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  Pharmaceutical Patents  

 

Title:  Antibody to BACE455, an alternative splice variant of the human beta-secretase
United States Patent: 
7,790,864
Issued: 
September 7, 2010

Inventors: 
Desire; Laurent (Paris, FR)
Assignee:
Exonhit Therapeutics SA (Paris, FR)
Appl. No.: 
10/578,493
Filed:
 November 5, 2004
PCT Filed:
 November 05, 2004
PCT No.: 
PCT/IB2004/003897
371(c)(1),(2),(4) Date: 
May 05, 2006
PCT Pub. No.:
 WO2005/045021
PCT Pub. Date:
 May 19, 2005


 

Training Courses -- Pharm/Biotech/etc.


Abstract

The present invention relates generally to the fields of genetics, biochemistry, medicinal chemistry and medicine. The present invention more particularly discloses the identification of a human gene variant involved in neuropathological conditions, and methods for the diagnosis, prevention and treatment of such diseases and related disorders, as well as for the screening of therapeutically active drugs. The present invention relates to catalytically active beta-secretase (Memapsin2, BACE) variants, and nucleic acids encoding them. The invention is useful in the identification of agents that inhibit the activity of a particular BACE isoform and thus agents and therapies affecting the genesis, development or progression of neuropathological conditions, including Alzheimer's disease and dementia.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention is drawn to compositions and methods related to the identification of a new isoform of human beta-secretase (BACE), which is selectively expressed by brain tissue from patients suffering from a neuropathological condition (e.g., the human AD brain), which is catalytically active and which is pharmacologically different from the existing other active native BACE isoform BACE501. This new neuropathological-specific isoform of BACE, termed BACE455, has been discovered to result from the deletion of exon 4 and the development of a novel nucleotide and corresponding amino acid sequence present in the junction of exon3 and exon5.

Based on the published crystal structure of native BACE, it is anticipated that the novel isoform BACE455 of the present invention has a similar 3D structure to BACE, with the two aspartate residues within the active site of the protease still facing each other. However, the active site of BACE455 is more open and accessible and is therefore likely to produce significantly increased levels of A.beta. peptide as compared to the native BACE molecule. In addition, BACE455 lacks an endoproteolytic site that is responsible for BACE inactivation and thus activity regulation in the native molecule. Therefore, compared to BACE and other known BACE isoforms, BACE455 lacks posttranslational regulation, has an altered 3-D structure, and is likely to produce increased levels of pathological A.beta. peptide. BACE455 is significantly different from BACE501 in terms of inhibitory profile. This is shown using a well described inhibitor. Thus, the deletion of exon4 in BACE455 generates a significant pharmacological difference when compared to other known active BACE isoforms.

It is postulated that the observed functional and pharmacological properties of this isoform which distinguish it from native BACE may contribute to and selectively drive pathological conditions in mammals, particularly in human subjects.

Isolated BACE455, as well as distinctive fragments thereof, and corresponding nucleic acids can be used for the diagnosis of neuropathological conditions which are associated with BACE455 and for the screening of drugs, especially inhibitors of BACE455, which are therapeutically active in the treatment of neurological disorders, particularly neuropathological conditions including neurodegenerative disorders and dementia, more preferably disorders now known to be related to A.beta. formation or accumulation such as Alzheimer's disease and related disorders.

In another embodiment of the invention are provided methods for the treatment of neuropathological conditions comprising the administration to affected tissue of an inhibitor of BACE455 transcription, translation, or activity.

By a "neuropathological condition" is meant one or more conditions including, but not limited to, Motor Neuron Disease (ALS), Down's syndrome, Parkinsonian Syndromes, multiple sclerosis, diffuse cerebral cortical atrophy, Lewy-body dementia, Pick disease, mesolimbocortical dementia, thalamic degeneration, bulbar palsy, Huntington chorea, cortical-striatal-spinal degeneration, cortical-basal ganglionic degeneration, cerebrocerebellar degeneration, familial dementia with spastic paraparesis, polyglucosan body disease, Shy-Drager syndrome, olivopontocerebellar atrophy, progressive supranuclear palsy, dystonia musculorum deformans, Hallervorden-Spatz disease, Meige syndrome, familial tremors, Gilles de la Tourette syndrome, acanthocytic chorea, Friedreich ataxia, Holmes familial cortical cerebellar atrophy, AIDS related dementia, Gerstmann-Straussler-Scheinker disease, progressive spinal muscular atrophy, progressive balbar palsy, maculopathies and retinal degeneration, such as Non-Exudative Age Related Macular Degeneration (ARMD), Exudative Age Related Macular Degeneration, primary lateral sclerosis, hereditary muscular atrophy, spastic paraplegia, peroneal muscular atrophy, hypertrophic interstitial polyneuropathy, heredopathia atactica polyneuritiformis, optic neuropathy, diabetic retinopathy, Alzheimer's disease and opthalmoplegia. Examples of ocular conditions include, but are not limited to, glaucoma, including open angle glaucoma, ocular hypertension, maculopathies and retinal degeneration, such as Non-Exudative Age Related Macular Degeneration (ARMD), Exudative Age Related Macular Degeneration (ARMD), Choroidal Neovascularization, Diabetic Retinopathy, Central Serous Chorioretinopathy, Cystoid Macular Edema, Diabetic Macular Edema, Myopic Retinal Degeneration; inflammatory diseases, such as Acute Multifocal Placoid Pigment Epitheliopathy, Behcet's Disease, Birdshot Retinochoroidopathy, Infectious (Syphilis, Lyme, Tuberculosis, Toxoplasmosis), Intermediate Uveitis (Pars Planitis), Multifocal Choroiditis, Multiple Evanescent White Dot Syndrome (MEWDS), Ocular Sarcoidosis, Posterior Scleritis, Serpiginous Choroiditis, Subretinal Fibrosis and Uveitis Syndrome, Vogt-Koyanagi-Harada Syndrome, Punctate Inner Choroidopathy, Acute Posterior Multifocal Placoid Pigment Epitheliopathy, Acute Retinal Pigement Epitheliitis, Acute Macular Neuroretinopathy; vascular and exudative diseases, such as Diabetic retinopathy, Central Retinal Arterial Occlusive Disease, Central Retinal Vein Occlusion, Disseminated Intravascular Coagulopathy, Branch Retinal Vein Occlusion, Hypertensive Fundus Changes, Ocular Ischemic Syndrome, Retinal Arterial Microaneurysms, Coat's Disease, Parafoveal Telangiectasis, Hemi-Retinal Vein Occlusion, Papillophlebitis, Central Retinal Artery Occlusion, Branch Retinal Artery Occlusion, Carotid Artery Disease (CAD), Frosted Branch Angiitis, Sickle Cell Retinopathy and other Hemoglobinopathies, Angioid Streaks, Familial Exudative Vitreoretinopathy; Eales Disease; traumatic, surgical and environmental disorders, such as Sympathetic Ophthalmia, Uveitic Retinal Disease, Retinal Detachment, Trauma, Retinal Laser, Photodynamic therapy, Photocoagulation, Hypoperfusion During Surgery, Radiation Retinopathy, Bone Marrow Transplant Retinopathy; proliferative disorders, such as Proliferative Vitreal Retinopathy and Epiretinal Membranes; infectious disorders, such as Ocular Histoplasmosis, Ocular Toxocariasis, Presumed Ocular Histoplasmosis Syndrome (POHS), Endophthalmitis, Toxoplasmosis, Retinal Diseases Associated with HIV Infection, Choroidal Disease Associate with HIV Infection, Uveitic Disease Associate with HIV Infection, Viral Retinitis, Acute Retinal Necrosis, Progressive Outer Retinal Necrosis, Fungal Retinal Diseases, Ocular Syphilis, Ocular Tuberculosis, Diffuse Unilateral Subacute Neuroretinitis, Myiasis; genetic disorders, such as Retinitis Pigmentosa, Systemic Disorders with Accosiated Retinal Dystrophies, Congenital Stationary Night Blindness, Cone Dystrophies, Stargardt's Disease And Fundus Flavimaculatus, Best's Disease, Pattern Dystrophy of the Retinal Pigmented Epithelium, X-Linked Retinoschisis, Sorsby's Fundus Dystrophy, Benign Concentric Maculopathy, Bietti's Crystalline Dystrophy, pseudoxanthoma elasticum; retinal injuries, such as Macular Hole, Giant Retinal Tear; retinal tumors, such as Retinal Disease Associated With Tumors, Congenital Hypertrophy Of The RPE, Posterior Uveal Melanoma, Choroidal Hemangioma, Choroidal Osteoma, Choroidal Metastasis, Combined Hamartoma of the Retina and Retinal Pigmented Epithelium, Retinoblastoma, Vasoproliferative Tumors of the Ocular Fundus, Retinal Astrocytoma, and Intraocular Lymphoid Tumors.

By "isolated" is meant a molecule existing in an environment other than that normally present in nature without human intervention. Thus, for example, "isolated BACE455" includes naturally-produced BACE455 contained in a cell lysate, purified or partially purified BACE455, recombinant BACE455, as well as BACE455 existing within a heterologous host cell or culture, such as tissue culture cell from any organism (including, without limitation, human, rat, monkey or other mammalian cells, bacterial or fungal cells, or insect cells).

In one embodiment of this invention, methods are described which involve the selective inhibition of BACE455 protein activity. Such methods can be used therapeutically to inhibit the progression of neuropathological conditions such as, without limitation, Alzheimer's disease, other dementia, glaucoma, Parkinson's disease, ALS, and the effects of stroke.

In another embodiment, this invention is drawn to the use of BACE455 mRNA encoding BACE455 or a distinctive fragment thereof, BACE455 protein, or any distinctive fragment of the BACE455 protein, to screen for molecules that inhibit the production of A.beta. peptide.

Preferably a fragment of a protein or polypeptide in accordance with the present invention comprises at least 10 amino acids comprising a contiguous amino acid sequence, more preferably at least 9 amino acids comprising a contiguous amino acid sequence, even more preferably at least 8 amino acids comprising a contiguous amino acid sequence, even more preferably at least 7 amino acids comprising a contiguous amino acid sequence, even more preferably at least 6 amino acids comprising a contiguous amino acid sequence, even more preferably at least 5 amino acids comprising a contiguous amino acid sequence.

Preferably a fragment of a nucleic acid or polynucleotide in accordance with the present invention comprises at least 30 nucleotides comprising a contiguous nucleotide sequence, more preferably at least 27 nucleotides comprising a contiguous nucleotide sequence, even more preferably at least 24 nucleotides comprising a contiguous nucleotide sequence, even more preferably at least 21 nucleotides comprising a contiguous nucleotide sequence, even more preferably at least 18 nucleotides comprising a contiguous nucleotide sequence, even more preferably at least 15 nucleotides comprising a contiguous nucleotide sequence.

By "distinctive" when used to describe a nucleic acid encoding the BACE455 protein or a fragment thereof, is meant that the nucleotide sequence of such nucleic acid comprises at least one codon derived from exon 3 immediately proximal to at least one codon derived from exon 5 of the BACE 455 DNA sequence, encodes a protein or fragment thereof that has the same amino acid sequence encoded by such a nucleic acid or fragment, or is exactly complementary to such nucleotide sequence. In a particular embodiment, a distinctive nucleic acid comprises a nucleotide sequence comprising at least 5 contiguous nucleotides present in the junction of exon3 and exon5 of BACE455.

By "distinctive" when used to describe the BACE455 protein or a fragment thereof, is meant a peptide, polypeptide or protein comprising an amino acid sequence encoded by a distinctive nucleic acid encoding the BACE455 protein or a fragment thereof.

The BACE455 protein, distinctive fragments thereof and nucleic acids encoding these molecules can be used by those skilled in the art to design new BACE455 inhibitors, using commercially available software programs and techniques familiar to those in organic chemistry and enzymology. Such inhibitors are both useful in the diagnosis and treatment and/or prevention of Alzheimer's disease and other neuropathological conditions. Methods for making BACE455 inhibitors may make use of techniques well known in the art, such as, without limitation, combinatorial and other chemical libraries, use of the isolated BACE455 protein or a distinctive fragment thereof in high throughput screening of chemical libraries, rational drug design using medicinal chemistry techniques based on structure-activity relationships, and the like. Polypeptides of the present invention may be employed in conventional low capacity screening methods and also in high-throughput screening (HTS) formats. Such HTS formats include, for example, 96- and, and 384-well microtiter plates (see Bennett, et al., J. Mol. Recognition, 8:52-58 (1995); and Johanson, et al., J. Biol. Chem., 270(16): 9459-9471 (1995)). Exemplary methods that can be used for making BACE inhibitors have been disclosed in WO 9967221, WO 9967220, WO 9967219, WO 9966934, WO 9932453, WO 9838177, WO 9828268, WO 9822494, WO 9822493, WO 9822441, WO 9822433, and WO 9822430. Methods for making combinatorial libraries of compounds are disclosed in references such as Turner et al., Biochemestry 40: 10001-10006 (2001) or Gruninger-Leicht et al., J. Biol. Chem. 277: 4687-4693 (2002).

BACE455 inhibitors may be chosen from small molecule inhibitors, peptides, antisense oligonucleotides, iRNA, and blocking antibodies. Thus, inhibition of BACE455 includes the use of agents which inhibit BACE455 transcription, splicing, translation, and/or activity in vivo.

The present invention is preferably, though not exclusively, drawn to BACE455 inhibitors that alternatively exhibit 2-fold, or 5-fold, or 10-fold, or 30-fold, or 100-fold, or 1000 fold, or >1000 fold selectivity in the inhibition of BACE455 as compared to the other known BACE isoforms. Such inhibitors have an improved utility, compared to compounds that lack such selectivity, or which preferably inhibit native BACE rather than BACE455. Selective BACE455 inhibitors having a 2-fold, 5-fold, 10-fold or greater selectivity at inhibiting BACE455 have a number of benefits including greater efficacy at inhibiting pathological disease progression, decreased side effects due to less inhibition of non-pathological A.beta. production, and improved safety due to the increased selectivity of such compounds.

The BACE455 protein, nucleic acid, distinctive fragments of these molecules, and BACE455 inhibitors can be used for the purpose of diagnosis of neuropathological conditions. Compounds specifically binding to BACE455 polypeptide or nucleic acid, or to a distinctive fragment thereof, can help identify individuals prone to develop AD. Additionally, BACE455 inhibitors can be used with therapeutic effect for treatment and/or prevention of Alzheimer's disease and conditions associated with elevated levels of A.beta. 40 or 42 peptide, and the accumulation of the peptide in amyloid plaques, as well as other neuropathological conditions.

In another aspect of this invention, the novel deletion of exon 4 and the novel junction of exons 3 and 5 may be used to diagnose and/or assess the actual or potential development of a neuropathological condition. For example, through the development of in vitro nucleic acid- and/or antibody-based assays, including quantitative assays, of human tissue or fluids, one can determine whether the patient suffers from, or is inclined to suffer from, neuropathological conditions that involve the production and expression of BACE455. Such assay methods may involve, without limitation, nucleic acid detection by, for example, Northern Blot, oligonucleotide-based junction array; nucleic acid amplification methods such as RT-PCR, quantitative PCR and ligation-PCR and other methods. These methods may include the use of an oligonucleotide probe capable of selectively or specifically detecting the region of the analyte comprising the new splice junction. BACE455 may also be directly detected using specific ligands thereof, for example, an antibody specifically recognizing the region of BACE455 comprising the new splice junction, as one example, the region comprising amino acid residues 190 to 235 of the BACE455 protein.

In yet another embodiment, the present invention includes bacterial, insect and mammalian cells or cell lines and transgenic non-human animals that specifically express the BACE455 isoform or a distinctive fragment thereof, in preference to the native BACE501 or other isoforms. Preferred host cells are mammalian cells. Non limiting examples of suitable mammalian cells include the NIH3T3 line of mouse embryo cultures (Jainchill et al. J. Virol. 4: 549-553 (1969)), Chinese hamster ovary cells (CHO), human embryonic kidney cell line 293 or neuroblastoma cell line SH-SY5Y cell line (Biedler et al. Cancer Res. 38: 3751-3757 (1978)). For example, cell lines specifically expressing the BACE455 isoform can be obtained by (a) transfecting a BACE455 expression vector, such as, for example, a pcDNA3 vector with BACE455 full length cDNA cloned into the expression cassette, into the cell type of interest and (b) selecting transfected cells using antibiotics or selection agents corresponding to the resistance gene encoded by the expression vector. Methods for the generation of clonal cell lines composed by stable transformant are disclosed, for example, in Wigler et al. (Cell 11: 223-232 (1977)); Kriegler (Gene Transfer and Expression. Stockton Press, New York, N.Y. (1990)) or Gramer & Goochee (Biotechnol. Prog. 9(4):366-73 (1993)). Alternatively, transfected cells can be used in transient transfection assays to monitor BACE455 activity without further selection. Transgenic animals may be obtained, as known in the art, by recombination techniques using methods well known in the art of molecular biology which are described in such reference as Sambrook et al. (Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor N.Y. (1988)) and Ausubel (Current Protocols in Molecular Biology (1989)).

Such cell lines represent an original screening tool for compounds preventing A.beta. processing, and non-human transgenic animals would be useful for studying the development of A.beta.-dependent dementia and for identifying compounds that inhibit A.beta. dependent disease progression in vivo.

DETAILED DESCRIPTION OF THE INVENTION

In a first embodiment, the present invention is drawn to a polypeptide comprising all or a distinctive fragment of the BACE455 polypeptide. The BACE455 amino acid sequence is depicted in FIG. 1 (see Original Patent). The term "BACE 455" or "BACE455 polypeptide" means any BACE polypeptide (preferably of human origin), that comprises a deletion of all or part of exon 4 of the gene encoding the wild-type BACE peptide.

Preferred examples of distinctive fragments of the BACE455 polypeptide are those comprising the amino acid sequence IARIIG (SEQ ID NO: 3). Further examples of such fragments are polypeptides comprising the amino acid sequence EIARIIG (SEQ ID NO: 4), typically a sequence selected from EIARIIGG (SEQ ID NO: 5), AEIARIIG (SEQ ID NO: 6), AEIARIIGG (SEQ ID NO: 7), AEIARIIGGI (SEQ ID NO: 8), YAEIARIIG (SEQ ID NO: 9), YAEIARIIGG (SEQ ID NO: 10) and YAEIARIIGGI (SEQ ID NO: 11). Most preferred fragments are at least 6, 7, 8, 9 or 10 amino acids long.

A polypeptide of the present invention comprising a distinctive BACE455 polypeptide fragment may comprise the entire amino acid sequence of BACE455 or a variant thereof. The term "variant" in this context designates any polypeptide comprising a distinctive fragment of the BACE455 polypeptide and further comprising a modified amino acid sequence as compared to the sequence depicted in FIG. 1 as a result of one or several amino acid mutation(s), substitution(s) and/or insertion(s). Typically, such BACE455 variants lack all of exon 4. Preferred variants are naturally-occurring variants, i.e., BACE polypeptides resulting from polymorphism, splicing, etc., which lack exon 4. Most preferred polypeptides of this invention are of human origin and/or retain one property of BACE455 of FIG. 1, in particular at least one BACE455-selective immunological property and/or a beta-secretase activity.

Such polypeptides may optionally comprise additional residues or functions, such as, without limitation, additional amino acid residues, chemical or biological groups, including labels, tags, stabilizer, targeting moieties, purification tags, secretory peptides, functionalizing reactive groups, etc. Such additional residues or functions may be chemically derivatized, added as an amino acid sequence region of a fusion protein, complexed with or otherwise either covalently or non-covalently attached. They may also contain natural or non-natural amino acids. The polypeptide may be in soluble form, or attached (or complexed with or embedded in) to a support, such as a matrix, a column, a bead, a membrane, a cell, a lipid or liposome, etc.

Certain polypeptides of the present invention may be used as such to cause production of A.beta. peptide in vitro or in vivo. They may also be used to design specific reagents such as peptides, antibodies (and derivatives thereof), antagonists, agonists, etc. that specifically detect, bind or affect expression or activity of a BACE455 polypeptide as defined above. The polypeptides may also be used as immunogens in vaccine compositions or to produce or detect or dose specific antibodies.

In yet another embodiment, the present invention comprises a polynucleotide comprising a nucleotide sequence encoding a BACE455 polypeptide as defined above, including a distinctive fragment thereof. The polynucleotide preferably encodes a polypeptide that comprises a fragment of mammalian memapsin 2 (BACE) protein, wherein said polypeptide lacks all of exon4 (encoded by amino acids 190 to 235 of full-length BACE protein). A typical example of such a polypeptide comprises all or a distinctive fragment of the nucleotide sequence set forth in FIG. 1 (SEQ ID NO: 2).

A particular embodiment of this invention also includes any polynucleotide comprising a nucleotide sequence that selectively hybridizes to a distinctive fragment of BACE 455 RNA (or its exact complement) under stringent conditions. More preferably, such selectively hybridizing polynucleotide encodes a polypeptide having beta-secretase activity. By stringent conditions is intended, for example, incubations of hybridization filters at about 42.degree. C. for about 2.5 hours in 2.times.SSC/0.1% SDS, followed by washing of the filters four times of 15 minutes in 1.times.SSC/0.1% SDS at 65.degree. C. Protocols used are described in such reference as Sambrook et al. (Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor N.Y. (1988)) and Ausubel (Current Protocols in Molecular Biology (1989)).

In a particularly preferred embodiment, the encoded BACE polypeptide comprises a distinctive fragment of a human BACE protein.

The nucleic acids, oligonucleotides and polynucleotides of the present invention may be DNA or RNA, such as genomic DNA, complementary DNA, synthetic DNA, mRNA, or analogs of these containing, for example, modified nucleotides such as 3' alkoxyribonucleotides, methylphosphanates, and the like, and peptide nucleic acids (PNAs), etc. The polynucleotide may be produced according to techniques well-known per se in the art, such as by chemical synthetic methods, in vitro transcription, or through recombinant DNA methodologies, using sequence information contained in the present application. In particular, the polynucleotide may be produced by chemical oligonucleotide synthesis, library screening, amplification, ligation, recombinant techniques, and combination(s) thereof.

A specific embodiment of this invention resides in a polynucleotide encoding a polypeptide comprising a distinctive fragment of BACE having the amino acid sequence set forth as SEQ ID NO 2.

Polynucleotides of this invention may comprise additional nucleotide sequences, such as regulatory regions, i.e., promoters, enhancers, silencers, terminators, and the like that can be used to cause or regulate expression of a BACE455 polypeptide.

Polynucleotides of this invention may be used to produce a recombinant polypeptide of this invention. They may also be used to design specific reagents such as primers, probes or antisense molecules (including antisense RNA, iRNA, aptamers, ribozymes, etc.), that specifically detect, bind or affect expression of a polynucleotide encoding a BACE455 polypeptide as defined above. They may also be used as therapeutic molecules (e.g., as part of an engineered virus, such as, without limitation, an engineered adenovirus or adeno-associated virus vector in gene therapy programs) or to generate recombinant cells or genetically modified non-human animals, which are useful, for instance, in screening compound libraries for agents that modulate the activity of BACE455.

A further aspect of this invention resides in a vector, such as an expression or reporter vector comprising a BACE455 polynucleotide as defined above. Such vectors may be selected from plasmids, recombinant viruses, phages, episomes, artificial chromosomes, and the like. Many such vectors are commercially available and may be produced according to recombinant techniques well known in the art, such as the methods set forth in manuals such as Sambrook et al., Molecular Cloning (2d ed. Cold Spring Harbor Press 1989), which is hereby incorporated by reference herein in its entirety.

A further aspect of this invention resides in a host cell transformed or transfected with a polynucleotide or a vector as defined above. The host cell may be any cell that can be genetically modified and, preferably, cultivated. The cell can be eukaryotic or prokaryotic, such as a mammalian cell, an insect cell, a plant cell, a yeast, a fungus, a bacterial cell, etc. Typical examples include mammalian primary or established cells (3T3, CHO, Vero, Hela, etc.), as well as yeast cells (e.g., Saccharomyces species, Kluyveromyces, etc.) and bacteria (e.g., E. Coli). It should be understood that the invention is not limited with respect to any particular cell type, and can be applied to all kinds of cells, following common general knowledge.

Nucleic Acid Probes

A specific type of polynucleotide of this invention is a nucleic acid probe that selectively hybridizes under stringent hybridization condition to a nucleic acid or fragment thereof encoding a distinctive fragment of the BACE455 polypeptide . . . . As is well-known in the art, "stringent hybridization conditions" depend upon the length of the probe and the ratio of guanine-cytosine pairs to thymine (uracil)-adenine pairs in the resulting hybrid.

Within the context of this invention, a "probe" refers to a nucleic acid or oligonucleotide having a polynucleotide sequence which is capable of selective hybridization with a distinctive fragment of BACE455 RNA (or the nucleotide sequence exactly complementary thereto), and which is suitable for detecting the presence of a BACE455 RNA (or nucleic acid having its exact complementary nucleic acid sequence) in any sample containing said RNA or complement. Probes are preferably perfectly complementary to a distinctive fragment of the BACE455 RNA. Probes typically comprise single-stranded nucleic acids of between 8 to 1400 nucleotides in length, for instance of between 10 and 1000, more preferably of between 15 and 800, typically of between 20 and 600. It should be understood that longer probes may be used as well. A preferred probe of this invention is a single stranded nucleic acid molecule of between 8 to 600 nucleotides in length, which can specifically hybridize to a distinctive fragment of BACE455 RNA.

A specific embodiment of this invention is a nucleic acid probe selective for an isoform of BACE which lacks exon 4, a nucleic acid probe that selective hybridizes to said isoform gene or RNA and does not substantially hybridize to at least one other BACE isoform containing a full length exon 4, and nucleic acid probes exactly complementary to these.

A further specific embodiment of this invention is a nucleic acid probe selective for BACE455 RNA, i.e., a nucleic acid probe that selectively hybridizes to said BACE455 gene or RNA and does not substantially hybridize to at least one other BACE isoform, and nucleic acids exactly complementary to these.

Selectivity, when used to denote nucleic acid hybridization, indicates that a probe is able to hybridization to the target sequence without substantially hybridizing to at least one other BACE-encoding nucleic acid.

Preferred probes of this invention comprise a sequence which is complementary to a distinctive fragment of the BACE455 RNA (or its exact complement). Specific examples of such probes comprise the nucleic acid sequence -- see Original Patent.

The sequence of the probes can be derived from the sequences of the BACE455 RNA as provided in the present application. Nucleotide substitutions may be performed, as well as chemical modifications of the probe. Such chemical modifications may be accomplished to increase the stability of hybrids (e.g., intercalating groups or modified nucleotides, such as 2' alkoxyribonucleotides) or to label the probe, as disclosed above. Typical examples of labels include, without limitation, radioactivity, fluorescence, luminescence, enzymatic labelling, and the like. The probe may be hybridized to the target nucleic acid in solution, suspension, or attached to a solid support, such as, without limitation, a bead, column, plate, substrate (to produce nucleic acid arrays or chips).

In one example, a BACE455-selective oligonucleotide probe of 15 by exactly complementary to a distinctive fragment of the BACE455 RNA is labeled with a chemiluminescent compound such as an N-hydroxysuccinimide (NHS) ester of acridinium (e.g., 4-(2-succinimidyloxycarbonyl ethyl) phenyl-10-methylacridinium 9-carboxylate fluorosulfonate) generally as described in Weeks et al., Clin. Chem. 29: 1474-1478 (1983), and Nelson et al., U.S. Pat. No. 5,658,737, both of which are hereby incorporated by reference herein. Reaction of the primary amine of the linker arm:hybridization probe conjugate with the selected NHS-acridinium ester is performed as follows. The oligonucleotide hybridization probe:linker arm conjugate synthesized as described above is vacuum-dried in a Savant SPEED-VAC.RTM. drying apparatus, then dissolved in 8 .mu.l of 0.125 M HEPES buffer (pH 8.0) in 50% (v/v) DMSO. To this solution is added 2 .mu.l of 25 mM of the desired NHS-acridinium ester. The solution is mixed and incubated at 37.degree. C. for 20 minutes.

An additional 3 .mu.l of 25 mM NHS-acridinium ester in DMSO is added to the solution and mixed gently, then 2 .mu.l of 0.1 M HEPES buffer (pH 8.0) is added, mixed, and the tube is allowed to incubate for an additional 20 minutes at 37.degree. C. The reaction is quenched with the addition of 5 .mu.l 0.125 M lysine in 0.1 M HEPES buffer (pH 8.0) in DMSO, which is mixed gently into the solution.

The labeled oligonucleotide is recovered from solution by the addition of 30 .mu.l 3 M sodium acetate buffer (pH 5.0), 245 .mu.l water, and 5 .mu.l of 40 mg/ml glycogen. Six hundred forty microliters of chilled 100% ethanol is added to the tube, and the tube is held on dry ice for 5 to 10 minutes. The precipitated labeled probe is sedimented in a refrigerated microcentrifuge at 15,000 rpm using a standard rotor head. The supernatant is aspirated off, and the pellet is redissolved in 20 .mu.l 0.1 M sodium acetate (pH 5.0) containing 0.1% (w/v) sodium dodecyl sulfate (SDS).

Eleven fmoles of the labeled probe is hybridized to various amounts (0.00, 0.01, 0.02, 0.05, 0.20, 0.50, 2, 5, 20, 50, 200, 500, 2000, and 5000 fmoles) of the target BACE455 RNA. Each set consisted of 100 .mu.l hybridization reactions containing 100 mM lithium succinate (pH 5.0), 8.5% (w/v) lithium lauryl sulfate, 1.5 mM EDTA, and 1.5 mM EGTA and each reaction mixture was incubated at 50.degree. C. for 50 minutes. Three hundred microliters of a solution containing 150 mM Na.sub.2B.sub.4O.sub.7 (pH 8.6) and 1% (v/v) TRITON.RTM.. X-100 were added to each reaction, and the mixtures incubated at 50.degree. C. for 11 minutes. The reaction mixtures were then placed into a LEADER.RTM. 50 luminometer (Gen-Probe, Inc.), and a chemiluminescent reaction initiated in each mixture upon the injection of 200 .mu.l 0.1% (v/v) H.sub.2O.sub.2 and 1 mM HNO.sub.3, followed by 200 .mu.l of 1.5 N NaOH. Chemiluminescence was read at a wavelength range from 300 to 650 nm for 2 seconds following the second injection and compared to a negative and positive control standard. Significant chemiluminescence above the negative control indicates the presence of a BACE455-selective hybrid.

Nucleic Acid Primers

Other selective polynucleotides of this invention are nucleic acid primers for amplifying a region comprising a distinctive fragment of a BACE455 RNA or its exact complement. Such primers are designed to amplify BACE455-selective nucleic acid fragments.

Particular primers of this invention are able to selectively hybridise with a portion of a BACE455 RNA or its exact complement that flanks an isoform-specific nucleic acid sequence region, more preferably a region of BACE455 RNA resulting from the newly created junction between exon 3 and exon 5., or its exact complement. The term "flanks" indicates that the portion should be located at a distance of the target region that is compatible with conventional polymerase activities, e.g., not above 300 by from the newly created junction, preferably not exceeding 200, 150, 100 or, further preferably, 50 by upstream from said junction. Examples of such primers comprise or are complementary to a portion of the sequences flanking nucleotide region 567-704 in SEQ ID NO: 1. Specific examples of such primers comprise the following sequences: AGGCATCCTG (SEQ ID NO: 13), GGGCTGGCCT (SEQ ID NO: 14), ATGCTGAGAT (SEQ ID NO: 15), TGCCAG (SEQ ID NO: 16), GATCAT (SEQ ID NO: 17), TGGAGGTATC (SEQ ID NO: 18), GACCACTCGC (SEQ ID NO: 19), TGTACACAGG (SEQ ID NO: 20) or CAGTCTCTGG (SEQ ID NO: 21).

Other particular primers of this invention are able to selectively hybridise with an isoform-specific region comprising a distinctive fragment of BACE455 RNA or its exact complement, more specifically a region of BACE455 resulting from a newly created junction between exon 3 and exon 5. Such primers are advantageous in that amplification occurs only when the template comprises the isoform-specific alteration. By using such primers, the detection of an amplification product indicates the presence of BACE455 RNA. In contrast, the absence of amplification product indicates that the specific alteration is not present in the sample. Examples of such primers comprise all or a portion of the following sequences -- see Original Patent.

A further aspect of this invention also includes at least one pair of nucleic acid primers, wherein said pair of primers comprise a sense and a reverse primer, and wherein said sense and reverse primer allows selective amplification of the BACE455 RNA or an isoform-specific portion thereof, or the exactly complementary sequence. A further embodiment of this invention is a pair of nucleic acid primers, wherein said pair comprises a sense and a reverse primer, and wherein said sense and reverse primers allow selective amplification of all or an isoform-specific portion of a BACE RNA isoform lacking all or part of exon4.

Typical primers of this invention are single-stranded nucleic acid molecules of about 5 to 60 nucleotides in length, more preferably of about 8 to about 35 nucleotides in length, further preferably of about 10 to 25 nucleotides in length. Their sequences can be derived or inferred directly from the nucleotide sequence of BACE455 as disclosed in this application. Perfect complementarity is preferred, to ensure high specificity. However, certain mismatch may be tolerated.

Inhibitory Nucleic Acids

The invention also relates to nucleic acid molecules that can specifically alter expression or activity of a BACE455 polypeptide or polypeptide comprising a fragment thereof (or of any BACE polypeptide lacking a functional exon4). Such inhibitory nucleic acids include antisense nucleic acids, iRNA, ribozymes, aptamers, and the like. These inhibitory nucleic acids comprise a sequence that is complementary to a portion of the target isoform gene or RNA, and cause a specific reduction in transcription or translation thereof. Absolute complementarity, although preferred, is not required.

Techniques for the production and use of such molecules are well known to those of skill in the art, and are succinctly described below. Oligonucleotides may be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Applied Biosystems, Inc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Matsukura et al. (Gene. 1988 72:343-7), etc.

Antisense deoxynucleotides have been widely used to study the effect of a given gene (for review, see Stein & Cheng, Science 261: 1004-12 (1993), and can be used on exon-exon junctions and target specific mRNA isoform (Sugi et al. Dev. Biol. 157: 28-37 (1993); Mahon et al. Exp Hematol. 23:1606-11 (1995); Desire et al. J. Neurochem. 75:151-163 (2000)).

Antisense oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, (selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof) to improve stability of the molecule, hybridization, etc. The oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane or the blood-brain barrier (Letsinger (1989) Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556), hybridization-triggered cleavage agents (Krol (1988) Bio. Techniques 6:958-976) or intercalating agents (Zon (1988) Ann. N.Y. Acad. Sci. 616:161-72 (1990)).

The antisense oligonucleotide can be an alpha.-anomeric oligonucleotide which forms specific double-stranded hybrids with complementary RNA in parallel strands (Gautier et al. (1987) Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a 2'-O-methylribonucleotide (Mayeda et al. J. Biochem. 108: 399-405 (1990)), or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).

Gene silencing can also be achieved using small interfering RNAs in mammalian cells (Elbashir et al. Nature 411: 494-498; Brummelkamp et al. Science 296: 550-553 (2003) and has successfully been used in the alternative splicing context to study the functional relevance of specific isoforms (Celotto & Graveley, RNA 8: 718-724 (2002)).

Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA can also be used to prevent translation of target gene mRNA and, therefore, expression of target gene product (Sarver et al. (1990) Science 247:1222-1225; Rossi (1994) Current Biology 4:469-471). The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by an endonucleolytic cleavage event. Hammerhead ribozymes are modified ribozymes which cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The construction and production of hammerhead ribozymes is well known in the art and is described more fully in Haseloff and Gerlach, 1988 (Nature) 334:585-591.

These inhibitory nucleic acids can be designed based on the sequences disclosed in the present application.

Specific Ligands

The invention also relates to ligands that selectively bind a BACE455 isoform or a distinctive fragment thereof, as disclosed above.

Different types of ligands may be contemplated, such as specific antibodies, synthetic molecules, aptamers, peptides, and the like.

In a specific embodiment, the ligand is an antibody, or a fragment or derivative thereof. Accordingly, a particular aspect of this invention resides in an antibody that specifically binds a BACE455-specific epitope, more preferably an epitope generated by the deletion of all or part of exon4 (encoded by amino acids 190 to 235 of SEQ ID NO 9).

Within the context of this invention, an antibody designates a polyclonal antibody, a monoclonal antibody, as well as fragments or derivatives thereof having substantially the same antigen specificity. Fragments include Fab, Fab'2, CDR regions, etc. Derivatives include single-chain antibodies, humanized antibodies, human antibodies, poly-functional antibodies, etc.

Antibodies against human BACE455 protein may be produced by procedures generally known in the art. For example, polyclonal antibodies may be produced by injecting the protein alone or coupled to a suitable protein into a non-human animal. After an appropriate period, the animal is bled, sera recovered and purified by techniques known in the art (see Paul, W. E. "Fundamental Immunology" Second Ed. Raven Press, NY, p. 176, 1989; Harlow et al "Antibodies: A laboratory Manual", CSH Press, 1988; Ward et al (Nature 341 (1989) 544). Monoclonal antibodies may be prepared, for example, by the Kohler-Millstein (2) technique (Kohler-Millstein, Galfre, G., and Milstein, C, Methods Enz. 73 p. 1 (1981)) involving fusion of an immune B-lymphocyte to myeloma cells. For example, antigen as described above can be injected into mice as described above until a polyclonal antibody response is detected in the mouse's serum. The mouse can be boosted again, its spleen removed and fusion with myeloma conducted according to a variety of methods. The individual surviving hybridoma cells are tested for the secretion of anti-BACE antibodies first by their ability to bind the immunizing antigen and then by their ability to immunoprecipitate BACE from cells.

An antibody "selective for a BACE455 polypeptide" designates an antibody that selectively binds a BACE455 polypeptide to a greater extent than other BACE isoforms, i.e., an antibody raised against a BACE455 polypeptide or an epitope-containing fragment thereof. Although non-specific binding towards other antigens may occur, binding to the target BACE455 polypeptide occurs with a higher affinity and can be reliably discriminated from non-specific binding. Preferred antibodies are selective for a BACE455 specific domain comprising the newly created junction region between exon 3 and exon 5. Antibodies selective for said domain allow the detection of the presence of BACE455 polypeptides in a sample. The ligand may be used in soluble form, or coated on a surface or support.

Specific examples of synthetic inhibitors include Gleevec and Brefeldin A. A specific example of a peptide inhibitor is BACE inhibitor III (SEQ ID NO: 3).

Detection and Diagnostics

The present invention allows the performance of detection or diagnostic assays that can be used, among other things, to detect the presence, absence, or amount of BACE455 or a corresponding nucleic acid in a sample or subject. The term "diagnostics" shall be construed as including methods of detecting the BACE455 isoform, corresponding nucleic acids, and fragments of these in mammalian (preferably human) samples, diagnostics (either qualitative and quantitative), pharmacogenomics, prognostic, and so forth.

In a particular aspect, the invention relates to a method of detecting a BACE455 nucleic acid or polypeptide, or fragments of these in a sample, preferably, a human tissue sample, comprising contacting said sample with a specific ligand thereof and determining the formation of a complex.

A particular object of this invention resides in a method of detecting the presence of or predisposition to a neurodegenerative disease or an associated disorder in a subject, the method comprising detecting the presence of a distinctive BACE455 nucleic acid or polypeptide in a sample from the subject, particularly a BACE isoform lacking all of exon4, even more preferably a BACE isoform having the polynucleotide or amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 2, respectively.

Another embodiment of this invention is directed to a method of assessing the response of a subject to a treatment of a neurodegenerative disease or an associated disorder, the method comprising detecting the presence of a distinctive BACE455 nucleic acid or polypeptide in a sample from the subject at different times before and during the course of treatment.

This invention also relates to a method of determining the efficacy of a treatment of a neurodegenerative disease or an associated disorder, the method comprising (i) providing a tissue sample from the subject during or after said treatment, (ii) determining the presence and/or abundance of a BACE455 nucleic acid or polypeptide, or distinctive fragment thereof, in said sample and (iii) comparing said presence and/or abundance to the amount of such nucleic acid, polypeptide, or fragment in a reference sample from said subject taken prior to or at an earlier stage of the treatment.

The presence (or increase) in a distinctive BACE455 polypeptide or nucleic acid in a sample is indicative of the presence, predisposition or stage of progression of a neurodegenerative disease or related disorders. Therefore, the invention allows the design of appropriate therapeutic intervention, which is more effective and customized. Also, this determination at the pre-symptomatic level allows a preventive regimen to be applied.

Determination of the presence, absence, or relative abundance of a distinctive BACE455 polypeptide or nucleic acid in a sample can be performed by a variety of techniques. More preferably, the determination comprises contacting the sample with BACE 455-selective reagents such as probes, primers or ligands, as defined above, and thereby detecting the presence, or measuring the amount, of BACE455 polypeptide or nucleic acids originally in the sample. Contacting may be performed in any suitable device, such as a plate, microtiter dish, test tube, well, glass, column, and so forth In specific embodiments, the contacting is performed on a substrate coated with the reagent, such as a nucleic acid array or a specific ligand array. The substrate may be a solid or semi-solid substrate such as any suitable support comprising glass, plastic, nylon, paper, metal, polymers and the like. The substrate may be of various forms and sizes, such as a slide, a membrane, a bead, a column, a gel, etc. The contacting may be made under any condition suitable for a detectable complex, such as a nucleic acid hybrid or an antibody-antigen complex, to be formed between the reagent and the nucleic acids or polypeptides of the sample.

In a specific embodiment, the method comprises contacting a sample from the subject with (a support coated with) a BACE 455 selective antibody, as described above, and determining the presence of an immune complex. Various well-known methods for detecting an immune complex can be used, such as ELISA, radio-immunoassays (RIA), and so forth.

In another specific embodiment, the method comprises contacting a sample from the subject with (a support coated with) a BACE 455-selective nucleic acid probe, as described above, under appropriate conditions allowing hybridization to occur, and determining the presence of a hybrid.

In another specific embodiment, the method comprises contacting a sample from the subject with a nucleic acid primer as defined above, under conditions allowing nucleic acid amplification to occur, and determining the presence of an amplification product ("amplicon"). Amplification may be performed according to various techniques known per se in the art, such as, without limitation, by polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA).

Suitable methods to detect nucleic acids in a sample include, without limitation, the following methods: allele-specific oligonucleotide (ASO), allele-specific amplification, Southern blot (for DNAs), Northern blot (for RNAs), single-stranded conformation analysis (SSCA), fluorescent in situ hybridization (FISH), gel migration, clamped denaturing gel electrophoresis, heteroduplex analysis, etc.

The diagnostic methods of the present invention can be performed in vitro, ex vivo or in vivo, preferably in vitro or ex vivo. The sample may be any biological sample derived from a subject, which contains nucleic acids or polypeptides, as appropriate. Examples of such samples include fluids, tissues, cell samples, organs, biopsies, etc. Most preferred samples are blood, plasma, saliva, urine, seminal fluid, and the like. Prenatal diagnosis may also be performed by testing fetal cells or placental cells, for instance, the sample may be collected according to conventional techniques and used directly for diagnosis or stored. The sample may be treated prior to performing the method, in order to render or improve availability of nucleic acids or polypeptides for testing. Treatments may include, for instance one or more of the following: cell lysis (e.g., mechanical, physical, chemical, etc.), centrifugation, extraction, column chromatography, and the like.

Therapeutics

In addition to cleaving APP-based substrates, recombinant human BACE also cleaves a substrate with the sequence LVNM/AEGD (SEQ ID NO: 35) (Lin et al. Proc Natl Acad Sci USA. 97(4):1456-1460 (2000)), a sequence which is the in vivo processing site sequence of human presenilins. Presenilin 1 and presenilin 2 are unstable proteins which are processed and subsequently stabilized by an unknown protease (Capell et al., J. Biol. Chem. 273, 3205 (1998); Thinakaran et at, Neurobiol. Dis. 4, 438 (1998)). It is known that presenilins control the formation of A-.beta. peptide by cleavage of APP at the gamma-secretase site, but also the activity of BACE. Presenilins therefore enhance the progression of Alzheimer's disease. Thus, the processing of presenilins by BACE would enhance the production of A-.beta. peptide and therefore, further stimulate the progress of Alzheimer's disease. Therefore, a BACE inhibitor would decrease the likelihood of developing or slow the progression of Alzheimer's disease by inhibiting APP cleavage at the beta-secretase site and/(or) by preventing the processing of presenilins, thus indirectly inhibiting APP cleavage at the gamma-secretase site.

A further object of this invention is a pharmaceutical composition comprising a BACE455 inhibitor, preferably a BACE455-selective inhibitor, and a pharmaceutically acceptable carrier or vehicle. In a specific embodiment, the invention relates to a pharmaceutical composition comprising (i) a specific ligand of BACE455 or a BACE455 inhibitory nucleic acid molecule as described above and (ii) a pharmaceutically acceptable carrier or vehicle.

The invention also relates to a method of treating or preventing neurodegenerative diseases or an associated disorder in a subject, the method comprising administering to said subject an effective amount of a BACE455-selective inhibitor.

Another embodiment of this invention resides in a method of treating or preventing production or accumulation of A.beta. peptide in a subject, the method comprising administering to said subject an effective amount of a BACE455-selective inhibitor.

The invention also relates, generally, to the use of a BACE455-selective inhibitor in the manufacture of a pharmaceutical composition for treating or preventing neurodegenerative diseases or an associated disorder in a subject.

As throughout this specification, most preferably the subject is a human subject.

The BACE455-selective inhibitor may be any agent, condition or treatment that reduces the expression or activity of a BACE455 polypeptide or a distinctive fragment thereof; or the expression, transcription or translation of a BACE455 nucleic acid or a distinctive fragment thereof in a subject. Most preferably, the BACE455-selective inhibitor is specific, i.e., preferentially alters the expression or activity of a BACE455 isoform and essentially does not directly alter expression of other BACE splicing isoforms. The inhibitors may, however, also affect wild-type BACE expression or activity to a greater or lesser extent.

BACE 455-selective inhibitors that exhibit 2-fold, or 5-fold, or 10-fold, or 30-fold, or 100-fold, and/or >1000 fold selectivity for inhibiting BACE455 activity vs. that of at least one other BACE isoform have an improved utility, compared to compounds that lack such selectivity or that significantly reduce the activity of wild-type BACE. Inhibitors that inhibit BACE455 activity and have a 2-fold or greater selectivity at inhibiting BACE455 as compared to wild type-BACE have a number of non-obvious benefits including (but not limited to) greater efficacy at inhibiting pathological disease progression, decreased side effects due to less inhibition of non-pathological A.beta. production, and improved safety due to increased selectivity of compounds. Inhibitors can be used for the purpose of prophylactic or curative treatment of conditions including, for example, Alzheimer's disease and other conditions associated with elevated levels of A.beta. 40 or 42 peptide, and the accumulation of the peptide in amyloid plaques. BACE455 inhibitors may be selected from peptides, proteins, nucleic acids, small drugs and the like. Typical examples include inhibitory nucleic acids and antibodies as disclosed above, as well as small drugs. Such drugs can be characterized or validated using screening approaches as disclosed below. They may be obtained from existing libraries of molecules or they can be designed using commercially available software programs and techniques familiar to those skilled in the art in organic chemistry and enzymology. Methods for making inhibitors may include (but may not limited to), combinatorial chemistry, screening of molecules libraries, rational drug design--these screening methods may employ BACE455 polypeptide or nucleic acids, or fragments of these, as elements of an assay for the selection of appropriate therapeutic candidates. Methods of inhibiting BACE455 may include, but not be limited to, the use of small molecule inhibitors, peptides, antisense oligonucleotides, iRNA, ribozymes, and blocking antibodies, i.e. agents that decrease BACE455 protein levels, activity or prevent cleavage of naturally occurring substrate in the brain.

In a particular embodiment, the BACE455 inhibitor is an antibody that binds BACE455. Antibodies can be produced by a variety of techniques that are known per se in the art. In particular, they may be produced by a method comprising immunizing a non-human animal with a BACE455 polypeptide or a distinctive fragment thereof, and collecting antibodies or antibody-producing cells from said animal. Antibodies may be monoclonals, polyclonals, as well as fragments or derivatives thereof having substantially the same antigen specificity (i.e., the ability to bind BACE455). Antibody fragments include Fab, Fab'2, CDR, etc. Antibody derivatives include single chain antibodies (ScFv), humanized antibodies, human antibodies, recombinant antibodies, bi-specific antibodies, etc. Techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Ward et al., Nature 334:544-54 (1989)) can be adapted to produce 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. Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skerra et al., Science 242:1038-1041 (1988)). In addition, techniques developed for the production of "chimeric antibodies" (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984); Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature 314:452-454 (1985)) by splicing 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 region derived from a murine mAb and a human immunoglobulin constant region, e.g., humanized antibodies.

Human antibodies are desirable for therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art including phage display methods (Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280 (1994); PCT publications WO 90/02809; WO 91/10737) using antibody libraries derived from human immunoglobulin sequences. Methods of producing such antibodies are well know in the literature, as illustrated in the following, non limiting references: Harlow et al (Antibodies: A laboratory Manual, CSH Press, 1988; Ward et al (Nature 341 (1989) 544)). Most preferred antibodies selectively bind BACE455, e.g., bind an epitope that is distinctive of BACE455 as compared to other BACE isoforms.

In an other particular embodiment, the BACE455 inhibitor is an inhibitory nucleic acid that binds BACE455 gene or RNA, preferably BACE455 RNA, and inhibits or reduces the transcription or translation thereof. Such inhibitory nucleic acids can be produced as disclosed above. They preferably comprise a sequence that hybridizes to a distinctive fragment of a BACE455 RNA molecule, as disclosed above.

In an other embodiment, the BACE455 inhibitor is a selective inhibitor such as BACE inhibitor III (SEQ ID NO: 3).

BACE455 Inhibitors may be formulated in any suitable diluent, excipient, carrier or vehicle, that is compatible for pharmaceutical use. In this regard, the invention also contemplates a method of making a composition comprising a BACE455 inhibitor, the method comprising: i) selecting a compound that inhibits BACE455, ii) producing said compound, and iii) mixing said compound with a pharmaceutically acceptable salt thereof.

Compound that inhibits BACE of the present invention can be administered orally using any pharmaceutically acceptable dosage form known in the art for such administration. The vehicle may be any solution, suspension, powder, gel, etc., including isotonic solution, buffered and saline solutions, such as syrups or aqueous suspensions, etc. The compounds may be administered by any suitable route, including systemic delivery, intra-venous, intra-arterial, intra-cerebral or intrathecal injections. Repeated injections may be performed, if desired. The dosage can vary within wide limits and will have to be adjusted to the individual requirements in each particular case, depending upon several factors known to those of ordinary skill in the art. Agents determining the dosage of dosage the active compounds can be the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the age, health and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment; the frequency of treatment; and the effect desired. A daily dosage of active ingredient can be expected to be about 0.001 to about 1000 milligrams per kilogram of body weight, with the preferred dose being about 0.1 to about 30 mg/kg. The daily oral dosage can vary from about 0.01 mg to 1000 mg, 0.1 mg to 100 mg, or 10 mg to 500 mg per day of a compound. The daily dose may be administered as single dose or in divided doses and, in addition, the upper limit can also be exceeded when this is found to be indicated.

The compounds of the present invention can be administered in such oral dosage forms as tablets, capsules (each of which can include sustained release or timed release formulations), pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. Likewise, they may also be administered in intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular form, all using dosage forms well known to those of ordinary skill in the pharmaceutical arts. An effective but non-toxic amount of the compound desired can be employed to prevent or treat neurological disorders related to .beta.-amyloid production or accumulation, such as Alzheimer's disease and Down's Syndrome.

The compounds can be administered alone, but is generally administered with a pharmaceutical carrier, with respect to standard pharmaceutical practice (such as described in Remington's Pharmaceutical Sciences, Mack Publishing).

Compound that inhibits BACE455 can be administered by any means that produces contact of the active agent with the agent's site of action in the body of a host, such as a human or a mammal. They can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic agents or in a combination of therapeutic agents, either administered alone, or administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.

The compound that inhibits BACE455 for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches wall known to those of ordinary skill in that art.

Oral administration in the form of a tablet or capsule containing the active compound can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, starch, sucrose, glucose, methyl callulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like; for oral administration in liquid form, the oral drug components can be combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents, and coloring agents can also be incorporated into the mixture. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as butylated hydroxyanisol or alpha-tocopherol. Suitable binders include starch, gelatin, natural sugars such as glucose or .beta.-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

Compound that inhibits BACE455 can also be administered in the form of liposomal particulate delivery systems, such as small unilamellar vesicles, large unilamallar vesicles, and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines. Alternatively, compounds of the present invention may also be coupled with soluble polymers as targetable drug carriers, such as polymers made of polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamide--phenol, polyhydroxyethylaspartamide--phenol, or polyethyleneoxide--polylysine substituted with palmitoyl residues. Polymers may also belong to the class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polycyanoacylates, etc. . . . or block copolymers of hydrogels.

Compounds for the present invention may be formulated into gelatin capsules with the addition of lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like as powdered carriers. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance. In general, water, a suitable oil, saline, aqueous dextrose (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions. Solutions for parenteral administration preferably contain a water soluble salt of the active ingredient, suitable stabilizing agents, and if necessary, buffer substances. Antioxidizing agents such as sodium bisulfate, sodium sulfite, or ascorbic acid, either alone or combined, are suitable stabilizing agents. Also used are citric acid and its salts and sodium EDTA. In addition, parenteral solutions can contain preservatives, such as benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. The therapeutic compositions and methods of this invention can be used to inhibit the progression of pathological diseases such as (but not limited to), Alzheimer's, dementia, glaucoma, Parkinson's, ALS, and stroke. Within the context of this invention, the term "treatment" designates preventive or curative treatments of neurological disorders, including at early or late stages of progression. Treatment includes delaying disease progression, reducing A.beta. peptide production or accumulation, ameliorating the patients' condition, etc. Treatment can be used alone or in combination with other active agents.

Drug Screening

In another embodiment, the present invention also provides novel targets and methods for the screening of drug candidates or leads. The methods include binding assays and/or functional (activity) assays, and may be performed in vitro, in cell systems, in animals, etc.

A particular object of this invention resides in a method of selecting, characterizing, screening or optimizing a biologically active compound, said method comprising contacting in vitro a test compound with a distinctive BACE455 nucleic acid, polypeptide, or fragment of one of these, and determining the ability of said test compound to bind and/or influence the activity of said BACE455 nucleic acid or polypeptide. Binding alone provides some indication as to the ability of the compound to modulate the activity of said target, (although non-binding allosteric inhibitors may also be useful in, and are included within the scope of, the therapeutic methods of the present invention) and thus to affect a pathway leading to neurodegenerative disorders. In a presently preferred embodiment, the method comprises contacting in vitro a test compound with a distinctive BACE455 polypeptide or fragment thereof and determining the ability of said test compound to bind said polypeptide or fragment. The fragment preferably comprises an isoform-specific domain and/or a catalytic domain.

The determination of binding may be performed by various techniques, such as by labelling of the test compound, by competition with a labelled reference ligand, or other direct or indirect means of detecting a binding complex.

A further embodiment of the present invention resides in a method of selecting, characterizing, screening or optimizing a biologically active compound, said method comprising contacting in vitro a test compound with a BACE455 polypeptide and determining the ability of said test compound to modulate the activity of said polypeptide. In a specific embodiment, the test compound is contacted with a BACE455 polypeptide in the presence of BACE substrate (e.g., APP or an appropriate fragment thereof), and any changes in BACE455 activity, for example, proteolytic activity) is observed. In a preferred embodiment, the method comprises determining whether the test compound selectively affects BACE-mediated hydrolysis of the substrate. Typically, the test is carried out using a cell (e.g., a recombinant host cell) that expresses a BACE455 polypeptide.

A further embodiment of this invention resides in a method of selecting, characterizing, screening and/or optimizing a biologically active compound, said method comprising contacting a test compound with a BACE455 RNA and determining the ability of said test compound to modulate the translation of said BACE455 RNA.

Selectivity of the compound may be assessed by determining the effect thereof on other BACE splice isoforms that contain a full length exon 4.

The above screening assays may be performed in any suitable device, such as plates, tubes, dishes, flasks, etc. Typically, the assay is performed in multi-well microtiter dishes. Using the present invention, several test compounds can be assayed in parallel. Furthermore, the test compound may be of various origin, nature and composition. It may be any organic or inorganic substance, such as a lipid, peptide, polypeptide, nucleic acid, small molecule, in isolated or in mixture with other substances. The compounds may be all or part of a combinatorial library of compounds, for instance.
 

Claim 1 of 1 Claim

1. An isolated monoclonal antibody, fragment or derivative thereof that specifically binds to a polypeptide consisting of the sequence of SEQ ID NO: 5, wherein said fragment is a Fab fragment, a Fab'2 fragment, or a CDR fragment, and wherein said derivative is a single chain antibody, human antibody, humanized antibody, or a recombinant antibody.

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