO:4 (mature INSP149), SEQ ID NO:6 (INSP149 extracellular region), SEQ ID NO:8 (mature INSP168-SV1), SEQ ID NO:10 (mature INSPl 69), SEQ ID NO:12 (INSP169 extracellular region), SEQ ID NO: 14 (LRR motif), SEQ ID NO:30 (foil length INSP168), SEQ ID NO:32 (foil length INSPl 49), SEQ ID NO:34 (foil length INSP168-SV1), SEQ ID NO:36 (foil length INSP169) or SEQ ID NO:67 (INSP169 cloned extracellular region) and a histidine tag.
  • the histidine tag is located at the C-terminus of the polypeptide.
  • the histidine tag comprises between 1 and 10 histidine residues (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues). More preferably, the histidine tag comprises 6 residues.
  • a polypeptide according to part (i) of the first aspect of the invention may comprise the amino acid sequence as recited in SEQ ID NO:16 (his tag mature INSP168), SEQ ID NO:18 (his tag mature INSPl 49), SEQ ID NO:20 (his tag INSP 149 extracellular region), SEQ ID NO:22 (his tag mature INSP168-SV1), SEQ ID NO:24 (his tag mature INSPl 69), SEQ ID NO:26 (his tag INSP169 extracellular region), SEQ ID NO:28 (his tag LRR motif), SEQ ID NO:38 (his tag full length INSP 168), SEQ ID NO:40 (his tag full length INSP 149), SEQ ID NO:42 (his tag full length INSP 168-S Vl) or SEQ ID NO:44 (his tag full length INSP 169).
  • the first aspect of the present invention provides a polypeptide which comprises or consists of the amino acid sequence as recited in SEQ ID NO:2 (mature INSP 168), SEQ ID NO:4 (mature INSP 149), SEQ ID NO:6 (INSP 149 extracellular region), SEQ ID NO:8 (mature INSP168-SV1), SEQ ID NO:10 (mature INSP169), SEQ ID NO:12 (INSP169 extracellular region), SEQ ID NO:14 (LRR motif), SEQ ID NO:16 (his tag mature INSP168), SEQ ID NO:18 (his tag mature INSP149), SEQ ID NO:20 (his tag INSP149 extracellular region), SEQ ID NO:22 (his tag mature INSP168-SV1), SEQ ID NO:24 (his tag mature INSP169), SEQ ID NO:26 (his tag INSP169 extracellular region), SEQ ID NO:28 (his tag LRR motif), SEQ ID NO:30 (full length INSP168), SEQ ID NO:32 (full length IN
  • the first aspect of the present invention also provides a polypeptide which is a fragment of such a polypeptide and which functions as a biologically active polypeptide and/or has an antigenic determinant in common with such a polypeptide or which is a functional equivalent of such a polypeptide.
  • the INSP 168 polypeptides and "an INSP 168 polypeptide” as used herein include polypeptides comprising or consisting of the amino acid sequence as recited in SEQ TD NO:2 (mature INSPl 68), such as polypeptides comprising or consisting of the amino acid sequence as recited in SEQ ID NO:4 (mature INSP149), SEQ ID NO:6 (INSP149 extracellular region), SEQ ID NO:8 (mature INSP168-SV1), SEQ ID NO:10 (mature INSP169), SEQ ID NO:12 (INSP169 extracellular region), SEQ ID NO:16 (his tag mature INSP168), SEQ ID NO:18 (his tag mature INSP149), SEQ ID NO:20 (his tag INSP149 extracellular region), SEQ ID NO:22 (his tag mature INSP168-SV1), SEQ ID NO:24 (his tag mature INSP169), SEQ ID NO:26 (his tag INSP169 extracellular region), SEQ ID NO:30 (full
  • INSP 149, INSP 168 and INSP 169 were a set of predictions representing splice variants of a leucine rich repeat-containing sequence with similarity to the retina- specific protein PAL.
  • INSP149 was a prediction for a 595 amino acid (1785 bp) ORF encoded in 5 exons.
  • INSP 168 was a prediction for a 197 amino acid (591 bp) ORF encoded in 3 exons.
  • INSP 169 was a prediction for a 679 amino acid (2037 bp) ORF encoded in 4 exons.
  • INSP 149 and INSP 169 were predicted to be type I transmembrane proteins comprising a leucine-rich repeat motif, an immunoglobulin domain and a fibronectin type 3 domain.
  • the INSP 169 polypeptide is a splice variant of the INSP 149 polypeptide that is identical to INSP 149 except for the longer final exon, which subsumes the final two exons of INSP149.
  • the longer sequence encoded by INSP169 has the same domain organisation as INSP 149 but has a low complexity insert between the Ig and FN III domains.
  • INSP 168 was essentially a truncated splice variant of INSP 149 and FNSP 169 and was predicted to represent a secreted protein.
  • the INSP168 polypeptide is a splice variant of the FNSP149 polypeptide that comprises a stop codon at the start of its third exon.
  • the INSP 168 polypeptide lacks the transmembrane region found in exon 5 of INSP 149. All 3 of the predictions contained 4 leucine rich repeat regions in the N-terminal portion (SEQ ID NO:14). INSP149 and INSP169 also contained an immunoglobulin domain and a fibronectin type III domain in the predicted extracellular regions. An alignment of the 3 predicted amino acid sequences is shown in Figure 1. As noted above, a signal peptide was predicted spanning from residues 1 to 19.
  • the open reading frame (ORF) of the INSP 168 prediction has been cloned using a pair of PCR primers (see Figures 2 and 3).
  • the primer pair was tested on selected cDNA libraries derived from brain and retina and on brain and eye cDNA templates using Platinum® Taq DNA Polymerase High Fidelity (HiFi).
  • HiFi Platinum® Taq DNA Polymerase High Fidelity
  • PCR products were cloned and sequenced and a clone was identified, amplified from brain cDNA, which contained the expected INSP 168 ORF.
  • a second clone was identified, also amplified from brain cDNA, which contained a splice variant of the INSP 168 ORP.
  • This clone contained a 32 amino acid insertion towards the 3' end of the INSPl 68 ORP which represented a new exon 3.
  • the insertion also caused a frameshift such that the new exon 4 contained an extra 6 amino acids compared with the original INSP 168 exon 3.
  • This clone was called INSP168-SV1, and is also shown in the Figure 1 alignment.
  • the INSP 168 polypeptides are structurally related to the Retinal Specific Protein PAL (SwissProt Ace. Code PALP_HUMAN) and to a nogo receptor homolog (SwissProt Ace. Code Q6X814).
  • An amino acid alignment between the INSP 168, INSP168-SV1, INSP 149 and INSP 169 polypeptides and PAL is shown in Figure 5, and the schematic representation of domains is shown in Figure 6.
  • the cloned extracellular region of INSP 169 differs from the predicted extracellular region of INSP 169 at amino acid 524 (see Example 7 and Figure 8).
  • PAL may be implicated in diseases of the retina, retinal pigment epithelium (RPE), and choroids (see for example JP2001128686). These include ocular neovascularization, ocular inflammation and retinal degenerations.
  • these disease states include diabetic retinopathy, chronic glaucoma, retinal detachment, sickle cell retinopathy, senile macular degeneration, retinal neovascularization, subretinal neovascularization; rubeosis ulceris inflammatory diseases, chronic posterior and pan uveitis, neoplasms, retinoblastoma, pseudoglioma, neovascular glaucoma; neovascularization resulting following a combined vitrectomy and lensectomy, vascular diseases retinal ischemia, choroidal vascular insufficiency, choroidal thrombosis, neovascularization of the optic nerve, diabetic macular edema, cystoid macular edema, retinitis pigmentosa, retinal vein occlusion, proliferative vitreoretinopathy, angioid streak, and retinal artery occlusion, and, neovascularization due to penetration of the
  • Additional relevant diseases include the neuropathies, such as Leber's, idiopathic, drug-induced, optic, and ischemic neropathies.
  • Nogo receptor-like proteins could be major inhibitors of CNS neuronal regeneration (Schwab ME. Curr Opin Neurobiol. 2004 Feb;14(l):l 18-24; Teng et al. J Neurochem. 2004 May;89(4):801-6).
  • Animals treated with antibodies targeted to Nogo-A always showed a higher degree of recovery in various behavioural tests (e.g. IN-I Fab' fragments or new purified IgGs against Nogo-A).
  • a Nogo-66 antagonistic peptide (NEP 1-40) effected significantly axon growth of the corticospinal tract and improved functional recovery in rats inflicted with mid-thoracic spinal cord hemisections.
  • Subcutaneous administration of NEP 1-40 in spinal cord lesioned animals resulted in extensive growth of corticospinal axons, sprouting of serotonergic fibres, synapse formation and enhanced locomotor recovery.
  • Soluble Fc fusion proteins of the Nogo receptor subunit NgR which blocks Nogo, significantly reduce the inhibitory activity of myelin. Similar results were obtained after Nogo gene deletions and blockade of the downstream messengers Rho-A and ROCK in animal models.
  • the leucine-rich repeat domain of SLIT proteins is sufficient for guiding both axon projection and neuronal migration in vitro (the LRR region of SLIT is structurally related to the LRR region of INSP 168, INSP168-SV1, INSP 149 and INSP 169). As such, the LRR region of INSP168, INSP168-SV1, INSP149 and INSP169 or fragments containing the LRR region might be useful in the treatment of the diseases listed herein.
  • SLIT-like proteins are thought to act as molecular guidance cue in cellular migration, and function appears to be mediated by interaction with roundabaout homolog receptors (bind ROBOl and ROBO2 with high affinity).
  • SLIT is involved in axonal navigation at the ventral midline of the neural tube and projection of axons to different regions.
  • spinal chord development SLIT may play a role in guiding commissural axons once they reached the floor plate by modulating the response to netrin.
  • SLIT may be implicated in spinal chord midline post-crossing axon repulsion.
  • SLIT appears to function as repellent for retinal ganglion axons by providing a repulsion that directs these axons along their appropriate paths prior to, and after passage through, the optic chiasm.
  • SLIT collapses and repels retinal ganglion cell growth cones.
  • SLIT seems to play a role in branching and arborization of CNS sensory axons, and in neuronal cell migration, hi vitro, Slit homolog 2 protein N- product, but not Slit homolog 2 protein C-product, repells olfactory bulb (OB) but not dorsal root ganglia (DRG) axons, induces OB growth cones collapse and induces branching of DRG axons.
  • SLIT seems to be involved in regulating leukocyte migration.
  • INSP168, INSP168-SV1, INSP149 and INSP169 and other INSP168 polypeptides and/or fragments and functional equivalents thereof can be useful in the diagnosis and/or treatment of diseases for which other (e.g. above mentioned PAL- and Nogo receptor-like proteins) structurally related proteins demonstrate therapeutic activity.
  • polypeptides of the invention consisting of and/or comprising of the mature (lacking a signal peptide) forms and/or cleaved forms of INSP 168, INSP 168- SVl and/or mature soluble forms of INSP 149 and/or INSP 169, and/or agonists thereof are useful for the diagnosis and/or treatment of diseases.
  • the soluble forms of INSP 149 and/or INSP 169 consist of the mature extracellular part and/or cleaved fragments of INSP 149 and/or INSP 169.
  • Antagonists of membrane bound INSP 149 and/or INSP 169, for example antibodies, are useful for the diagnosis and/or treatment of diseases.
  • the INSP168, INSP168-SV1, INSP149 and INSP169 polypeptides maybe implicated in diseases of the retina, spinal cord injuries (e.g. paraplegia) and neurodegenerative disorders. These include disorders of the central nervous system as well as disorders of the peripheral nervous system. Neurodegenerative disorders include, but are not limited to, brain injuries, cerebrovascular diseases and their consequences, Parkinson's disease, corticobasal degeneration, motor neuron disease (including amyotrophic lateral sclerosis, ALS), multiple sclerosis, traumatic brain injury, stroke, post-stroke, post- traumatic brain injury, and small-vessel cerebrovascular disease. Dementias, such as Alzheimer's disease, vascular dementia, dementia with Lewy bodies, frontotemporal dementia and Parkinsonism, frontotemporal dementias (including
  • the INSP168, INSP168-SV1, INSP149 and INSP169 polypeptides may be implicated in diseases of the retina, retinal pigment epithelium (RPE), and choroids; ocular neovascularization, ocular inflammation and retinal degenerations; diabetic retinopathy, chronic glaucoma, retinal detachment, sickle cell retinopathy, senile macular degeneration, retinal neovascularization, subretinaL neovascularization; rubeosis ulceris inflammatory diseases, chronic posterior and pan uveitis, neoplasms, retinoblastoma, pseudoglioma, neovascular glaucoma; neovascularization resulting following a combined vitrectomy and lensectomy, vascular diseases retinal ischemia, choroidal vascular insufficiency, choroidal thrombosis, neovascularization of the optic nerve, diabetic macular edema, cystoi
  • Neuro-inflammation is a common feature of several neurological diseases, traumatic situations (at central or peripheral level), stroke (brain, heart, renal), or infectious diseases (mediated by viral agents such as HIV or bacterial agents such as meningitis), leading to an excessive inflammatory response in central nervous system.
  • Many stimuli originated by neuronal or oligodendroglial cells suffering due to these various conditions, can trigger neuro-inflammation.
  • astrocytes can secrete various chemokines and cytokines, inducing a recruitment of additional leukocytes that in their turn will further stimulate astrocytes, leading to an exacerbated response.
  • MS multiple sclerosis
  • SMA spinal muscular atrophies
  • AD Alzheimer's disease
  • PD Parkinson's disease
  • HD Huntington's disease
  • ALS amylotrophic lateral sclerosis
  • axon regeneration and plasticity is central to the pathophysiology of a range of neurological disorders, including stroke, head trauma, multiple sclerosis, and neurodegenerative disease.
  • the polypeptides of the present invention are implicated in cancer through EGFR inhibition.
  • the cancer is lung cancer.
  • the biological properties of the INSP 168, INSP168-SV1, INSP 149 and INSP 169 polypeptides related to neuroprotection, maintenance of axonal integrity, myelination and re-/generation of myelin producing cells, can be tested in various assays involving cell lines.
  • the neuroimmunodulatory effects of a compound can be evaluated in U373, a human astroglioma cell line in which the nuclear translocation of specific regulatory proteins involved in cytokine/chemokine expression can be quantified (Le Roy E et al, J Virol. 1999, 73: 6582-9; Jin Y et al, J Infect Dis. 1998, 177: 1629-1638; Acevedo-Duncan M et al, Cell Growth Differ. 1995, 6: 1353-1365).
  • a polypeptide according to the first aspect of the invention may thus function as an activator of cell proliferation, as a neuromodulator (neuroimmunomodulator), as a modulator of the inflammatory response in the CNS 5 as a regulator of astrocyte proliferation or as a regulator of axon projection, neuronal migration or leukocyte recruitment or migration.
  • a neuromodulator neuroimmunomodulator
  • the activity of a polypeptide of the present invention can be confirmed in at least one of the following assays: a) in the maintenance of neuronal cell survival, for example in the regeneration of injured adult neurons, or b) in the modulation of neurite growth in animal models of spinal cord injury (Fouad et al, Brain.Res.Rev. 2001, Vol. 36, pp.204-212; Bareyre et al, J.Neurosci. 2002, Vol.22, ⁇ p.7097-7110; GrandPre et al Nature 2002, Vol.417, pp.547-551; Li and Strittmatter, J.Neurosci. 2003, Vol.23, ⁇ .4219- 4227; Liebscher et al.
  • axonal growth of many neuron types for example of corticospinal tract (CST) axons, corticofugal, retinal, superior cervical ganglion, spinal or hippocampal neurons, dorsal column axons, for example, in the modulation of axonal plasticity of unlesioned cortical neurons (with enhanced behavioural recovery), or d) in the translocation of Stat2 as described in Example 6, or e) in the up-regulation of growth factors or growth-related proteins, for example of Brain-derived neurotrophic factor (BDNF), Vascular Endothelial Growth Factor (VEGF) and/or Growth-associated protein 43 (GAP-43), or f) in the regeneration of nerve fibers, for example of raphespinal, rubrospinal or corticospinal fibers, or in the regeneration of injured optic nerve fibers (for example in an optic nerve crush model), or g) in the improvement of locomotor function, or
  • CST corticospinal tract
  • NgR Nogo receptor
  • MAG myelin-associated glycoprotein
  • Omgp oligodendrocyte myelin glycoprotein
  • polypeptides of the invention comprising and/or consisting of the LRR domain can at least display activity in one of the above-mentioned assays.
  • Polypeptides of the invention consisting of and/or comprising of the mature (lacking a signal peptide) forms and/or cleaved forms of INSP168, INSP168-SV1 and/or mature soluble forms of INSP 149 and/or INSP 169, and/or agonists thereof are preferably used in the above-mentioned assays.
  • the soluble forms of INSP 149 and/or INSP 169 consist of the mature extracellular part and/or cleaved fragments of INSP 149 and/or INSP 169.
  • antagonists of membrane bound INSP 149 and/or INSP 169, for example antibodies can be used in the above-mentioned assays.
  • Preferred epitopes of the polypeptides of the present invention can be detected by "affinity fingerprinting" as described in Schimmele and Pl ⁇ ckthun (Journal of Molecular Biology 2005, Vol.352, Issue 1, ⁇ .229-241).
  • Polypeptides of the present invention may undergo cleavage by metalloendopeptidase and/or proprotein convertases such as zinc metalloproteinases, N-Arginine dibasic (NDR) convertase or subtilisin-like proprotein convertases.
  • NDR cleavage sites and PCSK cleavage sites have been detected in the polypeptides of the present invention.
  • the skilled artisan will appreciate that such resulting cleaved fragments of the polypeptides of the present invention can be used for the diagnosis and/or treatment of diseases.
  • Cleavage of membrane-bound INSP 149 and/or INSP 169 can yield soluble N- and C- terminal fragments useful on their own or as components of fusion proteins such as Fc fusion.
  • a NDR cleavage site has been detected in the full length polypeptides of the present invention at position 70-72 (RRI), located after the first LRR motif.
  • PCSK cleavage sites have been detected at position 207-209 (KRT) of full length INSP168-SV1 and at positions 439-441 (KRS) and 449-451 (KRN) of Ml length membrane bound INSP 169.
  • KRT position 207-209
  • KRS 439-441
  • KRN 449-451
  • the PCSK cleavage site in INSP168-SV1 is located just after the LRR motifs and for INSP 169 between the LRR motifs and the fibronectin domain.
  • the polypeptides of the present invention also encompass the resulting cleaved N-fragments and/or C-fragments or mature forms thereof.
  • the resulting cleaved fragments are soluble fragments.
  • the resulting fragments consist of:
  • This aspect of the invention also includes fusion proteins that incorporate the polypeptides of the first aspect of the invention.
  • an “antigenic determinant” of the present invention may be a part of a polypeptide of the present invention, which binds to an antibody-combining site or to a T-cell receptor (TCR).
  • an "antigenic determinant” may be a site on the surface of a polypeptide of the present invention to which a single antibody molecule binds.
  • an antigen has several or many different antigenic determinants and reacts with antibodies of many different specificities.
  • the antibody is immunospecific to a polypeptide of the invention.
  • the antibody is immunospecific to a polypeptide of the invention, which is not part of a fusion protein.
  • the antibody is immunospecific to INSP168, INSP168-SV1, INSP 149 or INSP 169 or a fragment thereof.
  • Antigenic determinants usually consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and can have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • the "antigenic determinant” refers to a particular chemical group on a polypeptide of the present invention that is antigenic, i.e. that elicit a specific immune response.
  • polypeptides AAI04038 (SEQ ID NO:68), XP_853150 (SEQ ID NO:69) and ENSCAFPOOOOOO 16927 (SEQ ID NO:70), and their encoding nucleic acid sequences are specifically excluded from the scope of this invention.
  • the invention provides a purified nucleic acid molecule which encodes a polypeptide of the first aspect of the invention.
  • the term "purified nucleic acid molecule” preferably refers to a nucleic acid molecule of the invention that (1) has been separated from at least about 50 percent of proteins, lipids, carbohydrates, or other materials with which it is naturally found when total nucleic acid is isolated from the source cells, (2) is not linked to all or a portion of a polynucleotide to which the "purified nucleic acid molecule" is linked in nature, (3) is operably linked to a polynucleotide which it is not linked to in nature, or (4) does not occur in nature as part of a larger polynucleotide sequence.
  • the isolated nucleic acid molecule of the present invention is substantially free from any other contaminating nucleic acid molecule(s) or other contaminants that are found in its natural environment that would interfere with its use in polypeptide production or its therapeutic, diagnostic, prophylactic or research use.
  • genomic DNA are specifically excluded from the scope of the invention.
  • genomic DNA larger than 10 kbp (kilo base pairs), 50 kbp, 100 kbp, 150 kbp, 200 kbp, 250 kbp or 300 kbp are specifically excluded from the scope of the invention.
  • the "purified nucleic acid molecule" consists of cDNA only.
  • the purified nucleic acid molecule comprises or consists of the nucleic acid sequence as recited in the nucleic acid sequence as recited in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41 , SEQ ID NO: 43 and/or SEQ ID NO:66, or is a redundant equivalent or fragment of those sequences.
  • the purified nucleic acid molecule consists of the nucleic acid sequence as recited in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9,
  • SEQ ID NO:41 SEQ ID NO: 43 and/or SEQ ID NO:66, or is a redundant equivalent or fragment of those sequences.
  • the invention provides a purified nucleic acid molecule which hybridizes under high stringency conditions with a nucleic acid molecule of the second aspect of the invention.
  • High stringency hybridisation conditions are defined as overnight incubation at 42°C in a solution comprising 50% formamide, 5XSSC (15OmM NaCl, 15mM trisodium citrate), 5OmM sodium phosphate (pH7.6), 5x Denhardts solution, 10% dextran sulphate, and 20 microgram/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1 X SSC at approximately 65°C.
  • the invention provides a vector, such as an expression vector, that contains a nucleic acid molecule of the second or third aspect of the invention.
  • the invention provides a host cell transformed with a vector of the fourth aspect of the invention.
  • the invention provides a ligand which binds specifically to, and which preferably inhibits the activity of, a leucine-rich repeat motif containing polypeptide of the first aspect of the invention.
  • the invention provides a compound that is effective to alter the expression of a natural gene which encodes a polypeptide of the first aspect of the invention or to regulate the activity of a polypeptide of the first aspect of the invention.
  • Such compounds may be identified using the assays and screening methods disclosed herein.
  • a compound of the seventh aspect of the invention may either increase (agonise) or decrease (antagonise) the level of expression of the gene or the activity of the polypeptide.
  • the identification of the domain organisation and function of the INSP168, INSP168-SV1, INSP149 and INSP169 polypeptides allows for the design of screening methods capable of identifying compounds that are effective in the treatment and/or diagnosis of disease.
  • Ligands and compounds according to the sixth and seventh aspects of the invention may be identified using such methods. These methods are included as aspects of the present invention.
  • inhibitors or antagonists of INSP 168, INSP168-SV1, INSP 149 and INSP 169 such as, for example, monoclonal antibodies, which may be of use in modulating INSP168, INSP168-SV1, INSP149 and INSP169 activity in vivo in clinical applications.
  • Such compounds are likely to be useful in counteracting the biological activity of the INSP168, INSP168-SV1, INSP149 and INSP169 polypeptides.
  • Another aspect of this invention resides in the use of an INSP 168, INSP168-SV1, INSP 149 or INSP 169 gene or polypeptide as a target for the screening of candidate drug modulators, particularly candidate drugs active against leucine-rich repeat (LRR) motif containing protein related disorders.
  • LRR leucine-rich repeat
  • a further aspect of this invention resides in methods of screening of compounds for therapy of leucine-rich repeat (LRR) motif containing protein related disorders, comprising determining the ability of a compound to bind to an INSP 168, INSP 168- S V 1 , INSP 149 or INSP 169 gene or polypeptide, or a fragment thereof.
  • LRR leucine-rich repeat
  • a further aspect of this invention resides in methods of screening of compounds for therapy of leucine-rich repeat (LRR) motif containing protein related disorders, comprising testing for modulation of the activity of an INSP168, INSP168-SV1, INSP 149 or INSP 169 gene or polypeptide, or a fragment thereof.
  • the invention provides a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a host cell of the fifth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention, for use in therapy or diagnosis of diseases in which leucine-rich repeat motif containing proteins are implicated.
  • Such diseases include, but are not limited to, diseases of the retina, retinal pigment epithelium (RPE), and choroids; ocular neovascularization, ocular inflammation and retinal degenerations; diabetic retinopathy, chronic glaucoma, retinal detachment, sickle cell retinopathy, senile macular degeneration, retinal neovascularization, subretinal neovascularization; rubeosis ulceris inflammatory diseases, chronic posterior and pan uveitis, neoplasms, retinoblastoma, pseudoglioma, neovascular glaucoma; neovascularization resulting following a combined vitrectomy and lensectomy, vascular diseases retinal ischemia, choroidal vascular insufficiency, choroidal thrombosis, neovascularization of the optic nerve, diabetic macular edema, cystoid macular edema, retinitis pigmentosa, retinal
  • the moieties of the first, second, third, fourth, fifth, sixth or seventh aspect of the invention may also be used in the manufacture of a medicament for the treatment of such diseases.
  • the invention provides a method of diagnosing a disease in a patient, comprising assessing the level of expression of a natural gene encoding a polypeptide of the first aspect of the invention or the activity of a polypeptide of the first aspect of the invention in tissue from said patient and comparing said level of expression or activity to a control level, wherein a level that is different to said control level is indicative of disease.
  • a method will preferably be carried out in vitro.
  • Similar methods may be used for monitoring the therapeutic treatment of disease in a patient, wherein altering the level of expression or activity of a polypeptide or nucleic acid molecule over the period of time towards a control level is indicative of regression of disease.
  • a preferred method for detecting polypeptides of the first aspect of the invention comprises the steps of: (a) contacting a ligand, such as an antibody, of the sixth aspect of the invention with a biological sample under conditions suitable for the formation of a ligand-polypeptide complex; and (b) detecting said complex.
  • a number of different such methods according to the ninth aspect of the invention exist, as the skilled reader will be aware, such as methods of nucleic acid hybridization with short probes, point mutation analysis, polymerase chain reaction (PCR) amplification and methods using antibodies to detect aberrant protein levels. Similar methods may be used on a short or long term basis to allow therapeutic treatment of a disease to be monitored in a patient.
  • the invention also provides kits that are useful in these methods for diagnosing disease.
  • the disease diagnosed by a method of the ninth aspect of the invention is a disease in which leucine-rich repeat motif containing polypeptides are implicated, as described above.
  • the invention provides for the use of the polypeptide of the first aspect of the invention as an activator of cell proliferation, as a neuromodulator (neuroimmunomodulator), as a modulator of the inflammatory response in the CNS, as a regulator of astrocyte proliferation or as a regulator of axon projection, neuronal migration or leukocyte recruitment or migration.
  • INSP 168 and INSP168-SV1, or truncated fo ⁇ ns of INSP 149 and INSP 169 could be used as recombinant soluble antagonists of the endogenous activity of INSP 149 and INSP 169.
  • Another suitable use of the INSP 168 polypeptides is use in the screening of drug compounds that are effective against the diseases and conditions in which the INSP 168 polypeptides are implicated.
  • the invention provides a pharmaceutical composition comprising a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a host cell of the fifth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention, in conjunction with a pharmaceutically-acceptable carrier.
  • the present invention provides a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a host cell of the fifth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention, for use in the manufacture of a medicament for the diagnosis or treatment of a disease in which leucine-rich repeat motif containing polypeptides are implicated.
  • diseases include those described above in connection with the eighth aspect of the invention.
  • the invention provides a method of treating a disease in a patient comprising administering to the patient a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a host cell of the fifth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention.
  • the polypeptide, nucleic acid molecule, vector, host cell, ligand or compound administered to the patient should be an agonist.
  • the polypeptide, nucleic acid molecule, vector, host cell, ligand or compound administered to the patient should be an antagonist.
  • the invention provides transgenic or knockout non-human animals that have been transformed to express higher, lower or absent levels of a polypeptide of the first aspect of the invention.
  • Such transgenic animals are very useful models for the study of disease and may also be used in screening regimes for the identification of compounds that are effective in the treatment or diagnosis of such a disease.
  • “functional equivalent” refers to a protein or nucleic acid molecule that possesses functional or structural characteristics that are substantially similar to a polypeptide or nucleic acid molecule of the present invention.
  • a functional equivalent of a protein may contain modifications depending on the necessity of such modifications for the performance of a specific function.
  • the term “functional equivalent” is intended to include the fragments, mutants, hybrids, variants, analogs, or chemical derivatives of a molecule.
  • the "functional equivalent” may be a protein or nucleic acid molecule that exhibits any one or more of the functional activities of the polypeptides of the present invention.
  • the "functional equivalent” may be a protein or nucleic acid molecule that displays substantially similar activity compared with INSP168, INSP168-SV1, INSP 149 or INSP 169 or fragments thereof in a suitable assay for the measurement of biological activity or function.
  • the "functional equivalent” may be a protein or nucleic acid molecule that displays identical or higher activity compared with INSP168, INSP168-SV1, INSP149 or INSP169 or fragments thereof in a suitable assay for the measurement of biological activity or function.
  • the "functional equivalent” may be a protein or nucleic acid molecule that displays 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 100% or more activity compared with INSP168, INSP168-SV1, INSP149 or INSP169 or fragments thereof in a suitable assay for the measurement of biological activity or function.
  • the "functional equivalent” may be a protein or polypeptide capable of exhibiting a substantially similar in vivo or in vitro activity as the polypeptides of the invention.
  • the "functional equivalent” may be a protein or polypeptide capable of interacting with other cellular or extracellular molecules in a manner substantially similar to the way in which the corresponding portion of the polypeptides of the invention would.
  • a "functional equivalent” would be able, in an immunoassay, to diminish the binding of an antibody to the corresponding peptide (i.e., the peptide the amino acid sequence of which was modified to achieve the "functional equivalent") of the polypeptide of the invention, or to the polypeptide of the invention itself, where the antibody was raised against the corresponding peptide of the polypeptide of the invention.
  • An equimolar concentration of the functional equivalent will diminish the aforesaid binding of the corresponding peptide by at least about 5%, preferably between about 5% and 10%, more preferably between about 10% and 25%, even more preferably between about 25% and 50%, and most preferably between about 40% and 50%.
  • functional equivalents can be fully functional or can lack function in one or more activities.
  • variations can affect the function, for example, of the activities of the polypeptide that reflect its possession of a leucine- rich repeat (LRR) motif.
  • polypeptide includes any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e. peptide isosteres. This term refers both to short chains (peptides and oligopeptides) and to longer chains (proteins).
  • the polypeptide of the present invention may be in the form of a mature protein or may be a pre-, pro- or prepro- protein that can be activated by cleavage of the pre-, pro- or prepro- portion to produce an active mature polypeptide.
  • the pre-, pro- or prepro- sequence may be a leader or secretory sequence or may be a sequence that is employed for purification of the mature polypeptide sequence.
  • the polypeptide of the first aspect of the invention may form part of a fusion protein.
  • the mature polypeptide may be fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol).
  • Polypeptides of the invention are useful on their own, as components of fusion proteins such as Fc fusion, and/or in combination with another agent.
  • the Fc fusion comprises the mature form of INSP 168, the mature form of INSP168-SV1 or the mature form of the extracellular part of INSP 169 or INSP 149.
  • the agent is selected among interferon-beta, soluble NgR (e.g. Nogo-66), antibodies targeted to NgR, antibodies targeted to myelin inhibitors (e.g. Nogo, MAG or Omgp), CXCLlO, agonists of serotonin receptors (e.g. 5-HT1A/2A/7), LIF, EGFR blockers such as Erlotinib, and/or methylprednisolone.
  • a polypeptide of the invention that may comprise a sequence having at least 85% of homology with INSP 168, INSP 168-S Vl , INSP 149 or INSP 169, is a fusion protein.
  • fusion proteins can be obtained by cloning a polynucleotide encoding a polypeptide comprising a sequence having at least 85% of homology with INSP 168, INSP168-SV1, INSP149 or INSP169 in frame to the coding sequences for a heterologous protein sequence.
  • heterologous when used herein, is intended to designate any polypeptide other than a human INSP 168, INSPl 68-S Vl, INSP 149 or INSP 169 polypeptide.
  • heterologous sequences that can be comprised in the fusion proteins either at the N- or C-terminus, include: extracellular domains of membrane-bound protein, immunoglobulin constant regions (Fc regions), multimerization domains, domains of extracellular proteins, signal sequences, export sequences, and sequences allowing purification by affinity chromatography.
  • heterologous sequences are commercially available in expression plasmids since these sequences are commonly included in fusion proteins in order to provide additional properties without significantly impairing the specific biological activity of the protein fused to them (Terpe K, 2003, Appl Microbiol Biotechnol, 60:523-33).
  • additional properties are a longer lasting half-life in body fluids, the extracellular localization, or an easier purification procedure as allowed by the a stretch of Histidines forming the so-called "histidine tag" (Gentz et at.
  • the heterologous sequence can be eliminated by a proteolytic cleavage, for example by inserting a proteolytic cleavage site between the protein and the heterologous sequence, and exposing the purified fusion protein to the appropriate protease.
  • the INSP 168, INSP168-SV1, INSP 149 or INSP 169 polypeptide may be purified by means of a hexa-histidine peptide fused at the C-terminus of INSP 168, INSP168-SV1, INSP 149 or INSP 169.
  • the fusion protein comprises an immunoglobulin region
  • the fusion may be direct, or via a short linker peptide which can be as short as 1 to 3 amino acid residues in length or longer, for example, 13 amino acid residues in length.
  • Said linker may be a tripeptide of the sequence E-F-M (Glu-Phe-Met), for example, or a 13 -amino acid linker sequence comprising Glu-Phe- Gly-Ala-Gly-Leu-Val-Leu-Gly-Gly-Gln-Phe-Met (SEQ ID NO:71) introduced between the sequence of the substances of the invention and the immunoglobulin sequence.
  • the resulting fusion protein has improved properties, such as an extended residence time in body fluids (i.e. an increased half-life), increased specific activity, increased expression level, or the purification of the fusion protein is facilitated.
  • the protein is fused to the constant region of an Ig molecule.
  • it is fused to heavy chain regions, like the CH2 and CH3 domains of human IgGl, for example.
  • Other isoforms of Ig molecules are also suitable for the generation of fusion proteins according to the present invention, such as isoforms IgG2 or IgG4, or other Ig classes, like IgM or IgA, for example. Fusion proteins may be monomelic or multimeric, hetero- or homomultimeric.
  • the functional derivative comprises at least one moiety attached to one or more functional groups, which occur as one or more side chains on the amino acid residues.
  • the moiety is a polyethylene (PEG) moiety. PEGylation may be carried out by known methods, such as the ones described in WO99/55377, for example.
  • Polypeptides may contain amino acids other than the 20 gene-encoded amino acids, modified either by natural processes, such as by post-translational processing or by chemical modification techniques which are well known in the art.
  • modifications which may commonly be present in polypeptides of the present invention are glycosylation, lipid attachment, sulphation, gamma-carboxylation, for instance of glutamic acid residues, hydroxylation and ADP-ribosylation.
  • Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini.
  • blockage of the amino or carboxyl terminus in a polypeptide, or both, by a covalent modification is common in naturally-occurring and synthetic polypeptides and such modifications may be present in polypeptides of the present invention.
  • the modifications that occur in a polypeptide often will be a function of how the polypeptide is made.
  • the nature and extent of the modifications in large part will be determined by the post-translational modification capacity of the particular host cell and the modification signals that are present in the amino acid sequence of the polypeptide in question. For instance, glycosylation patterns vary between different types of host cell.
  • polypeptides of the present invention can be prepared in any suitable manner.
  • Such polypeptides include isolated naturally-occurring polypeptides (for example purified from cell culture), recombinantly-produced polypeptides (including fusion proteins), synthetically-produced polypeptides or polypeptides that are produced by a combination of these methods.
  • the functionally-equivalent polypeptides of the first aspect of the invention may be polypeptides that are homologous to the INSP 168 polypeptides.
  • Two polypeptides are said to be "homologous", as the term is used herein, if the sequence of one of the polypeptides has a high enough degree of identity or similarity to the sequence of the other polypeptide. "Identity” indicates that at any particular position in the aligned sequences, the amino acid residue is identical between the sequences. "Similarity” indicates that, at any particular position in the aligned sequences, the amino acid residue is of a similar type between the sequences.
  • Homologous polypeptides therefore include natural biological variants (for example, allelic variants or geographical variations within the species from which the polypeptides are derived) and mutants (such as mutants containing amino acid substitutions, insertions or deletions) of the INSP 168 polypeptides.
  • Such mutants may include polypeptides in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code.
  • Such substitutions are among Ala, VaI, Leu and He; among Ser and Thr; among the acidic residues Asp and GIu; among Asn and GIn; among the basic residues Lys and Arg; or among the aromatic residues Phe and Tyr.
  • Particularly preferred are variants in which several, i.e. between 5 and 10, 1 and 5, 1 and 3, 1 and 2 or just 1 amino acids are substituted, deleted or added in any combination.
  • silent substitutions, additions and deletions which do not alter the properties and activities of the protein. Also especially preferred in this regard are conservative substitutions.
  • Such mutants also include polypeptides in which one or more of the amino acid residues includes a substituent group.
  • any substitution should be preferably a "conservative” or “safe” substitution, which is commonly defined a substitution introducing an amino acids having sufficiently similar chemical properties ⁇ e.g. a basic, positively charged amino acid should be replaced by another basic, positively charged amino acid), in order to preserve the structure and the biological function of the molecule.
  • non-conservative mutations can be also introduced in the polypeptides of the invention with different purposes. Mutations reducing the affinity of the protein may increase its ability to be reused and recycled, potentially increasing its therapeutic potency (Robinson CR, 2002). Immunogenic epitopes eventually present in the polypeptides of the invention can be exploited for developing vaccines (Stevanovic S, 2002), or eliminated by modifying their sequence following known methods for selecting mutations for increasing protein stability, and correcting them (van den Burg B and Eijsink V, 2002; WO 02/05146, WO 00/34317, WO 98/52976). Preferred alternative, synonymous groups for amino acids derivatives included in peptide mimetics are those defined in Table 2.
  • amino acid derivatives also include aminoisobutyric acid (Aib), hydroxyproline (Hyp), 1. ,2,3, 4- tetrahydro-isoquinoline-3-COOH, indoline-2carboxylic acid, 4-difluoro-proline, L- thiazolidine-4-carboxylic acid, L-homoproline, 3,4-dehydro-proline, 3,4-dihydroxy- phenylalanine, cyclohexyl-glycine, and phenylglycine.
  • amino acid derivative is intended an amino acid or amino acid-like chemical entity other than one of the 20 genetically encoded naturally occurring amino acids.
  • the amino acid derivative may contain substituted or non-substituted, linear, branched, or cyclic alkyl moieties, and may include one or more heteroatoms.
  • the amino acid derivatives can be made de novo or obtained from commercial sources (Calbiochem-Novabiochem AG, Switzerland; Bachem, USA).
  • Various methodologies for incorporating unnatural amino acids derivatives into proteins, using both in vitro and in vivo translation systems, to probe and/or improve protein structure and function are disclosed in the literature (Dougherty DA, 2000).
  • Such mutants also include polypeptides in which one or more of the amino acid residues includes a substituent group.
  • polypeptides of the first aspect of the invention typically have a degree of sequence identity with the INSP 168 polypeptides, or with active fragments thereof, of greater than 80%. More preferred polypeptides have degrees of identity of greater than 90%, 95%, 98% or 99%, respectively.
  • the functionally-equivalent polypeptides of the first aspect of the invention may also be polypeptides which have been identified using one or more techniques of structural alignment.
  • the Inpharmatica Genome Threader technology that forms one aspect of the search tools used to generate the Biopendium search database may be used (see PCT application published as WO 01/69507) to identify polypeptides of presently-unknown function which, while having low sequence identity as compared to the INSP 168 polypeptides, are predicted to be cell surface recognition molecules by virtue of sharing significant structural homology with the INSP168 polypeptide sequences.
  • significant structural homology is meant that the Inpharmatica Genome Threader predicts two proteins to share structural homology with a certainty of 10% and above.
  • polypeptides of the first aspect of the invention also include fragments of the INSP 168 polypeptides and fragments of the functional equivalents of these polypeptides, provided that those fragments retain the biological activity of the INSP 168 polypeptides, or have an antigenic determinant in common with these polypeptides.
  • fragment refers to a polypeptide having an amino acid sequence that is the same as part, but not all, of the amino acid sequence of the INSP 168 polypeptides or one of their functional equivalents.
  • the fragments should comprise at least n consecutive amino acids from the sequence and, depending on the particular sequence, n preferably is 7 or more (for example, 8, 10, 12, 14, 16, 18, 20 or more). Small fragments may form an antigenic determinant.
  • Nucleic acids according to the invention are preferably 10-2000 nucleotides in length, preferably 100-1750 nucleotides, preferably 500-1500, preferably 600-1200, preferably 750-1000 nucleotides in length.
  • Polypeptides according to the invention are preferably 10-700 amino acids in length, preferably 50-600, preferably 100-500, preferably 200-400, preferably 300-375 amino acids in length. Fragments of the full length INSP168, INSP168-SV1, INSP149 and INSP169 exon polypeptides and the INSP168, INSP168-SV1, INSP149 and INSP169 polypeptides may consist of combinations of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 of neighbouring exon sequences in the INSP168, INSP168-SV1, INSP149 and INSP169 polypeptides, respectively. Such fragments may be "free-standing", i.e.
  • the fragment of the invention most preferably forms a single continuous region.
  • certain preferred embodiments relate to a fragment having a pre- and/or pro- polypeptide region fused to the amino terminus of the fragment and/or an additional region fused to the carboxyl terminus of the fragment.
  • several fragments may be comprised within a single larger polypeptide.
  • polypeptides of the present invention or their immunogenic fragments can be used to generate ligands, such as polyclonal or monoclonal antibodies, that are immunospecific for the polypeptides.
  • ligands such as polyclonal or monoclonal antibodies
  • Such antibodies may be employed to isolate or to identify clones expressing the polypeptides of the invention or to purify the polypeptides by affinity chromatography.
  • the antibodies may also be employed as diagnostic or therapeutic aids, amongst other applications, as will be apparent to the skilled reader.
  • immunospecif ⁇ c means that the antibodies have substantially greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the prior art.
  • antibody refers to intact molecules as well as to fragments thereof, such as Fab, F(ab')2 and Fv, which are capable of binding to the antigenic determinant in question. Such antibodies thus bind to the polypeptides of the first aspect of the invention.
  • substantially greater affinity we mean that there is a measurable increase in the affinity for a polypeptide of the invention as compared with the affinity for known cell-surface receptors.
  • the affinity is at least 1.5-fold, 2-fold, 5-fold 10-fold, 100-fold, 10 3 -fold, 10 4 -fold, 10 5 -fold, 10 6 -fold or greater for a polypeptide of the invention than for known cell surface recognition molecules.
  • LRR leucine-rich repeat
  • a selected mammal such as a mouse, rabbit, goat or horse
  • a polypeptide of the first aspect of the invention may be immunised with a polypeptide of the first aspect of the invention.
  • the polypeptide used to immunise the animal can be derived by recombinant DNA technology or can be synthesized chemically.
  • the polypeptide can be conjugated to a carrier protein.
  • Commonly used carriers to which the polypeptides may be chemically coupled include bovine serum albumin, thyroglobulin and keyhole limpet haemocyanin.
  • the coupled polypeptide is then used to immunise the animal. Serum from the immunised animal is collected and treated according to known procedures, for example by immunoaffinity chromatography.
  • Monoclonal antibodies to the polypeptides of the first aspect of the invention can also be readily produced by one skilled in the art.
  • the general methodology for making monoclonal antibodies using hybridoma technology is well known (see, for example, Kohler, G. and Milstein, C, Nature 256: 495-497 (1975); Kozbor et al, Immunology Today 4: 72 (1983); Cole et al, 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985).
  • Panels of monoclonal antibodies produced against the polypeptides of the first aspect of the invention can be screened for various properties, i.e., for isotype, epitope, affinity, etc.
  • Monoclonal antibodies are particularly useful in purification of the individual polypeptides against which they are directed.
  • genes encoding the monoclonal antibodies of interest may be isolated from hybridomas, for instance by PCR techniques known in the art, and cloned and expressed in appropriate vectors.
  • Chimeric antibodies in which non-human variable regions are joined or fused to human constant regions (see, for example, Liu et al, Proc. Natl. Acad. Sci. USA, 84, 3439 (1987)), may also be of use.
  • the antibody may be modified to make it less immunogenic in an individual, for example by humanisation (see Jones et al, Nature, 321, 522 (1986); Verhoeyen et al, Science, 239, 1534 (1988); Kabat et al, J. Immunol., 147, 1709 (1991); Queen et al, Proc. Natl Acad. Sci. USA, 86, 10029 (1989); Gorman et al, Proc. Natl Acad. Sci.
  • humanised antibody refers to antibody molecules in which the CDR amino acids and selected other amino acids in the variable domains of the heavy and/or light chains of a non-human donor antibody have been substituted in place of the equivalent amino acids in a human antibody.
  • the humanised antibody thus closely resembles a human antibody but has the binding ability of the donor antibody.
  • the antibody may be a "bispecific" antibody, that is an antibody having two different antigen-binding domains, each domain being directed against a different epitope.
  • Phage display technology may be utilised to select genes which encode antibodies with binding activities towards the polypeptides of the invention either from repertoires of PCR amplified V-genes of lymphocytes from humans screened for possessing the relevant antibodies, or from naive libraries (McCafferty, J. et al, (1990), Nature 348, 552-554; Marks, J. et al, (1992) Biotechnology 10, 779-783).
  • the affinity of these antibodies can also be improved by chain shuffling (Clackson, T. et al, (1991) Nature 352, 624-628).
  • Antibodies generated by the above techniques have additional utility in that they may be employed as reagents in immunoassays, radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA).
  • the antibodies can be labelled with an analytically-detectable reagent such as a radioisotope, a fluorescent molecule or an enzyme.
  • Preferred nucleic acid molecules of the second and third aspects of the invention are those which encode a polypeptide sequence as recited in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44 and/or SEQ ID NO:67, and functionally equivalent polypeptides.
  • nucleic acid molecules may be used in the methods and applications described herein.
  • the nucleic acid molecules of the invention preferably comprise at least n consecutive nucleotides from the sequences disclosed herein where, depending on the particular sequence, n is 10 or more (for example, 12, 14, 15, 18, 20, 25, 30, 35, 40 or more).
  • nucleic acid molecules of the invention also include sequences that are complementary to nucleic acid molecules described above (for example, for antisense or probing purposes).
  • Nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance cDNA, synthetic DNA or genomic DNA. Such nucleic acid molecules may be obtained by cloning, by chemical synthetic techniques or by a combination thereof. The nucleic acid molecules can be prepared, for example, by chemical synthesis using techniques such as solid phase phosphoramidite chemical synthesis, from genomic or cDNA libraries or by separation from an organism. RNA molecules may generally be generated by the in vitro or in vivo transcription of DNA sequences. The nucleic acid molecules may be double-stranded or single-stranded. Single- stranded DNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand.
  • nucleic acid molecule also includes analogues of DNA and RNA, such as those containing modified backbones, and peptide nucleic acids (PNA).
  • PNA peptide nucleic acids
  • PNAs may be pegylated to extend their lifespan in a cell, where they preferentially bind complementary single stranded DNA and RNA and stop transcript elongation (Nielsen, P.E. et al.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 2 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:1.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:4 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:3.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:6 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO.5.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 8 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:7.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 10 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:9.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 12 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:11.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:14 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 13.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 16 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 15.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 18 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 17.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:20 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 19.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:22 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:21.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:24 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:23.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:26 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:25.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:28 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:27.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:30 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:29.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:32 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:31.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:34 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:33.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:36 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:35.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:38 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:37.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:40 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:39.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:42 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:41.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:44 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:43.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:67 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:66.
  • These molecules also may have a different sequence which, as a result of the degeneracy of the genetic code, encodes a polypeptide of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44 or SEQ ID NO:67.
  • nucleic acid molecules may include, but are not limited to, the coding sequence for the mature polypeptide by itself; the coding sequence for the mature polypeptide and additional coding sequences, such as those encoding a leader or secretory sequence, such as a pro-, pre- or prepro- polypeptide sequence; the coding sequence of the mature polypeptide, with or without the aforementioned additional coding sequences, together with further additional, non- coding sequences, including non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription (including termination signals), ribosome binding and mRNA stability.
  • the nucleic acid molecules may also include additional sequences which encode additional amino acids, such as those which provide additional functionalities.
  • nucleic acid molecules of the second and third aspects of the invention may also encode the fragments or the functional equivalents of the polypeptides and fragments of the first aspect of the invention.
  • a nucleic acid molecule may be a naturally- occurring variant such as a naturally-occurring allelic variant, or the molecule may be a variant that is not known to occur naturally.
  • non-naturally occurring variants of the nucleic acid molecule may be made by mutagenesis techniques, including those applied to nucleic acid molecules, cells or organisms.
  • variants in this regard are variants that differ from the aforementioned nucleic acid molecules by nucleotide substitutions, deletions or insertions. The substitutions, deletions or insertions may involve one or more nucleotides.
  • the variants may be altered in coding or non-coding regions or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or insertions.
  • the nucleic acid molecules of the invention can also be engineered, using methods generally known in the art, for a variety of reasons, including modifying the cloning, processing, and/or expression of the gene product (the polypeptide).
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides are included as techniques which may be used to engineer the nucleotide sequences.
  • Site-directed mutagenesis may be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations and so forth.
  • Nucleic acid molecules which encode a polypeptide of the first aspect of the invention may be ligated to a heterologous sequence so that the combined nucleic acid molecule encodes a fusion protein.
  • Such combined nucleic acid molecules are included within the second or third aspects of the invention.
  • a fusion protein that can be recognised by a commercially-available antibody.
  • a fusion protein may also be engineered to contain a cleavage site located between the sequence of the polypeptide of the invention and the sequence of a heterologous protein so that the polypeptide may be cleaved and purified away from the heterologous protein.
  • the nucleic acid molecules of the invention also include antisense molecules that are partially complementary to nucleic acid molecules encoding polypeptides of the present invention and that therefore hybridize to the encoding nucleic acid molecules (hybridization).
  • antisense molecules such as oligonucleotides, can be designed to recognise, specifically bind to and prevent transcription of a target nucleic acid encoding a polypeptide of the invention, as will be known by those of ordinary skill in the art (see, for example, Cohen, J.S., Trends in Pharm. Sci., 10, 435 (1989), Okano, J. Neurochem. 56, 560 (1991); O'Connor, J. Neurochem 56, 560 (1991); Lee et ah, Nucleic Acids Res 6, 3073 (1979); Cooney et al, Science 241, 456 (1988); Dervan et al, Science 251, 1360 (1991).
  • hybridization refers to the association of two nucleic acid molecules with one another by hydrogen bonding. Typically, one molecule will be fixed to a solid support and the other will be free in solution. Then, the two molecules may be placed in contact with one another under conditions that favour hydrogen bonding. Factors that affect this bonding include: the type and volume of solvent; reaction temperature; time of hybridization; agitation; agents to block the non-specific attachment of the liquid phase molecule to the solid support (Denhardt's reagent or BLOTTO); the concentration of the molecules; use of compounds to increase the rate of association of molecules (dextran sulphate or polyethylene glycol); and the stringency of the washing conditions following hybridization (see Sambrook et al. [supra]).
  • the inhibition of hybridization of a completely complementary molecule to a target molecule may be examined using a hybridization assay, as known in the art (see, for example, Sambrook et al [supra]).
  • a substantially homologous molecule will then compete for and inhibit the binding of a completely homologous molecule to the target molecule under various conditions of stringency, as taught in Wahl, G.M. and SX. Berger (1987; Methods Enzymol. 152:399-407) and Kimmel, A.R. (1987; Methods Enzymol. 152:507-511).
  • Stringency refers to conditions in a hybridization reaction that favour the association of very similar molecules over association of molecules that differ.
  • High stringency hybridisation conditions are defined as overnight incubation at 42°C in a solution comprising 50% formamide, 5XSSC (15OmM NaCl, 15mM trisodium citrate), 5OmM sodium phosphate (pH7.6), 5x Denhardts solution, 10% dextran sulphate, and 20 microgram/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1X SSC at approximately 65°C.
  • Low stringency conditions involve the hybridisation reaction being carried out at 35°C (see Sambrook et al. [supra]).
  • the conditions used for hybridization are those of high stringency.
  • Preferred embodiments of this aspect of the invention are nucleic acid molecules that are at least 70% identical over their entire length to nucleic acid molecules encoding the INSP 168 polypeptides (such as SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO: 44, or SEQ ID NO:67) and nucleic acid molecules that are substantially complementary to such nucleic acid molecules.
  • the INSP 168 polypeptides such as SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12,
  • a nucleic acid molecule according to this aspect of the invention comprises a region that is at least 80% identical over its entire length to the nucleic acid molecules having the sequence recited in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43 or SEQ ID NO:66 or a nucleic acid molecule that is complementary thereto.
  • nucleic acid molecules at least 90%, preferably at least 95%, more preferably at least 98% or 99% identical over their entire length to the same are particularly preferred.
  • Preferred embodiments in this respect are nucleic acid molecules that encode polypeptides which retain substantially the same biological function or activity as the INSP 168 polypeptides.
  • the invention also provides a process for detecting a nucleic acid molecule of the invention, comprising the steps of: (a) contacting a nucleic probe according to the invention with a biological sample under hybridizing conditions to form duplexes; and (b) detecting any such duplexes that are formed.
  • a nucleic acid molecule as described above may be used as a hybridization probe for RNA, cDNA or genomic DNA, in order to isolate full- length cDNAs and genomic clones encoding the INSP 168 polypeptides and to isolate cDNA and genomic clones of homologous or orthologous genes that have a high sequence similarity to the gene encoding this polypeptide.
  • the following techniques among others known in the art, may be utilised and are discussed below for purposes of illustration. Methods for DNA sequencing and analysis are well known and are generally available in the art and may, indeed, be used to practice many of the embodiments of the invention discussed herein.
  • Such methods may employ such enzymes as the Klenow fragment of DNA polymerase I, Sequenase (US Biochemical Corp, Cleveland, OH), Taq polymerase (Perkin Elmer), thermostable T7 polymerase (Amersham, Chicago, IL), or combinations of polymerases and proof-reading exonucleases such as those found in the ELONGASE Amplification System marketed by Gibco/BRL (Gaithersburg, MD).
  • the sequencing process may be automated using machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno, NV), the Peltier Thermal Cycler (PTC200; MJ Research, Watertown, MA) and the ABI Catalyst and 373 and 377 DNA Sequencers (Perkin Elmer).
  • One method for isolating a nucleic acid molecule encoding a polypeptide with an equivalent function to that of the INSP 168 polypeptides is to probe a genomic or cDNA library with a natural or artificially-designed probe using standard procedures that are recognised in the art (see, for example, "Current Protocols in Molecular Biology", Ausubel et al. (eds). Greene Publishing Association and John Wiley Interscience, New York, 1989,1992).
  • Probes comprising at least 15, preferably at least 30, and more preferably at least 50, contiguous bases that correspond to, or are complementary to, nucleic acid sequences from the appropriate encoding gene (such as SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43 or SEQ ID NO:66) are particularly useful probes.
  • the appropriate encoding gene such as SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:15,
  • Such probes may be labelled with an analytically-detectable reagent to facilitate their identification.
  • Useful reagents include, but are not limited to, radioisotopes, fluorescent dyes and enzymes that are capable of catalysing the formation of a detectable product.
  • the ordinarily skilled artisan will be capable of isolating complementary copies of genomic DNA, cDNA or RNA polynucleotides encoding proteins of interest from human, mammalian or other animal sources and screening such sources for related sequences, for example, for additional members of the family, type and/or subtype.
  • isolated cDNA sequences will be incomplete, in that the region encoding the polypeptide will be cut short, normally at the 5' end.
  • telomere shortening uses universal primers to retrieve unknown nucleic acid sequence adjacent a known locus.
  • Inverse PCR may also be used to amplify or to extend sequences using divergent primers based on a known region (Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186).
  • capture PCR involves PCR amplification of DNA fragments adjacent a known sequence in human and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods Applic, 1, 111-119).
  • Another method which may be used to retrieve unknown sequences is that of Parker, J.D. et al. (1991); Nucleic Acids Res. 19:3055- 3060). Additionally, one may use PCR, nested primers, and PromoterFinderTM libraries to walk genomic DNA (Clontech, Palo Alto, CA). This process avoids the need to screen libraries and is useful in finding intron/exon junctions.
  • libraries that have been size-selected to include larger cDNAs.
  • random-primed libraries are preferable, in that they will contain more sequences that contain the 5' regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA.
  • Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
  • the nucleic acid molecules of the present invention may be used for chromosome localisation.
  • a nucleic acid molecule is specifically targeted to, and can hybridize with, a particular location on an individual human chromosome.
  • the mapping of relevant sequences to chromosomes according to the present invention is an important step in the confirmatory correlation of those sequences with the gene-associated disease. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found in, for example, V. McKusick, Mendelian Inheritance in Man (available on-line through Johns Hopkins University Welch Medical Library).
  • the relationships between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes). This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the disease or syndrome has been crudely localised by genetic linkage to a particular genomic region, any sequences mapping to that area may represent associated or regulatory genes for further investigation.
  • the nucleic acid molecule may also be used to detect differences in the chromosomal location due to translocation, inversion, etc. among normal, carrier, or affected individuals.
  • the nucleic acid molecules of the present invention are also valuable for tissue localisation.
  • Such techniques allow the determination of expression patterns of the polypeptide in tissues by detection of the mRNAs that encode them.
  • These techniques include in situ hybridization techniques and nucleotide amplification techniques, such as PCR. Results from these studies provide an indication of the normal functions of the polypeptide in the organism.
  • comparative studies of the normal expression pattern of mRNAs with that of mRNAs encoded by a mutant gene provide valuable insights into the role of mutant polypeptides in disease. Such inappropriate expression maybe of a temporal, spatial or quantitative nature.
  • RNA interference (Elbashir, SM et al., Nature 2001, 411, 494-498) is one method of sequence specific post-transcriptional gene silencing that may be employed. Short dsRNA oligonucleotides are synthesised in vitro and introduced into a cell. The sequence specific binding of these dsRNA oligonucleotides triggers the degradation of target mRNA, reducing or ablating target protein expression.
  • the vectors of the present invention comprise nucleic acid molecules of the invention and may be cloning or expression vectors.
  • the host cells of the invention which may be transformed, transfected or transduced with the vectors of the invention may be prokaryotic or eukaryotic.
  • polypeptides of the invention may be prepared in recombinant form by expression of their encoding nucleic acid molecules in vectors contained within a host cell.
  • expression methods are well known to those of skill in the art and many are described in detail by Sambrook et al (supra) and Fernandez & Hoeffler (1998, eds. "Gene expression systems. Using nature for the art of expression”. Academic Press, San Diego, London, Boston, New York, Sydney, Tokyo, Toronto).
  • any system or vector that is suitable to maintain, propagate or express nucleic acid molecules to produce a polypeptide in the required host may be used.
  • nucleotide sequence may be inserted into an expression system by any of a variety of well-known and routine techniques, such as, for example, those described in Sambrook et al., (supra).
  • the encoding gene can be placed under the control of a control element such as a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator, so that the DNA sequence encoding the desired polypeptide is transcribed into RNA in the transformed host cell.
  • suitable expression systems include, for example, chromosomal, episomal and virus-derived systems, including, for example, vectors derived from: bacterial plasmids, bacteriophage, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses such as baculoviruses, papova viruses such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, or combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, including cosmids and phagemids.
  • Human artificial chromosomes may also be employed to deliver larger fragments of DNA than can be contained and expressed in a plasmid.
  • the pCR4-TOPO-INSP168, pCR4-TOPO-INSP168-SVl, pEAK12d_INSP168-6HIS, pENTR_INSP168-6HIS, and ⁇ DEST12.2_INSP168-6HIS vectors are preferred examples of suitable vectors for use in accordance with this invention.
  • Particularly suitable expression systems include microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (for example, baculovirus); plant cell systems transformed with virus expression vectors (for example, cauliflower mosaic virus,
  • CaMV tobacco mosaic virus
  • TMV tobacco mosaic virus
  • bacterial expression vectors for example, Ti or pBR322 plasmids
  • nucleic acid molecules encoding a polypeptide of the present invention into host cells can be effected by methods described in many standard laboratory manuals, such as Davis et ah, Basic Methods in Molecular Biology (1986) and Sambrook et al., [supra]. Particularly suitable methods include calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection (see Sambrook et ah, 1989 [supra]; Ausubel et ah, 1991 [supra]; Spector, Goldman & Leinwald, 1998). In eukaryotic cells, expression systems may either be transient (for example, episomal) or permanent (chromosomal integration) according to the needs of the system.
  • the encoding nucleic acid molecule may or may not include a sequence encoding a control sequence, such as a signal peptide or leader sequence, as desired, for example, for secretion of the translated polypeptide into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment.
  • a control sequence such as a signal peptide or leader sequence
  • These signals may be endogenous to the polypeptide or they may be heterologous signals.
  • Leader sequences can be removed by the bacterial host in post-translational processing.
  • regulatory sequences that allow for regulation of the expression of the polypeptide relative to the growth of the host cell.
  • regulatory sequences are those which cause the expression of a gene to be increased or decreased in response to a chemical or physical stimulus, including the presence of a regulatory compound or to various temperature or metabolic conditions.
  • Regulatory sequences are those non-translated regions of the vector, such as enhancers, promoters and 5' and 3 1 untranslated regions. These interact with host cellular proteins to carry out transcription and translation. Such regulatory sequences may vary in their strength and specificity. Depending on the vector system and host utilised, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used.
  • inducible promoters such as the hybrid lacZ promoter of the Bluescript phagemid (Stratagene, LaJoIIa, CA) or pSportlTM plasmid (Gibco BRL) and the like may be used.
  • the baculovirus polyhedrin promoter may be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (for example, heat shock, RUBISCO and storage protein genes) or from plant viruses (for example, viral promoters or leader sequences) may be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of the sequence, vectors based on SV40 or EBV may be used with an appropriate selectable marker.
  • An expression vector is constructed so that the particular nucleic acid coding sequence is located in the vector with the appropriate regulatory sequences, the positioning and orientation of the coding sequence with respect to the regulatory sequences being such that the coding sequence is transcribed under the "control" of the regulatory sequences, i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence. In some cases it may be necessary to modify the sequence so that it may be attached to the control sequences with the appropriate orientation; i.e., to maintain the reading frame.
  • the control sequences and other regulatory sequences may be ligated to the nucleic acid coding sequence prior to insertion into a vector. Alternatively, the coding sequence can be cloned directly into an expression vector that already contains the control sequences and an appropriate restriction site.
  • cell lines which stably express the polypeptide of interest may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media.
  • the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells that successfully express the introduced sequences.
  • Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type.
  • Mammalian cell lines available as hosts for expression are known in the art and include many immortalised cell lines available from the American Type Culture Collection (ATCC) including, but not limited to, Chinese hamster ovary (CHO), HeLa, baby hamster kidney (BHK), monkey kidney (COS), C127, 3T3, BHK, HEK 293, Bowes melanoma and human hepatocellular carcinoma (for example Hep G2) cells and a number of other cell lines.
  • ATCC American Type Culture Collection
  • the materials for baculovirus/insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego CA (the "MaxBac” kit). These techniques are generally known to those skilled in the art and are described fully in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Particularly suitable host cells for use in this system include insect cells such as Drosophila S2 and Spodoptera Sf9 cells.
  • all plants from which protoplasts can be isolated and cultured to give whole regenerated plants can be utilised, so that whole plants are recovered which contain the transferred gene.
  • Practically all plants can be regenerated from cultured cells or tissues, including but not limited to all major species of sugar cane, sugar beet, cotton, fruit and other trees, legumes and vegetables.
  • Examples of particularly preferred bacterial host cells include streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis cells.
  • Examples of particularly suitable host cells for fungal expression include yeast cells (for example, S. cerevisiae) and Aspergillus cells.
  • any number of selection systems are known in the art that may be used to recover transformed cell lines. Examples include the herpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell 11 :223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1980) Cell 22:817-23) genes that can be employed in tk- or aprt ⁇ cells, respectively. Also, antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dihydrofolate reductase (DHFR) that confers resistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl.
  • DHFR dihydrofolate reductase
  • npt which confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin, F. et al (1981) J. MoI. Biol. 150:1-14) and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. Additional selectable genes have been described, examples of which will be clear to those of skill in the art.
  • marker gene expression suggests that the gene of interest is also present, its presence and expression may need to be confirmed.
  • a marker gene can be placed in tandem with a sequence encoding a polypeptide of the invention under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
  • host cells that contain a nucleic acid sequence encoding a polypeptide of the invention and which express said polypeptide may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassays, for example, fluorescence activated cell sorting (FACS) or immunoassay techniques (such as the enzyme-linked immunosorbent assay [ELISA] and radioimmunoassay [RIA]), that include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein (see Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS Press, St Paul, MN) and Maddox, D.E. et al. (1983) J. Exp. Med, 158, 1211-1216).
  • FACS fluorescence activated cell sorting
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • Means for producing labelled hybridization or PCR probes for detecting sequences related to nucleic acid molecules encoding polypeptides of the present invention include oligolabelling, nick translation, end-labelling or PCR amplification using a labelled polynucleotide.
  • sequences encoding the polypeptide of the invention may be cloned into a vector for the production of an mRNA probe.
  • RNA polymerase such as T7, T3 or SP6 and labelled nucleotides. These procedures may be conducted using a variety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo, MI); Promega (Madison WI); and U.S. Biochemical Corp., Cleveland, OH)).
  • Suitable reporter molecules or labels include radionuclides, enzymes and fluorescent, chemiluminescent or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • Nucleic acid molecules according to the present invention may also be used to create transgenic animals, particularly rodent animals. Such transgenic animals form a further aspect of the present invention. This may be done locally by modification of somatic cells, or by germ line therapy to incorporate heritable modifications. Such transgenic animals may be particularly useful in the generation of animal models for drug molecules effective as modulators of the polypeptides of the present invention.
  • the polypeptide can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulphate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography is particularly useful for purification. Well known techniques for refolding proteins may be employed to regenerate an active conformation when the polypeptide is denatured during isolation and or purification.
  • Specialised vector constructions may also be used to facilitate purification of proteins, as desired, by joining sequences encoding the polypeptides of the invention to a nucleotide sequence encoding a polypeptide domain that will facilitate purification of soluble proteins.
  • purification-facilitating domains include metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilised metals, protein A domains that allow purification on immobilised immunoglobulin, and the domain utilised in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, WA).
  • cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen, San Diego, CA) between the purification domain and the polypeptide of the invention may be used to facilitate purification.
  • One such expression vector provides for expression of a fusion protein containing the polypeptide of the invention fused to several histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification by IMAC (immobilised metal ion affinity chromatography as described in Porath, J. et al. (1992), Prot. Exp. Purif.
  • the polypeptide is to be expressed for use in screening assays, generally it is preferred that it be secreted into the culture medium of the host cell in which it is expressed.
  • the polypeptides of the invention may be purified from the culture medium may be harvested prior to use in the screening assay, for example using standard protein purification techniques such as gel exclusion chromatography, ion-exchange chromatography or affinity chromatography. Examples of suitable methods of protein purification are provided in the Examples herein. If polypeptide is produced intracellularly, the cells must first be lysed before the polypeptide is recovered.
  • polypeptides of the invention be expressed as cell-surface fusion proteins.
  • the host cells may be harvested prior to use in the screening assay, for example using techniques such as fluorescence activated cell sorting (FACs) or immunoaffinity techniques.
  • FACs fluorescence activated cell sorting
  • the present invention also provides novel targets and methods for the screening of drug candidates or leads. These screening methods include binding assays and/or functional assays, and may be performed in vitro, in cell systems or in animals.
  • a particular object of this invention resides in the use of an INSP 168 polypeptide as a target for screening candidate drugs for treating or preventing disorders in which leucine-rich repeat (LRR) motif containing proteins are implicated.
  • LRR leucine-rich repeat
  • Another object of this invention resides in methods of selecting biologically active compounds, said methods comprising contacting a candidate compound with a INSP 168 gene or polypeptide, and selecting compounds that bind said gene or polypeptide.
  • a further other object of this invention resides in methods of selecting biologically active compounds, said method comprising contacting a candidate compound with recombinant host cell expressing a INSP 168 polypeptide with a candidate compound, and selecting compounds that bind said INSPl 68 polypeptide at the surface of said cells and/or that modulate the activity of the INSP 168 polypeptide.
  • a “biologically active” compound denotes any compound having biological activity in a subject, preferably therapeutic activity, more preferably a compound that can be used for treating disorders in which leucine-rich repeat (LRR) motif containing proteins are implicated, or as a lead to develop drugs for treating disorders in which leucine-rich repeat (LRR) motif containing proteins are implicated.
  • a “biologically active” compound preferably is a compound that modulates the activity of a INSP 168 polypeptide.
  • the above methods may be conducted in vitro, using various devices and conditions, including with immobilized reagents, and may further comprise an additional step of assaying the activity of the selected compounds in a model of a disorder in which leucine-rich repeat (LRR) motif containing proteins are implicated, such as an animal model.
  • LRR leucine-rich repeat
  • Binding to a target gene or polypeptide provides an indication as to the ability of the compound to modulate the activity of said target, and thus to affect a pathway leading to disorder in a subject.
  • the determination of binding may be performed by various techniques, such as by labelling of the candidate compound, by competition with a labelled reference ligand, etc.
  • the polypeptides may be used in essentially pure form, in suspension, immobilized on a support, or expressed in a membrane (intact cell, membrane preparation, liposome, etc.). Modulation of activity includes, without limitation, stimulation of the surface expression of a receptor, and modulation of multimerization of said receptor ⁇ e.g., the formation of multimeric complexes with other sub-units), etc.
  • the cells used in the assays may be any recombinant cell ⁇ i.e., any cell comprising a recombinant nucleic acid encoding a INSPl 68 polypeptide) or any cell that expresses an endogenous INSP 168 polypeptide.
  • examples of such cells include, without limitation, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.).
  • E.coli E.coli, Pichia pastoris, Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces or Saccharomyces yeasts, mammalian cell lines ⁇ e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.) as well as primary or established mammalian cell cultures (e.g., produced from fibroblasts, embryonic cells, epithelial cells, nervous cells, adipocytes, etc.).
  • mammalian cell lines ⁇ e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.
  • primary or established mammalian cell cultures e.g., produced from fibroblasts, embryonic cells, epithelial cells, nervous cells, adipocytes, etc.
  • Preferred selected compounds are agonists of INSP168, i.e., compounds that can bind to INSP 168 and mimic the activity of an endogenous ligand thereof.
  • a further object of this invention resides in a method of selecting biologically active compounds, said method comprising contacting in vitro a test compound with a INSP 168 polypeptide according to the present invention and determining the ability of said test compound to modulate the activity of said INSP 168 polypeptide.
  • a further object of this invention resides in a method of selecting biologically active compounds, said method comprising contacting in vitro a test compound with a INSP 168 gene according to the present invention and determining the ability of said test compound to modulate the expression of said INSP 168 gene, preferably to stimulate expression thereof.
  • this invention relates to a method of screening, selecting or identifying active compounds, the method comprising contacting a test compound with a recombinant host cell comprising a reporter construct, said reporter construct comprising a reporter gene under the control of a INSP 168 gene promoter, and selecting the test compounds that modulate ⁇ e.g. stimulate or reduce, preferably stimulate) expression of the reporter gene.
  • the polypeptide of the invention can be used to screen libraries of compounds in any of a variety of drug screening techniques. Such compounds may activate (agonise) or inhibit (antagonise) the level of expression of the gene or the activity of the polypeptide of the invention and form a further aspect of the present invention. Preferred compounds are effective to alter the expression of a natural gene which encodes a polypeptide of the first aspect of the invention or to regulate the activity of a polypeptide of the first aspect of the invention.
  • Agonist or antagonist compounds may be isolated from, for example, cells, cell-free preparations, chemical libraries or natural product mixtures. These agonists or antagonists may be natural or modified substrates, ligands, enzymes, receptors or structural or functional mimetics. For a suitable review of such screening techniques, see Coligan et ah, Current Protocols in Immunology l(2):Chapter 5 (1991).
  • Compounds that are most likely to be good antagonists are molecules that bind to the polypeptide of the invention without inducing the biological effects of the polypeptide upon binding to it.
  • Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to the polypeptide of the invention and thereby inhibit or extinguish its activity. In this fashion, binding of the polypeptide to normal cellular binding molecules may be inhibited, such that the normal biological activity of the polypeptide is prevented.
  • the polypeptide of the invention that is employed in such a screening technique may be free in solution, affixed to a solid support, borne on a cell surface or located intracellularly.
  • screening procedures may involve using appropriate cells or cell membranes that express the polypeptide that are contacted with a test compound to observe binding, or stimulation or inhibition of a functional response.
  • the functional response of the cells contacted with the test compound is then compared with control cells that were not contacted with the test compound.
  • Such an assay may assess whether the test compound results in a signal generated by activation of the polypeptide, using an appropriate detection system.
  • Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist in the presence of the test compound is observed.
  • a particular example is cotransfecting a construct expressing a polypeptide according to the invention, or a fragment such as the LBD, in fusion with the GAL4 DNA binding domain, into a cell together with a reporter plasmid, an example of which is pFR-Luc (Stratagene Europe, Amsterdam, The Netherlands).
  • This particular plasmid contains a synthetic promoter with five tandem repeats of GAL4 binding sites that control the expression of the luciferase gene. When a potential ligand is added to the cells, it will bind the GAL4-polypeptide fusion and induce transcription of the luciferase gene.
  • the level of the luciferase expression can be monitored by its activity using a luminescence reader (see, for example, Lehman et al. JBC 270, 12953, 1995; Pawar et al. JBC, 277, 39243, 2002).
  • a preferred method for identifying an agonist or antagonist compound of a polypeptide of the present invention comprises:
  • a further preferred method for identifying an agonist or antagonist of a polypeptide of the invention comprises:
  • a method such as FRET detection of a ligand bound to the polypeptide in the presence of peptide co-activators might be used.
  • the general methods that are described above may further comprise conducting the identification of agonist or antagonist in the presence of labelled or unlabelled ligand for the polypeptide.
  • the method for identifying agonist or antagonist of a polypeptide of the present invention comprises: determining the inhibition of binding of a ligand to cells which have a polypeptide of the invention on the surface thereof, or to cell membranes containing such a polypeptide, in the presence of a candidate compound under conditions to permit binding to the polypeptide, and determining the amount of ligand bound to the polypeptide.
  • a compound capable of causing reduction of binding of a ligand is considered to be an agonist or antagonist.
  • the ligand is labelled.
  • a method of screening for a polypeptide antagonist or agonist compound comprises the steps of:
  • step (c) adding a candidate compound to a mixture of labelled ligand and the whole cell or the cell membrane of step (a) and allowing the mixture to attain equilibrium;
  • step (d) measuring the amount of labelled ligand bound to the whole cell or the cell membrane after step (c);
  • step (e) comparing the difference in the labelled ligand bound in step (b) and (d), such that the compound which causes the reduction in binding in step (d) is considered to be an agonist or antagonist.
  • polypeptides may be found to modulate a variety of physiological and pathological processes in a dose-dependent manner in the above-described assays.
  • the "functional equivalents" of the polypeptides of the invention include polypeptides that exhibit any of the same modulatory activities in the above-described assays in a dose-dependent manner.
  • the degree of dose-dependent activity need not be identical to that of the polypeptides of the invention, preferably the "functional equivalents" will exhibit substantially similar dose-dependence in a given activity assay compared to the polypeptides of the invention.
  • simple binding assays may be used, in which the adherence of a test compound to a surface bearing the polypeptide is detected by means of a label directly or indirectly associated with the test compound or in an assay involving competition with a labelled competitor
  • competitive drug screening assays may be used, in which neutralising antibodies that are capable of binding the polypeptide specifically compete with a test compound for binding. In this manner, the antibodies can be used to detect the presence of any test compound that possesses specific binding affinity for the polypeptide.
  • Assays may also be designed to detect the effect of added test compounds on the production of mRNA encoding the polypeptide in cells.
  • an ELISA may be constructed that measures secreted or cell-associated levels of polypeptide using monoclonal or polyclonal antibodies by standard methods known in the art, and this can be used to search for compounds that may inhibit or enhance the production of the polypeptide from suitably manipulated cells or tissues. The formation of binding complexes between the polypeptide and the compound being tested may then be measured.
  • Another technique for drug screening which may be used provides for high throughput screening of compounds having suitable binding affinity to the polypeptide of interest (see International patent application WO84/03564).
  • This method large numbers of different small test compounds are synthesised on a solid substrate, which may then be reacted with the polypeptide of the invention and washed.
  • One way of immobilising the polypeptide is to use non-neutralising antibodies.
  • Bound polypeptide may then be detected using methods that are well known in the art.
  • Purified polypeptide can also be coated directly onto plates for use in the aforementioned drug screening techniques.
  • Assay methods that are also included within the terms of the present invention are those that involve the use of the genes and polypeptides of the invention in overexpression or ablation assays.
  • Such assays involve the manipulation of levels of these genes/polypeptides in cells and assessment of the impact of this manipulation event on the physiology of the manipulated cells. For example, such experiments reveal details of signalling and metabolic pathways in which the particular genes/polypeptides are implicated, generate information regarding the identities of polypeptides with which the studied polypeptides interact and provide clues as to methods by which related genes and proteins are regulated.
  • Another technique for drug screening which may be used provides for high throughput screening of compounds having suitable binding affinity to the polypeptide of interest (see International patent application WO84/03564). hi this method, large numbers of different small test compounds are synthesised on a solid substrate, which may then be reacted with the polypeptide of the invention and washed.
  • One way of immobilising the polypeptide is to use non-neutralising antibodies. Bound polypeptide may then be detected using methods that are well known in the art. Purified polypeptide can also be coated directly onto plates for use in the aforementioned drug screening techniques.
  • the polypeptide of the invention may be used to identify membrane-bound or soluble receptors, through standard receptor binding techniques that are known in the art, such as ligand binding and crosslinking assays in which the polypeptide is labelled with a radioactive isotope, is chemically modified, or is fused to a peptide sequence that facilitates its detection or purification, and incubated with a source of the putative receptor (for example, a composition of cells, cell membranes, cell supernatants, tissue extracts, or bodily fluids).
  • a source of the putative receptor for example, a composition of cells, cell membranes, cell supernatants, tissue extracts, or bodily fluids.
  • the efficacy of binding may be measured using biophysical techniques such as surface plasmon resonance (supplied by Biacore AB,
  • Binding assays may be used for the purification and cloning of the receptor, but may also identify agonists and antagonists of the polypeptide, that compete with the binding of the polypeptide to its receptor. Standard methods for conducting screening assays are well understood in the art.
  • this invention relates to the use of a INSP 168, INSP168-SV1, INSP 149 or INSP 169 polypeptide or fragment thereof, whereby the fragment is preferably a INSP168, INSP168-SV1, INSP149 or INSP169 gene-specific fragment, for isolating or generating an agonist or stimulator of the INSP 168, INSP168-SV1, INSP 149 or INSP 169 polypeptide for the treatment of a disorder, wherein said agonist or stimulator is selected from the group consisting of:
  • a specific antibody or fragment thereof including: a) a chimeric, b) a humanized or c) a fully human antibody, as well as;
  • an antibody-mimetic such as a) an anticalin or b) a fibronectin-based binding molecule (e.g. trinectin or adnectin).
  • Anticalins are also known in the art (Vo gt et ah, 2004). Fibronectin-based binding molecules are described in US6818418 and WO2004029224.
  • test compound may be of various origin, nature and composition, such as any small molecule, nucleic acid, lipid, peptide, polypeptide including an antibody such as a chimeric, humanized or fully human antibody or an antibody fragment, peptide- or non-peptide mimetic derived therefrom as well as a bispecific or multispecific antibody, a single chain (e.g. scFv) or single domain antibody or an antibody-mimetic such as an anticalin or fibronectin-based binding molecule (e.g. trinectin or adnectin), etc., in isolated form or in mixture or combinations.
  • an antibody such as a chimeric, humanized or fully human antibody or an antibody fragment, peptide- or non-peptide mimetic derived therefrom as well as a bispecific or multispecific antibody, a single chain (e.g. scFv) or single domain antibody or an antibody-mimetic such as an anticalin or fibronectin-based binding molecule (e.g. trinectin
  • the invention also includes a screening kit useful in the methods for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, that are described above.
  • the invention includes the agonists, antagonists, ligands, receptors, substrates and enzymes, and other compounds which modulate the activity or antigenicity of the polypeptide of the invention discovered by the methods that are described above.
  • the various moieties of the invention i.e. the polypeptides of the first aspect of the invention, a nucleic acid molecule of the second or third aspect of the invention, a vector of the fourth aspect of the invention, a host cell of the fifth aspect of the invention, a ligand of the sixth aspect of the invention, a compound of the seventh aspect of the invention
  • the various moieties of the invention may be useful in the therapy or diagnosis of diseases.
  • one or more of the following assays may be carried out.
  • test compound refers to the test compound as being a protein/polypeptide
  • a person skilled in the art will readily be able to adapt the following assays so that the other moieties of the invention may also be used as the "test compound”.
  • the invention also provides pharmaceutical compositions comprising a polypeptide, nucleic acid, ligand or compound of the invention in combination with a suitable pharmaceutical carrier. These compositions may be suitable as therapeutic or diagnostic reagents, as vaccines, or as other immunogenic compositions, as outlined in detail below.
  • a composition containing a polypeptide, nucleic acid, ligand or compound [X] is "substantially free of impurities [herein, Y] when at least 85% by weight of the total X+Y in the composition is X.
  • X comprises at least about 90% by weight of the total of X+Y in the composition, more preferably at least about 95%, 98% or even 99% by weight.
  • the pharmaceutical compositions should preferably comprise a therapeutically effective amount of the polypeptide, nucleic acid molecule, ligand, or compound of the invention.
  • therapeutically effective amount refers to an amount of a therapeutic agent needed to treat, ameliorate, or prevent a targeted disease or condition, or to exhibit a detectable therapeutic or preventative effect.
  • the therapeutically effective dose can be estimated initially either in cell culture assays, for example, of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • an effective amount for a human subject will depend upon the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. This amount can be determined by routine experimentation and is within the judgement of the clinician. Generally, an effective dose will be from 0.01 mg/kg to 50 mg/kg, preferably 0.05 mg/kg to 10 mg/kg. Compositions may be administered individually to a patient or may be administered in combination with other agents, drugs or hormones.
  • a pharmaceutical composition may also contain a pharmaceutically acceptable carrier, for administration of a therapeutic agent.
  • a pharmaceutically acceptable carrier for administration of a therapeutic agent.
  • Such carriers include antibodies and other polypeptides, genes and other therapeutic agents such as liposomes, provided that the carrier does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity.
  • Suitable carriers may be large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles.
  • Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulphates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulphates, and the like
  • organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • compositions of therapeutic compositions may additionally contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such compositions. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient. Once formulated, the compositions of the invention can be administered directly to the subject.
  • the subjects to be treated can be animals; in particular, human subjects can be treated.
  • compositions utilised in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal or transcutaneous applications (for example, see WO98/20734), subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal or rectal means.
  • Gene guns or hyposprays may also be used to administer the pharmaceutical compositions of the invention.
  • the therapeutic compositions may be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.
  • Direct delivery of the compositions will generally be accomplished by injection, subcutaneously, intraperitoneally, intravenously or intramuscularly, or delivered to the interstitial space of a tissue.
  • the compositions can also be administered into a lesion. Dosage treatment may be a single dose schedule or a multiple dose schedule. If the activity of the polypeptide of the invention is in excess in a particular disease state, several approaches are available.
  • One approach comprises administering to a subject an inhibitor compound (antagonist) as described above, along with a pharmaceutically acceptable carrier in an amount effective to inhibit the function of the polypeptide, such as by blocking the binding of ligands, substrates, enzymes, receptors, or by inhibiting a second signal, and thereby alleviating the abnormal condition.
  • such antagonists are antibodies. Most preferably, such antibodies are chimeric and/or humanised to minimise their immunogenicity, as described previously.
  • polypeptide that retain binding affinity for the ligand, substrate, enzyme, receptor, in question, may be administered.
  • polypeptide may be administered in the form of fragments that retain the relevant portions.
  • expression of the gene encoding the polypeptide can be inhibited using expression blocking techniques, such as the use of antisense nucleic acid molecules (as described above), either internally generated or separately administered.
  • Modifications of gene expression can be obtained by designing complementary sequences or antisense molecules (DNA, RNA, or PNA) to the control, 5' or regulatory regions (signal sequence, promoters, enhancers and introns) of the gene encoding the polypeptide.
  • inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules.
  • the complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Such oligonucleotides may be administered or may be generated in situ from expression in vivo.
  • Ribozymes are catalytically active RNAs that can be natural or synthetic (see for example Usman, N, et al., Curr. Opin. Struct. Biol (1996) 6(4), 527-33). Synthetic ribozymes can be designed to specifically cleave mRNAs at selected positions thereby preventing translation of the mRNAs into functional polypeptide. Ribozymes may be synthesised with a natural ribose phosphate backbone and natural bases, as normally found in RNA molecules. Alternatively the ribozymes may be synthesised with non-natural backbones, for example, 2'-O-methyl RNA, to provide protection from ribonuclease degradation and may contain modified bases.
  • RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of non-traditional bases such as inosine, queosine and butosine, as well as acetyl-, methyl-, thio- and similarly modified forms of adenine, cytidine, guanine, thymine and uridine which are not as easily recognised by endogenous endonucleases.
  • One approach comprises administering to a subject a therapeutically effective amount of a compound that activates the polypeptide, i.e., an agonist as described above, to alleviate the abnormal condition.
  • a therapeutic amount of the polypeptide in combination with a suitable pharmaceutical carrier may be administered to restore the relevant physiological balance of polypeptide.
  • Gene therapy may be employed to effect the endogenous production of the polypeptide by the relevant cells in the subject. Gene therapy is used to treat permanently the inappropriate production of the polypeptide by replacing a defective gene with a corrected therapeutic gene. Gene therapy of the present invention can occur in vivo or ex vivo.
  • Ex vivo gene therapy requires the isolation and purification of patient cells, the introduction of a therapeutic gene and introduction of the genetically altered cells back into the patient. In contrast, in vivo gene therapy does not require isolation and purification of a patient's cells.
  • the therapeutic gene is typically "packaged" for administration to a patient.
  • Gene delivery vehicles may be non-viral, such as liposomes, or replication-deficient viruses, such as adenovirus as described by Berkner, K.L., in Curr. Top. Microbiol. Immunol., 158, 39-66 (1992) or adeno-associated virus (AAV) vectors as described by Muzyczka, N., in Curr. Top. Microbiol.
  • a nucleic acid molecule encoding a polypeptide of the invention may be engineered for expression in a replication-defective retroviral vector.
  • This expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding the polypeptide, such that the packaging cell now produces infectious viral particles containing the gene of interest.
  • producer cells may be administered to a subject for engineering cells in vivo and expression of the polypeptide in vivo (see Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, (and references cited therein) in Human Molecular Genetics (1996), T Strachan and A P Read, BIOS Scientific Publishers Ltd).
  • Another approach is the administration of "naked DNA" in which the therapeutic gene is directly injected into the bloodstream or muscle tissue.
  • the invention provides that they can be used in vaccines to raise antibodies against the disease causing agent.
  • Vaccines according to the invention may either be prophylactic (ie. to prevent infection) or therapeutic (ie. to treat disease after infection).
  • Such vaccines comprise immunising antigen(s), immunogen(s), polypeptide(s), protein(s) or nucleic acid, usually in combination with pharmaceutically-acceptable carriers as described above, which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Additionally, these carriers may function as immunostimulating agents ("adjuvants").
  • the antigen or immunogen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, H. pylori, and other pathogens.
  • vaccines comprising polypeptides are preferably administered parenterally (for instance, subcutaneous, intramuscular, intravenous, or intradermal injection).
  • parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents.
  • the vaccine formulations of the invention may be presented in unit-dose or multi- dose containers.
  • sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use.
  • the dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
  • jet injection may also be useful in the formulation of vaccine compositions.
  • This invention also relates to the use of nucleic acid molecules according to the present invention as diagnostic reagents. Detection of a mutated form of the gene characterised by the nucleic acid molecules of the invention which is associated with a dysfunction will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, or susceptibility to a disease, which results from under-expression, over- expression or altered spatial or temporal expression of the gene. Individuals carrying mutations in the gene may be detected at the DNA level by a variety of techniques. Nucleic acid molecules for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy material.
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR, ligase chain reaction (LCR), strand displacement amplification (SDA), or other amplification techniques (see Saiki et ah, Nature, 324, 163-166 (1986); Bej, et ah, Grit Rev. Biochem. Molec. Biol., 26, 301-334 (1991); Birkenmeyer et al., J. Virol. Meth., 35, 117-126 (1991); Van Brunt, J., Bio/Technology, 8, 291-294 (1990)) prior to analysis.
  • LCR ligase chain reaction
  • SDA strand displacement amplification
  • this aspect of the invention provides a method of diagnosing a disease in a patient, comprising assessing the level of expression of a natural gene encoding a polypeptide according to the invention and comparing said level of expression to a control level, wherein a level that is different to said control level is indicative of disease.
  • the method may comprise the steps of: a) contacting a sample of tissue from the patient with a nucleic acid probe under stringent conditions that allow the formation of a hybrid complex between a nucleic acid molecule of the invention and the probe; b) contacting a control sample with said probe under the same conditions used in step a); c) and detecting the presence of hybrid complexes in said samples; wherein detection of levels of the hybrid complex in the patient sample that differ from levels of the hybrid complex in the control sample is indicative of disease.
  • a further aspect of the invention comprises a diagnostic method comprising the steps of: a) obtaining a tissue sample from a patient being tested for disease; b) isolating a nucleic acid molecule according to the invention from said tissue sample; and c) diagnosing the patient for disease by detecting the presence of a mutation in the nucleic acid molecule which is associated with disease.
  • an amplification step for example using PCR, may be included. Deletions and insertions can be detected by a change in the size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labelled RNA of the invention or alternatively, labelled antisense DNA sequences of the invention. Perfectly-matched sequences can be distinguished from mismatched duplexes by RNase digestion or by assessing differences in melting temperatures.
  • the presence or absence of the mutation in the patient may be detected by contacting DNA with a nucleic acid probe that hybridises to the DNA under stringent conditions to form a hybrid double-stranded molecule, the hybrid double-stranded molecule having an unhybridised portion of the nucleic acid probe strand at any portion corresponding to a mutation associated with disease; and detecting the presence or absence of an unhybridised portion of the probe strand as an indication of the presence or absence of a disease-associated mutation in the corresponding portion of the DNA strand.
  • Point mutations and other sequence differences between the reference gene and "mutant" genes can be identified by other well-known techniques, such as direct DNA sequencing or single-strand conformational polymorphism, (see Orita et al., Genomics, 5, 874-879 (1989)).
  • a sequencing primer may be used with double-stranded PCR product or a single-stranded template molecule generated by a modified PCR.
  • the sequence determination is performed by conventional procedures with radiolabeled nucleotides or by automatic sequencing procedures with fluorescent-tags.
  • Cloned DNA segments may also be used as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR.
  • point mutations and other sequence variations, such as polymorphisms can be detected as described above, for example, through the use of allele-specific oligonucleotides for PCR amplification of sequences that differ by single nucleotides.
  • DNA sequence differences may also be detected by alterations in the electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing (for example, Myers et al, Science (1985) 230:1242). Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and Sl protection or the chemical cleavage method (see Cotton et al., Proc. Natl. Acad. Sci. USA (1985) 85: 4397-4401).
  • mutations such as microdeletions, aneuploidies, translocations, inversions, can also be detected by in situ analysis (see, for example, Keller et ah, DNA Probes, 2nd Ed., Stockton Press, New York, N.Y., USA (1993)), that is, DNA or RNA sequences in cells can be analysed for mutations without need for their isolation and/or immobilisation onto a membrane.
  • FISH Fluorescence in situ hybridization
  • an array of oligonucleotide probes comprising a nucleic acid molecule according to the invention can be constructed to conduct efficient screening of genetic variants, mutations and polymorphisms.
  • Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability (see for example: M.Chee et ah, Science (1996), VoI 274, pp 610-613).
  • the array is prepared and used according to the methods described in PCT application WO95/11995 (Chee et ah); Lockhart, D. J. et a (1996) Nat. Biotech. 14: 1675-1680); and Schena, M. et a (1996) Proc. Natl. Acad. Sci. 93: 10614-10619).
  • Oligonucleotide pairs may range from two to over one million.
  • the oligomers are synthesized at designated areas on a substrate using a light-directed chemical process.
  • the substrate may be paper, nylon or other type of membrane, filter, chip, glass slide or any other suitable solid support.
  • an oligonucleotide may be synthesized on the surface of the substrate by using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application W095/25116 (Baldeschweiler et at), hi another aspect, a "gridded" array analogous to a dot (or slot) blot may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures.
  • An array such as those described above, may be produced by hand or by using available devices (slot blot or dot blot apparatus), materials (any suitable solid support), and machines (including robotic instruments), and may contain 8, 24, 96, 384, 1536 or 6144 oligonucleotides, or any other number between two and over one million which lends itself to the efficient use of commercially-available instrumentation.
  • diseases may be diagnosed by methods comprising determining, from a sample derived from a subject, an abnormally decreased or increased level of polypeptide or mRNA. Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, nucleic acid amplification, for instance PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.
  • nucleic acid amplification for instance PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.
  • Assay techniques that can be used to determine levels of a polypeptide of the present invention in a sample derived from a host are well-known to those of skill in the art and are discussed in some detail above (including radioimmunoassays, competitive- binding assays, Western Blot analysis and ELISA assays).
  • This aspect of the invention provides a diagnostic method which comprises the steps of: (a) contacting a ligand as described above with a biological sample under conditions suitable for the formation of a ligand-polypeptide complex; and (b) detecting said complex.
  • Protocols such as ELISA, RIA, and FACS for measuring polypeptide levels may additionally provide a basis for diagnosing altered or abnormal levels of polypeptide expression.
  • Normal or standard values for polypeptide expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably humans, with antibody to the polypeptide under conditions suitable for complex formation The amount of standard complex formation may be quantified by various methods, such as by photometric means.
  • Antibodies which specifically bind to a polypeptide of the invention may be used for the diagnosis of conditions or diseases characterised by expression of the polypeptide, or in assays to monitor patients being treated with the polypeptides, nucleic acid molecules, ligands and other compounds of the invention.
  • Antibodies useful for diagnostic purposes may be prepared in the same manner as those described above for therapeutics. Diagnostic assays for the polypeptide include methods that utilise the antibody and a label to detect the polypeptide in human body fluids or extracts of cells or tissues.
  • the antibodies may be used with or without modification, and may be labelled by joining them, either covalently or non-covalently, with a reporter molecule.
  • a wide variety of reporter molecules known in the art may be used, several of which are described above.
  • a diagnostic kit of the present invention may comprise:
  • a diagnostic kit may comprise a first container containing a nucleic acid probe that hybridises under stringent conditions with a nucleic acid molecule according to the invention; a second container containing primers useful for amplifying the nucleic acid molecule; and instructions for using the probe and primers for facilitating the diagnosis of disease.
  • the kit may further comprise a third container holding an agent for digesting unhybridised RNA.
  • a diagnostic kit may comprise an array of nucleic acid molecules, at least one of which may be a nucleic acid molecule according to the invention.
  • a diagnostic kit may comprise one or more antibodies that bind to a polypeptide according to the invention; and a reagent useful for the detection of a binding reaction between the antibody and the polypeptide.
  • kits will be of use in diagnosing a disease or susceptibility to disease, particularly diseases in which leucine-rich repeat motif containing proteins are implicated.
  • diseases include, but are not limited to, diseases of the retina, retinal pigment epithelium (RPE), and choroids; ocular neovascularization, ocular inflammation and retinal degenerations; diabetic retinopathy, chronic glaucoma, retinal detachment, sickle cell retinopathy, senile macular degeneration, retinal neovascularization, subretinal neovascularization; rubeosis ulceris inflammatory diseases, chronic posterior and pan uveitis, neoplasms, retinoblastoma, pseudoglioma, neovascular glaucoma; neovascularization resulting following a combined vitrectomy and lensectomy, vascular diseases retinal ischemia, choroidal vascular insufficiency, choroidal thrombosis, neovascularization
  • Figure 1 Amino acid alignment of INSP168, INSP168-SV1, INSP149 and INSP169 ORFs. The predicted transmembrane region is in bold. The predicted internal LRR region is boxed.
  • Figure 2 Nucleotide sequence with translation of the PCR product INSP168 cloned using primers INSPl 68-CP 1 and INSP168-CP2. The predicted signal peptide is in bold. The predicted internal LRR region is boxed. The position and sense of the primers are indicated by arrows.
  • Figure 3 Nucleotide sequence with translation of the PCR product INSP168-SV1 cloned using primers INSPl 68-CP 1 and INSPl 68-CP2. The predicted signal peptide is in bold. The predicted internal LRR region is boxed. The position and sense of the primers are indicated by arrows.
  • Figure 4 Genomic organisation of the PCR product INSP168-SV1.
  • Figure 5 Amino acid alignment between INSP168, INSP168-SV1, INSP149 and INSP 169 and Retinal Specific Protein PAL (SwissProt Ace. Code PALP_HUMAN).
  • Figure 6 Schematic domain representation of INSP 168, INSP168-SV1, INSP 149, INSP 169, Retinal Specific Protein PAL (SwissProt Ace. Code PALP HUMAN) and nogo receptor homolog (SwissProt Ace. Code Q6X814).
  • Figure 7 Effect of INSP 168 on Stat-2 nuclear translocation in U373 in two distinct experiments.
  • the two left hand columns illustrate the effect of addition of medium only, the two middle columns illustrate the effect of addition of the positive control IFN-beta, and the two right hand columns illustrate the effect of addition of INSP 168 to the medium.
  • Figure 8 cDNA coding sequence and deduced peptide sequence of the cloned INSP 169 extracellular domain.
  • the position and orientation of primers used for cloning and sequencing are indicated by arrows.
  • the sequence in bold corresponds to the centrally located BamHI site used in the assembly of the full length clone.
  • Figure 9 Nucleotide sequence of the cloned INSP 169 extracellular domain.
  • Figure 10 Peptide sequence of the cloned cDNA from the N terminus to the TM domain. Signal sequence is in italic; the leucine rich repeat region is in bold; the immunoglubulin C-2 domain is underlined; the fibronectin type3 domain is in bold italic. TABLEl
  • First strand cDNA was prepared from a variety of normal human tissue total RNA samples (Clontech, Stratagene, Ambion, Biochain Institute and in-house preparations) using Superscript II RNase H- Reverse Transcriptase (Invitrogen) according to the manufacturer's protocol.
  • Oligo (dT)15 primer (l ⁇ l at 500 ⁇ g/ml) (Promega)
  • 2 ⁇ g human total RNA 1 ⁇ l 10 mM dNTP mix (10 mM each of dATP, dGTP, dCTP and dTTP at neutral pH) and sterile distilled water to a final volume of 12 ⁇ l were combined in a 1.5 ml Eppendorf tube, heated to 65 °C for 5 min and then chilled on ice.
  • coli RNase H (Invitrogen) was added and the reaction mixture incubated at 37 °C for 20 min. The final 21 ⁇ l reaction mix was diluted by adding 179 ⁇ l sterile water to give a total volume of 200 ⁇ l.
  • the cDNA templates used for the amplification of INSP 168 were derived from brain and eye RNA. cDNA libraries
  • Human cDNA libraries (in bacteriophage lambda ( ⁇ ) vectors) were purchased from Stratagene or Clontech or prepared at the Serono Pharmaceutical Research Institute in ⁇ ZAP or ⁇ GTlO vectors according to the manufacturer's protocol (Stratagene). Bacteriophage ⁇ DNA was prepared from small scale cultures of infected E. coli host strain using the Wizard Lambda Preps DNA purification system according to the manufacturer's instructions (Promega, Corporation, Madison WL). cDNA library templates used for the amplification of INSP 168 were derived from brain, fetal brain, retina, and a mixed brain-lung-testis library. Gene specific cloning primers for PCR
  • PCR primers having a length of between 18 and 25 bases was designed for amplifying the predicted coding sequence of the virtual cDNA using Primer Designer Software (Scientific & Educational Software, PO Box 72045, Durham, NC 27722- 2045, USA). PCR primers were optimized to have a Tm close to 55 + 10 0 C and a GC content of 40-60%. Primers were selected which had high selectivity for the target sequence (INSP 168) with little or no none specific priming. PCR amplification of INSP 168 from human cDNA templates
  • Gene-specific cloning primers (INSPl 68-CP 1 and INSP168-CP2, Figure 2, Figure 3 and Table 3) were designed to amplify a cDNA fragment of 614 bp covering the entire of the predicted INSP 168 cds.
  • the primer pair was used with the human cDNA samples and cDNA libraries listed above as PCR templates.
  • PCR was performed in a final volume of 50 ⁇ l containing IX Platinum® Taq High Fidelity (HiFi) buffer, 2 mM MgSO4, 200 ⁇ M dNTPs, 0.2 ⁇ M of each cloning primer, 1 unit of Platinum® Taq DNA Polymerase High Fidelity (HiFi) (Invitrogen), and approximately 20 ng of template cDNA.
  • HiFi IX Platinum® Taq High Fidelity
  • Cycling was performed using an MJ Research DNA Engine, programmed as follows: 94 0 C, 2 min; 40 cycles of 94 °C, 30 sec, 55 0 C, 30 sec, and 68 0 C, 1 min; followed by 1 cycle at 68 0 C for 7 min and a holding cycle at 4 0 C.
  • PCR products were subcloned into the topoisomerase I modified cloning vector (pCR4-TOPO) using the TA cloning kit purchased from the Invitrogen Corporation using the conditions specified by the manufacturer. Briefly, 4 ⁇ l of gel purified PCR product was incubated for 15 min at room temperature with 1 ⁇ l of TOPO vector and 1 ⁇ l salt solution. The reaction mixture was then transformed into E. coli strain TOPlO (Invitrogen) as follows: a 50 ⁇ l aliquot of One Shot TOPlO cells was thawed on ice and 2 ⁇ l of TOPO reaction was added. The mixture was incubated for 15 min on ice and then heat shocked by incubation at 42 0 C for exactly 30 s.
  • TOPO E. coli strain TOPO
  • Colonies were inoculated into 50 ⁇ l sterile water using a sterile toothpick. A 10 ⁇ l aliquot of the inoculum was then subjected to PCR in a total reaction volume of 20 ⁇ l containing IX AmpliTaqTM buffer, 200 ⁇ M dNTPs, 20 pmoles of T7 primer, 20 pmoles of T3 primer, 1 unit of AmpliTaqTM (Applied Biosystems) using an MJ Research DNA Engine. The cycling conditions were as follows: 94 0 C, 2 min; 30 cycles of 94 0 C, 30 sec, 48 °C, 30 sec and 72 °C for 1 min. Samples were maintained at 4 °C (holding cycle) before further analysis.
  • PCR reaction products were analyzed on 1 % agarose gels in 1 X TAE buffer. Colonies which gave PCR products of approximately the expected molecular weight (614 bp or 267 bp + 105 bp due to the multiple cloning site (MCS)) were grown up overnight at 37 0 C in 5 ml L-Broth (LB) containing ampicillin (100 ⁇ g /ml), with shaking at 220 rpm. Plasmid DNA preparation and sequencing
  • Miniprep plasmid DNA was prepared from the 5 ml culture using a Biorobot 8000 robotic system (Qiagen) or Wizard Plus SV Minipreps kit (Promega cat. no. 1460) according to the manufacturer's instructions. Plasmid DNA was eluted in 80 ⁇ l of sterile water. The DNA concentration was measured using a Spectramax 190 photometer (Molecular Devices). Plasmid DNA (200-500 ng) was subjected to DNA sequencing with the T7 and T3 primers using the BigDye Terminator system (Applied Biosystems cat. no. 4390246) according to the manufacturer's instructions. The primer sequences are shown in Table 3. Sequencing reactions were purified using Dye-Ex columns (Qiagen) or Montage SEQ 96 cleanup plates (Millipore cat. no. LSKS09624) then analyzed on an Applied Biosystems 3700 sequencer.
  • Sequence analysis identified a clone, amplified from brain cDNA, which matched the expected INSP 168 sequence.
  • the plasmid map of the cloned PCR product is pCR4- TOPO-INSP168.
  • the nucleotide sequence with translation of the PCR product INSPl 68 is shown in Figure 2.
  • a second clone was identified, also amplified from brain, which contained the INSP 168 cds with a 77 bp insertion towards the 3' end of the sequence. This led to an insertion of 32 amino acids and a frameshift such that an ORF of 229 amino acids was produced.
  • the insertion represented an additional exon, giving an ORF encoded in 4 exons.
  • a stop codon was not identified but one present 2 bp downstream of the 3' end of the new sequence in genomic DNA was assumed to be functional.
  • the nucleotide sequence with translation of the PCR product INSPl 68-SV 1 is shown in Figure 3.
  • This clone was named pCR4-TOPO-INSP168-SVl.
  • the genomic organisation of the INSPl 68-SV 1 cds is shown in Figure 4.
  • the plasmid map of the cloned PCR product is pCR4-TOPO-INSP168-SVl.
  • the position of the INSPl 68-CP 1 amplification primer meant that the final base of the cds was missing - this base was added during transfer into the Gateway entry vector pDONR 221 (see below).
  • the first stage of the Gateway cloning process involves a two step PCR reaction which generates the ORF of INSP 168 flanked at the 5' end by an attBl recombination site and Kozak sequence, and flanked at the 3' end by a sequence encoding an in frame 6 histidine (6HIS) tag, a stop codon and the attB2 recombination site (Gateway compatible cDNA).
  • 6HIS in frame 6 histidine
  • the first PCR reaction (in a final volume of 50 ⁇ l) contains respectively: 1 ⁇ l (40 ng) of plasmid pCR4-TOPO-INSP168, 1.5 ⁇ l dNTPs (10 mM), 10 ⁇ l of 1OX Pfx polymerase buffer, 1 ⁇ l MgSO4 (50 mM), 0.5 ⁇ l each of gene specific primer (100 ⁇ M) (INSP168-EX1 and INSP168-EX2), and 0.5 ⁇ l Platinum Pfx DNA polymerase (Invitrogen).
  • the PCR reaction was performed using an initial denaturing step of 95 °C for 2 min, followed by 12 cycles of 94 0 C for 15 s; 55 °C for 30 s and 68 °C for 2 min; and a holding cycle of 4 0 C.
  • the amplification product was directly purified using the Wizard PCR Preps DNA Purification System (Promega) and recovered in 50 ⁇ l sterile water according to the manufacturer's instructions.
  • the second PCR reaction (in a final volume of 50 ⁇ l) contained 10 ⁇ l purified PCR 1 product, 1.5 ⁇ l dNTPs (10 mM), 5 ⁇ l of 1OX Pfx polymerase buffer, 1 ⁇ l MgSO4 (50 mM), 0.5 ⁇ l of each Gateway conversion primer (100 ⁇ M) (GCP forward and GCP reverse) and 0.5 ⁇ l of Platinum Pfx DNA polymerase.
  • the conditions for the 2nd PCR reaction were: 95 °C for 1 min; 4 cycles of 94 °C, 15 sec; 50 °C, 30 sec and 68 0 C for 2 min; 25 cycles of 94 0 C, 15 sec; 55 0 C , 30 sec and 68 °C, 2 min; followed by a holding cycle of 4 °C.
  • PCR product was visualized on 0.8 % agarose gel in 1 X TAE buffer (Invitrogen) and the band migrating at the predicted molecular mass (661 bp) was purified from the gel using the Wizard PCR Preps DNA Purification System (Promega) and recovered in 50 ⁇ l sterile water according to the manufacturer's instructions.
  • the second stage of the Gateway cloning process involves subcloning of the Gateway modified PCR products into the Gateway entry vector pDONR221 (Invitrogen) as follows: 5 ⁇ l of purified product from PCR2 were incubated with 1.5 ⁇ l pDONR221 vector (0.1 ⁇ g/ ⁇ l), 2 ⁇ l BP buffer and 1.5 ⁇ l of BP clonase enzyme mix (Invitrogen) in a final volume of 10 ⁇ l at RT for 1 h. The reaction was stopped by addition of proteinase K 1 ⁇ l (2 ⁇ g/ ⁇ l) and incubated at 37 °C for a further 10 min. An aliquot of this reaction (1 ⁇ l) was used to transform E.
  • pDONR221 Invitrogen
  • coli DHlOB cells by electroporation as follows: a 25 ⁇ l aliquot of DHlOB electrocompetent cells (Invitrogen) was thawed on ice and 1 ⁇ l of the BP reaction mix was added. The mixture was transferred to a chilled 0.1 cm electroporation cuvette and the cells electroporated using a BioRad Gene-PulserTM according to the manufacturer's recommended protocol. SOC media (0.5 ml) which had been pre- warmed to room temperature was added immediately after electroporation. The mixture was transferred to a 15 ml snap-cap tube and incubated, with shaking (220 rpm) for 1 h at 37 °C.
  • Plasmid mini-prep DNA was prepared from 5 ml cultures from 6 of the resultant colonies using a Qiaprep BioRobot 8000 system (Qiagen). Plasmid DNA (150-200 ng) was subjected to DNA sequencing with 21M13 and M13Rev primers using the BigDyeTerminator system (Applied Biosystems cat. no. 4336919) according to the manufacturer's instructions. The primer sequences are shown in Table 3. Sequencing reactions were purified using Montage SEQ 96 cleanup plates (Millipore cat. no. LSKS09624) then analyzed on an Applied Biosystems 3700 sequencer.
  • Plasmid eluate (2 ⁇ l or approx. 150 ng) from one of the clones which contained the correct sequence (pENTR_INSP168-6HIS) was then used in a recombination reaction containing 1.5 ⁇ l of either pEAK12d vector or pDEST12.2 vector (0.1 ⁇ g / ⁇ l), 2 ⁇ l LR buffer and 1.5 ⁇ l of LR clonase (Invitrogen) in a final volume of 10 ⁇ l.
  • the mixture was incubated at RT for 1 h, stopped by addition of proteinase K (2 ⁇ g) and incubated at 37 0 C for a further 10 min. An aliquot of this reaction (1 ul) was used to transform E.
  • coli DHlOB cells by electroporation as follows: a 25 ⁇ l aliquot of DHlOB electrocompetent cells (Invitrogen) was thawed on ice and 1 ⁇ l of the LR reaction mix was added. The mixture was transferred to a chilled 0.1 cm electroporation cuvette and the cells electroporated using a BioRad Gene-PulserTM according to the manufacturer's recommended protocol. SOC media (0.5 ml) which had been pre-warmed to room temperature was added immediately after electroporation. The mixture was transferred to a 15 ml snap-cap tube and incubated, with shaking (220 rpm) for 1 h at 37 °C. Aliquots of the transformation mixture (10 ⁇ l and 50 ⁇ l) were then plated on L-broth (LB) plates containing ampicillin (100 ⁇ g/ml) and incubated overnight at 37 °C.
  • LB L-broth
  • Plasmid mini-prep DNA was prepared from 5 ml cultures from 6 of the resultant colonies subcloned in each vector using a Qiaprep Bio Robot 8000 (Qiagen). Plasmid DNA (200-500 ng) in the pEAK12d vector was subjected to DNA sequencing with pEAK12F and pEAK12R primers as described above. Plasmid DNA (200-500 ng) in the pDEST12.2 vector was subjected to DNA sequencing with 21M13 and M13Rev primers as described above. Primer sequences are shown in Table 3.
  • CsCl gradient purified maxi-prep DNA was prepared from a 500 ml culture of the sequence verified clone (pEAK12d_INSP168-6HIS) using the method described by Sambrook J. et al., 1989 (in Molecular Cloning, a Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press). Plasmid DNA was resuspended at a concentration of 1 ⁇ g/ ⁇ l in sterile water (or 10 mM Tris-HCl pH 8.5) and stored at -20 °C.
  • Endotoxin-free maxi-prep DNA was prepared from a 500 ml culture of the sequence verified clone ( ⁇ DEST12.2_INSP168-6HIS) using the EndoFree Plasmid Mega kit (Qiagen) according to the manufacturer's instructions. Purified plasmid DNA was resuspended in endotoxin free TE buffer at a final concentration of at least 3 ⁇ g/ ⁇ l and stored at -20 0 C.
  • Example 3 Tissue Distribution and Expression Levels of INSP 168 Further experiments may now be performed to determine the tissue distribution and expression levels of the INSP 168 polypeptide in vivo, on the basis of the nucleotide and amino acid sequence disclosed herein.
  • the presence of the transcripts for INSP 168 may be investigated by PCR of cDNA from different human tissues.
  • the INSP 168 transcripts may be present at very low levels in the samples tested. Therefore, extreme care is needed in the design of experiments to establish the presence of a transcript in various human tissues as a small amount of genomic contamination in the RNA preparation will provide a false positive result. Thus, all RNA should be treated with DNAse prior to use for reverse transcription. In addition, for each tissue a control reaction may be set up in which reverse transcription was not undertaken (a -ve RT control).
  • RNA from each tissue may be used to generate cDNA using Multiscript reverse transcriptase (ABI) and random hexamer primers.
  • ABSI Multiscript reverse transcriptase
  • PCR reactions are set up for each tissue on the reverse transcribed RNA samples and the minus RT controls.
  • INSP168-specific primers may readily be designed on the basis of the sequence information provided herein. The presence of a product of the correct molecular weight in the reverse transcribed sample together with the absence of a product in the minus RT control may be taken as evidence for the presence of a transcript in that tissue.
  • Any suitable cDNA libraries may be used to screen for the INSP 168 transcripts, not only those generated as described above. The tissue distribution pattern of the INSP 168 polypeptides will provide further useful information in relation to the function of those polypeptides.
  • Human Embryonic Kidney 293 cells expressing the Epstein-Barr virus Nuclear Antigen (HEK293-EBNA, Invitrogen) are maintained in suspension in Ex-cell VPRO serum-free medium (seed stock, maintenance medium, JRH). Sixteen to 20 hours prior to transfection (Day-1), cells are seeded in 2x T225 flasks (50ml per flask in
  • plasmid DNA is co-transfected with GFP (fluorescent reporter gene) DNA.
  • GFP fluorescent reporter gene
  • the transfection mix is then added to the 2xT225 flasks and incubated at 37°C (5%CO 2 ) for 6 days. Confirmation of positive transfection may be carried out by qualitative fluorescence examination at day 1 and day 6 (Axiovert 10 Zeiss).
  • Scale-up batches may be produced by following the protocol called "PEI transfection of suspension cells", referenced BP/PEI/HH/02/04, with PolyEthylenelmine from Polysciences as transfection agent.
  • the culture medium sample containing the recombinant protein with a C-terminal 6His tag is diluted with cold buffer A (5OmM NaH 2 PO 4 ; 60OmM NaCl; 8.7 % (w/v) glycerol, pH 7.5).
  • the sample is filtered then through a sterile filter (Millipore) and kept at 4 0 C in a sterile square media bottle (Nalgene).
  • the purification is performed at 4°C on the VISION workstation (Applied Biosystems) connected to an automatic sample loader (Labomatic).
  • the purification procedure is composed of two sequential steps, metal affinity chromatography on a Poros 20 MC (Applied Biosystems) column charged with Ni ions (4.6 x 50 mm, 0.83ml), followed by gel filtration on a Sephadex G-25 medium (Amersham Pharmacia) column (1,0 x 10cm).
  • the metal affinity column is regenerated with 30 column volumes of EDTA solution (10OmM EDTA; IM NaCl; pH 8.0), recharged with Ni ions through washing with 15 column volumes of a 10OmM NiSO 4 solution, washed with 10 column volumes of buffer A, followed by 7 column volumes of buffer B (5OmM NaH 2 PO 4 ; 60OmM NaCl; 8.7 % (w/v) glycerol, 40OmM; imidazole, pH 7.5), and finally equilibrated with 15 column volumes of buffer A containing 15mM imidazole.
  • EDTA solution 10OmM EDTA; IM NaCl; pH 8.0
  • the sample is transferred, by the Labomatic sample loader, into a 200ml sample loop and subsequently charged onto the Ni metal affinity column at a flow rate of lOml/min.
  • the column is washed with 12 column volumes of buffer A, followed by 28 column volumes of buffer A containing 2OmM imidazole. During the 2OmM imidazole wash loosely attached contaminating proteins are eluted from the column.
  • the recombinant His-tagged protein is finally eluted with 10 column volumes of buffer B at a flow rate of 2ml/min, and the eluted protein is collected.
  • the Sephadex G-25 gel-filtration column is regenerated with 2ml of buffer D (1.137M NaCl; 2.7mM KCl; 1.5mM KH 2 PO 4 ; 8mM Na 2 HPO 4 ; pH 7.2), and subsequently equilibrated with 4 column volumes of buffer C (137mM NaCl; 2.7mM KCl; 1.5mM KH 2 PO 4 ; 8mM Na 2 HPO 4 ; 20% (w/v) glycerol; pH 7.4).
  • buffer D (1.137M NaCl; 2.7mM KCl; 1.5mM KH 2 PO 4 ; 8mM Na 2 HPO 4 ; pH 7.2
  • buffer C 137mM NaCl; 2.7mM KCl; 1.5mM KH 2 PO 4 ; 8mM Na 2 HPO 4 ; 20% (w/v) glycerol; pH 7.4
  • the peak fraction eluted from the Ni-column is automatically loaded onto the Sephadex G-25 column through the integrated sample loader on the VISION and the protein is eluted with buffer C at a flow rate of 2 ml/min.
  • the fraction was filtered through a sterile centrifugation filter (Millipore), frozen and stored at -80°C.
  • An aliquot of the sample is analyzed on SDS-PAGE (4-12% NuPAGE gel; Novex) Western blot with anti-His antibodies.
  • the NuPAGE gel may be stained in a 0.1 % Coomassie blue R250 staining solution (30% methanol, 10% acetic acid) at room temperature for Ih and subsequently destained in 20% methanol, 7.5% acetic acid until the background is clear and the protein bands clearly visible.
  • the proteins are electrotransferred from the gel to a nitrocellulose membrane.
  • the membrane is blocked with 5% milk powder in buffer E
  • the membrane is washed with buffer E (3 x lOmin), and then incubated with a secondary HRP-conjugated anti-rabbit antibody (DAKO, HRP 0399) diluted 1/3000 in buffer E containing 2.5% milk powder for 2 hours at room temperature. After washing with buffer E (3 x 10 minutes), the membrane is developed with the ECL kit (Amersham Pharmacia) for 1 min. The membrane is subsequently exposed to a Hyperfilm (Amersham Pharmacia), the film developed and the western blot image visually analysed.
  • DAKO secondary HRP-conjugated anti-rabbit antibody
  • the protein concentration may be determined using the BCA protein assay kit (Pierce) with bovine serum albumin as standard.
  • BCA protein assay kit Pieris
  • Example 5 Biological Significance of INSP168. INSP168-SV1. INSP149 and INSP 169 As explained above, INSP168, INSP168-SV1, INSP149 and INSP169 are structurally related to the Retinal Specific Protein PAL (SwissProt Ace. Code PALP_HUMAN) and to a nogo receptor homolog (SwissProt Ace. Code Q6X814).
  • An amino acid alignment between INSP168, INSP168-SV1, INSP149 and INSP169 and PAL is shown in Figure 5, and the schematic representation of domains is shown in Figure 6.
  • PAL may be implicated in diseases of the retina, retinal pigment epithelium (RPE), and choroids (see for example JP2001128686).
  • ocular neovascularization include diabetic retinopathy, chronic glaucoma, retinal detachment, sickle cell retinopathy, senile macular degeneration, retinal neovascularization, subretinal neovascularization; rubeosis ulceris inflammatory diseases, chronic posterior and pan uveitis, neoplasms, retinoblastoma, pseudoglioma, neovascular glaucoma; neovascularization resulting following a combined vitrectomy and lensectomy, vascular diseases retinal ischemia, choroidal vascular insufficiency, choroidal thrombosis, neovascularization of the optic nerve, diabetic macular edema, cystoid macular edema, retinitis pigmentosa, retinal vein occlusion, proliferative vitreoretinopathy, angioid streak,
  • Additional relevant disease include the neuropathies, such as Leber's, idiopathic, drug-induced, optic, and ischemic neropathies.
  • Nogo receptor-like proteins could be major inhibitors- of CNS neuronal regeneration (Schwab ME. Curr Opin Neurobiol. 2004 Feb;14(l):l 18-24; Teng et al. J Neurochem. 2004 May;89(4):801-6).
  • Animals treated with antibodies targeted to Nogo-A always showed a higher degree of recovery in various behavioural tests (e.g. IN-I Fab' fragments or new purified IgGs against Nogo-A).
  • a Nogo-66 antagonistic peptide (NEP 1-40) effected significantly axon growth of the corticospinal tract and improved functional recovery in rats inflicted with mid-thoracic spinal cord hemisections.
  • Subcutaneous administration of NEP 1-40 in spinal cord lesioned animals resulted in extensive growth of corticospinal axons, sprouting of serotonergic fibres, synapse formation and enhanced locomotor recovery.
  • Soluble Fc fusion proteins of the Nogo receptor subunit NgR which blocks Nogo, significantly reduce the inhibitory activity of myelin. Similar results were obtained after Nogo gene deletions and blockade of the downstream messengers Rho-A and ROCK in animal models.
  • the leucine-rich repeat domain of SLIT proteins is sufficient for guiding both axon projection and neuronal migration in vitro (the LRR region of SLIT is structurally related to the LRR region of INSP168, INSP168-SV1, INSP149 and INSP169).
  • SLIT- like proteins are thought to act as molecular guidance cue in cellular migration, and function appears to be mediated by interaction with roundabaout homolog receptors (bind ROBOl and ROBO2 with high affinity).
  • SLIT are involved in axonal navigation at the ventral midline of the neural tube and projection of axons to different regions.
  • SLIT may play a role in guiding commissural axons once they reached the floor plate by modulating the response to netrin.
  • SLIT may be implicated in spinal chord midline post-crossing axon repulsion.
  • In the developing visual system appears to function as repellent for retinal ganglion axons by providing a repulsion that directs these axons along their appropriate paths prior to, and after passage through, the optic chiasm, hi vitro, SLIT collapses and repels retinal ganglion cell growth cones.
  • SLIT seems to play a role in branching and arborization of CNS sensory axons, and in neuronal cell migration.
  • Slit homolog 2 protein N-product but not Slit homolog 2 protein C-product, repells olfactory bulb (OB) but not dorsal root ganglia (DRG) axons, induces OB growth cones collapse and induces branching of DRG axons.
  • SLIT seems to be involved in regulating leukocyte migration.
  • INSPl 68, INSP168-SV1, INSP 149 and INSP 169 and/or fragments thereof can be useful in the diagnosis and/or treatment of diseases for which other (e.g. above mentioned PAL- and Nogo receptor-like proteins) structurally related proteins demonstrate therapeutic activity.
  • INSP168, INSP168-SV1, INSP149 and INSP169 may be implicated in diseases of the retina, spinal cord injuries (e.g. paraplegia) and neurodegenerative disorders. These include disorders of the central nervous system as well as disorders of the peripheral nervous system.
  • Neurodegenerative disorders include, but are not limited to, brain injuries, cerebrovascular diseases and their consequences, Parkinson's disease, corticobasal degeneration, motor neuron disease (including amyotrophic lateral sclerosis, ALS), multiple sclerosis, traumatic brain injury, stroke, post-stroke, post- traumatic brain injury, and small-vessel cerebrovascular disease.
  • Dementias such as Alzheimer's disease, vascular dementia, dementia with Lewy bodies, frontotemporal dementia and Parkinsonism, frontotemporal dementias (including Pick's disease), progressive nuclear palsy, corticobasal degeneration, Huntington's disease, thalamic degeneration, Creutzfeld-Jakob dementia, HTV dementia, schizophrenia with dementia, and Korsakoffs psychosis, as well as stroke and trauma.
  • INSP 149, INSP 168, INSPl 68-SV1 and INSP 169 may be implicated in diseases of the retina, retinal pigment epithelium (RPE), and choroids; ocular neovascularization, ocular inflammation and retinal degenerations; diabetic retinopathy, chronic glaucoma, retinal detachment, sickle cell retinopathy, senile macular degeneration, retinal neovascularization, subretinal neovascularization; rubeosis ulceris inflammatory diseases, chronic posterior and pan uveitis, neoplasms, retinoblastoma, pseudoglioma, neovascular glaucoma; neovascularization resulting following a combined vitrectomy and lensectomy, vascular diseases retinal ischemia, choroidal vascular insufficiency, choroidal thrombosis, neovascularization of the optic nerve, diabetic macular edema, cystoid
  • Leber's, idiopathic, drug-induced, optic, and ischemic neropathies spinal cord injuries, paraplegia, neurodegenerative disorders, disorders of the central nervous i system, disorders of the peripheral nervous system, brain injuries, cerebrovascular diseases, Parkinson's disease, corticobasal degeneration, motor neuron disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis, traumatic brain injury, stroke, post-stroke, post- traumatic brain injury, small-vessel cerebrovascular disease, dementias, Alzheimer's disease, vascular dementia, dementia with Lewy bodies, frontotemporal dementia, Parkinsonism, frontotemporal dementias, Pick's disease, progressive nuclear palsy, corticobasal degeneration, Huntington's disease, thalamic degeneration, Creutzfeld-Jakob dementia, HIV dementia, schizophrenia with dementia, Korsakoffs psychosis, stroke and trauma.
  • Example 6 Neuroprotective activities of INSP168
  • Neuro-inflammation is a common feature of several neurological diseases, traumatic situations (at central or peripheral level), stroke (brain, heart, renal), or infectious diseases (mediated by viral agents such as HIV or bacterial agents such as meningitis), leading to an excessive inflammatory response in central nervous system.
  • Many stimuli originated by neuronal or oligodendroglial cells suffering due to these various conditions, can trigger neuro-inflammation.
  • astrocytes can secrete various chemokines and cytokines, inducing a recruitment of additional leukocytes that in their turn will further stimulate astrocytes, leading to an exacerbated response.
  • MS multiple sclerosis
  • SMA spinal muscular atrophies
  • AD Alzheimer's disease
  • PD Parkinson's disease
  • HD Huntington's disease
  • ALS amylotrophic lateral sclerosis
  • the biological properties of INSP 168 related to neuroprotection, maintenance of axonal integrity, myelination and re-/generation of myelin producing cells, can be tested in various assays involving cell lines.
  • the neuroimmunodulatory effects of a compound can be evaluated in U373, a human astroglioma cell line in which the nuclear translocation of specific regulatory proteins involved in cytokine/chemokine expression can be quantified (Le Roy E et al, J Virol. 1999, 73: 6582-9; Jin Y et al, J Infect Dis. 1998, 177: 1629-1638; Acevedo-Duncan M et al, Cell Growth Differ. 1995, 6: 1353-1365).
  • a series of assays was performed on the human astroglioma cell line U373 to check whether INSP 168 can affect the translocation of transcription factors such as Stat-2 (Signal transducer and activator of transcription-2, a transcription factor induced by cytokines and modulating IFNbeta response; Banninger G and Reich NC, J Biol Chem. 2004, 279: 39199-39206; Leonard WJ, Int J Hematol. 2001, 73: 271-277) from the cytoplasm to the nucleus.
  • U373 cells (ECACC ref no: 89081403) were seeded at the density of 4000 cells/well in 96-well-plates (Packard ViewPlate-96, black; Cat. No.
  • K01-0003-1 for Stat-2, Cellomics Stat-2 activation HitKit, Cat. No. K01-0005-1) according to the manufacturer's instructions. After staining, plates were read on an image analysis system (ArrayScan II HCS System; Cellomics).
  • Results were expressed as "nuclear translocation units".
  • the nuclear translocation unit is the measure of the fluorescence intensity of the target in the nuclear region minus that of the cytoplasm region, reported as an average for all analyzed cells in the well (approx. 100 cells/well). In order to compare several experiments, results were also expressed as the percentage of maximal stimulation calculated with the positive control (IFNbeta). Statistics were performed using Student's T test or measure analysis of variance (ANOVA) and one-way ANOVA, followed by Dunnett's test depending of the number of groups per experiments. The level of significance was set at p ⁇ 0.05. The results were expressed as mean ⁇ standard error of the mean (s.e.m.).
  • INSP 169 is a re-prediction of INSP 149 which encodes a protein of 679 amino acids spanning 4 coding exons.
  • a transmembrane (TM) domain is predicted near the C- terminus.
  • the N-terminal extracellular domain extends over 580 amino acids and includes a cluster of 4 Leucine rich repeats (LRR) flanked by Cys-rich domains, an IgC-2 domain and a fibronectin type 3 domain. The extracellular domain of this prediction has been cloned.
  • LRR Leucine rich repeats
  • the cloning strategy used was to prepare an initial pool of RNAs from a wide variety of human tissues (see below) and from this to make a single preparation of multi- tissue poly A + mRNA as template for reverse transcription.
  • Gene specific cDNA primers were designed for a small set of the predictions (typically 5-10 sequences), and aliquots of the resulting cDNA mix provided templates for separate PCR reactions using primers designed to obtain the corresponding coding region. Amplified fragments were then purified by gel electorphoresis and cloned into the Bluescript cloning vector by virtue of specific restriction sites added to the ends of the PCR primers.
  • a two step strategy was employed which makes use of a unique BamHI restriction site in the central part of this sequence.
  • a first RT-PCR was performed to obtain the 1063nt sequence between the TM domain to the BamHI site; a second RT-PCR was performed to obtain the remaining 683nt sequence upstream of the BamHI site as far as the initiator methionine codon. These two fragments were then assembled following BamHI digestion and ligation.
  • RNA was prepared by mixing approximately 10 ⁇ g total RNA from each of the following sources: Brain (Clontech), Heart (Clontech), Kidney (Clontech), Liver (Clontech), Lung (Clontech), Placenta (Clontech), Skeletal Muscle (Clontech), Small Intestine (Clontech), Spleen (Clontech), Thymus (Clontech), Uterus (Clontech) Bone Marrow (Clontech) Thyroid (Clontech), Ovary (Ambion), Testis (Ambion), Prostate (Ambion), Skin (Resgen), Pancreas (Clontech), Salivary gland (BD Biosciences), Adrenal gland (BD Biosciences), Breast (Ambion), Pituitary gland (BioChain Institut), Stomach (Ambion), Mammary gland (Clontech), Lymph Node (BioChain Institut), Adipose tissue (BioChain
  • RNA was fractionated by chromatography on a pre-packed oligo-dT column (Stratagene) according to the protocol supplied by the manufacturer. Approximately 400 ⁇ g total RNA yielded 16 ⁇ g polyA+ mRNA which was aliquotted and frozen at -80 0 C.
  • Top strand (AS503) and bottom strand (AS504) PCR primers were designed to span the predicted coding sequence between an internal BamHI site and the TM domain. EcoRI restrictions sites were added at the 5' end of each primer.
  • a reaction mixture was set up containing 1 x PCR buffer, 0.2mM each dNTP, 0.5 ⁇ M each PCR primer, 5 ⁇ l cDNA above, and the PCR reaction was initiated by addition of 5U PfuTurbo (Stratagene).
  • Cycling conditions for 'touchdown' PCR were: 94 °C 2 min (I cycle); 94 °C 30 sec, 64 °C (decreasing by 1 0 C each cycle) 30 sec, 72 0 C 90 sec (14 cycles); 94 °C 30 sec, 50 °C 30 sec, 72 0 C 90 sec (25 cycles); 72 0 C 7 min (1 cycle).
  • An aliquot of the PCR reaction was analysed by electrophoresis in a 0.8% agarose gel and the remainder was purified using the Wizard PCR Cleanup System (Promega) as recommended by the manufacturer, prior to subcloning of the PCR products.
  • the ligation mixture was then used to transform E. coli strain JMlOl as follows: 50 ⁇ l aliquots of competent JMlOl cells were thawed on ice and l ⁇ l or 5 ⁇ l of the ligation mixture was added. The cells were incubated for 40 min on ice and then heat shocked by incubation at 42 °C for 2min. 1ml of warm (room temperature) L-Broth (LB) was added and samples were incubated for a further 1 h at 37 0 C. The transformation mixture was then plated on LB plates containing ampicillin (100 ⁇ g/ml) IPTG (0.1 ⁇ M) and X-gal (50 ⁇ g/ml) and incubated overnight at 37 0 C. Single white colonies were chosen for plasmid isolation.
  • Miniprep plasmid DNA was prepared from 5 ml cultures using a Biorobot 8000 robotic system (Qiagen) according to the manufacturer's instructions. Plasmid DNA was eluted in 80 ⁇ l sterile water. The DNA concentration was measured using an Eppendorf BO photometer or Spectramax 190 photometer (Molecular Devices).
  • the predicted mRNA coding sequence for the region spanning the internal BainHI site to the TM domain was confirmed.
  • the DNA miniprep #14 was taken as a representative clone for further work.
  • a gene specific cDNA primer for INSP 169 (AS515) located immediately downstream of the internal BamHI site, was pooled with gene specific cDNA primers for 6 other predictions, each at a final concentration of IpM.
  • the pooled cDNA primer set was diluted 10 fold into 40 ⁇ l of a mixutre containing 1 x RT buffer, 500 ⁇ M each dNTPs, lOU/ ⁇ l RNAguard (Pharmacia) and l ⁇ g denatured polyA + RNA prepared as described above.
  • cDNA synthesis was initiated by addition of 1OU Omniscript reverse transcriptase (Qiagen) and allowed to proceed for Ih at 37 °C. At the end of the reaction, 5 ⁇ l of the cDNA mix was used for PCR amplification as described below.
  • Top strand (AS516) and bottom strand (AS517) PCR primers were designed to span the predicted coding sequence between the initiator methionine and the internal BamHI site.
  • a BamHI restriction site was added at the 5' end of AS516.
  • a reaction mixture was set up containing 1 x PCR buffer, 0.2mM each dNTP, 0.5 ⁇ M each PCR primer, 5 ⁇ l cDNA above, and the PCR reaction was initiated by addition of 5U PfuTurbo (Stratagene).
  • Cycling conditions for 'touchdown' PCR were: 94 °C 2 min (I cycle); 94 0 C 30 sec, 64 °C (decreasing by 1 °C each cycle) 30 sec, 72 °C 80 sec (14 cycles); 94°C 30 sec, 50 °C 30 sec, 72 0 C 80 sec (25 cycles); 72 °C 7 min (1 cycle).
  • An aliquot of the PCR reaction was analysed by electrophoresis in a 0.8% agarose gel and the remainder was purified using the Wizard PCR Cleanup System (Promega) as recommended by the manufacturer, prior to subcloning of the PCR products.
  • the ligation mixture was then used to transform E. coli strain JMlOl as follows: 50 ⁇ l aliquots of competent JMlOl cells were thawed on ice and l ⁇ l or 5 ⁇ l of the ligation mixture was added. The cells were incubated for 40 min on ice and then heat shocked by incubation at 42 0 C for 2min. 1ml of warm (room temperature) L-Broth (LB) was added and samples were incubated for a further 1 h at 37 °C. The transformation mixture was then plated on LB plates containing ampicillin (100 ⁇ g/ml) IPTG
  • Miniprep plasmid DNA was prepared from 5 ml cultures using a Biorobot 8000 robotic system (Qiagen) according to the manufacturer's instructions. Plasmid DNA was eluted in 80 ⁇ l sterile water. The DNA concentration was measured using an Eppendorf BO photometer or Spectramax 190 photometer (Molecular Devices).
  • Transformation of competent JMl 01 with aliquots of the ligation mix, plasmid isolation and sequence analysis were performed as described in sections 7.2.1.3 and 7.2.1.4 above.
  • DNA miniprep #11 was selected and the complete sequence was verified using sequencing primers T3, T7, AS515 and AS599 (see Table 4). Compared to the prediction, a single SNP was found, G524A, which results in a codon change from Ser>Asn (underlined in Fig 8).
  • Table 4 Primers used for cloning and sequencing of INSP169ec

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Abstract

L'invention concerne des protéines, à savoir INSP168, INSP168-SV1, INSP149 et INSP169, identifiées en tant que protéines contenant des motifs répétés riches en leucine (LRR), et leur utilisation ainsi que des séquences d'acides nucléiques du gène de codage lors du diagnostic, de la prévention et du traitement d'une maladie.
PCT/GB2005/004390 2004-11-15 2005-11-15 Proteines contenant des motifs repetes riches en leucine (lrr) Ceased WO2006051333A2 (fr)

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CN116036239A (zh) * 2023-03-28 2023-05-02 中国人民解放军军事科学院军事医学研究院 Nep1-40在制备特异性抑制幻觉作用的药物中的应用
WO2025096678A1 (fr) * 2023-10-30 2025-05-08 The Board Of Regents Of The University Of Texas System Nouveaux peptides de signal ciblant des parties saillantes de cellule et leurs utilisations

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WO2003054166A2 (fr) * 2001-12-20 2003-07-03 Incyte Genomics, Inc. Polymorphisme nucleotidiques associes a l'osteoarthrite
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CN116036239A (zh) * 2023-03-28 2023-05-02 中国人民解放军军事科学院军事医学研究院 Nep1-40在制备特异性抑制幻觉作用的药物中的应用
WO2025096678A1 (fr) * 2023-10-30 2025-05-08 The Board Of Regents Of The University Of Texas System Nouveaux peptides de signal ciblant des parties saillantes de cellule et leurs utilisations

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