WO2005098433A2 - Diagnostic assays for alzheimer’s disease - Google Patents

Abstract

The invention provides a method of diagnosing Alzheimer’s Diseases (AD) in a human patient by measuring the amount of Ankyrin G present in a biological sample from a patient who may have AD relative to the amount of Ankyrin G in a control sample from an unaffected human. Also included in the invention are antibodies, diagnostic kits for AD and methods of screening for effective therapeutics for AD.

Description

DIAGNOSTIC ASSAYS FOR ALZHEIMER'S DISEASE

Background of the Invention

[0001] This invention relates to diagnosis of Alzheimer's Disease (AD).

[0002] Senile (πeuritic) plaques and neurofibrillary tangles are histopathological features of AD. Neuritic plaques consist of microglia, reactive astrocytes and a subset of histologically defined degenerative swollen axons and nerve terminals termed dystrophic neurites surrounding a core of extracellular amyloid. See Selkoe, Physiol Rev,Λ/o\. 81, No. 2, pp. 741-766 (2001). The appearance of dystrophic neurites occurs before the onset of dementia and is assumed to be associated with neurotoxic forms of AB peptide, the major constituent of amyloid in the brain. Most if not all transgenic mouse models of AD engineered to overexpress Aβ develop dystrophic neuritis [see Hock and Lamb, Trends Genet, Vol. 17, pp. S7-S12 (2001)], supporting a critical relationship between neuronal expression of AD^' and neurodegeneration.

[0003] Molecular and cellular hallmarks of neurodegeneration, especially those found in vulnerable regions of the brain such as the hippocampus- and entorhinal cortex, are highly relevant to understanding the progression of AD. One of the most prominent^' cellular markers in the diagnosis of AD is the presence of neurofibrillary tangles (NFTs), composed of highly-phosphorylated forms of the microtubule associated protein tau. See Iqbal et al. (1989). NFT pathology evolves in a well-defined sequence that permits AD affected brain to be distinguished from normal brain. See Braak, Braak and Mandelkow, Acta Neuropathol (Bed), Vol. 87, No. 6, pp. 554-567 (1994); and Braak and Braak, Acta Neuropathol (Berl), Vol. 82, No. 4, pp. 239-259 (1991). However, the mere presence of NFTs alone does not distinguish AD from other neurological diseases, since NFTs are common in many neurodegenerative diseases, such as the tauopathies. See Lee, Goedert and Trojanowski, Annu Rev Neυrosci, Vol. 24, pp. 1121-1159 (2001).

[0004] Accumulation of neurofilaments within neurites is another important pathological indicator in AD brain. Changes in the phosphorylation pattern of neurofilaments represent one of the earliest known cytoskeletal alterations associated with neurodegeneration in AD. See Dickson, King, McCormack and Vickers, Exp Neurol, Vol. 156, No. 1, pp. 100-110 (1999); Su, Cummings and Cotman, Acta Neuropathol (Berl), Vol. 96, pp. 463-471 (1998); and Vickers et al., Exp Neurol, Vol. 141 , No. 1, pp. 1-11 (1996). Before the onset of dementia, dystrophic neurites with hyperphosphorylated forms of neurofilament are found in the absence of tau pathology. See Dickson, King, McCormack and Vickers (1999), supra. While, in advanced cases of AD, subpopulations of dystrophic neurites contain ring-like neurofilament staining surrounding tau positive centers. This suggests a temporal and spatial relation with neurofilament alterations preceding changes in tau. However, as is the case with tau, neurofilament alterations are not confined to AD. Aberrant neurofilament accumulation in cell bodies and axons of motor neurons is a prominent pathological feature of several motor neuron diseases, including sporadic and familial Amyotrophic Lateral Sclerosis [See Julien and Beaulieu, J Neurol Sci, Vol. 180, Nos. 1-2, pp. 7-14 (2000) and aggregated neurofilaments are a major constituent of Lewy body containing neurons in Parkinson's disease (PD) and in Lewy body dementia. See Trojanowski, Goedert, Iwatsubo and Lee, Cell Death Differ, Vol. 5, No. 10, pp. 832-837 (1998).

[0005] Ankyrin G is known to be an intracellular adaptor protein which targets proteins to specialized membrane domains. For example, Ankyrin G enriches voltage-gated sodium channels at initiation zones and nodes of Ranvier. See Bouzidi et al., J Biol Chem, Vol. 277, No. 32, pp. 28996-29004 (2002); Malhotra, Kazen-Gillespie, Hortsch and Isom, J Biol Chem, Vol. 275, No. 15, pp. 11383-11388 (2000); and Zhou et al., J Cell Biol, Vol. 143, No. 5, pp. 1295-1304 (1998). Voltage-gated sodium channel clustering at excitable membranes is necessary for axonal depolarization during saltatory conduction and its binding to Ankyrin G has recently been shown to be mediated through a conserved domain. See Garrido et al., Science, Vol. 300, No. 5628, pp. 2091-2094 (2O03); and Lemaillet, Walker and Lambert, J Biol Chem, Vol. 278, No. 30, pp. 27333-27339 (2003). Neural cell adhesion molecules, such as CD44 and LICAMs are also targeted within neurons by Ankyrin G. See Tuvia, Garver and Bennett, Proc NatlAcad Sci U S A, Vol. 94, No. 24, pp. 12957-12962 (1997); and Zhou et al. (1998), supra. The regulated targeting and concentration of these cell adhesion proteins help join cell processes, e.g., dendrites, axons and synaptic elements, with the extracellular environment including the processes of other cells.

[0006] Ankyrin G expression at sites of neuronal degeneration has been documented outside of the brain. In the peripheral nervous system, early after nerve cell injury, Ankyrin G is concentrated in nerve terminals at sites of physical injury. See Kretschmer et al., Neurosci Lett, Vol. 323, No. 2, pp. 151-155 (2002); and Kretsc mer et al., J Neurosurg, Vol. 97, No. 6, pp. 1424-1431 (2002). This response is thought to reflect a regenerative attempt of damaged nerve terminals through activation of pathways involved in growth and remodeling. Indeed, amyloid has been shown to be associated with a regenerative sprouting response in neurons reorganizing their cytoskeleton. See Vickers et al. (1996), supra; and Vickers, Neuroscience, Vol. 78, No. 3, pp. 629-639 (1997).

[0007] The Caenorhabditis elegans (C. elegans) ankyrin homolog, unc-44, was identified in a genetic screen for suppressors of an axon guidance phenotype [see Colavita and Culotti, Dev Biol, Vol. 19 -, No. 1, pp. 72-85 (1998)] and is required for proper axonal growth and retraction during neural development. See Otsuka et al., J Cell Biol, Vol. 129, No. 4, pp. 1081-1092 (1995). Interestingly, unc-44 and mammalian Ankyrin G have the unique domain composition of a death domain and a ZU5 domain (which is found in netrin receptors) suggesting that Ankyrin G, like its C. elegans homolog, may participate in axonal remodeling in response to extracellular signals.

[0008] The cause of AD is not known, nor is there treatment or accurate diagnostic methods for AD. Currently, medication is prescribed to AD patients to control symptoms of the disease, such as agitation, depression, hallucinations or delusion. Identification of biological molecules and pathways which can be used in quantitative assessments would be essential to understanding critical pathways involved in the progression of the disease and will provide a means to treat the cause, not just the symptoms of AD. Thus there is a continuing need to identify methods to diagnose AD or identify diagnostic markers associated with AD to elucidate the mechanisms causing AD.

Summary of the Invention

[0009] We have shown that Ankyrin G is specifically associated in neuritic plaques in the brain of AD patients. We have analyzed its expression patterns in tissues from neurodegenerative disease and age-matched normal patients, as well as from affected tissues from PD and Amyotrophic Lateral Sclerosis (ALS) donors. Our results show selective altered expression of Ankyrin G in AD brain and its localization within neuritic plaques. Thus, detection and quantification of an Ankyrin G protein is useful for diagnosing AD, pathways involved in AD or proteins expressed or associated with Ankyrin G. [0010] In one aspect, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes an Ankyrin G polypeptide. The isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, and alternatively at least about 99% nucleic acid sequence identity to: (a) a nucleotide sequence encoding Ankyrin G polypeptide having the sequence of SEQ ID NOs: 1-8, a mature polypeptide thereof; or (b) the complement of the nucleic sequence of (a).

[0011] In another aspect, the invention provides nucleic acid sequence fragments encoding Ankyrin G polypeptide coding sequences that may find use as, e.g., hybridization probes or that may optionally encode a polypeptide comprising a binding site for an anti- Ankyrin G antibody. Such nucleic acid fragments are usually at least about 20- 50nucleotides in length, alternatively at least about 30 nucleotides in length, alternatively at least about 70 nucleotides in length, alternatively at least about 80 nucleotides in length, alternatively at least about 90 nucleotides in length, alternatively at least about 200 nucleotides in length, wherein in this context the term "about" means the referenced nucleotide sequence length plus or minus 10% of that referenced length. In a preferred embodiment, the nucleotide sequence fragment is derived from any coding region of nucleotide sequences SEQ ID NO: 1, 3, 5 or 7. It is noted that novel fragments encoding Ankyrin G polypeptide sequences may be determined in a routine manner by aligning Ankryin G nucleotide sequence s with other known nucleotide sequences using any of a number of well-known sequence alignment programs and determining which nucleotide sequence fragment(s) encoding a region of Ankyrin G protein are novel without undue experimentation. Also contemplated are Ankyrin G polypeptide fragments encoded by these nucleotide sequence fragments, preferably those that comprise a binding site for an anti- Ankyrin G antibody.

[0012] In another aspect, the invention provides isolated Ankyrin G polypeptides encoded by any of the isolated nucleic acid sequences hereinabove identified inclusive of the polypeptide comprising amino acid sequences SEQ ID NO: 2, 4, 6 or 8. [0013] The invention further provides a method for producing an Ankyrin G polypeptide by providing a cell containing an Ankyrin G nucleic acid, e.g., a vector that includes an Ankyrin G nucleic acid, and culturing trie cell under conditions sufficient to express Ankryin G polypeptide encoded by the nucleic acid. The expressed Ankyrin G polypeptide is then recovered from the cell. Preferably, the cell produces little or no endogenous Ankryin G polypeptide. The cell can be, e.g., a prokaryotic cell or eukaryotic cell.

[0014] In another aspect, the invention provides antibodies that bind to Ankyrin G and can target an Ankyrin G-expressing cell in vivo or in vitro. Also contemplated by the invention are antibodies that compete for binding to the same epitope as the epitope bound by any of the antibodies of the invention or inhibits or alters the growth of Ankyrin G expressing cells, or is cytotoxic in vivo to such cells.

[0015] The invention also provides methods of identifying Ankyrin G polypeptide or nucleic acid in a sample by contacting the sample "with a compound that specifically binds to the polypeptide or nucleic acid, and detecting complex formation with Ankyrin G, if present.

[0016] In other aspects, the invention provides methods of identifying Ankyrin G modulators that modulate expression of Ankyrin G by contacting Ankyrin G with a substance that inhibits or stimulates Ankyrin G expression and determining whether expression of Ankyrin G polypeptide or nucleic acid nucleic acid molecules encoding an Ankryin G polypeptide are modified. In one aspect, an Ankyrin G modulator may also affect expression of beta (β)-amyloid plaques wherein Ankyrin G is localized or co-expressed.

[0017] In another aspect, the invention provides methods of screening for Ankyrin G modulators known or not yet known to affect Ankyrin G expression by a sample in which Ankyrin G is present with a substance that alters vnkyrin G expression and determining the expression level of Ankyrin G polypeptide or nucleic acid molecules encoding Ankyrin G. In an aspect, an Ankyrin G modulator may also affect expression of β-amyloid plaques, wherein Ankyrin G is localized or expressed.

[0018] The invention is also directed to compounds that modulate Ankyrin G expression by contacting Ankyrin G polypeptides with a compound and determining whether the compound modifies expression or function of Ankyrin G polypeptides, binds to Ankryin G polypeptide, or binds to a nucleic acid molecule encoding an Ankryin G polypeptide. [0019] In another aspect, the invention provides a method of determining the presence of or predisposition of an Ankyrin G-associated disorders in a subject. The method includes providing a sample from the subject and measuring the amount of Ankyrin G polypeptide in the subject sample. The amount of Ankyrin G polypeptide in the subject sample is then compared to the amount of Ankyrin G polypeptide in a control sample. An alteration in the amount of Ankyrin G polypeptide in the subject protein sample relative to the amount of Ankyrin G polypeptide in the control protein sample indicates the subject has an AD-associated condition. A control sample is preferably taken from a matched individual, i.e., an individual of similar age, sex or other general condition but who is not suspected of having an Ankyrin G related disorder. In another aspect, the control sample may be taken from the subject at a time when the subject is not suspected of having a condition or disorder associated with abnormal expression of Ankyrin G, such as AD.

[0020] In another aspect, the invention features a method for measuring the ability of a candidate compound to modulate the level of Ankyrin C3. In this method, a cell that expresses Ankyrin G is contacted with a candidate compound and the level of Ankyrin G in the cell is determined. This determination of Ankyrin G levels may be made using any of the above-described immunoassays or techniques disclosed herein. The cell can be Ankyrin G transfected cells taken from a brain tissue biopsy from a patient, or any other Ankyrin G expressing cell type.

[0021] In another aspect, the invention features a pharmaceutical composition that includes an Ankyrn G nucleic acid and a pharmaceutically acceptable carrier or diluent.

[0022] In other aspects, the invention provides a kit for in vitro diagnosis of AD by detection of Ankyrin G in a biological sample from a pati ent. A kit for detecting Ankyrin G may include: (1) a primary antibody capable of binding to Ankyrin G; and (2) a secondary antibody conjugated to a signal-producing label, the secondary antibody being capable of binding to Ankryin G, but to a site different from, i.e., spaced from, that to which the first monoclonal antibody binds.

[0023] Such antibodies can be prepared by methods well-known in the art. This kit is most suitable for carrying out a two-antibody sandwiclh immunoassay, e.g., two-antibody sandwich ELISA. Brief Description of the Figures

[0024] Figure 1. Illustrates Ankyrin G expression in the hippocampal formation; Figure 1 (A) - Protein domains present in the large neuron specific 4-80 kDa isoform of Ankyrin G include a Λ/-terminal ANK repeat domain, a spectrin-binding domain within which a ZU5 domain resides, a Serine/Threonine (ST-Rich) and a C-termin al death domain (DD); Figure 1 (B) - 4G3F8 antibody directed against the spectrin-binding domain of Ankyrin G stains axonal fibers of perforant pathway projections within hippocampus area CA3 (arrowheads); Figure 1 (C) -Ankyrin G expression within cell bodies and axon initial segments of entorhinal cortex layer II (ECL II); Figure 1 (D) - Entorhϊnal cortex layer VI (ECL VI) neurons; and Figure 1 (E) - White matter tissue was generally unstained.

[0025] Figure 2. Illustrates Ankyrin G expression in neuritic plaques of AD brain; Figure 2 (A and B): Intense staining of neuritic plaques in hippocampus area CA3 [see Figure 2 (A)] and dentate gyrus [see Figure 2 (B)]; Figure 2 (C) - Higher magnification of the neuritic staining in Figure 2 (B) showing the focal staining surrounding a core of amyloid; Figure 2 (D) - In some neuritic plaques, antibody 4G3F8 staining was clearly within dystrophic cells with swollen processes; and Figure 2 (E) - Representative staining with 4G3F8 in dystrophic neurites of an ECL II neuritic plaque.

[0026] Figure 3. Illustrates that Ankyrin G expression is not altered in PD and ALS tissues. In PD brain, de-pigmented [see Figures 3 (A and B)] and pigmented [see Figure 3 (C)] neurons containing Lewy bodies (LB) of the substantia nigra express little if any Ankyrin G. Low magnification of ALS spinal cord tissue [see Figure 3 (D)] and the Nucleus of Clarke [see Figure 3 (E)] probed with 4G3F8. Overall the staining pattern observed with tissues from PD and ALS donors was similar to the respective tissues from normal unaffected donors (data not shown).

[0027] Figure 4. Illustrates Aβ42 and Ankyrin G labeling in human [see Figure 4 (A-F)] and APP23 mouse [see Figure 4 (G-L)] hippocampus. Low-powered [see Figure 4 (A)] and high-powered [see Figure 4 (B)] magnification of anti-Aβ42 staining of hippocampus area CA3 plaques and deposition on capillary walls [see Figure 4 (C)]. Figure 4 (D-F) - Double-labeling with anti-Aβ42 and 4G3F8 in the hi ppocampus. 4G3F8 positive dystrophic structures (arrowheads) are associated with Aβ42 deposition but do not colocalize within the same cellular or extracellular space. Figure 4 CG-L) - Adjacent serial sagittal brain sections from APP23 mice were stained with either anti-Aβ42 [see Figure 4 (G-l)] or 4G3F8 [see Figure 4 (J-L)] antibodies. Slides of the adjacent tissue sections are presented with the anti-Aβ42 stained slide directly above its neighboring tissue section stained with 4G3F8. 4G3F8 immunoreactive structures resembling dystrophic neurites (arrowheads) are seen peripheral to a central core of Aβ42 deposition.

[0028] Figure 5. Illustrates double-labeling of plaque associated dystroplnic neurites with phosphorylated neurofilament (SMI312) and Ankyrin G (4G3F8) antibodies. Figure 5 (A and B) - Adjacent serial section of a hippocampus area CA4 plaque staining with SMI312 [see Figure 5 (A), black arrowheads] and with 4G3F8 [see Figure 5 (B), white arrowheads]. In addition to staining a subset of dystrophic structures, 4G3F8 immunoreactivity surrounds amyloid deposition. Notice the unstained amyloid [see Figure 5 (A), white arrowheads] which is clearly distinguished by 4G3F8 [see Figure 5 (B), white arrowheads]. Figure 5 (C and D)] - Double-labeling with SMI312 and 4G3F8. 4G3F8 stains the periphery of the amyloid core within a neuritic structures while SMI312 staining is seen in plaque associated dystrophic neurites.

Detailed Description of the Invention

[0029] All patent applications, patents, literature and website references cited herein are hereby incorporated by reference in their entirety.

[0030] In practicing the present invention, many conventional techniques in molecular biology and recombinant DNA are used. These techniques are well-known and are explained in, e.g., Current Protocols in Molecular Biology, Vols. I-III, Ausubel, Ed. (1997); Sambrook et al., Molecular Cloning: A Laboratory Manual, 2^nd Edition, Cold Spri ng Harbor Laboratory Press, Cold Spring Harbor, NY (1989); DNA Cloning: A Practical Approach, Vols. I and II, Glover, Ed. (1985); A Practical Guide to Molecular Cloning; the series, Methods in Enzymology, Academic Press, Inc.; Gene Transfer Vectors for Mammalian Cells, Miller and Calos, Eds., Cold Spring Harbor Laboratory (1987); and Methods in Enzymology Vols. 154 and 155, Wu and Grossman, and Wu, Eds., respectively.

[0031] As used herein, the term "Abeta" (Aβ) refers to β-amyloid peptide, which is a short (40-42 amino acid) peptide produced by proteolytic cleavage of APP by β and gamma secretases. It is the primary component of amyloid depositions, the hallmark of AD and the cause of neuronal cell death and degeneration. Aβ peptide of the present invention includes, but is not limited to, peptides of 40 and 42 amino acids and are referred to, respectively, as Aβ40 and Aβ42.

[0032] As used herein, "Ankyrin G" refers to human Ankyrin G Genbank NM_020987, as well as the mouse ortholog of this protein Genbank NM_1707^"28. Included in the definition are any and all forms of this protein including, but not limited to, variants, partial forms, isoforms, precursor forms, full-length polypeptides, fusion proteins or fragments of any of the above, from human or any other species. Variants of -Ankyrin G include: name, Genbank Accession No. NMJD01149, NM_009670, NM 460O5, NM_170687, NM 70688, NM_170689, NM_170690, NM_170729 and NM_1 70730. Homologs of Ankyrin G, which would be apparent to one of skill in the art, are also meant to be included in this definition. It is also contemplated that the term refers to Artkyrin G isolated from naturally-occurring sources of any species, such as genomic DMA libraries, as well as genetically-engineered host cells comprising expression systems, or roduced by chemical synthesis using, i.e., automated peptide synthesizers or a combination of such methods. Means for isolating and preparing such polypeptides are well-understood in the art.

[0033] The term "sample", as used herein, is used in its broadest sense. A biological sample from a subject may comprise blood, cerebrospinal fluid or other biological material with which Ankyrin G gene expression may be assayed. A biological sample may include cerebrospinal fluid from which total RNA may be purified for gene expression profiling using conventional glass chip microarray technologies, such as Affymetrix chips, RT-PCR or other conventional methods.

[0034] As used herein, "isolated" refers to a polypeptide or nucleic act d molecule that has been identified and separated and/or recovered from a component of its natural environment. Preferably, the isolated polypeptide or nucleic acid molecule is free of association with all components with which it is naturally associated. Contam inant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. An isolated nucleic acid molecule is free of association with all components with which it is naturally associated. [0035] As used herein, the following and related phrases, pathological conditions associated with abnormalities in the APP pathway, conditions associated with abnormal regulation of the APP pathway, conditions related to AD, pathological conditions associated with defects in the APP pathway, all include, but are not limited to, AD, and include those conditions characterized by degeneration and eventual death of neurons in brain clusters controlling memory, cognition and behavior.

[0036] As used herein, the phrase "specifically (or selectively) binds to an antibody", or "specifically (or selectively) immunoreactive with", when referring to a protein or peptide, refers to a binding reaction which is determinative of the presence of the protein in the presence of a heterogenous population of proteins and other biologies. Thus, under designated immunoassay conditions, specific antibodies bind to a particular protein and do not bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein or region of a protein. For example, antibodies raised to the protein with the amino acid sequence encoded by the Ankyrin G proteins of the invention can be selected to obtain antibodies specifically immunoreactive with that protein and not with other proteins except for polymorphic variants. In one aspect, the Ankyrin G antibody of the present invention binds to SEQ ID NO: 2, 4, 6, or 8 or fragments. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays, Western blots or immunohistochemistry are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow and Lange, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, NY (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.' Typically a specific or selective reaction will be at least twice background signal or noise and more typically more than 10-100 times background.

[0037] The ability of a substance or compound to "modulate" Ankyrin G, e.g., a Ankyrin G modulator, includes, but is not limited to, the ability of a substance to inhibit or stimulate expression of Ankyrin G or localization of Ankyrin G in neuritic plaques, such as β-amyloid plaques, e.g., by acting directly on Ankyrin G and/or by inhibiting or stimulating Ankyrin G gene expression or localization. Modulation could also involve effecting the ability of other proteins to interact directly (physical interaction) or indirectly (in the same signal transduction pathway via upstream or down stream effects) with Ankyrin G, e.g., related regulatory proteins or other proteins that are modified by or modify Ankyrin G or proteins associated or co-localized with Anykyrin G. Specifically, the Ankryin G nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., Ankyrin G localization, mislocalization or association with plaques and NFT in brain cells.

[0038] Modulators also include, but are not limited to, antagonists or agonists of Ankyrin G, as well as small molecules, antisense oligonucleotides, triple helix DNA, RNA aptamers, siRNA, ribozymes and double-stranded RNA directed to a nucleic acid sequence of Ankyrin G.

[0039] The term "antagonist of Ankyrin G", as it is used herein, refers to a molecule which decreases the amount or expression of Ankyrin G. Antagonists can include, but are not limited to, peptides, proteins, nucleic acids, carbohydrates, antibodies or any molecules which decrease the expression Ankyrin G.

[0040] An "agonist" of Ankyrin G refers to a substance that has affinity for and stimulates expression of Ankyrin G and can thus trigger a biochemical or physically measurable response.

[0041] "Nucleic acid sequence", as used herein, refers to an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin that may be single- or double-stranded, and represent the sense or antisense strand.

[0042] The term "antisense", as used herein, refers to nucleotide sequences which are complementary to a specific DNA or RNA sequence. The term "antisense strand" is used in reference to a nucleic acid strand that is complementary to the "sense" strand. Antisense molecules may be produced by any method, including synthesis by ligating the gene(s) of interest in a reverse orientation to a viral promoter which permits the synthesis of a complementary strand. Once introduced into a cell, this transcribed strand combines natural sequences produced by the cell to form duplexes. These duplexes then block either the further transcription or translation. The designation "negative" is sometimes used in reference to the antisense strand, and "positive" is sometimes used in reference to the sense strand. [0043] As contemplated herein, antisense oligonucleotides, triple helix DNA, RNA aptamers, ribozymes and double-stranded RNA are directed to a nucleic acid sequence of Ankyrin G such that introduction of these molecules will produce gene-specific inhibition of Ankyrin G gene expression. For example, knowledge of the Ankyrin G nucleotide sequence may be used to design an antisense molecule which gives strong inhibition to mRNA. Similarly, ribozymes can be synthesized to recognize specific nucleotide sequences of Ankyrin G and cleave it. See Cech, JAMA, Vol. 260, No. 20, pp. 3030-3034 (1988). Techniques for the design of such molecules for use in targeted inhibition of gene expression are well-known to one of skill in the art.

[0044] As used herein, the term "antibody" refers to intact molecules, as well as fragments thereof, such as Fa, F(ab')₂ and Fv, which are capable of binding the epitopic determinant. Antibodies that bind Ankyrin G polypeptides can be prepared using intact polypeptides or fragments containing small peptides of interest as the immunizing antigen, such as fragments comprising SEQ ID NO. 4, 6 or 8. The polypeptides or peptides used to immunize an animal can be derived from the translation of RNA or synthesized-chemically, and can be conjugated to a carrier protein, if desired. Commonly used carriers that are chemically coupled to peptides include bovine serum albumin and thyroglobulin. The coupled peptide is then used to immunize an animal, e.g., a mouse, a rat or a rabbit.

[0045] The term "humanized antibody", as used herein, refers to antibody molecules in which amino acids have been replaced in the non-antigen binding regions in order to more closely resemble a human antibody, while still retaining the original binding ability.

[0046] "Therapeutically-effective amount" refers to that amount of active ingredient, e.g., compounds or gene products which ameliorate the symptoms of the condition being treated.

[0047] "Related regulatory proteins" and "related regulatory polypeptides", as used herein, refer to polypeptides involved in the regulation of Ankyrin G which may be identified by one of skill in the art using conventional methods.

[0048] "Subject" refers to any human or non-human organism.

[0049] As used herein, the term "substantially pure" describes a compound, e.g., a protein or polypeptide, e.g., an Ankyrin G protein or fragment thereof which has been separated from components which naturally accompany it. Typically, a compound is substantially pure when at least 10%, more preferably at least 20%, more preferably at least 50%, more preferably at least 60%, more preferably at least 75%, more preferably at least 90%, and most preferably at least 99%, of the total material (by volume, by wet or dry weight, or by mole percent or mole fraction) in a sample is the compound of interest. Purity can be measured by any appropriate method, e.g., in the case of polypeptides by column chromatography, polyacrylamide gel electrophoresis or HPLC analysis. A compound, e.g., a protein, is also substantially purified when it is essentially free of naturally-associated components or when it is separated from the native contaminants which accompany it in its natural state.

[0050] As used herein, the phrase "stringent hybridization conditions" refers to conditions under which a probe, primer or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength and pH. The T_m is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at T_m, 50% of the probes are occupied at equilibrium. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01-1.0 M sodium ion (or other salts) at pH 7.0-8.3 and the temperature is at least about 30°C for short probes, primers or oligonucleotides, e.g., 10-50 nt, and at least about 60°C for longer probes, primers and oligonucleotides. Stringent conditions may also be achieved with the addition of de-stabilizing agents, such as formamide.

[0051] Stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY, pp. 6.3.1-6.3.6 (1989). Preferably, the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other. A non-limiting example of stringent hybridization conditions is hybridization in a high- salt buffer comprising 6 x SSC, 50 mM Tris-HCI (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA and 500 mg/mL denatured salmon sperm DNA at 65°C. This hybridization is followed by one or more washes in 0.2 x SSC, 0.01% BSA at 50°C. An isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO: 1 , 3, 5 or 7 corresponds to a naturally-occurring nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature, e.g., encodes a natural protein.

[0052] The invention is based on the surprising discovery that Ankyrin G is associated with β-amyloid plaques in AD brain. The human Ankyrin G gene, identified as a modifier in the fly screen in co-pending U.S. Patent Application No. 09/964,899 is a useful drug target for the development of therapeutics for the treatment and diagnosis of conditions or disorders associated with abnormal Ankyrin G expression, such as in AD, an association not previously known to involve Ankyrin G.

[0053] We have shown that Ankyrin G is misexpressed in brain tissue from AD patients relative to controls. Thus, the invention provides substantially pure preparations of Ankryin G and fragments thereof.

[0054] In one aspect, an Ankryin G nucleic acid sequence is provided in SEQ ID NO: 1, 3, 5 or 7, fragments or complements thereof. The invention includes mutant or variant nucleic acids of SEQ ID NO: 1 , 3, 5 or 7, fragments or complements thereof, any of whose bases may be changed from the corresponding bases shown in SEQ ID NO: 1 , 3, 5 of 7, while still encoding an Ankyrin G protein or biologically active fragment thereof. Fragments may be derived from any contiguous portion of a nucleic acid or amino acid sequence of choice and may be used as primers or probes. Fragments provided herein are defined as sequences of at least 10-25, 7-19, at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, respectively, and are at most some portion less than a full-length sequence. The invention further includes derivatives, analogs and homologs of an Ankyrin G nucleic acid sequence. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications.

[0055] A nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to Ankyrin G nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.

[0056] Another aspect of the invention pertains to isolated Ankyrin G proteins, variants, biologically active portions thereof, or derivatives, fragments, analogs or homologs thereof. Also provided are polypeptide fragments suitable for use as immunogens to raise anti-Ankyrin G antibodies. In one embodiment, native Ankyrin G proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, Ankyrin G proteins are produced by recombinant DNA techniques. Alternative to recombinant expression, a Ankyrin G protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.

[0057] The isolated nucleic acid molecules of the invention can be used to express Ankyrin G protein, e.g., via a recombinant expression vector in a host cell in gene therapy applications; to detect Ankyrin G mRNA, e.g., in a biological sample; to modulate Ankyrin G expression; or to identify proteins associated with Ankyrin G expression, as well as functions of such association as described further below. In addition, the Ankyrin G proteins can be used to screen drugs or compounds that modulate the Ankyrin G protein expression, as well as to treat disorders characterized by insufficient or excessive production of Ankyrin G protein or production of Ankyrin G protein forms that have decreased or aberrant activity compared to Ankyrin G wild-type protein. In addition, the anti-Ankyrin G antibodies of the invention can be used to detect and isolate Ankyrin G proteins and modulate Ankyrin G expression.

[0058] The invention further relates to novel agents identified by the screening assays described herein and uses thereof for treatments as described herein.

[0059] In one aspect, the present invention is directed to methods of identifying Ankyrin G modulators that alter expression of Ankyrin G poplypeptides or nucleic acid sequences encoding the polypeptide. Identification of Ankyrin G modulators has particular use for identifying agonists or antagonists of Ankyrin G and to eludicate mechanisms or conditions related to defects in the APP pathway including AD.

[0060] For example, Ankyrin G can be used in methods for screening for compounds which modulate Ankyrin G expression or may be useful as therapeutic agents for AD. The screening assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays and cell-based assays, which are well-characterized in the art.

[0061] In one aspect, screening of Ankyrin G modulators may be carried out as follows. First, candidate modulators are tested in cultured cells. Second, candidate modulators which test positive in the cultured cells are tested in an animal model system. In the cell culture assay, cells that express Ankyrin G, such as PC12 transfected as in Examples below, are cultured in the presence of the candidate modulator. The level of Ankyrin G expression in the cells can be determined by methods known in the art and described herein.

[0062] Candidate modulators for analysis according to the methods disclosed herein include chemical compounds which are known to possess Ankyrin G modulatory activity, as well as compounds whose effects on these proteins at any level have yet to be characterized. Compounds known to possess modulatory activity could be directly assayed in the animal AD models described in co-pending U.S. Application No. 09/964,899 or in clinical trials.

[0063] Candidate modulators may also be small molecule compounds or drug candidates that mimic an Ankyrin G polypeptide (agonists) or inhibit expression or localization of Ankryin G polypeptides (antagonists). Screening assays for antagonist drug candidates are designed to identify compounds that bind or complex with Ankryin G polypeptides encoded by the genes identified herein, or otherwise interfere with the interaction of the encoded polypeptides with other cellular proteins, such as β-amyloid proteins. Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates.

[0064] Assays for antagonists are common in that they call for contacting a drug candidate with an Ankyrin G polypeptide encoded by a nucleic acid identified herein under conditions and for a time sufficient to allow these two components to interact.

[0065] To assay for antagonists, Ankyrin G may be added to a cell along with a compound to be screened for a particular activity and the ability of the compound to inhibit the activity of interest in the presence of the Ankyrin G polypeptide indicates that the compound is an antagonist to Ankyrin G polypeptide. [0066] In binding assays, the interaction is binding and the complex formed can be isolated or detected in the reaction mixture. In a particular embodiment, an Ankyrin G polypeptide encoded by the gene identified herein or a drug candidate is immobilized on a solid phase, e.g., on a microtiter plate, by covalent or non-covalent attachments. Non- covalent attachment generally is accomplished by coating the solid surface with a solution of an Ankryin G polypeptide and drying. Alternatively, an immobilized antibody, e.g., a monoclonal antibody, specific for an Ankyrin G polypeptide to be immobilized can be used to anchor it to a solid surface. The assay is performed by adding the non-immobilized component, which may be labeled by a detectable label, to the immobilized component, e.g., the coated surface containing the anchored component. When the reaction is complete, the non-reacted components are removed, e.g., by washing, and complexes anchored on the solid surface are detected. When the originally non-immobilized component carries a detectable label, the detection of label immobilized on the surface indicates that complexing occurred. Where the originally non-immobilized component does not carry a label, complexing can be detected, e.g., by using a labeled antibody specifically binding the immobilized complex.

[0067] If the candidate compound interacts with but does not bind to a particular Ankyrin G polypeptide encoded by a gene identified herein, its interaction with that polypeptide can be assayed by methods well-known for detecting protein-protein interactions. Such assays include traditional approaches, such as, e.g., cross-linking, co- immunoprecipitation and co-purification through gradients or chromatographic columns. In addition, protein-protein interactions can be monitored by using a yeast-based genetic system described by Fields and co-workers [see Fields and Song, Nature (London), Vol. 340, pp. 245-246 (1989); and Chien, Bartel, Sternglanz and Fields, Proc NatlAcad Sci U S A, Vol. 88, No. 21, pp. 9578-9582 (1991)] as disclosed by Chevray and Nathans, Proc NatlAcad Sci U S A, Vol. 89, No. 13, pp. 5789-5793 (1991). Many transcriptional activators, such as yeast GAL4, consist of two physically discrete modular domains, one acting as the DNA-binding domain, the other one functioning as the transcription-activation domain. The yeast expression system described in the foregoing publications (generally referred to as the "two-hybrid system") takes advantage of this property, and employs two hybrid proteins, one in which the target protein is fused to the DNA-binding domain of GAL4, and another, in which candidate activating proteins are fused to the activation domain. The expression of a GAL1-lacZ reporter gene under control of a GAL4-activated promoter depends on reconstitution of GAL4 activity via protein-protein interaction. Colonies containing interacting polypeptides are detected with a chromogenic substrate for β-galactosidase. A complete kit (MATCHMAKER™) for identifying protein-protein interactions between two specific proteins using the two-hybrid technique is commercially-available from Clontech. This system can also be extended to map protein domains involved in specific protein interactions, as well as to pinpoint amino acid residues that are crucial for these interactions.

[0068] Compounds that interfere with the interaction of a gene encoding an Ankyrin G polypeptide identified herein and other intra- or extracellular components can be tested as follows. Usually a reaction mixture is prepared containing the product of the gene and the intra- or extracellular component under conditions and for a time allowing for the interaction and binding of the two products. To test the ability of a candidate compound to inhibit binding, the reaction is run in the absence and in the presence of the test compound. In addition, a placebo may be added to a third reaction mixture, to serve as positive control. The binding (complex formation) between the test compound and the intra- or extracellular component present in the mixture is monitored as described hereinabove. The formation of a complex in the control reaction(s) but not in the reaction mixture containing the test compound indicates that the test compound interferes with the interaction of the gene product, Ankyrin G polypeptide and its reaction partner.

[0069] In traditional immunoassay approaches, binding between Ankyrin G and an Ankyrin G modulator can be determined by analysis of enzymatic, chromodynamic, radioactive or luminescent labels that are attached to either the antibody which binds Ankyrin G or to a secondary antibody which recognizes the antibody which binds to Ankyrin G. Immunoassays which may be used include, but are not limited to, ELISA, inhibition ELISA, Western blots, immunoprecipitation, slot or dot blot assays, immunostaining, RIA, fluorescent immunoassays using antibody conjugates or antigen conjugates of fluorescent substances, such as fluorescein or rhodamine, Ouchterlony double-diffusion analysis and immunoassays employing an avidin-biotin or streptavidin-biotin detection system.

[0070] Detection of the antibodies described herein may be achieved using standard ELISA, FACS analysis and neuroimaging techniques used in vitro or in vivo. Detection can be facilitated by coupling, i.e., physically linking, the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase (β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include ¹²⁵l, ¹³¹l ³⁵S or ³H.

[0071] Particularly preferred, for ease of detection, is the sandwich assay, of which a number of variations exist, all of which are intended to be encompassed by the present invention. For example, in a typical forward assay, unlabeled antibody is immobilized on a solid substrate and the sample to be tested brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-antigen binary complex, a second antibody, labeled with a reporter molecule capable of inducing a detectable signal, is added and incubated, allowing time sufficient for the formation of a ternary complex of antibody-antigen-labeled antibody. Any unreacted material is then washed away, and the presence of the antigen is determined by observation of a signal, or may be quantitated by comparing with a control sample containing known amounts of antigen. Variations on the forward assay include the simultaneous assay, in which both sample and antibody are added simultaneously to the bound antibody, or a reverse assay in which the labeled antibody and sample to be tested are first combined, incubated and added to the unlabeled surface bound antibody. These techniques are well- known to those skilled in the art, and the possibility of minor variations will be readily apparent. As used herein, "sandwich assay" is intended to encompass all variations on the basic two-site technique. For the immunoassays of the present invention, the only limiting factor is that the labeled antibody be an antibody which is specific for Ankyrin G polypeptide or related regulatory protein, or fragments thereof.

[0072] The most commonly used reporter molecules are either enzymes, fluorophore- or radionuclide-containing molecules. In the case of an enzyme immunoassay an enzyme is conjugated to the second antibody, usually by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different ligation techniques exist, which are well-known to the skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose oxidase, β-galactosidase and alkaline phosphatase, among others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable color change. For example, p-nitrophenyl phosphate is suitable for use with alkaline phosphatase conjugates; for peroxidase conjugates, 1 ,2-phenylenediamine or toluidine are commonly used. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. A solution containing the appropriate substrate is then added to the tertiary complex. The substrate reacts with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an evaluation of the amount of polypeptide or polypeptide fragment of interest which is present in the serum sample.

[0073] Alternately, fluorescent compounds, such as fluorescein and rhodamine, may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labeled antibody absorbs the light energy, inducing a state of excitability in the molecule, followed by emission of the light at a characteristic longer wavelength. The emission appears as a characteristic color visually detectable with a light microscope. Immunofluorescence and EIA techniques are both very well-established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotopes, chemiluminescent or bioluminescent molecules may also be employed. It will be readily apparent to the skilled artisan how to vary the procedure to suit the required use.

[0074] These methods allow detection of Ankyrin G present in biological samples which, for the purpose of AD diagnosis in symptomatic patients may include, but are not limited to, lumbar cerebral spinal fluid (CSF), ventricular CSF, brain tissue homogenates or thin sections obtained from the patient. These methods also allow identification of other Ankyrin G-associated disorders that occur concurrently with AD or as yet are not elucidated, as well as the ability to distinguish AD from other neurodegenerative disorders.

[0075] It is contemplated herein that monitoring Ankyrin G protein levels and/ or detecting Ankyrin G gene expression (mRNA levels) in addition to other proteins known to be associated with AD, such as tau, can be used as part of a clinical testing procedure, e.g., to determine the efficacy of a given compound treatment regimen or to increase the sensitivity of typical clinical testing procedures. For example, patients to whom medicine for AD has been administered would be evaluated and the clinician would be able to identify those patients in whom Ankyrin G protein levels, gene expression levels differ from levels in control patients or patients who are asymptomatic for AD. Based on these data, the clinician could then adjust the dosage, administration regimen or type of medicine prescribed. Therein, the clinician can get an idea of the effectiveness of particular AD medication by monitoring qualitative diagnosis, such as questions about the person's general health, past medical problems, and the history of any difficulties the person has carrying out daily activities, tests of memory, problem solving, attention, counting and language.

[0076] Any of the immunoassays described herein can be used to analyze lumbar CSF obtained from patients by standard methods [see The Merck Manual, 12^th Edition, Holvey, Ed., Merck Sharp and Dohme Research Labs Publishing, NJ, pp. 1746-1748 (1972) or ventricular CSF [see Appleyard et al., Brain, Vol. 110, Pt. 5, pp. 1309-1322 (1987); and Wester et al., J Neurochem, Vol. 54, No. 4, pp. 1148-1156 (1990)] and brain homogenates. See Takeuchi, Saito and Nixon, J Neurochem, Vol. 58, No. 4, pp. 1526-1532 (1992).

[0077] The antibodies useful for the methods of the invention include those which are specific in their binding to Ankyrin G; preferably, the antibodies 4Gϊ 3F8 may be used in the methods and preparations of the invention. Prefereably, the antibodies are monoclonal.

[0078] Monoclonal antibodies, which are homogeneous populations of antibodies to a particular antigen, may be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique of Kohler and Milstein, Nature, Vol. 256, No. 5517, pp. 495-497 (1975); and U.S. Patent No. 4,376,110; the human B-cell hybridoma technique [see Kosbor et al., Immunol Today, Vol. 4, pp. 72 (1983); and Cole et al., Proc Nat/Acad Sci U S A, Vol. 80, pp. 2026-2030 (1983); and the EBV-hybridoma technique. See Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 (1985). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently preferred method of production.

[0079] Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen, such as target gene product, or an antigenic functional derivative thereof. For the production of polyclonal antibodies, host animals, such as those described above, may be immunized by injection with the polypeptides, or a portion thereof, supplemented with adjuvants as also described above. [0080] In addition, techniques developed for the production of "chimeric antibodies" [see Morrison, Johnson, Herzenberg and Oi, Proc NatlAcad Sci U S A, Vol. 81, No. 21, pp. 6851-6855 (1984); Neuberger, Williams and Fox, Nature, Vol. 312, No. 5995, pp. 604-608 (1984); Takeda et al., Nature, Vol. 314, No. 6010, pp. 452-454 (1985)] by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable or hypervariable region derived from a murine mAb and a human immunoglobulin constant region.

[0081] Alternatively, techniques described for the production of single-chain antibodies [see U.S. Patent No. 4,946,778; Bird, Science, Vol. 242, pp. 423-426 (1988); Huston et al., Proc NatlAcad Sci U S A, Vol. 85, No. 16, pp. 5879-5883 (1988); and Ward et al., Nature, Vol. 334, pp. 544-546 (1989)] can be adapted to produce differentially-expressed gene single-chain antibodies . Single-chain antibodies are formed by linking the heavy- and light-chain fragments of the Fv region via an amino acid bridge, resulting in a single-chain polypeptide.

[0082] Most preferably, techniques useful for the production of "humanized antibodies" can be adapted to produce antibodies to the polypeptides, fragments, derivatives and functional equivalents disclosed herein. Such techniques are disclosed in U.S. Patent Nos. 5,932, 448; 5,693,762; 5,693,761 ; 5,585,089; 5,530,101; 5,910,771; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,545,580; 5,661 ,016; and 5,770,429, the disclosures of all of which are incorporated by reference herein in their entirety.

[0083] Antibody frag ents that recognize specific epitopes may be generated by known techniques. For example, such fragments include, but are not limited to, the F(ab')₂ fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab')₂ fragments. Alternatively, Fab expression libraries may be constructed [see Huse et al., Scfence, Vol. 246, No. 4935, pp. 1275-1281 (1989)] to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.

[0084] It is understood that some of the antibodies of the invention may recognize proteins in addition to Ankyrin G. In the cases of such antibodies, immunoassays that allow differentiation of protein size and weight are most useful. [0085] The useful antibodies described above can also be used for postmortem diagnosis to quantitate levels of Ankyrin G in a subject or identify Ankyrin-G associated disorders. For example, Ankyrin G expression levels and localization would indicate AD but may also be useful in distinguishing AD-like conditions from other neurodegenerative diseases, such as ALS or PD as described herein. Such information would also be useful to identify or predict subsets of AD patients that may not respond to therapeutic treatment. Additionally, the antibodies may be used in assays to identify any other diseases or conditions that are associated with abnormal Ankyrin G expression, preferably in pathological conditions associated with defects in the APP pathway. Immunostaining of sectioned brain tissue can be carried out according to methods described in Immunohistochemistry, Cuello, Ed., John Wiley and Sons, NY, p. 501 (1983).

[0086] Portions or fragments of the cDNA sequences identified herein (and the corresponding complete gene sequences) can be used as polynucleotide reagents. By way of example, and not of limitation, these sequences can be used to: (i) identify an individual from a minute biological sample (tissue typing); (ii) aid in forensic identification of a biological sample; and (iii) as molecular weight makers for protein electrophoresis purposes.

[0087] Ankyrin G sequences of the invention can be used to identify individuals from minute biological samples. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification. The sequences of the invention are useful as additional DNA markers for restriction fragment length polymorphisms (RFLP), described in U.S. Patent No. 5,272,057.

[0088] Furthermore, the sequences of the invention can be used to provide an alternative technique that determines the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the Ankyrin G sequences described herein can be used to prepare two PCR primers from the 5'- and 3'-termini of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.

[0089] Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences. The sequences of the invention can be used to obtain such identification sequences from individuals and from tissue. The Ankyrin G sequences of the invention uniquely represent portions of the human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases. Much of the allelic variation is due to single nucleotide polymorphisms (SNPs), which include RFLPs.

[0090] Ankyrin G polypeptides or nucleic acid sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences can comfortably provide positive individual identification with a panel of perhaps 10-1 ,000 primers that each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences are used, a more a ppropriate number of primers for positive individual identification would be 500-2,000 primers.

[0091] Another aspect of the invention provides methods for determining Ankyrin G protein, nucleic acid or protein expression or activity in an individual to thereby select appropriate therapeutic or prophylactic agents for that individual, referred to herein as "pharmacogenomics". Pharmacogenomics allows for the selection of agents, e.g., drugs, for therapeutic or prophylactic treatment of an individ ual based on the genotype of the individual, e.g., the genotype of the individual exa ined to determine the ability of the individual to respond to a particular agent.

[0092] Thus, administering compounds w-hich affect Ankyring G expression levels or association with β-amyloid proteins may be useful in blocking accumulation of neuritic plaques or β-toxicity components of AD.

[0093] In addition, Ankyrin G proteins and nucleic acid sequences can be used as control standards in the diagnostic methods and kits of the invention. The present invention relates to a diagnostic kit which comprises: (a) a polynucleotide of Ankyrin G or a fragment thereof; (b) a nucleotide sequence complementary to that of (a); (c) an Ankyrin G polypeptide, or a fragme nt thereof; or (d) an antibody to Ankyrin G polypeptide. [0094] It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component. It is also contemplated that said kit could comprise components (a)-(d) designed to detect levels of -Ankyrin G-related regulatory proteins or proteins modified by Ankryin G as discussed herein.

[0095] In another aspect, the invention relates to a method to treat or ameliorate AD comprising administering to a subject in need thereof a pharmaceutical composition comprising an effective amount of an Ankyrin G modulator. Such modulators include agonists, antagonists or antibodies directed to Ankyrin G polypeptide or fragments thereof. In certain particularly preferred embodiments, the pharmaceutical composition comprises antibodies that are highly selective for human Ankyrin G polypeptides or fragments thereof. Antibodies to Ankyrin G may cause the aggregation of these proteins in a subject and thus inhibit or reduce the expression of these proteins. Such antibodies may also inhibit or decrease Ankyrin G association with neuritic plaques or β-amyloid portein, or NFTs, e.g., by blocking its interaction with components which cause Ankyrin G to accumulate in plaques associated with neuritic structures, or by interacting directly with active sites or by blocking access of substrates to active sites producing A-nkyrin G expression. Ankyrin G antibodies may also be used to inhibit localization or association of Ankyrin G with β-amyloid by preventing protein-protein interactions that may be involved in the formation of β-amyloid plaques. Antibodies with inhibitory activity, such as described herein, can be produced and identified according to standard assays familiar to one of skill in the art.

[0096] The pharmaceutical compositions of the present invention may also comprise substances that alter the expression of Ankyrin G at the nucleic acid level. Such molecules include ribozymes, antisense oligonucleotides, triple helix DNA, RNA aptamers and/or double-stranded RNA directed to an appropriate nucleotide sequence of Ankyrin G nucleic acid. These molecules may be created using conventional techniques by one of skill in the art without undue burden or experimentation. For example, modifications, e.g., inhibition, of gene expression can be obtained by designing antisense molecules, DNA or RNA, to the important regions of the genes encoding the polypeptides discussed herein. For a mRNA of 3,000 nucleotides, there are approximately 30,000 different 10-20-mers possible. It is not possible to predict a priori how many and which of the 30,000 possible 10-20-mer antisense oligonucleotides for a mRNA will have useful activity; experience shows that a small number will be active. The surest method to identify the best oligonucleotide would be to synthesize all 30,000 sequences and test them individually. There are numerous reports in literature of a small selection of between 10-30 different antisense sequences providing suitable compounds. It would be helpful to be able to predict which regions of the target RNA are open to binding of oligonucleotides to determine whether a given region of a mRNA is accessible are available and are being developed and improved. Computer programs to predict secondary interactions of RNA are also available, despite their relatively primitive methods in providing reliable predictions for long polynucleotides, e.g., greater than a few hundred nucleotides. The use of enzyme mapping experiments [see Lima, Monia, Ecker and Freier, Biochemistry, Vol. 31, No. 48, pp. 12055-12061 (1992)] to reveal single and double-stranded regions of a RNA have been applied, but are tedious; multiple incubations with various enzymes and several polyacrylamide gels are required- Mapping of the RNA accessible regions with a combinatorial library of short DNA-oligo nucleotides is a recently introduced technique. See Lima et al., J Biol Chem, Vol. 272, No. 1 , pp. 626-638 (1997). After hybridization of members of the library with the RNA, RNase hi is introduced to give cleavage of the formed duplexes. These regions are subsequently identified on a polyacrylamide gel. This method has its limitations in the degree oi resolution of the gel, and use of the information is subject to the errors of supposing that single-stranded regions located by short oligonucleotides would also offer good binding sites to longer antisense sequences. Scanning array technology gives a direct readout of the binding capacity of a complete set of antisense oligonucleotides, and appears to be a big step forward in identifying accessible regions of a RNA target for high affinity duple-x formation by antisense oligonucleotides. See Southern et al., Nucleic Acids Res, Vol. 22, Mo. 8, pp. 1368-1373 (1994). The method itself is quite straightforward and is described in detail in a number of peer-reviewed publications. See, e.g., Southern et al. (1994), supra; and Milner, Mir and Southern, Nat Biotechnol, Vol. 15, No. 6, pp. 537-541 (1997); Soha il and Southern, Mol Cell Biol Res Commun, Vol. 3, No. 2, pp. 67-72 (2000); and WO 95/117-48.

[0097] Once one or more target sites have been identified, oligonucleotides are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect, using, e.g., any of the techniques mentioned above.

[0098] Similarly, inhibition of nueritic plaque formation and β-amyloid toxicity may 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. Recent therapeutic advances using triplex DNA have been described in the literature. See Gee et al., Molecular and Immunologic Approaches, Huber and Carr, Eds., Futura Publishing Co., Mt. KLisco, NY (1994). These molecules may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.

[0099] Ribozymes, enzymatic RNA molecules, may also be used to in ibit gene expression by catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples which may be used i clude engineered "hammerhead" or "hairpin" motif ribozyme molecules that can be designed to specifically and efficiently catalyze endonucleolytic cleavage of gene sequences, e.g., the gene for Ankyrin G.

[0100] Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which i nclude the following sequences: GUA, GUU and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for secondary structural features which may render the oligonucleotide inoperable. The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.

[0101] Ribozyme methods include exposing a cell to ribozymes or inducing expression in a cell of such small RNA ribozyme molecules. See Grassi and arini, Ann Med, Vol, 28, No. 6, pp. 499-510 (1996); Gibson, Cancer Metastatis Rev, Vol. 15, No. 3, pp. 287-299 (1996). Intracellular expression of hammerhead and hairpin ribozymes targeted to mRNA corresponding to at least one of the genes, discussed herein, can be utilized to inhibit protein encoded by the gene.

[0102] Ribozymes can either be delivered directly to cells, in the form of RNA oligonucleotides incorporating ribozyme sequences, or introduced into the cell as an expression vector encoding the desired ribozymal RNA. Ribozymes can be routinely expressed in vivo in sufficient number to be catalytically effective in cleaving mRNA, and thereby modifying mRNA abundance in a cell. See Cotton and Birnstiel, EME30 J, Vol. 8, No. 12, pp. 3861-3866 (1989). In particular, a ribozyme coding DNA sequence, designed according to conventional, well-known rules and synthesized, e.g., by standard phosphoramidite chemistry, can be ligated into a restriction enzyme site in the antlcodon stem and loop of a gene encoding a tRNA, which can then be transformed into an d expressed in a cell of interest by methods routine in the art. Preferably, an inducible promoter, e.g., a glucocorticoid or a tetracycline response element, is also introduced into this construct so that ribozyme expression can be selectively controlled. For saturating use, a highly and constituently active promoter can be used. tDNA genes, i.e., genes encoding tRNAs, are useful in this application because of their small size, high rate of transcription and ubiquitous expression in different kinds of tissues.

[0103] Therefore, ribozymes can be routinely designed to cleave virtually any mRNA sequence, and a cell can be routinely transformed with DNA coding for such riboz:yme sequences such that a controllable and catalytically effective amount of the ribozyme is expressed. Accordingly the abundance of virtually any RNA species in a cell can be modified or perturbed.

[0104] Ribozyme sequences can be modified in essentially the same manner as described for antisense nucleotides, e.g., the ribozyme sequence can comprise a modified base moiety.

[0105] RNA aptamers can also be introduced into or expressed in a cell to modify RNA abundance or activity. RNA aptamers are specific RNA ligands for proteins-, such as for Tat and Rev RNA [see Good et al., Gene Ther, Vol. 4, No. 1, pp. 45-54 (1997)] that can specifically inhibit their translation.

[0106] Gene specific inhibition of gene expression may also be achieved using conventional double-stranded RNA technologies. A description of such technology may be found in WO 99/32619, as well as Harborth et al., J Cell Sci, Vol. 114, Pt. 24, pp. 4557-4565 (2001), and the entire contents of both references are hereby incorporated by reference.

[0107] Antisense molecules, triple helix DNA, RNA aptamers, ribozymes and double- stranded RNA of the present invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the genes of the polypeptides discussed herein. Such D sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters, such as T7 or SP6. Alternatively, cDNA constructs that synthesize antisense RNA constitutively or inducibly can be introduced into cell lines, cells or tissues.

[0108] Vectors may be introduced into cells or tissues by many available means, and may be used in vivo, in vitro or ex vivo. For ex vivo therapy, vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. Delivery by transfection and by liposome injections may be achieved using methods that are well-known in the art.

[0109] In addition to the above described methods for inhibiting the gene expression of Ankyrin G, it is contemplated herein that one could identify and employ small molecules or other natural products to inhibit the transcription in vivo of the polypeptides discussed herein including, but not limited to, Ankyrin G. For example, one of skill in the art could establish an assay for Ankyrin G or proteins which localize with Ankyrin G that can be easily applied to samples from the culture media of a cell line using conventional methods. Using this assay, cell lines would be screened to find ones that express such proteins. These cell lines would likely be of neuronal origin and would be cultured in, e.g., 96-well plates.

[0110] In addition, the cDNA and/or protein of Ankyrin G can be used to identify other molecules, e.g., promoters or receptors, that modify Ankyrin G in neurons from brain cells or tissues in which plaques are found. Proteins thus identified can be used for drug screening to treat Ankyrin G-associated disorders. To identify these genes that are downstream of Ankryin G, it is contemplated, e.g., that one could use conventional methods to treat animals in AD models with a specific Ankyrin G inhibitor, sacrifice the animals, remove brain sections/slices and isolate total RNA from these cells and employ standard microarray assay technologies to identify message levels that are altered relative to a control animal (animal to whom no drug has been administered).

[0111] Based on the knowledge that Ankyrin G is localized in neuritic plaques in AD, conventional in vitro or in vivo assays may be used to identify possible genes that lead to over expression of Ankyrin G. These related regulatory proteins encoded by genes thus identified can be used to screen drugs that might be potent therapeutics for the treatment of AD. For example, a conventional reporter gene assay could be used in which a promoter region of Ankyrin G is placed upstream of a reporter gene, the construct transfected into a suitable neuronal cell and using conventional techniques, the cells assayed for an upstream gene that causes activation of Ankyrin G promoter by detection of the expression of the reporter gene.

[0112] The pharmaceutical compositions disclosed herein useful for treating and/or ameliorating Ankyrin G associated disorders, preferably AD, are to be administered to a patient at therapeutically-effective doses to treat or ameliorate symptoms of such disorders. A therapeutically-effective dose refers to that amount of the compound sufficient to result in amelioration of pain symptoms of chronic pain based on, e.g., use of the McGill pain score. See Melzack, Pain, Vol. 1, No. 3, pp. 277-299 (1975).

[0113] The inhibitory/stimulatory substances of the present invention can be administered as pharmaceutical compositions. Such pharmaceutical compositions for use in accordance with the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients.

[0114] Thus, the compounds and their physiologically acceptable salts and solvates may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or topical, oral, buccal, parenteral or rectal administration.

[0115] For oral administration, the pharmaceutical compositions may take the form of, e.g., tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients, such as binding agents, e.g., pre-gelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose; fillers, e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate; lubricants, e.g., magnesium stearate, talc or silica; disintegrants, e.g., potato starch or sodium starch glycolate; or wetting agents, e.g., sodium lauryl sulphate. The tablets may be coated by methods well-known in the art. Liquid preparations for oral administration may take the form of, e.g., solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents, e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats; emulsifying agents, e.g., lecithin or acacia; non-aqueous vehicles, e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils; and preservatives, e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid. The preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate. [0116] Preparations for oral administration may be suitably formulated to give controlled-release of the active compound._,

[0117] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0118] For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base, such as lactose or starch.

[0119] The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents, such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0120] The compounds may also be formulated in rectal compositions, such as suppositories or retention enemas, e.g., containing conventional suppository bases, such as cocoa butter or other glycerides.

[0121] In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long-acting formulations may be administered by implantation, e.g., subcutaneously or intramuscularly, or by intramuscular injection. Thus, e.g., the compounds may be formulated with suitable polymeric or hydrophobic materials, e.g., as an emulsion in an acceptable oil; or ion exchange resins, or as sparingly soluble derivatives, e.g., as a sparingly soluble salt. [0122] The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may, e.g., comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.

[0123] Pharmaceutical compositions suitable for use in the invention include compositions, wherein the active ingredients are contained in an effective amount to achieve the intended purpose. The determination of an effective dose is well within the capability of those skilled in the art.

[0124] For any compound, the therapeutically-effective dose can be estimated initially either in cell culture assays, e.g., 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. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the concentration of the test compound that achieves a half-maximal inhibition of symptoms (IC₅₀). Such information can then be used to determine useful doses and routes for administration in humans.

[0125] A therapeutically-effective dose refers to that amount of active ingredient, e.g., antisense oligonucleotides, triple helix DNA, ribozymes, RNA aptamer and double- stranded RNA designed to inhibit Mob-5 gene expression, antibodies to Mob-5 or related regulatory proteins or fragments thereof, useful to treat and/or ameliorate the pathological effects of chronic pain. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., the dose therapeutically-effective in 50% of the population (ED₅₀) and the dose lethal to 50% of the population (LD₅₀). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD₅₀ ED₅o. Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient and the route of administration. [0126] The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors that may be taken into account include 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. Long-acting pharmaceutical compositions may be administered every 3-4 days, every week or once every 2 weeks depending on half-life and clearance rate of the particular formulation.

[0127] Normal dosage amounts may vary from 0.1-100,000 mg, up to a total dose of about 1 g, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc. Pharmaceutical formulations suitable for oral administration of proteins are described, e.g., in U.S. Patent Nos. 5,008,114; 5,505,962; 5,641,515; 5,681,811; 5,700,486; 5,766,633; 5,792,451; 5,853,748; 5,972,387; 5,976,569; and 6,051 ,561.

[0128] For the production of antibodies to Ankyrin G polypeptides discussed herein, various host animals may be immunized by injection with the polypeptides, or a portion thereof. Such host animals may include, but are not limited to, rabbits, mice and rats. Various adjuvants may be used to increase the immunological response, depending on the host species including, but not limited to, Freund's (complete and incomplete); mineral gels, such as aluminum hydroxide; surface active substances, such as l solecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin and dinitrophenol; and potentially useful human adjuvants, such as bacille Calmette-Guerin (BCG) and Corynebacterium parvum.

[0129] The following examples are meant to illustrate, but not limit, the methods and compositions of the present invention. Other suitable modifications and adaptations of the variety of conditions and parameters normally encountered in immunodiagnostics which are obvious to those skilled in the art are within the spirit and scope of the present invention. EXAMPLES

[0130] The following procedures are performed to conduct the examples:

[0131] Antibodies: 4G3F8 is a mouse monoclonal which binds to a region of the spectrin-binding domain of human Ankyrin G (Zymed, catalog No. 33-8800). SMI312 is a mixture of mouse monoclonals against phosphorylated NF-M and NF-H (Sternberger Monoclonals, Inc., catalog No. SMI312). Rabbit polyclonal anti-Aβ42 is specific for the C-terminus of Aβ42 and does not cross-react with other species of Aβ or full-length APP (Signet Laboratories, catalog No. 9134). 22C11 is a mouse monoclonal that recognizes full- length APP (Chemicon, catalog No. MAB3480). Anti-βlll Tubulin is from Promega, catalog No. G712A. Biotinylated secondary antibodies for immunohistochemistry may be either anti- mouse (Vector Laboratories, catalog No. BA-2000) or anti-rabbit (Vector Laboratories, catalog No. BA-1000). HRP-conjugated anti-mouse secondary antibodies are obtained from Amersham, catalog No. NA93W.

[0132] Immunohistochemistry: Post mortem brain tissue are obtained from the Human Brain and Spinal Fluid Resource Center (Los Angeles, CA), Case Western Reserve University, Institute of Pathology (Cleveland, OH) and Lifespan Biosciences, Inc. Tissue and Disease Repository (Seattle, WA). Table 1 summarizes the neuropathologically classified normal, AD, PD and ALS tissues used in this study.

Table 1. Summary of Donors Neuropath Primary Pathological Diagnosis Age/Sex PMI (h) Diagnosis Normal 74/M 3.0 COPD End Stage Normal 78/F 10.8 Unavailable Normal 70/M 24.8 Abdominal aortic aneurysm Normal 58/F 8.9 Emphysema Normal 37/F 17.3 Histiocytic Sarcoma Normal 60/F 16.2 Unavailable AD 78/F 24.0 AD AD 79/M 18.3 Ruptured abdominal aortic aneurysm AD 79/F 8.0 Hemorrhagic shock AD 73/F 18.4 Atheromatous Arteriosclerosis PD 79/F 29.7 PD PD 73/F 16.3 PD ALS 61/M 18.0 ALS ALS 52/F 14.3 ALS [0133] Paraffin-embedded formalin-fixed tissues are cut at 4 microns on a Leica Rotary microtome, floated in a water bath at 37°C and mounted. Slides are deparaffinized by xylene and alcohol, rehydrated and then subjected to the steam method of target retrieval. Automated staining procedures are performed with an DAKO Autostainer as follows.

[0134] The slides are incubated for 20 minutes with blocking reagent (DAKO, catalog No. X0909-30), primary antibodies are applied for 45 minutes at room temperature followed by a TBS rinse. Biotinylated secondary antibodies are applied at 5 μlJmL for 30 minutes at room temperature, rinsed in TBS and developed with Vector Laboratories Vectastain ABC- AP kit (catalog No. AK-5000) and Vector Red substrate (catalog No. SK-5100) giving a red colored reaction. For double-labeling experiments, DAKO LSAB2 kit (catalog No. K0675) and a DAKO DAB+ chromogen substrate (catalog No. K3468) is used to produce a brown colored reaction. Slides are counterstained with hematoxylin prior to coverslipping. Titration experiments are conducted for each antibody to establish dilutions resulting in minimal background staining. The following dilutions may be used: 1 :50 (4G3F8); 1:25 (Aβ42); 1:10,000 (SMI312); 1 :250 (βlll tubulin). Slides are imaged with a DVC 1310C digital camera coupled to a Nikon microscope. Images are stored as TIFF files in Adobe Photoshop. Mouse brain slides from APP23 mice expressing human APP with the Swedish double mutations [see Sturchler-Pierrat et al., Proc NatlAcad Sci U S A, Vol. 94, No. 24, pp. 13287- 13292 (1997)] and control littermates are processed the same as human tissues except fixation may be carried out in 4% formaldehyde. Negative control slides are obtained by performing the immunohistochemistry procedure described above on adjacent serial sections in the absence of primary antibody.

[0135] Aβ peptide preparation: Synthetic Aβ1 -40, Aβ1-42 and a control peptide Aβ42-1 , having the reversed amino acid sequence of Aβ1-42 (Biosource Inc.), are resuspended in 1 ,1 ,1,3,3,3-hexafluoro-2-propanol (Sigma, catalog No. H-8508), aliqouted into 1.5 mL siliconized tubes and dried under a gentle stream of nitrogen gas. The desiccated peptide film is resuspended in DMSO, sonicated for 10 minutes in a Branson model 1510 water bath and diluted to 100 μM in deionized water. The solution is aged overnight at 25°C. [0136] Cell Culture: PC 12 cells (ATCC CRL-1721) are maintained at 37°C, 5% CO₂ in RPM1 1640 media containing 25 mM HEPES, 0.5 mM EGTA, 10% horse serum and 5% fetal bovine serum. SH-SY5Y cells (ATCC CRL-2266) are maintained at 37°C, 5% 0O in DMEM media containing 20% fetal bovine serum. PC12 cells are differentiated in collagen I coated plates with 50 ng/mL NGF (Sigma, catalog No. N2513). The first day following the addition of NGF is considered differentiation day 1. For Aβ treatment of differentiated PC12 cells, serum supplemented media is carefully removed and replaced with N2 supplemented (Invitrogen, catalog No. 17502-048) media containing serial dilutions of water prepared Aβ1-40, Aβ1-42 and Aβ42-1. After a 20-hour incubation with Aβ-peptides, cells are washed in PBS prior to protein extraction.

[0137] Protein Extraction and Gel Electrophoresis: Cells are washed with 1 x PBS and lysed in Cell lytic-M reagent (Sigma, catalog No. C-2978) plus a protease inhibitor cocktail (Sigma, catalog No. P-8340) by gentle shaking for 10 minutes at 4°C. Cell lysates cleared by a 14,000 g spin for 10 minutes. Tissue protein is extracted on ice using a PowerGen 35 homogenizer equipped with a flat bottom generator and RIPA buffer containing 50 mM Tris-HCL, pH 8.0, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate plus EDTA-free protease inhibitor cocktail (Roche, catalog No. 1873580). Tissue Lysates are cleared by a 3,000 g spin for 10 minutes followed by a 30,000 g spin for 30 minutes at 4°C. Protein concentration is determined by Coomassie Plus Protein Assay (Pierce, catalog No. 1856210). Samples are electrophoresed on 4-20% gradient Tris-HCL gels (Bio-Rad) and transferred onto 0.45 μM nitrocellulose membranes for 1.5 hours at 240 miiliamps. To check for consistent transfer, membranes are stained with a 0.1% Ponceau S solution (Sigma, catalog No. P-7170) prior to blocking in 5% milk TBS/0.05% Tween20 (TBST). Primary antibodies are added at the following dilutions: 1 :200 (4G3F8); 1 :4000 (βlll tubulin); 1:1000 (22C1 1). Following primary antibody incubation, membranes are washed 6 times for 10 minutes with 1 x TBST. HRP-conjugated secondary antibody was incubated for 1 hour and washed 6 times for 10 minutes with 1 x TBST and membranes imaged using ECL chemiluminescence autoradiography.

[0138] Cloning of Hippocampal isoforms of Ankyrin G: An oligo(dT) and random primed human hippocampus 5' stretch plus λgtl 1 cDNA library (Clontech, catalog No. HL3023b, lot 32186) is triple-screened with a ³²P-labeled Ankyrin3 cDNA probe. The probe is generated by PCR amplification from the same hippocampus cDNA library using Ankyrin3 specific (5'-CCAGGGCGTTTATTTTCG-3') and λgtl 1 -specific (5'-ATGGTACCGACTCCTGGAGCCCG-3') primers and subcloned into pSTBIue-1 (Novagen). The probe fragment corresponding to basepairs 3713-4414 within the spectrin- binding domain of the Ankyrin G470 transcript (Genbank Accession No. NM_020987) is isolated following XCMI/ECORI double digestion. Hybridization and post-hybridization washes are done at 65°C in 0.2 x SSC. cDNA inserts are subcloned into the pCR2.1 vector (Invitrogen) and sequenced (ABI model 3100).

Example 1

Ankyrin G expression in AD brain [0139] The domain structure of Ankyrin G is described in Figure 1. To examine the distribution of Ankyrin G, monoclonal antibody 4G3F8 raised against a region of the spectrin- binding domain [see Kordeli, Lambert and Bennett, J Biol Chem, Vol. 270, No. 5, pp. 2352- 2359 (1995)] is used to stain thin sections of normal brain tissues. This antibody selectively binds the cell bodies, axon initial segments and axons of many neurons in the entorhinal cortex layers, subiculum and hippocampus, including the granule cells of the dentate gyrus [see FIG. 1 (B-D)]. In the CA3 region of the hippocampus, bundles of axonal fibers resembling afferent projections of perforant pathway neurons are identified [see Figure 1 (B), arrowheads]. In white matter tissues, some neuronal cells did stain [see Figure 1 (E)] while no staining is observed in astrocytes, microglial or oligodendrocytes. Adjacent choroid plexus and ependymal cells are also negative (data not shown). In general, the staining spectrum of 4G3F8 in neuropathologically normal human brain is similar to previous reports in rat hippocampus. See Kordeli, Lambert and Bennett (1995), supra. [0140] By contrast, robust staining of neuritic plaques is observed in all neuropathologically classified AD brain used in this study (see FiguVe 2 and data not shown). This staining pattern is particularly prominent in the hippocampus area CA3 [see Figure 2 (A)] and dentate gyrus [see Figure 2 (B)] while generally less intense Ankyrin G positive plaques are noted in the subiculum and entorhinal cortex layers [see Figure 2 (E) and data not shown]. In many plaques, the neuritic staining appears to surround a central core of amyloid [see Figure 2 (A-C) and (E)]. The intensity and spatial distribution of the staining suggests that Ankyrin G accumulates within dystrophic neural structures associated with amyloid and possibly within extracellularly deposited material. However, in some instances, neuritic structures resembling degenerating neurons with swollen axons are clearly visible [see Figure 2 (D)]. Other neuropathological hallmarks of AD, including neurofibrillary tangles and granulovacuolar degenerative changes are generally weakly stained or unstained (data not shown). [0141] Within the same AD donor tissues exhibiting intense Ankyrin G positive neuritic plaques, significantly decreased Ankyrin G expression as compared to normal age- matched donors is seen in neurons and bundles of axons not within neuritic plaques [compare Figures 1 (B) and 2 (A)]. This reduction is noted throughout areas CA1 , CA3 and CA4, the granular layer, subiculum, white matter neurons, and entorhinal cortex layers. Antibodies against either neurofilament (see below) or βlll tubulin (data not shown) did not exhibit reduced staining of non-plaque associated neurons in AD versus normal donor tissues. Thus, the overall reduced neuronal expression of Ankyrin G throughout the hippocampus, subiculum and entorhinal cortex and its focal intense neuritic plaque staining of dystrophic plaque associated neurites suggests that during the pathogenesis of AD the neuronal distribution or subcellular localization of Ankyrin G is changed. [0142] Importantly, reductions in Ankyrin G expression is particularly noticeable within perforant path neural projections which largely consist of closely packed axons. The entorhinal cortex neurons which give rise to these projections are known to be one of the earliest lost in AD brain, coincident with synapse loss in the hippocampus. See Braak and Braak, Acta Neuropathol (Berl), Vol. 80, No. 5, pp. 479-486 (1990); Braak, Braak and Bohl, Eur Neurol, Vol. 33, No. 6, pp. 403-408 (1993); Cabalka et al., Neurobiol Aging, Vol. 13, No. 2, pp. 283-291 (1992); and Hyrnan, Van Hoesen and Damasio, Neurology, Vol. 40, No. 11, pp. 1721-1730 (1990). Thus, the low levels of Ankyrin G expression in non-plaque associated neurons, including perforant pathway axonal bundles, and its concentration within plaque associated dystrophic neurites in the hippocampus could reflect early degenerative changes in this neural network. Further, it may also s^'uggest that the distribution of plasma membrane bound proteins localized by Ankyrin G, such as ion channels and cell adhesion molecules might be altered in AD brain. [0143] Ankyrin G expression is also examined in two additional neurodegenerative diseases. Examination of brain tissue from the substantia nigra and locus coeruleus of PD stained with 4G3F8 and compared to age-matched normal control tissues is shown in Figure 3. Interestingly, the intense neuritic staining seen in AD brain is not observed in any of the PD tissues examined. Pigmented and non-pigmented neurons containing Lewy body (LB) deposits remain unstained or weakly stained with 4G3F8 [see Figure 3 (A-C)]. Similar to the case with PD tissues, the staining pattern of spinal cord tissues from ALS donors is indistinguishable from control tissues [see Figure 3 (D and E) and data not shown]. The lack of changes in PD and ALS suggests that Ankyrin G distribution is selectively altered in AD.

Example 2

Assocation between Ankyrin G expression and Aβ42 deposition [0144] To determine whether Ankyrin G staining is colocalized with Aβ amyloid deposition, human tissue sections from normal and AD donors are stained with both 4G3F8 and anti-Aβ42 antibodies (see Figure 4). The anti-Aβ42 antibody identified numerous diffuse and well defined senile plaques in AD hippocampus [see Figure 4 (A and B)] while little to no staining is noted in normal age-matched control tissues (data not shown). The known staining pattern of anti-Aβ42 antibody suggests that it is indeed identifying Aβ amyloid. Double-labeling experiments using 4G3F8 and anti-Aβ42 antibodies show Ankyrin G positive dystrophic structures (see Figure 4 arrowheads) in close proximity to Aβ42 positive deposits, however, co-localization is not observed [see Figure 4 (D-F)]. [0145] These observations are extended into the APP23 mouse model overexpressing human APP with the familial 'Swedish' double mutations. See Sturchler- Pierrat (1997), supra. Neuronal expression of this familial form of APP leads to large, clearly identifiable extracellular deposits of amyloid. See Sturchler-Pierrat (1997), supra. Adjacent serial brain sections of 1.5 year old hemizygous APP23 mice are stained with either anti- Aβ42 or 4G3F8 antibodies. Anti-Aβ42 antibodies reveals numerous well-defined amyloid plaques in the neocortex, olfactory cortex and hippocampus [see Figure 4 (G-l)]. Staining of the adjacent slide with 4G3F8 shows staining of spherical plaque associated structures localized peripheral to amyloid deposition [see Figure 4 (J-L)]. This pattern is reminiscent of ft previous histological studies on the morphology of dystrophic neurites in proximity to amyloid accumulation in these mice. See Bomemann and Staufenbiel, Ann N YAcad Sci, Vol. 908, pp. 260-266 (2000). Thus, the staining pattern with 4G3F8 and anti-Aβ42 antibodies in APP23 mice exhibit a spatial association between Ankyrin G and Aβ42 deposition similar to that seen in human neuritic plaques and also suggests that Ankyrin G is expressed in plaque associated dystrophic neurites. Example 3

Phosphorylated neurofilament and Ankyrin G staining in AD hippocampus [0146] Neurofilaments are type IV intermediate filaments critical for the development and maintenance of axonal caliber and conduction velocity of neurons. Phosphorylation of the C-terminal side arms of middle and heavy neurofilaments (NF-M and NF-H) regulate cytoskeletal stability in mature axons. See Ackerley et al., J Cell Biol, Vol. 161 , No. 3, pp. 489-495 (2003). SMI312, an antibody cocktail that detects phosphorylated NF-H and NF-M neurofilaments, is a well-characterized and commonly-used marker of dystrophic neuritis. See Dickson, King, McCormack and Vickers (1999), supra; and Masliah et al., Am J Pathol, Vol. 142, No. 3, pp. 871-882 (1993). [0147] To compare Ankyrin G expression with the staining pattern of SMI312, adjacent serial sections of AD hippocampus are stained with either antibody alone [see Figure 5 (A and B), showing dystrophyic neuritis with black arrows] or double-stained with both antibodies [see Figure 5 (C and D)]. Agreeing with previously published reports on the staining spectrum of SMI312, dystrophic neurites are preferentially stained within senile plaques of AD [see Figure 5 (A), region of amyloid deposition is shown by white arrowheads]. 4G3F8 strongly stains the same neuritic plaques as SMI312. However, Ankyrin G is, in general, more tightly associated with amyloid deposition than SMI312 [compare 4G3F8 staining in Figure 5 (B) with that of SMI312 in Figure 5 (A)]. The distinct staining pattern between 4G3F8 and SMI312 antibodies is also pronounced in double- labeling experiments in which 4G3F8 positive material surrounds an amyloid core while SMI312 stained peripheral structures [see Figure 5 (C and D)]. This pattern suggests that Ankyrin G may be present within a subset of dystrophic neuronal projections unstained by SMI312 or in some instances deposited extracellularly. Importantly, 4G3F8 and SMI312 together clearly distinguish AD specific pathology including amyloid deposition, dystrophic structures and neuritic plaque pathology.

Example 4

Presence of small isoforms of Ankyrin G in the hippocampus and neuronal cell lines [0148] 4G3F8 is raised against a region of the spectrin-binding domain not conserved within other Ankyrins and recognizes neuron-specific isoforms of 270 kDa and 480 kDa. See Kordeli et al. (1995), supra. However, since alternative transcripts of Ankyrin G are known to exist within the same tissue and cell types, Western blot analysis of protein lysates from dissected hippocampal tissue and neuronal cell lines is performed to check for additional isoforms of Ankyrin G detected by antibody 4G3F8. Two small isoforms at approximately 60 kDa and 110 kDa are detected in human, rat and mouse hippocampus cell lysate. Isoforms at approximately the same molecular weights are also observed in neuronal cell lines, such as SH-SY5Y and to a lesser extent in undifferentiated PC12 cells but are generally absent in non-neuronal cells (data not shown). Upon NGF mediated differentiation of PC12 cells, levels of the small isoforms increase, favoring the 60 kDa form. Whereas, there is little change in the abundance of the larger (110 kDa) form. The increase in relative expression levels following differentiation in PC 12 cells is similar to axonally expressed βlll tubulin and what has been previously reported for tau. See Drubin, Feinstein, Shooter and Kirschner, J Cell Biol, Vol. 101 , No. 5, Pt. 1, pp. 1799-1807 (1985). Further experimentation is planned to characterize the isoforms and their association with Ankyrin G expression and amyloid in AD tissues. [0149] The overall results suggest that Ankyrin G may not only participate in neurodegenerative processes at work in neuronal networks central to the pathogenesis of AD but may also be an important histopathological marker due to its AD specific expression pattern. Expanded studies with additional donors may establish whether the extent or severity of Ankyrin G neuritic pathology correlates with disease progression. Interestingly, Ankyrin G is located on chromosome 10 within a genetic interval linked to late-onset AD. See Bertram et al., Science, Vol. 290, No. 5500, pp. 2302-2303 (2000); Ertekin-Taner et al., Science, Vol. 290, No. 5500, pp. 2303-2304 (2000); and Myers et al., Science, Vol. 290, No. 5500, pp. 2304-2305 (2000). Genetic association studies with populations shown to have linkage on chromosome 10 could establish whether the presence of particular allelic variations of Ankyrin G may influence susceptibility to AD.

Other Embodiments [0150] The above specific examples are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. [0151] From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. All publications cited herein are fully incorporated by reference herein in their entirety. [0152] Other embodiments are in the following claims.

Claims

What is claimed is:

1. A method of diagnosing conditions associated with abnormal APP regulation in a subject comprising detecting expression of proteins selected from the group consisting of Ankyrin G, Aβ40, Aβ42 and tau and determining whether said protein is over- or under- expressed relative to a control sample from a subject to determine whether said subject has a condition associated with abnormal APP regulation.

2. The method according to Claim 1 , wherein said condition includes Alzheimer's Disease (AD).

3. The method according to Claim 1 , wherein said sample is brain tissue homogenate.

4. A method to diagnose subjects suffering from conditions associated with abnormal regulation of the APP pathway comprising measuring the mRNA level and/or the level or activity of polypeptides encoded by proteins selected from the group consisting of Aβ40, Aβ42 or Ankyrin G in a biological sample from a subject, wherein an abnormal level relative to the level thereof in a control subject is diagnostic of said condition.

5. The method according to Claim 4, wherein said conditions include AD.

6. The method according to Claim 4, wherein said biological sample is brain tissue homogenate.

7. The method according to Claim 6, wherein said brain tissue is hippocampal brain tissue.

8. A method of detecting AD in a human patient, said method comprising using an antibody that specifically recognizes Ankyrin G in an immunoassay to measure the amount of Λnkyrin G present in a biological sample of said patient relative to the amount of Ankyrin G in a control sample from an unaffected human, a relative level of Ankyrin G in said sample from said patient 50% above the level of Ankyrin G in said control sample indicating a diagnosis of AD in said patient.

9. The method according to Claim 8, wherein said antibody binds to Ankyrin G proteins selected from the group consisting of SEQ ID NOs: 2, 4, 6 and 8.

10. The method according to Claim 8, wherein said biological sample is selected from the group consisting of a lumbar cerebral spinal fluid (CSF) sample, a ventricular CSF sample, a brain tissue sample and a hippocampal tissue sample of said patient.

11. The method according to Claim 8, wherein said immunoassay is Western blot analysis.

12. A method for the treatment, prevention or amelioration of conditions associated with abnormal regulation of the APP pathway comprising administering to a subject in need thereof a therapeutically-effective amount of a substance that inhibits or promotes the expression or function Ankyrin G.

13. The method according to Claim 12, wherein said substance is selected from the group consisting of compounds, triple helix DNA, antisense oligonucleotides, double- stranded RNA molecules and ribozymes, and wherein said substances are designed to inhibit expression of Ankyrin G or fragments thereof.

14. The method according to Claim 12, wherein said substance comprises any one or more antibodies and/or fragments thereof directed to the polypeptide encoded by SEQ ID NO: 1 or fragments th reof.

15. The method according to Claim 12, wherein said conditions include AD.

16. A method to treat, prevent or ameliorate conditions associated with abnormal regulation of the APP pathway comprising: (a) assaying for mRNA and/or protein levels of Ankyrin G in a subject; and (b) administering to a subject with abnormal mRNA and/or protein levels compared to control subjects a substance in an amount sufficient to treat or ameliorate the pathological effects of said condition.

17. The method according to Claim 16, wherein said condition is AD.

18. A method of screening for a compound which modulates Ankyrin G expression in a sample from a subject comprising: (a) exposing Ankyrin G in said sample to a compound; and (b) determining whether said compound modulates Ankyrin G expression to determine whether said subject has a condition associated with abnormal regulation of proteins expressed in an APP pathway.

19. The method according to Claim 18, wherein said substance is selected from the group consisting of compounds, triple helix DNA, antisense oligonucleotides, double- stranded RNA molecules and ribozymes, and wherein said substances are designed to inhibit expression of Ankyrin G or fragments thereof.

20. The method according to Claim 18, wherein said substance comprises any one or more antibodies and/or fragments thereof directed to the polypeptide encoded by SEQ ID NOs: 1, 3, 5 and T or fragments thereof.

21. The method according to Claim 18, wherein said sample is obtained from the group consisting of CSF, ventricular tissue or brain tissue.

22. The method according to Claim 21, wherein said sample is hippocampal brain tissue.

23. The method according to Claim 18, wherein said Ankyrin G is expressed in a neuritic plaque.

24. A diagnostic kit for detecting mRNA levels and/or protein levels of in a biological sample, said kit comprising: (a) a polynucleotide selected from the group consisting of Aβ40, Aβ42 or Ankyrin G or fragments thereof; (b) a nucleotide sequence complementary to that of (a); (c) a pol peptide encoded by polynucleotides of (a) or (b), or fragments thereof; or (d) an antibody to said polypeptides, wherein components (a), (b), (c) or (d) may comprise a substantial component.

25. An antibody or antibody fragment which recognizes and binds to one or more proteins encoded by SEQ ID NOs: 1, 3, 5 and 7 or fragments thereof .

26. An isolated nucleic acid sequence comprising any one of the following: (a) a nucleic acid sequence encoding a polypeptide of SEQ ID NO: 2, 4, 6 or 8; (b) a nucleic acid sequence at least 90% identical to the nucleic acid sequence of (a); or (c) a nucleic acid fragment of (a) or (b) wherein the fragment comprises at least about 20 contiguous nucleotides in length.

27. The nucleic acid sequence according to Claim 26, wherein said nucleic acid is selected from the group consisting of DNA and RNA.

28. The nucleic acid sequence of Claim 26, wherein said nucleic acid comprises an open-reading frame that encodes a polypeptide of SEQ ID NO: 2, 4, 6 or 8 or its complement, or a mutant or variant thereof.

28. A vector comprising the nucleic acid of Claim 26.

29. A cell comprising the vector of Claim 26.

30. A pharmaceutical composition comprising the nucleic acid of Claim 26 and a pharmaceutically acceptable carrier.

31. A process for producing a polypeptide encoded by the nucleic acid of Claim 26, said process comprising: (a) culturing a cell under conditions sufficient to express said polypeptide; and (b) recovering said polypeptide, thereby producing said polypeptide.

32. The process according to Claim 31 , wherein said cell is a prokaryotic or eukaryotic cell.