Background to the Invention Neurodegenerative diseases are a group of disorders characterised by changes in normal neuronal function, leading in the majority of cases to neuronal dysfunction and even cell death. Currently, it is estimated that there are in excess of one hundred neurodegenerative diseases, however, we still have very little understanding of the etiological cause of these diseases. The most consistent risk factor for the development of a neurodegenerative disease such as, for example, Alzheimer's disease or Parkinson's disease, is age of onset (Tanner, 1992, Neurol. Clin. 10: 317-329). In this respect, such diseases are more prevalent in aged or aging persons, with a doubling of risk every five years after the age of 65.

Over the past century the growth rate of the population aged 65 and beyond in industrialized countries has far exceeded that of the population as a whole.

Accordingly, it is anticipated that, over the next generations, the proportion of elderly citizens will double, and, with this, the proportion of persons suffering from a neurodegenerative disease.

Two of the most common and most studied forms of age-related neurodegenerative disease are Alzheimer's disease and Parkinson's disease. Currently, it is estimated that there are 4.5 million cases of Alzheimer's disease and 1.2 million cases of Parkinson's disease in the US alone. It is estimated that in the period from 2001 to 2010 an additional 1. 5 million Alzheimer's disease cases will be diagnosed in the US, while

currently there are approximately 480 new cases of Parkinson's disease per million people per year diagnosed. Alzheimer's disease alone is the third most expensive disease in the United States, and costs approximately US$100 billion each year.

Alzheimer's disease is a complex multi-genic neurodegenerative disorder characterized by progressive impairments in memory, behaviour, language, and visio-spatial skills, ending ultimately in death.

As used herein, the term"Alzheimer's disease"shall be taken to mean a neurodegenerative disease characterised by progressive impairments in memory, behaviour, language and/or visio-spatial skills. The term"Alzheimer's disease"shall be taken to include early onset Alzheimer's disease (ie. with an onset earlier than the sixth decade of life), a late onset Alzheimer's disease (ie. with an onset later than, or in, the sixth decade of life), a juvenile onset Alzheimer's disease, and a sporadic or idiopathic Alzheimer's disease.

Hallmark pathologies of Alzheimer's disease include granulovascular neuronal degeneration, extracellular neuritic plaques with p-amyloid deposits, intracellular neurofibrillary tangles and neurofibrillary degeneration, synaptic loss, and extensive neuronal cell death. It is now known that these histopathologic lesions of Alzheimer's disease correlate with the dementia observed in many elderly people.

Alzheimer's disease is commonly diagnosed using clinical evaluation including physical and psychological assessment, an electroencephalography (EEG), a computerized tomography (CT) scan and an electrocardiogram. This form of testing is performed to eliminate some possible causes of dementia other than Alzheimer's disease such as, for example, a stroke. Following elimination of possible causes of dementia, Alzheimer's disease is diagnosed. Accordingly, current diagnostic approaches for Alzheimer's disease are not only unreliable and subjective, but merely diagnose the onset of dementia of unknown cause.

Furthermore, not all causes of dementia are easily detectable by methods currently used for the diagnosis of Alzheimer's disease. Accordingly, a subject who has suffered an ischemic, metabolic, toxic, infectious or traumatic insult to the brain may also present with dementia and, as a consequence, be incorrectly diagnosed with Alzheimer's disease. In fact, the NIH estimates that up to 45% of subjects diagnosed with

Alzheimer's disease actually suffer from another form of dementia, not necessarily caused by a neurodegenerative disease.

Genetic studies of subjects with a family history of Alzheimer's disease indicate that mutations in genes such as, for example, amyloid precursor protein gene, presenillin-1 and presenillin-2 cause some forms of this disease. However, these forms of Alzheimer's disease represent less than 5% of total cases of the disease.

Studies to identify polymorphisms and alleles that confer susceptibility to Alzheimer's disease have identified a large number of polymorphisms and mutations (reviewed in Rocchi et al., Brain Res. Bull., 61 : 1-24,2003). The most widely studied of these is the s4 isoform of the apolipoprotein E gene. A number of studies have shown an association between apolipoprotein E e4 (ApoE-s4) and late onset familial and sporadic forms of Alzheimer's disease (for example, Corder et al., Science 1993,261 : 261-263).

However, less than 50% of non-familial Alzheimer's disease sufferers are carriers of the ApoE-E4 isoform (Corder et al., Science 1993,261 : 261-263).

Parkinson's Disease (PD) is a common progressive neurodegenerative disorder that is characterised clinically by a combination of motor symptoms including bradykinesia, resting tremor, muscular rigidity and disturbance of postural reflexes (Sethi, Curr Ops Neurol 2002,15 : 457-460). The term"Parkinson's disease"shall be understood to encompass early onset Parkinson's disease and late onset Parkinson's disease, juvenile onset Parkinson's disease, idiopathic Parkinson's disease and monogenic Parkinson's disease.

Neuropathologically, there is a selective degeneration of dopaminergic neurons of the substantia nigra, which leads to a deficiency of dopamine in the striatal projection areas of these neurons. Eosinophilic intracellular inclusions termed Lewy bodies are sometimes found in the surviving dopaminergic neurons (Talkahashi and Wakabayashi, Neuropathology, 2001,21 : 315-322). In rare dominantly inherited forms of PD, mutations have been detected in three genes, Parkin, a-synuclein and the ubiquitin C- terminal hydrolase gene (Lansbury and Brice, Curr Op Genet Devel, 2002,12 : 299- 306). However, for the majority of sporadic and late-onset PD cases, the mode of inheritance is unclear and multiple susceptibility genes and environmental factors are thought to contribute to the disease process (Shastry, Neurosci Res 2001,41 : 5-12).

Parkinson's disease is diagnosed by clinical evaluation of a subject. Subjects that have two or more of the principal symptoms, one of which is resting tremor or bradykinesia, are diagnosed as suffering from Parkinson's disease. Positron-emission tomography (PET-scan) using radio-labeled dopa is helpful in confirming a diagnosis in difficult cases. However, this test is not widely available. A SPECT-scan is a simpler test using a variety of different isotopes and is widely available but is less reliable in confirming PD. Magnetic resonance imaging (MRI) is useful in excluding other conditions such as tumors, strokes, and hydrocephalus. However, MRI cannot confirm PD.

As with Alzheimer's disease, diagnosis of Parkinson's disease is only accurate, or even possible, following onset of the disease. Accordingly, methods currently in use for diagnosis of these diseases are of no use in determining a predisposition to a neurodegenerative disease, or in a prophylactic method of treatment for a neurodegenerative disease.

The observation of subjects with a family history of Parkinson's disease has provoked a considerable research effort to determine genes, alleles of genes or mutations that cause neurodegenerative disease or are associated with a susceptibility to a neurodegenerative disease.

The MAPT gene codes for Tau, a phosphorylated, microtubule-binding protein that assists in the stabilisation of the cytoskeleton (Shahani and Brandt, Cell Mol Life Sci 2002, 59: 1668-1680). Within the MAPT gene, two conserved haplotypes H1 and H2, comprising at least 8 single nucleotide polymorphisms (SNPs) and a microsatellite marker, extend over the entire coding sequence of the gene (Baker and Litvan, Hum Mol Genet 1999,8 : 711-715). There is an established association between the more common H1 haplotype and late-onset and sporadic PD cases (Martin et al., J Am Med Assoc 2001,286 : 2245-2250; Maraganore et al., Ann Neurol 2001,50 : 658-661; Golbe et al., Movement Disorder 2001,16 : 442-447; Pastor et al., Ann Neurol 2000,47 : 242- 245), as well as two tauopathies, cortico-basal degeneration (CBD) and progressive supranuclear palsy (PSP) (Baker et al., Hum Mol Genet 1999,8 : 711-715; De Maria et al., Ann Neurol 2000,47 : 374-377; Higgins et al., Neurology 2000,55 : 1364-1367; Ezquerra et al., Neurosci Lett 1999,275 : 183-186 ; Houlden et al., Neurology 2001, 56: 1702-1706; De Silva et al., Neurosci Lett 2001,311 : 145-148). However, the polymorphisms that constitute the haplotypes do not alter the amino acid composition

or appear to affect any functional domains of MAPT, such as splice sites. Thus, the exact pathogenic mechanism of the association between MAPT and PD is unclear.

There is a clear need to develop improved diagnostic methods for determining a predisposition to a neurodegenerative disease in a subject, and for the early diagnosis of these diseases. Diagnostic assays that rapidly and reliably diagnose a neurodegenerative disease prior to onset of the disease are particularly desirable as are indicators of whether or not a subject will respond to a particular treatment.

Summary of the Invention The present inventors have now identified a series of novel biallelic variants in the regulatory region of the MAPT gene. These variants form distinct promoter ("regulatory region") haplotypes that have different strengths at driving transcription of a reporter gene. Moreover, the haplotype associated with the strongest promoter strength was found at higher frequencies in a cohort of PD patients compared with normal controls. This provides clear evidence that the biological mechanism behind the association MAPT and PD and other tauopathies is increased levels of expression of Tau protein.

Accordingly, the present invention provides a method of diagnosing a neurodegenerative disorder or a predisposition to a neurodegenerative disorder in a subject, the method comprising analysing a biological sample derived from the subject for the presence of at least one polymorphism located in (i) a regulatory region of a MAPT gene upstream of a nucleotide equivalent to nucleotide 1386 of SEQ ID NO: 1; or (ii) within exon 1 of the MAPT gene.

In one example of the present invention, the neurodegenerative disorder is a dementing neurodegenerative disorder. Preferably, the neurodegenerative disorder is a FTDP-17 disorder or a tauopathy. Further preferably, the FTDP-17 disorder or tauopathy is frontotemporal dementia, pallido-ponto-nigral degeneration, cortico-basal degeneration, progressive supranuclear palsy, familial progressive subcortical gliosis, a familial multisystem tauopathy, or any other tauopathy. In another embodiment, the neurodegenerative disorder is Alzheimer's disease or Parkinson's disease.

As used herein, the term"diagnosis", and variants thereof such as, but not limited to, "diagnose","diagnosed"or"diagnosing"shall not be limited to a primary diagnosis of a clinical state, but should be taken to include any primary diagnosis or prognosis of a clinical state.

As used herein, the term"predisposition to a neurodegenerative disorder"shall be taken to mean that a subject is susceptible to a form of a neurodegenerative disorder or is more likely to develop a neurodegenerative disorder than a normal subject or a normal population of subjects. In this regard a polymorphism that is indicative of a predisposition to a neurodegenerative disorder may itself cause a neurodegenerative disorder or, alternatively, be correlated with the development of a neurodegenerative disorder.

The polymorphism may be a variable nucleotide type polymorphism, a restriction fragment length polymorphism, a single nucleotide polymorphism (SNP), a deletion, an insertion, a duplication, a translocation, a transition, or a transversion. Such a polymorphism may be detected by isolating and sequencing DNA fragments from the MAPT gene regulatory region. Polymorphisms may also be detected by hybridisation using discriminating oligonucleotide probes, by amplification procedures using discriminating oligonucleotide primers, or by single-strand conformation polymorphism analysis.

As used herein, the term"single nucleotide polymozphism"or"SNP"shall be taken to mean that a specific nucleic acid in the genome of a subject or an expression product thereof (the SNP may also be transcribed) may be any of two possible nucleic acid bases, or any of three nucleic acid bases, or any of four nucleic acid bases.

Preferably, the presence of the polymorphism is determined by comparing the MAPT gene regulatory region or a portion thereof of the subject to the equivalent region of the MAPT gene of a healthy or normal subject.

In the present context, the term"healthy subject"shall be taken to mean an subject who is known not to suffer from a neurodegenerative disorder, such knowledge being derived from clinical data on the subject. As the present invention is particularly useful for the early detection of neurodegenerative disorders, it is preferred that the healthy

subject is asymptomatic with respect to the early symptoms associated with a neurodegenerative disorder.

The term"normal subject"shall be taken to mean a subject having a normal level of MAPT expression in a particular sample derived from said subject. As will be known to those skilled in the art, data obtained from a sufficiently large sample of the population will normalize, allowing the generation of a data set for determining the average level of a particular parameter. Accordingly, the level of expression of MAPT can be determined for any population of subjects, for subsequent comparison to MAPT expression levels determined for a sample being assayed. Where such normalized data sets are relied upon, internal controls are preferably included in each assay conducted to control for variation.

In one example, the regulatory region of the MAPT gene of a normal or healthy subject has a sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 3.

In a preferred example of the present invention, the method comprises analysing a biological sample derived from a subject for the presence of at least one polymorphism in a regulatory region of a MAPT gene upstream of a nucleotide equivalent to nucleotide 1386 of SEQ ID NO: 1. Preferably, the method comprises analysing a biological sample derived from the subject for the presence of at least one polymorphism located at a position equivalent to any one of nucleotides 763-767; 794; 817; 1011 ; 1036 ; 1159; and 1340 of SEQ ID NO: 1.

In a further preferred example, the polymorphism is located within a transcription factor binding site. Preferred transcription factor binding sites are those located at a position equivalent to nucleotide 763-767 or 1011 of SEQ ID NO: 1.

In a further preferred example, any one of the following polymorphisms are indicative of a neurodegenerative disorder or predisposition to a neurodegenerative disorder: (i) AATTT at a position equivalent to nucleotides 763-767 of SEQ ID NO: 1; (ii) TT at a position equivalent to nucleotides 793 and 794 of SEQ ID NO: 1; (iii) A at a position equivalent to nucleotide 817 of SEQ ID NO: 1 ; (iv) G at a position equivalent to nucleotide 1011 of SEQ ID NO : 1 ; (v) T at a position equivalent to nucleotide 1036 of SEQ ID NO : 1 ;

(vi) C at a position equivalent to nucleotide 1159 of SEQ ID NO: 1 ; (vii) A at a position equivalent to nucleotide 1340 of SEQ ID NO: 1; and (viii) any combination of the above polymorphisms.

In another example of the present invention, the method comprises analysing a biological sample derived from a subject for the presence of at least one polymorphism in exon 1 of a MAPT gene. Preferably, the method comprises analysing a biological sample derived from the subject for the presence of at least one polymorphism located at a position equivalent to nucleotide 1492 and/or nucleotide 1593 of SEQ ID NO: 1.

More preferably, the polymorphism (s) are 1492 (C) and/or 1583 (C).

In a preferred example of the present invention, the polymorphism is in the homozygous form. As used herein, the term"homozygous form"is intended to mean that the same form of the polymorphism occurs at the same locus on homologous chromosomes. For example, the same polymorphism is located within the MAPT gene on both copies of chromosome 17.

In one example, the polymorphism is detected by hybridizing a nucleic acid probe or primer to a nucleic acid that comprises the regulatory region of the MAPT gene in a sample derived from a subject and detecting the hybridization by a detection means, wherein hybridization of the probe or primer indicates that the subject being tested is predisposed to or suffers from a neurodegenerative disease.

In an alternative example, the polymorphism is detected by amplifying (eg using PCR, RT-PCR, NASBA, TMA, ligase chain reaction etc) a portion of the regulatory region of the MAPT gene that comprises at least one polymorphism. In accordance with this embodiment, a probe or primer (or two or more probes or primers) that flank or abut the portion of the regulatory region of the MAPT gene that comprises at least one polymorphism are hybridized to a nucleic acid linked to the polymorphism in a biological sample. The polymorphism is then detected using an amplification or primer extension protocol.

The present invention also provides a method of diagnosing a neurodegenerative disorder or a predisposition to a neurodegenerative disorder in a subject, the method comprising analysing a sample derived from the subject for levels of expression of a MAPT gene product, wherein increased levels of expression of the MAPT gene product

compared to a normal or healthy subject is indicative of neurodegenerative disorder or a predisposition to a neurodegenerative disorder.

The present invention also provides a method for monitoring the efficacy of a therapeutic or prophylactic treatment of a neurodegenerative disorder in a subject, the method comprising analysing a sample derived from a subject undergoing treatment for a neurodegenerative disorder for levels of expression of a MAPT gene product, wherein decreased levels of expression of the MAPT gene product compared to levels of expression of the MAPT gene product in a sample derived from the subject prior to treatment is indicative of efficacy of the treatment.

It will be appreciated by those skilled in the art that levels of expression of the MAPT gene can be determined by analysis of levels of MAPT mRNA and/or cDNA derived therefrom in the sample. For example, the method of determining the level of expression of the MAPT gene may comprise hybridizing a probe or primer to the MAPT mRNA, or cDNA derived therefrom, in a sample derived from a subject under at least moderate, or preferably high stringency hybridization conditions and detecting the level of hybridization by a detection means. Preferably, the detection means is a nucleic acid hybridization or amplification reaction.

"Stringency"of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures.

Hybridization generally depends on the ability of denatured DNA to re-anneal when complementary strands are present in an environment below their melting temperature.

The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature which can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).

"Stringent conditions"or"high stringency", as defined herein, may be identified by those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0. 1% sodium dodecyl sulfate at 50°C ; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0. 1% Ficoll/0. 1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C ; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6. 8), 0. 1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 llg/mlj, 0.1% SDS, and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2 x SSC (sodium chloride/sodium citrate) and 50% formamide at 55°C, followed by a high- stringency wash consisting of 0.1 x SSC containing EDTA at 55°C.

"Moderate stringency"may be identified as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and <BR> <BR> include the use of washing solution and hybridization conditions (e. g. , temperature, ionic strength and % SDS) less stringent than those described above. An example of moderately stringent conditions is overnight incubation at 37°C in a solution comprising: 20% formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 gel denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 35°C-50°C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.

In another example, the MAPT gene expression product is a polypeptide, ie. a Tau polypeptide. For example, the method of determining the level of expression of a MAPT gene expression product may comprise contacting a biological sample derived from a subject with an antibody and/or ligand capable of binding to the Tau polypeptide for a time and under conditions sufficient for an antibody (or ligand)/Tau polypeptide complex to form and detecting the complex, wherein detection of an enhanced level of the complex when compared with the level of the complex detected in a biological sample derived from a healthy or normal subject indicates that the subject suffers from a neurodegenerative disease and/or is predisposed to a neurodegenerative disease.

As used herein the term"antibody"refers to intact monoclonal or polyclonal antibodies, immunoglobulin (IgA, IgD, IgG, IgM, IgE) fractions, humanized

antibodies, or recombinant single chain antibodies, as well as fragments thereof such as, for example Fab, F (ab) 2, and Fv fragments.

The sample can be any nucleated cell from the subject. For assay of genomic DNA, virtually any biological sample (other than pure red blood cells) is suitable. For example, convenient samples include whole blood, nucleated blood cells, brain, semen, saliva, tears, urine, faecal material, sweat, skin, testis, placenta, kidney and hair. In a particularly preferred example, the sample comprises brain tissue or lymphocytes.

The present invention also provides a method of screening for a therapeutic for a neurodegenerative disorder, the method comprising determining the level of expression of a MAPT gene product in the presence and absence of a candidate compound, wherein decreased expression of the MAPT gene product in the presence of the compound indicates that the compound is a candidate therapeutic for a neurodegenerative disorder.

It is preferred that the method of screening is performed under conditions of relatively high levels of expression of the MAPT gene products.

Accordingly, in one preferred example, the regulatory region of the MAPT gene has one or more of the following polymorphisms: (i) AATTT at a position equivalent to nucleotides 763-767 of SEQ ID NO: 1; (ii) TT at a position equivalent to nucleotides 793 and 794 of SEQ ID NO: 1; (iii) A at a position equivalent to nucleotide 817 of SEQ ID NO: 1; (iv) G at a position equivalent to nucleotide 1011 of SEQ ID NO: 1; (v) T at a position equivalent to nucleotide 1036 of SEQ ID NO: 1; (vi) C at a position equivalent to nucleotide 1159 of SEQ ID NO: 1; or (vii) A at a position equivalent to nucleotide 1340 of SEQ ID NO: 1.

In another example, the MAPT gene has at least one polymorphism located in exon 1.

Preferably, the polymorphism is located at positions equivalent to nucleotide 1492 or 1583 of SEQ ID NO: 1. Preferably, the polymorphisms are 1492 (C) and/or 1583 (C).

In another example, the regulatory region of the MAPT gene has the sequence as shown in SEQ ID NO: 1.

In another example, the method of screening involves exposing a translation system capable of expressing MAPT to a candidate compound and comparing the levels of expression of MAPT in the presence of the compound to the levels achieved under similar conditions but in the absence of the compound. The translation system may be a cell-free translation system. Alternatively, the translation system may comprise eukaryotic or prokaryotic cells.

The present invention also provides a method of screening for a therapeutic for a neurodegenerative disorder, the method comprising determining the transcriptional activity of the MAPT regulatory region in the presence and absence of a candidate compound, wherein decreased transcriptional activity in the presence of the compound indicates that the compound is a candidate therapeutic for a neurodegenerative disorder.

In a preferred example of this method of screening, the MAPT regulatory region is operably linked to a reporter gene. Preferably, the reporter gene is chloramphenicol acetyltransferase, B-galactosidase, B-glucuronidase, luciferase, green fluorescent protein, red fluorescent protein, placental alkaline phosphatase, or secreted embryonic alkaline phosphatase.

In a further example, the present invention provides a method of screening for a therapeutic for a neurodegenerative disorder, the method comprising determining the ability of a candidate compound to modulate the binding of a transcription factor to the regulatory region of the MAPT gene, wherein a decreased level of binding of the transcription factor to the regulatory region in the presence of the compound indicates that the compound is a candidate therapeutic for a neurodegenerative disorder.

In one particular example, the transcription factor is S8 or Myc.

In a further example of the present invention provides a process for identifying or determining a candidate compound, said method comprising: (i) performing a screening method as described herein to thereby identify or determine a compound for the treatment of a neurodegenerative disease; (ii) optionally, determining the structure of the compound; and (iii) providing the compound or the name or structure of the compound such as, for example, in a paper form, machine-readable form, or computer- readable form.

In a preferred embodiment, the compound or the name or structure of the compound is provided with an indication as to its use e. g. , as determined by a screen described herein.

The present invention also provides a process for producing a compound, said process comprising: (i) performing a screening method as described herein to thereby identify or determine a compound for the treatment of a neurodegenerative disease; (ii) optionally, determining the structure of the compound; (iii) optionally, providing the name or structure of the compound such as, for example, in a paper form, machine-readable form, or computer-readable form; and (iv) providing the compound.

Preferably, the synthesized compound or the name or structure of the compound is provided with an indication as to its use e. g. , as determined by a screen described herein.

The present invention also provides a method of manufacturing a compound for the treatment of a neurodegenerative disease comprising: (i) determining a candidate compound for the treatment of a neurodegenerative disease; and (ii) using the compound in the manufacture of a therapeutic or prophylactic for the treatment of a neurodegenerative disease.

In a further example of the present invention, a candidate compound identified by a screening method of the present invention is formulated into a pharmaceutically acceptable composition.

The present invention also provides a method of treating a neurodegenerative disorder, the method comprising administering to a subject in need thereof an agent that reduces the level of MAPT gene product in the subject.

Preferably, the agent is provided in a dosage form such as solid preparations, e. g. tablets, troches, pills, powders, fine powders, granules, and capsules; liquid

preparations, e. g. solutions, suspensions, emulsions, syrups, and elixirs; or injections which may be made available in such forms as solutions, suspensions and emulsions.

The term"treating"as it pertains to administering the vectors containing the nucleic acid constructs of the present invention or agents to subjects in need thereof is understood to include administration to patients for the purpose of prophylaxis and amelioration of the neurodegenerative disorder.

In one example, the agent reduces levels of expression of the MAPT gene. The agent may reduce levels of expression of the MAPT gene by, for example, interfering with the binding of a transcription factor, such as S8 or Myc, to the regulatory region of the MAPT gene.

The present invention also provides a polynucleotide probe when used in the method according to the invention.

In a preferred example, the polynucleotide probe is labelled with a detectable marker.

Preferably, the detectable marker is selected from the group consisting of proteins, enzymes, radionuclides, fluorophores, luminophores, enzyme inhibitors, coenzymes, luciferins, paramagnetic metals and spin labels.

The present invention also provides a kit for diagnosis of a neurodegenerative disorder or diagnosis of a predisposition to a neurodegenerative disorder comprising at least one polynucleotide probe according to the invention. Preferably, the diagnostic kit includes instructions for its use.

Preferably, the diagnostic kit further comprises at least one additional reagent selected from the group consisting of a lysing buffer for lysing cells contained in a sample; enzyme amplification reaction components; dNTPs; NTPs ; reaction buffer; amplifying enzyme; and a combination of additional reagents.

The present invention also provides a vector comprising the regulatory region of the MAPT gene operably linked to a reporter gene. In another preferred embodiment of this aspect, the regulatory region has a sequence as shown in SEQ ID NO: 1.

The present invention also provides a host cell comprising a vector of the invention. In preferred embodiment of this aspect, the host cell is a SK-N-MC (ATCC HTB 10) cell or an embryonic kidney 293 (ATCC CRL 1573) cell.

Throughout this specification the word"comprise", or variations such as"comprises"or "comprising"will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of this application.

Brief description of the accompanying Figures Figure 1 shows A) Biallelic polymorphisms (vertical arrows) flanking the promoter region of MAPT are shown for the three haplotypes. Putative binding sites of transcription factors are shown on the H1 haplotype: SP1 (open circle), EF1 (close circle), AP1 (open square), OCT (close square), myc (open diamond), myb (close diamond), S8 (open triangle) and MZF (close triangle). Binding sites that are predicted to be affected by the promoter polymorphisms are indicated with asterisks. B) Electropherograms of nucleotide sequence of the MAPT promoter in two homozygous subjects using the Prom2R primer. C) Promoter strength assays using the Luciferase reporter gene system. Values and error bars were derived from six separate transfections into SK-N-MC cells. Fluorescence units for each promoter haplotype were normalised to the values for the pGL3 vector (vec) for each transfection.

Statistical analyses were performed by pair-wise student t tests between the H1 and the other two promoter haplotypes (* = p< 0.05, ** = p < 0.005).

Figure 2 provides A) A schematic diagram of a transcript map showing known genes which are adjacent to MAPT; oxysterol binding protein-like 7 (OSBPL7), Corticotropin releasing hormone receptor 1 (CRHR1), N-ethylmaleimide-sensitive factor (NSF) and Wingless-type MMTV integration site family, member 3 (WNT3). Relative positions

of the SNPs used to construct a common haplotype are indicated (vertical arrows). B) A common haplotype was constructed from the SNPs. Each haplotype from a panel of 12 subjects were examined to determine whether recombinations have occurred. The frequency of hapotypes which are contiguous with the MAPT haplotype is charted against relative positions of the SNPs.

Figure 3 provides a bar graphs showing relative levels of Tau transcripts in brain tissue samples of known genotype. Each brain sample was genotyped for the Tau promoter polymorphism (-373 T/C) to infer the relevant haplotype.

Key to Sequence Listing SEQ ID NO: 1 is a genomic DNA sequence of the regulatory region of the MAPT gene (Haplotype 1); SEQ ID NO: 2 is a genomic DNA sequence of the regulatory region of the MAPT gene (Haplotype 1') ; and SEQ ID NO: 3 is a genomic DNA sequence of the regulatory region of the MAPT gene (Haplotype 2).

SEQ ID NOs: 4 and 5 are a pair of primers for amplification of a region upstream of the transcription site of the MAPT gene.

SEQ ID NOs: 6 and 7 are a pair of primers for amplification of a region upstream of the transcription site of the MAPT gene.

SEQ ID NOs: 8 and 9 are a pair of primers for amplification of a region upstream of the transcription site of the MAPT gene.

SEQ ID NOs: 10 and 11 are a pair of primers for amplification of a region upstream of the transcription site of the MAPT gene.

SEQ ID NOs: 12 and 13 are a pair of primers for amplification of a region upstream of the transcription site of the MAPT gene.

SEQ ID NOs: 14 and 15 are a pair of primers for amplification of a region upstream of the transcription site of the MAPT gene.

SEQ ID NOs: 16 and 17 are a pair of primers for amplification of a region upstream of the transcription site of the MAPT gene.

SEQ ID NOs: 18 and 19 are a pair of primers for amplification of a region upstream of the transcription site of the MAPT gene.

SEQ ID NOs: 20 and 521 are a pair of primers for amplification of a region upstream of the transcription site of the MAPT gene.

SEQ ID NOs: 22 and 23 are a pair of primers for amplification of a region upstream of the transcription site of the MAPT gene.

SEQ ID NOs: 24 and 25 are a pair of primers which flank the Tau exon 9 sequence useful for amplification of Tau transcripts.

Detailed Description of the Preferred Embodiments 1. Diagnosis of a neurodegenerative disorder or a predisposition for a neurodegenerative disorder 1. 1 Analysis ofpolymorphisms within the MAPT regulatory region In one aspect, the present invention relates to a method for predicting the likelihood that a subject will have a neurodegenerative disorder, or for aiding in the diagnosis of a neurodegenerative disorder, comprising the steps of obtaining a sample from the subject to be assessed and analyzing the regulatory region of the gene encoding MAPT for the presence of at least one polymorphism.

In a preferred example of the present invention, the method comprises analysing a biological sample derived from a subject for the presence of at least one polymorphism in a regulatory region of a MAPT gene upstream of a nucleotide equivalent to nucleotide 1386 of SEQ ID NO: 1. Preferably, the method comprises analysing a biological sample derived from the subject for the presence of at least one

polymorphism located at a position equivalent to any one of nucleotides 763-767; 794; 817 ; 1011; 1036; 1159; and 1340 of SEQ ID NO: 1.

In a further preferred example, the polymorphism is located within a transcription factor binding site. Preferred transcription factor binding sites are those located at a position equivalent to nucleotide 763-767 or 1011 of SEQ ID NO: 1.

In a further preferred example, the following polymorphisms are indicative of a neurodegenerative disorder or predisposition to a neurodegenerative disorder: (i) AATTT at a position equivalent to nucleotides 763-767 of SEQ ID NO: 1; (ii) TT at a position equivalent to nucleotides 793 and 794 of SEQ ID NO: 1; (iii) A at a position equivalent to nucleotide 817 of SEQ ID NO: 1; (iv) G at a position equivalent to nucleotide 1011 of SEQ ID NO: 1; (v) T at a position equivalent to nucleotide 1036 of SEQ ID NO: 1; (vi) C at a position equivalent to nucleotide 1159 of SEQ ID NO: 1; (vii) A at a position equivalent to nucleotide 1340 of SEQ ID NO : 1; and (viii) any combination of the above polymorphisms.

In another example of the present invention, the method comprises analysing a biological sample derived from a subject for the presence of at least one polymorphism in exon 1 of a MAPT gene. Preferably, the method comprises analysing a biological sample derived from the subject for the presence of at least one polymorphism located at a position equivalent to nucleotide 1492 and/or nucleotide 1593 of SEQ ID NO: 1.

More preferably, the polymorphism (s) are 1492 (C) and/or 1583 (C).

Such polymorphism (s) may be determined using a positive or negative read-out assay.

As used herein, the term"positive read-out assay"shall be taken to mean that a positive result in an assay indicates that a biological sample comprises at least one polymorphism in the regulatory region of the gene encoding MAPT.

As used herein, the term"negative read-out assay"shall be taken to mean that a negative result in an assay indicates that a biological sample comprises at least one polymorphism in the regulatory region of the MAPT gene.

For example, a negative read-out assay for the detection of at least one polymorphism in the regulatory region of the MAPT gene comprises hybridizing a probe or primer to a portion of the regulatory region of the MAPT gene, wherein the probe or primer is specific for a corresponding normal portion of the regulatory region of the MAPT gene.

As will be apparent to the person skilled in the art, such a probe or primer will only hybridize to a form of the regulatory region of the MAPT gene that does not comprise the at least one polymorphism. Accordingly, an assay that fails to detect hybridization of the probe or primer to nucleic acid in the sample (or detects a reduced amount of the hybridization compared to a suitable control) indicates the presence of the at least one polymorphism in the regulatory region of the MAPT gene.

The detection of the hybridization of the probe or primer to the nucleic acid in the sample is detected using any method known in the art (e. g. such as described herein).

As will be apparent to the person skilled in the art, a negative read-out assay for the detection of the at least one polymorphism in the regulatory region of the MAPT gene may also include a positive control. Such a control is useful as failure to detect the polymorphism may indicate that the detection reaction has failed. Such a positive control may involve, for example, using a probe or primer that hybridizes to a portion of the regulatory region of the MAPT gene that comprises the at least one polymorphism.

Alternatively, a positive control may comprise a probe or primer that hybridizes to a portion of nucleic acid other than the regulatory region of the MAPT gene (that is known to be expressed in the biological sample) to determine the presence of nucleic acid in a biological sample. Examples of such nucleic acids includes the gene for actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), (32 microglobulin, hydroxy- methylbilane synthase, hypoxanthine phosphoribosyl-transferase 1 (HPRT), ribosomal protein L13c, succinate dehydrogenase complex subunit A and TATA box binding protein (TBP) etc.

The nucleotide (s) which occupies the polymorphic site of interest can be identified by a variety methods, such as Southern analysis of genomic DNA; direct mutation analysis by restriction enzyme digestion; Northern analysis of RNA; denaturing high pressure liquid chromatography (DHPLC); gene isolation and sequencing; hybridisation of an allele-specific oligonucleotide with amplified gene products; exon trapping, single base

extension (SBE); or analysis of MAPT expression levels. Examples of suitable procedures are discussed below.

1. 2 Hybridization based assays, amplification based assays and restriction endonuclease based assays Methods for detecting nucleic acids are known in the art and include for example, hybridization based assays, amplification based assays and restriction endonuclease based assays. For example, a change in the sequence of a region of the genome or an expression product thereof such as, for example, an insertion, a deletion, a transversion, a transition, alternative splicing or a change in the preference of or occurrence of a splice form of a gene is detected using a method such as, polymerase chain reaction (PCR), strand displacement amplification, ligase chain reaction, cycling probe technology or a DNA microarray chip amongst others. Methods of detecting SNPs are known in the art and reviewed, for example, in Landegren et al., Genome Research, 1998,8 : 769-776.

Methods of PCR are known in the art and described, for example, in Dieffenbach and Dveksler (Eds) (In : PCR Primer: A Laboratory Manual, Cold Spring Harbour Laboratories, NY, 1995). Generally, for PCR two non-complementary nucleic acid primer molecules comprising at least about 20 nucleotides in length, and'more preferably at least 30 nucleotides in length, are hybridized to different strands of a nucleic acid template molecule, and specific nucleic acid molecule copies of the template are amplified enzymatically. PCR products may be detected using electrophoresis and detection with a detectable marker that binds nucleic acids.

Alternatively, one or more of the oligonucleotides are labelled with a detectable marker (e. g. a fluorophore) and the amplification product detected using, for example, a lightcycler (Perkin Elmer, Wellesley, MA, USA). Allele specific PCR (as described, for example, In Liu et al., Genome Research, 1997,7 : 389-398) is useful for determining the presence of one or other allele of a polymorphism such as a SNP. In one example, an oligonucleotide is designed in which the most 3'base of the oligonucleotide hybridizes with the SNP. During a PCR reaction, if the 3'end of the oligonucleotide does not hybridize to a target sequence, little or no PCR product is produced, indicating that a base other than that present in the oligonucleotide is present at the site of SNP in the sample. PCR products may then be detected using, for example, gel or capillary electrophoresis or mass spectrometry. Clearly, the present

invention also encompasses quantitative forms of PCR such as, for example, a Taqman assay.

Strand displacement amplification (SDA) utilises oligonucleotides, a DNA polymerase and a restriction endonuclease to amplify a target sequence. The oligonucleotides are hybridized to a target nucleic acid and the polymerase used to produce a copy of this region. The duplexes of copied nucleic acid and target nucleic acid are then nicked with an endonuclease that specifically recognises a sequence of nucleotides at the beginning of the copied nucleic acid. The DNA polymerase recognises the nicked DNA and produces another copy of the target region at the same time displacing the previously generated nucleic acid. The advantage of SDA is that it occurs in an isothermal format, thereby facilitating high-throughput automated analysis.

Ligase chain reaction (described in, for example, EU 320,308 and US 4,883, 750) uses two or more oligonucleotides that bind to a target nucleic acid in such a way that they abut. A ligase enzyme is then used to link the oligonucleotides. Using thermocycling the ligated oligonucleotides then become a target for further oligonucleotides. The ligated fragments are then detected, for example, using electrophoresis, or matrix- assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Alternatively, or in addition, one or more of the probes is labelled with a detectable marker, thereby facilitating rapid detection.

Cycling Probe Technology uses a chimeric synthetic probe that comprises DNA-RNA- DNA that is capable of hybridizing to a target sequence. Upon hybridization to a target sequence the RNA-DNA duplex formed is a target for RNase H that cleaves the probe.

The cleaved probe is then detected using, for example, electrophoresis or MALDI-TOF MS.

Methods of RT-PCR are known in the art and described, for example, in Dieffenbach and Dveksler (Eds) (In : PCR Primer: A Laboratory Manual, Cold Spring Harbour Laboratories, NY, 1995).

Other suitable amplification methods include transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA, 1989,86, 1173), self-sustained sequence replication (Guatelli et al., Proc. Nat. Acad. Sci. USA, 1990,87, 1874) and nucleic acid based sequence amplification (NASBA). The latter two amplification methods involve

isothermal reactions based on isothermal transcription, which produce both single stranded RNA (ssRNA) and double stranded DNA (dsDNA) as the amplification products in a ratio of about 30 or 100 to 1, respectively.

Methods of TMA or self-sustained sequence replication (3SR) use two or more oligonucleotides that flank a target sequence, a RNA polymerase, RNase H and a reverse transcriptase. One oligonucleotide (that also comprises a RNA polymerase binding site) hybridizes to an RNA molecule that comprises the target sequence and the reverse transcriptase produces cDNA copy of this region. RNase H is used to digest the RNA in the RNA-DNA complex, and the second oligonucleotide used to produce a copy of the cDNA. The RNA polymerase is then used to produce a RNA copy of the cDNA, and the process repeated.

NASBA systems rely on the simultaneous activity of three enzymes (a reverse transcriptase, RNase H and RNA polymerase) to selectively amplify target mRNA sequences. The mRNA template is transcribed to cDNA by reverse transcription using an oligonucleotide that hybridizes to the target sequence and comprises a RNA polymerase binding site at its 5'end. The template RNA is digested with RNase H and double stranded DNA is synthesised. The RNA polymerase then produces multiple RNA copies of the cDNA and the process is repeated.

Nickerson, D. A. et al., (Proc. Natl. Acad. Sci. USA, 1990,87 : 8923-8927) have described a nucleic acid detection assay that combines attributes of PCR and Oligonucleotide Ligation Assay. In this method, PCR is used to achieve the exponential amplification of target DNA, which is then detected using OLA. The Landegren et al., (Genome Research, 1998, 8 (8): 769-776) protocol uses two oligonucleotides which are designed to be capable of hybridizing to abutting sequences of a single strand of a target. One of the oligonucleotides is biotinylated, and the other is detectably labeled. If the precise complementary sequence is found in a target molecule, the oligonucleotides will hybridize such that their termini abut, and create a ligation substrate. Ligation then permits the labeled oligonucleotide to be recovered using avidin, or another biotin ligand.

Variable number tandem repeats arise from spontaneous tandem duplications of di-or tri-nucleotide repeated motifs of nucleotides (Weber, J. L. , U. S. Pat. No. 5,075, 217;<BR> Armour et al., FEBS Lett. , 1992,307 : 113-115; Jones. et al., Eur. J. Haematol., 1987,

39: 144-147; Horn. et al., PCT Application W091/14003 ; Jeffreys, A. J. , European<BR> Patent Application 370,719 ; Jeffreys, A. J. , U. S. Pat. No. 5,175, 082; Jeffreys et al.,<BR> Amer. J, Hum. Genet. , 1986,39 : 11-24; Jeffreys. etal., Nature, 1985,316 : 76-79; Gray<BR> et al., Proc. R. Acad. Soc. Lond. , 1991,243 : 241-253; Moore et al., Genomics, 1991,<BR> 10: 654-660; Jeffreys et al., Anim, Genet., 1987, 18 : 1-15 ; Hillel et al., Anim. Genet. ,<BR> 1989,20 : 145-155; Hillel et al., Genet. , 1990,124 : 783-789). If such a variation alters the lengths of the fragments that are generated by restriction endonuclease cleavage, the variations are referred to as restriction fragment length polymorphisms ("RFLPs").

RFLPs have been widely used in human and animal genetic analyses (Glassberg, J., UK patent application 2135774; Skolnick et al., Cytogen, Cell Genet., 1982, 32: 58-67; Botstein et al., Ann. J. Hum. Genet., 1980, 32: 314-331 ; Fischer et al., PCT Application W090/13668 ; Uhlen, M. , PCT Application W090/11369).

Clearly, the hybridization to and/or amplification of a portion of the regulatory region of the MAPT gene comprising at least one polymorphism using any of these methods is detectable using, for example, electrophoresis and/or mass spectrometry. In this regard, one or more of the probes/primers and/or one or more of the nucleotides used in an amplification reaction may be labelled with a detectable marker to facilitate rapid detection of a marker, for example, a fluorescent label (e. g. Cy5 or Cy3) or a radioisotope (e. g. 32p).

1. 3 Probe/Primer Design As will be apparent to the person skilled in the art, the specific probe or primer used in a method of the present invention will depend upon the assay format used. Clearly, a probe or primer that is capable of specifically hybridizing to or detecting the polymorphism (s) of interest is preferred. Methods of designing probes and/or primers for, for example, PCR or hybridization are known in the art and described, for example, in Dieffenbach and Dveksler (Eds) (In: PCR Primer: A Laboratory Manual, Cold Spring Harbour Laboratories, NY, 1995). Furthermore, several software packages are publicly available that design optimal probes and/or primers for a variety of assays, e. g.

Primer 3 available from the Center for Genome Research, Cambridge, MA, USA.

Probes and/or primers useful for detection of a marker associated with a neurodegenerative disease are assessed to determine those that do not form hairpins, self-prime or form primer dimers (e. g. with another probe or primer used in a detection assay).

Furthermore, a probe or primer (or the sequence thereof) is assessed to determine the temperature at which it denatures from a target nucleic acid (i. e. the melting temperature of the probe or primer, or Tm). Methods of determining Tm are known in the art and described, for example, in Santa Lucia, Proc. Natl. Acad. Sci. USA, 1995, 95: 1460-1465 and Bresslauer et al., Proc. Natl. Acad. Sci. USA, 1986,83 : 3746-3750.

A primer or probe useful for detecting a polymorphism in an allele specific PCR assay or a ligase chain reaction assay is designed such that the 3'terminal nucleotide hybridizes to the site of the polymorphism. The 3'terminal nucleotide may be any of the nucleotides known to be present at the site of the polymorphism. When complementary nucleotides occur in both the probe or primer and at the site of the polymorphism the 3'end of the probe or primer hybridizes completely to the polymorphism of interest and facilitates, for example, PCR amplification or ligation to another nucleic acid. Accordingly, a probe or primer that completely hybridizes to the target nucleic acid produces a positive result in an assay.

In another example, a primer useful for a primer extension reaction is designed such that it specifically hybridizes to a region adjacent to a specific nucleotide of interest, eg a SNP. While the specific hybridization of a probe or primer may be estimated by determining the degree of homology of the probe or primer to any nucleic acid using software such as, for example, BLAST, the specificity of a probe or primer can be determined empirically using methods known in the art.

A locked nucleic acid (LNA) or protein-nucleic acid (PNA) probe or a molecular beacon useful, for example, for detection of a polymorphism by hybridization is at least about 8 to 12 nucleotides in length. Preferably, the nucleic acid, or derivative thereof, that hybridizes to the site of the polymorphism is positioned at approximately the centre of the probe, thereby facilitating selective hybridization and accurate detection.

Fluorescently labelled locked nucleic acid (LNA) molecules or fluorescently labelled protein-nucleic acid (PNA) molecules are useful for the detection of polymorphisms (as described in Simeonov and Nikiforov, Nucleic Acids Research, 2002,30 (17): 1-5).

LNA and PNA molecules bind, with high affinity, to nucleic acid, in particular, DNA.

Flurophores (in particular, rhodomine or hexachlorofluorescein) conjugated to the LNA or PNA probe fluoresce at a significantly greater level upon hybridization of the probe

to target nucleic acid. However, the level of increase of fluorescence is not enhanced to the same level when even a single nucleotide mismatch occurs. Accordingly, the degree of fluorescence detected in a sample is indicative of the presence of a mismatch between the LNA or PNA probe and the target nucleic acid such as, in the presence of a SNP or other polymorphism. Preferably, fluorescently labelled LNA or PNA technology is used to detect a single base change in a nucleic acid that has been previously amplified using, for example, an amplification method known in the art and/or described herein.

As will be apparent to the person skilled in the art, LNA or PNA detection technology is amenable to a high-throughput detection of one or more markers by immobilising an LNA or PNA probe to a solid support, as described in Orum et al., Clin. Chem. 1999, 45: 1898-1905.

Molecular beacons are also useful for detecting polymorphisms directly in a sample or in an amplified product (see, for example, Mhlanga and Malmberg, Methods, 2001, 25 : 463-471). Molecular beacons are single stranded nucleic acid molecules with a stem- and-loop structure. The loop structure is complementary to the region surrounding the SNP of interest. The stem structure is formed by annealing two"arms, " complementary to each other, that are on either side of the probe (loop). A fluorescent moiety is bound to one arm and a quenching moiety to the other arm, that suppresses any detectable fluorescence when the molecular beacon is not bound to a target sequence. Upon binding of the loop region to its target nucleic acid the arms are separated and fluorescence is detectable. However, even a single base mismatch significantly alters the level of fluorescence detected in a sample. Accordingly, the presence or absence of a particular base at the site of a polymorphism is determined by the level of fluorescence detected.

Methods of producing/synthesising probes and/or primers useful in the present invention are known in the art. For example, oligonucleotide synthesis is described, in Gait (Ed) (In: Oligonucleotide Synthesis: A Practical Approach, IRL Press, Oxford, 1984); LNA synthesis is described, for example, in Nielsen et al, J. Chem. Soc. Perkin <BR> <BR> Trans. , 1997,3423 ; Singh and Wengel, Chem. Commun. 1998, 1247, and PNA<BR> synthesis is described, for example, in Egholm et al., Am. Chem. Soc. , 1992,114 :<BR> 1895; Egholm et al., Nature, 1993,365 : 566 and Orum et al., Nucl. Acids Res. , 1993, 21: 5332.

The design and use of allele-specific probes/primers for analyzing polymorphisms is described by e. g. , Saiki et al., Nature, 1986,324, 163-166; Dattagupta, EP 235,726, Saiki, WO 89/11548. Allele-specific probes can be designed that hybridize to a segment of target DNA from one subject but do not hybridize to the corresponding segment from another subject due to the presence of different polymorphic forms in the respective segments from the two subjects. Hybridisation conditions should be sufficiently stringent that there is a significant difference in hybridisation intensity between alleles, and preferably an essentially binary response, whereby a probe hybridizes to only one of the alleles. Hybridisations are usually performed under stringent conditions, for example, at a salt concentration of no more than 1 M and a temperature of at least 25°C. For example, conditions of 5xSSPE (750 mM NaCI, 50 mM NaPhosphate, 5 mM EDTA, pH 7.4) and a temperature of 25-30°C, or equivalent conditions, are suitable for allele-specific probe hybridisations. Equivalent conditions can be determined by varying one or more of the parameters as known in the art, while maintaining a similar degree of identity or similarity between the target nucleotide sequence and the primer or probe used.

Some probes are designed to hybridize to a segment of target DNA such that the polymorphic site aligns with a central position (e. g. , in a 15-mer at the 7 position; in a 16-mer, at either the 8 or 9 position) of the probe. This design of probe achieves good discrimination in hybridisation between different allelic forms.

In a preferred embodiment the probe is a nucleic acid probe. Nucleic acid probes can comprise inosine, adenine, guanine, thymidine, cytidine or uracil residues or functional analogues or derivatives thereof that are capable of being incorporated into a polynucleotide molecule, provided that the resulting probe is capable of hybridizing under at least low stringency conditions to the regulatory region.

Whilst the probes may comprise double-stranded or single-stranded nucleic acid, single-stranded probes are preferred because they do not require melting prior to use in hybridisations. On the other hand, longer probes are also preferred because they can be used at higher hybridisation stringency than shorter probes and may produce lower background hybridisation than shorter probes.

So far as shorter probes are concerned, single-stranded, chemically-synthesized oligonucleotide probes are particularly preferred by the present invention. To reduce the noise associated with the use of such probes during hybridisation, the nucleotide sequence of the probe is carefully selected to maximize the Tm at which hybridisations can be performed, reduce non-specific hybridisation, and to reduce self-hybridisation.

Such considerations may be particularly important for applications involving high throughput screening using microarray technology. In general, this means that the nucleotide sequence of an oligonucleotide probe is selected such that it is unique to the regulatory region, has a low propensity to form secondary structure, low self- complementary, and is not highly A/T-rich.

The only requirement for the probes is that they cross-hybridize to nucleic acid comprising the MAPT gene regulatory region or exon 1 or the complementary nucleotide sequence thereto and are sufficiently unique in sequence to generate high signal: noise ratios under specified hybridisation conditions. As will be known to those skilled in the art, long nucleic acid probes are preferred because they tend to generate higher signal: noise ratios than shorter probes and/or the duplexes formed between longer molecules have higher melting temperatures (i. e. Tm values) than duplexes involving short probes. Accordingly, full-length DNA or RNA probes are contemplated by the present invention, as are specific probes comprising the sequence of the 3'- untranslated region or complementary thereto.

It is preferred that the nucleotide sequence of an oligonucleotide probe has the following properties: it comprises less than ten (10) A residues; it comprises less than ten (10) T residues; it comprises less than nine (9) C residues; it comprises less than nine (9) G residues; it comprises less than seven (7) A residues in any window consisting of 8 nucleotides; it comprises less than seven (7) T residues in any window consisting of 8 nucleotides; it comprises less than eight (8) C residues in any window consisting of 8 nucleotides; it comprises less than eight (8) G residues in any window consisting of 8 nucleotides;

it comprises less than six (6) consecutive A residues; it comprises less than six (6) consecutive T residues; it comprises less than five (5) consecutive C residues; and it comprises less than five (5) consecutive G residues.

Additionally, the self-complementarity of a nucleotide sequence can be determined by aligning the sequence with its reverse complement, wherein detectable regions of identity are indicative of potential self-complementarity. As will be known to those skilled in the art, such sequences may not necessarily form secondary structures during hybridisation reaction and, as a consequence, successfully identify a target nucleotide sequence. It is also known to those skilled in the art that, even where a sequence does form secondary structures during hybridisation reactions, reaction conditions can be modified to reduce the adverse consequences of such structure formation. Accordingly, a potential for self-complementarity should not necessarily exclude a particular candidate oligonucleotide from selection. In cases where it is difficult to determine nucleotide sequences having no potential self-complementarity, the uniqueness of the sequence should outweigh a consideration of its potential for secondary structure formation.

In a preferred example, the probe comprises a nucleotide sequence that is complementary to the regulatory region of the MAPT gene.

In another example, the probe comprises a nucleotide sequence that is within or complementary to exon 1 of the MAPT gene.

Preferably, the probe comprises a nucleotide sequence that corresponds with at least one polymorphism of the invention.

Preferred probes for detecting MAPT polymorphisms comprise a nucleotide sequence selected from the group consisting of : (i) a nucleotide sequence having at least 80% identity to SEQ ID NO: 1; (ii) a nucleotide sequence comprising at least 20 contiguous nucleotides of SEQ ID NO : 1; and (iii) a complement of either (i) or (ii) hereinabove.

Recommended pre-requisites for selecting oligonucleotide probes, particularly with respect to probes suitable for microarray technology, are described in detail by Lockhart etal. (NatureBiotech., 1996,14 : 1675-1680).

Allele-specific probes are often used in pairs, one member of a pair showing a perfect match to a reference form of a target sequence and the other member showing a perfect match to a variant form. Several pairs of probes can then be immobilized on the same support for simultaneous analysis of multiple polymorphisms within the same target sequence.

An allele-specific primer hybridizes to a site on target DNA overlapping a polymorphism and only primes amplification of an allelic form to which the primer exhibits perfect complementarity (see Gibbs, Nucleic Acid Res., 1989, 17,2427-2448).

This primer is used in conjunction with a second primer which hybridizes at a distal site. Amplification proceeds from the two primers, resulting in a detectable product which indicates the particular allelic form is present. A control is usually performed with a second pair of primers, one of which shows a single base mismatch at the polymorphic site and the other of which exhibits perfect complementarity to a distal site. The single-base mismatch prevents amplification and no detectable product is formed. The method works best when the mismatch is included in the 3'-most position of the oligonucleotide aligned with the polymorphism because this position is most destabilizing to elongation from the primer (see, e. g. , WO 93/22456).

1. 4 Tiling Arrays The polymorphisms can also be identified by hybridisation to nucleic acid arrays, some examples of which are described in WO 95/11995. WO 95/11995 also describes sub- arrays that are optimized for detection of a variant form of a pre-characterized polymorphism. Such a sub-array contains probes designed to be complementary to a second reference sequence, which is an allelic variant of the first reference sequence.

The second group of probes is designed by the same principles, except that the probes exhibit complementarity to the second reference sequence. The inclusion of a second group (or further groups) can be particularly useful for analyzing short sub-sequences of the primary reference sequence in which multiple mutations are expected to occur within a short distance commensurate with the length of the probes (e. g. , two or more mutations within 9 to 21 bases).

1. 5 Direct-Sequencing/Micro-Sequefacing/Mini-Sequencing The direct analysis of the sequence of polymorphisms of the present invention can be accomplished using either the dideoxy chain termination method or the Maxam-Gilbert method (see Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd Ed., CSHP, New York 1989); Zyskind et al., Recombinant DNA Laboratory Manual, (Acad. Press, 1988)).

Primer-guided nucleotide incorporation procedures for assaying polymorphic sites in DNA have been described (Komher et al., Nucl. Acids. Res. , 1989,17 : 7779-7784;<BR> Sokolov, B. P. , Nucl. Acids Res. , 1990,18 : 3671; Syvanen et al., Genomics, 1990, 8: 684-692; Kuppuswamy et al., Proc. Natl. Acad. Sci. USA, 1991,88 : 1143-1147; Prezant et al., Hum. Mutat. , 1992 1: 159-164; Ugozzoli et al., GATA, 1992,9 : 107-112;<BR> Nyren et al., Anal. Biochem. , 1993 208: 171-175). These methods rely on the incorporation of labeled deoxynucleotides to discriminate between bases at a polymorphic site. In such a format, since the signal is proportional to the number of deoxynucleotides incorporated, polymorphisms that occur in runs of the same nucleotide can result in signals that are proportional to the length of the run (Syvanen et al., Amer. J. Hum. Genet. , 1993 52: 46-59).

The present invention also extends to high-throughput forms of primer extension analysis such as, for example, mini-sequencing (Sy Vamen et al., Genomics, 1995, 9 : 341-342). In such a method, a probe or primer (or multiple probes or primers) are immbolized on a solid support (e. g. a glass slide). A biological sample comprising nucleic acid is then brought into direct contact with the probe/s or primers, and a primer extension protocol performed with each of the free nucleotide bases labelled with a different detectable marker. The nucleotide present at a polymorphic site is then determined by determining the detectable marker bound to each probe and/or primer.

1. 6 Denaturing Gradient Gel Electrophoresis Amplification products generated using the polymerase chain reaction can be analyzed by the use of denaturing gradient gel electrophoresis. Different alleles can be identified based on the different sequence-dependent melting properties and electrophoretic migration of DNA in solution (Erlich, ed. , PCR Technology, Principles and

Applications for DNA Amplification, (W. H. Freeman and Co, New York, 1992), Chapter 7).

1. 7 Exon trapping Exon trapping (exon amplification) is a rapid and efficient means of finding expressed DNA sequences in a genome sequence and is based on selection for functional splice sites in genomic DNA. The advantages of exon trapping are that it does not require any prior knowledge about tissue-specific gene expression and can easily be performed on complex genomes. It can identify constitutive exons as well as alternative exons but cannot be used to identify intronless genes.

Exon trapping requires a special type of"exon trap"vector e. g. pSPL1 that contains an artificial mini gene consisting of : (i) a segment of the simian virus 40 (SV40) genome which contains an origin of replication and a powerful promoter sequence; (ii) two splicing-competent exons flanking an intron sequence which contains a multiple cloning site (MCS); and (iii) an SV40 polyadenylation site.

The recombinant DNA is inserted into a restriction site in the MCS and the vector is transfected into a mammalian cell line e. g. monkey COS7 cells. Transcription occurs from the SV40 promoter and the RNA undergoes splicing under the control of the host cell's RNA splicing machinery. The result is that any exon contained in the genomic fragment becomes attached between the upstream and downstream minigene exons.

RT-PCR with primers specific for the minigene exons produces a PCR product containing the genomic DNA which can be seen on agarose gel electrophoresis as products with increasing sizes when compared to vector alone. As the minigene sequence is already known, the nucleotide positions at which the inserted exon starts can be determined by sequencing of the RT-PCR product.

Depending on the size and number of fragments obtained after RT-PCR, several further experiments can be performed. Sequencing, followed by database searches is the most direct way to obtain information from the trapped sequences. Trapped exons are also putative cDNAs and can be used as probes on cDNA libraries or as hybridisation probes in Southern blots, Northern blots or FISH (fluorescence in situ hybridisation).

1. N Single-Strand Conformation Polymorphism Analysis Alleles of target sequences can be differentiated using single-strand conformation polymorphism analysis, which identifies base differences by alteration in electrophoretic migration of single stranded PCR products, as described in Orita et al., Proc. Nat. Acad. Sci. USA, 1989, 86,2766-2770. Amplified PCR products can be generated as described above, and heated or otherwise denatured, to form single stranded amplification products. Single-stranded nucleic acids may refold or form secondary structures which are partially dependent on the base sequence. The different electrophoretic mobilities of single-stranded amplification products can be related to base-sequence differences between alleles of target sequences.

1. 9 Dnase I FootprintingAnalysis Dnase I Footprinting analysis may be performed to detect possible changes in the binding pattern of transcription factors due to the mutations found in the promoter region of the MAPT gene as described in US 6,265, 172.

1. 10 MALDI-TOF mass spectrometry analysis MALDI-TOF MS may be used for typing of single nucleotide polymorphisms using single nucleotide primer extension. Mass spectrometry is also useful for detecting the molecular weight of a short amplified product, wherein a nucleotide change causes a change in molecular weight of the nucleic acid molecule (such a method is also described, for example, in US 6,574, 700).

PCR reactions are performed as described below and the amplicons are further processed in a primer extension reaction.

The extension products can be spotted on microchips and analysed in a MALDI-TOF Mass Spectrometer. Preferably, a laser fires on the spotted products on the microchip and the DNA is accelerated in a vacuum to a detector. Smaller molecules are faster than larger molecules and are detected earlier. The mass of every extended product is determined and can be"translated"into one allele of the polymorphism.

1. 11 Primer Extension Reaction In this reaction, an oligonucleotide is designed to bind directly to the 5'end of the identified polymorphism. Dideoxy nucleotides (ddNTPs), which cannot be elongated, are substituted for one of the 4 deoxy nucleotides (dNTPs) in the reaction mix.

Therefore, if a polymorphism is present the complementary ddNTP will be incorporated and different allele specific fragments will be created. The two extension products therefore have different masses.

1. 12 Extension in solution orsolid-phase using ddNTPs Cohen et al. (French Patent 2,650, 840; WO 91/02087) discuss a solution-based method for determining the identity of the nucleotide of a polymorphic site. As in the Mundy method (i. e. the exonuclease-resistant nucleotide derivatization method) of U. S. Pat.

No. 4,656, 127, a primer is employed that is complementary to allelic sequences immediately 3'to a polymorphic site. The method determines the identity of the nucleotide of that site using labeled dideoxynucleotide derivatives, which, if complementary to the nucleotide of the polymorphic site will become incorporated onto the terminus of the primer.

The solid-phase method described by Goelet et al. (WO 92/15712) uses mixtures of labeled terminators and a primer that is complementary to the sequence 3'to a polymorphic site. The labeled terminator that is incorporated is thus determined by, and complementary to, the nucleotide present in the polymorphic site of the target molecule being evaluated. In contrast to the method of Cohen et al. (French Patent 2,650, 840; WO 91/02087), the method of Goelet. et al. is preferably a heterogeneous phase assay, in which the primer or the target molecule is immobilized to a solid phase. It is thus easier to perform, and more accurate than the method discussed by Cohen et al. (French Patent 2,650, 840; WO 91/02087).

The present invention also encompasses other methods of detecting a polymorphism that is within a regulatory region of the MAPT gene and associated with a neurodegenerative disease such as, for example, SNP microarrays (available from Affymetrix, and/or described, for example, in US 6,468, 743 or Hacia et al., Nature Genetics, 1996,14 : 441), Taqman assays (as described in Livak et al., Nature Genetics, 1995,9 : 341-342), solid phase mini-sequencing (as described in Syvamen et al.,

Genomics, 1992,13 : 1008-1017), mini-sequencing with FRET (as described in Chen and Kwok, Nucleic Acids Res. , 1997,25 : 347-353) or pyromini-sequencing (as<BR> reviewed in Landegren et al. Genome Res. , 1998, 8 (8) : 769-776).

1. 13 Biological samples As methods of the present invention are based upon detection of at least one polymorphism in the regulatory region of the MAPT gene, any cell or sample that comprises genomic DNA is useful for determining a neurodegenerative disease and/or a predisposition to a neurodegenerative disease. Preferably, the cell or sample is derived from a human.

In one example, the method is performed using genomic DNA derived from a biological sample. In another embodiment, the method is performed using mRNA or cDNA derived from the biological sample. In a still further embodiment, the method of the present invention is performed using protein derived from the biological sample.

As the present invention also includes detection of at least one polymorphism in the regulatory region of the MAPT gene that is associated with a neurodegenerative disease in a cell (eg. using immunofluorescence), the term"biological sample"also includes samples that comprise a cell or several cells, whether processed for analysis or not.

As will be apparent to the person skilled in the art, the size of a biological sample will depend upon the detection means used. For example, an assay such as PCR or single nucleotide primer extension may be performed on a sample comprising a single cell, although greater numbers of cells are preferred. Alternative forms of nucleic acid detection may require significantly more cells than a single cell. A person skilled in the art will appreciate that protein-based assays require sufficient cells to provide sufficient protein for an antigen based assay.

The genetic material to be assessed can be obtained from any nucleated cell from the subject. For assay of genomic DNA, virtually any biological sample (other than pure red blood cells) is suitable. For example, convenient samples include whole blood, leukocytes, semen, saliva, tears, urine, faecal material, sweat, skin, testis, placenta, kidney and hair.

The biological sample may also be one or more cells isolated using a method selected from the group consisting of amniocentesis, chorionic villus sampling, fetal blood sampling (e. g. cordocensesis or percutaneous umbilical blood sampling and other fetal tissue sampling (e. g. fetal skin biopsy). Such biological samples are useful for determining the predisposition of a developing embryo to a neurodegenerative disease.

1. 14 Diagnostic aalysis of transcriptional activity of the MAPT gene In another aspect, the present invention relates to a method for predicting the likelihood that a subject will have a neurodegenerative disorder, or for aiding in the diagnosis of a neurodegenerative disorder, comprising the steps of obtaining a biological sample from the subject to be assessed and analyzing the sample for levels of expression of MAPT gene products. Increased levels of expression of one or more MAPT gene products are indicative of a neurodegenerative disorder or predisposition to a neurodegenerative disorder.

The MAPT gene product may be MAPT (mRNA) or cDNA derived therefrom, or Tau protein.

Methods for detecting nucleic acids such as MAPT mRNA or cDNA are known in the art and include for example, hybridization based assays, amplification based assays and restriction endonuclease based assays.

Methods of PCR are known in the art and described, for example, in Dieffenbach and Dveksler (Eds) (In : PCR Primer: A Laboratory Manual, Cold Spring Harbour Laboratories, NY, 1995). Generally, for PCR two non-complementary nucleic acid primer molecules comprising at least about 20 nucleotides in length, and more preferably at least 30 nucleotides in length are hybridized to different strands of a nucleic acid template molecule, and specific nucleic acid molecule copies of the template are amplified enzymatically. PCR products may be detected using electrophoresis and detection with a detectable marker that binds nucleic acids.

Alternatively, one or more of the oligonucleotides are labelled with a detectable marker (e. g. a fluorophore) and the amplification product detected using, for example, a lightcycler (Perkin Elmer, Wellesley, MA, USA). Clearly, the present invention also encompasses quantitative forms of PCR such as, for example, a Taqman assay.

MAPT mRNA may also be measured utilizing DNA array or well-known methods such as the Northern blot method, as well as the RT-PCR method that utilizes oligonucleotides having nucleotide sequences complementary to the nucleotide sequence of the applicable MAPT mRNA.

Levels of Tau protein may be measured by implementing well-known methods such as the Western blot method utilizing an anti-Tau antibody.

In vitro techniques for detection of the Tau protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitation and immunofluorescence. Furthermore, in vivo techniques for detection of protein include introducing into a subject a labeled anti-protein antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

In one example, increased Tau protein production is detected by contacting a sample derived from a subject with a ligand capable of specifically binding to a Tau protein for a time and under conditions sufficient for an ligand/Tau protein complex to form and then detecting the complex wherein increased detection of the complex in the sample compared to a normal control indicates that the subject being tested is predisposed to or suffers from a neurodegenerative disease.

As used herein the term"ligand"shall be taken in its broadest context to include any antibody or fragment thereof, chemical compound, polynucleotide, peptide, protein, lipid, carbohydrate, small molecule, natural product, polymer, etc. that is capable of selectively binding, whether covalently or not, to one or more specific sites on a Tau polypeptide. The ligand may bind to its target via any means including hydrophobic interactions, hydrogen bonding, electrostatic interactions, van der Waals interactions, pi stacking, covalent bonding, or magnetic interactions amongst others.

Preferably the ligand is an anti-Tau antibody. High titer antibodies are preferred, as these are more useful commercially in kits for analytical, diagnostic and/or therapeutic applications. By"high titer"is meant a titer of at least about 1: 103 or 1: 104 or 1: 105.

Methods of determining the titer of an antibody will be apparent to the person skilled in the art. For example, the titer of an antibody in purified antiserum may be determined using an ELISA assay to determine the amount of IgG in a sample. Typically an anti-

IgG antibody or Protein G is used in such an assay. The amount detected in a sample is compared to a control sample of a known amount of purified and/or recombinant IgG.

Alternatively, a kit for determining antibody may be used, e. g. the Easy TITER kit from Pierce (Rockford, IL, USA).

Antibodies directed against the Tau protein may be prepared by any of a variety of techniques known to those of ordinary skill in the art, and described, for example in, Harlow and Lane (In : Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). In one such technique, an immunogen comprising the Tau polypeptide or an immunogenic fragment thereof is initially injected into any one of a wide variety of animals (e. g. , mice, rats, rabbits, sheep, humans, dogs, pigs, chickens and goats). The immunogen may be derived from a natural source, produced by recombinant expression means, or artificially generated, such as by chemical synthesis (e. g. , BOC chemistry or FMOC chemistry).

Monoclonal antibodies specific for the Tau polypeptide may be prepared, for example, using the technique of Kohler and Milstein, Eur. J. Immunol. , 1976,6 : 511-519, and improvements thereto. Briefly, these methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity (i. e. , reactivity with the polypeptide of interest). Such cell lines may be produced, for example, from spleen cells obtained from an animal immunized as described herein. The spleen cells are immortalized by, for example, fusion with a myeloma cell fusion partner, preferably one that is syngenic with the immunized animal. A variety of fusion techniques may be employed, for example, the spleen cells and myeloma cells may be combined with a nonionic detergent or electrofused and then grown in a selective medium that supports the growth of hybrid cells, but not myeloma cells. A preferred selection technique uses HAT (hypoxanthine, aminopterin, thymidine) selection. After a sufficient time, usually about 1 to 2 weeks, colonies of hybrids are observed. Single colonies are selected and growth media in which the cells have been grown is tested for the presence of binding activity against the polypeptide (immunogen). Hybridomas having high reactivity and specificity are preferred.

Monoclonal antibodies are isolated from the supernatants of growing hybridoma colonies using methods such as, for example, affinity purification as described herein.

In addition, various techniques may be employed to enhance the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such

as a mouse. Monoclonal antibodies are then harvested from the ascites fluid or the blood of such an animal subject. Contaminants are removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation, and/or extraction. The marker associated with neurodegeneration of this invention may be used in the purification process in, for example, an affinity chromatography step.

It is preferable that an immunogen used in the production of an anti-Tau antibody is one which is sufficiently antigenic to stimulate the production of high titer antibodies that will bind to the Tau protein. In one embodiment, an immunogen may be the entire Tau protein.

In another embodiment, an immunogen consists of a peptide representing a fragment of the Tau polypeptide. Preferably an antibody raised to such an immunogen also recognizes the full-length Tau protein such as, for example, in its native state or having native conformation.

Alternatively, or in addition, an antibody raised against a peptide immunogen will recognise the full-length Tau protein when the protein is denatured. By"denatured"is meant that conformational epitopes of the protein are disrupted under conditions that retain linear epitopes of the protein. As will be known to a person skilled in the art linear epitopes and conformational epitopes may overlap.

In one embodiment, the method used to determine the amount or level of Tau protein is a semi-quantitative assay.

In another embodiment, the method used to determine the amount or level of Tau protein in a quantitative assay.

As will be apparent from the preceding description, such an method may require the use of a suitable control, e. g. a normal subject or a typical population.

Preferably, the amount of antibody or ligand bound is determined using an immunoassay. Preferably, using an assay selected from the group consisting of, immunohistochemistry, immunofluorescence, ELISA, Western blotting, RIA, a biosensor assay, a protein chip assay, a mass spectrometry assay, a fluorescence

resonance energy transfer assay and an immunostaining assay (e. g. immunofluorescence).

Standard solid-phase ELISA formats are particularly useful in determining the concentration of a protein from a variety of samples.

In one form such an assay involves immobilising a biological sample onto a solid matrix such as, for example a polystyrene or polycarbonate microwell or dipstick, a membrane, or a glass support (e. g. a glass slide).

The antibody is generally labeled with a detectable reporter molecule, such as for example, a fluorescent label (e. g. FITC or Texas Red), a fluorescent semiconductor nanocrystal (as described in US 6,306, 610) or an enzyme (e. g. horseradish peroxidase (HRP), alkaline phosphatase (AP) or ß-galactosidase), alternatively a second labeled antibody can be used that binds to the first antibody. Following washing to remove any unbound antibody the label is detected either directly, in the case of a fluorescent label, or through the addition of a substrate, such as for example hydrogen peroxide, TMB, or toluidine, or 5-bromo-4-chloro-3-indol-beta-D-galaotopyranoside (X Ga in the case of an enzymatic label.

Such ELISA based systems are particularly suitable for quantification of the amount of a protein in a sample, by calibrating the detection system against known amounts of a protein standard to which the antibody binds, such as for example, an isolated recombinant phosphorylation site of a myosin heavy chain polypeptide.

It will be apparent to the skilled person that the assay formats described herein are amenable to high throughput formats such as, for example automation of screening processes, or a microarray format as described in Mendoza et al. (Biotechniques, 1999, 27 (4): 778-788). Furthermore, variations of the above-described assay will be apparent to those skilled in the art such as, for example, a competitive ELISA.

Biosensor devices generally employ an electrode surface in combination with current or impedance measuring elements to be integrated into a device in combination with the assay substrate (such as that described in U. S. Patent No. 5,567, 301). An antibody/ligand that specifically binds to a protein of interest is preferably incorporated onto the surface of a biosensor device and a biological sample contacted to said device.

A change in the detected current or impedance by the biosensor device indicates protein binding to said antibody. Some forms of biosensors known in the art also rely on surface plasmon resonance to detect protein interactions, whereby a change in the surface plasmon resonance surface of reflection is indicative of a protein binding to a ligand or antibody (U. S. Patent No. 5,485, 277 and 5,492, 840).

Biosensors are of particular use in high throughput analysis due to the ease of adapting such systems to micro-or nano-scales. Furthermore, such systems are conveniently adapted to incorporate several detection reagents, allowing for multiplexing of diagnostic reagents in a single biosensor unit. This permits the simultaneous detection of several proteins or peptides in a small amount of body fluids.

Evanescent biosensors are also preferred as they do not require the pre-treatment of a biological sample prior to detection of a protein of interest. An evanescent biosensor generally relies upon light of a predetermined wavelength interacting with a fluorescent molecule such as, for example, a fluorescent antibody attached near the probe's surface, to emit fluorescence at a different wavelength upon binding of the phosphorylation site of, for example, a myosin heavy chain polypeptide to the antibody or ligand.

Micro-or nano-cantilever biosensors are also preferred as they do not require the use of a detectable label. A cantilever biosensor utilizes a ligand and/or antibody capable of specifically detecting the analyte of interest that is bound to the surface of a deflectable arm of a micro-or nano-cantilever. Upon binding of the analyte of interest the deflectable arm of the cantilever is deflected in a vertical direction (i. e. upwards or downwards). The change in the deflection of the deflectable arm is then detected by any of a variety of methods such as, for example, atomic force microscopy, a change in oscillation of the deflectable arm or a change in piezoresistivity. Exemplary micro- cantilever sensors are described in USSN 20030010097.

Alternatively, a biosensor that utilizes a lipid membrane is used. Such a biosensor uses a lipid membrane that incorporates a lipid bilayer that comprises an ion channel or ionophore, wherein the lipid bilayer is tethered to a metal electrode (such biosensors are described in AU 623,747, US 5,234, 566 and USSN 20030143726). One form of such a biosensor involves two receptors or antibodies that bind to each other being incorporated into a lipid bilayer. One of these receptors/antibodies is bound to an ion channel or ionophore that spans the outer half of the membrane, and this

membrane/antibody is also capable of binding to the analyte of interest. The second receptor/antibody is tethered to a membrane molecule (i. e. not the ionophore or ion channel). When the receptors/antibodies are not bound to each other, the ion channel aligns with another half membrane spanning ionophore (i. e. an ionophore that spans the inner half of the membrane) thereby facilitating detectable ion transmission across the membrane. However, when the two receptors/antibodies bind each other, the outer membrane ionophore is displaced thereby disrupting membrane conductivity. The analyte of interest competes with the second receptor/antibody for the binding site on the first receptor/antibody. The presence of the analyte breaks the bond between the two receptors/antibodies and allows the half membrane ionophores to align and provide an ion conductive path.

To produce protein chips, the proteins, peptides, polypeptides, antibodies or ligands that are able to bind specific antibodies or proteins of interest are bound to a solid support such as for example glass, polycarbonate, polytetrafluoroethylene, polystyrene, silicon oxide, metal or silicon nitride. This immobilization is either direct (e. g. by covalent linkage such as, for example, Schiff s base formation, disulfide linkage, or amide or urea bond formation) or indirect. Methods of generating a protein chip are known in the art and are described in for example U. S. Patent Application Nos.

20020136821, 20020192654,20020102617 and U. S. Patent No. 6,391, 625. To bind a protein to a solid support it is often necessary to treat the solid support so as to create chemically reactive groups on the surface such as, for example, with an aldehyde- containing silane reagent. Alternatively, an antibody or ligand may be captured on a microfabricated polyacrylamide gel pad and accelerated into the gel using microelectrophoresis as described in, Arenkov et al. (Anal. Biochem. , 2000, 278 : 123- 131).

A protein chip may comprise only one protein, ligand or antibody, and be used to screen one or more patient samples for the presence of one polypeptide of interest.

Such a chip may also be used to simultaneously screen an array of patient samples for a polypeptide of interest.

Preferably, a protein sample to be analysed using a protein chip is attached to a reporter molecule such as, for example, a fluorescent molecule, a radioactive molecule, an enzyme, or an antibody that is detectable using methods well known in the art.

Accordingly, by contacting a protein chip with a labeled sample and subsequently

washing to remove any unbound proteins the presence of a bound protein is detected using methods well known in the art such as, for example, using a DNA microarray reader.

Alternatively, biomolecular interaction analysis-mass spectrometry (BIA-MS) is used to rapidly detect and characterise a protein present in complex biological samples at the low-to sub-fmole level (Nelson et al., Electrophoresis, 2000,21 : 1155-1163). One technique useful in the analysis of a protein chip is surface enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS) technology to characterise a protein bound to the protein chip. Alternatively, the protein chip is analysed using ESI as described in U. S. Patent Application 20020139751.

The invention also encompasses kits for detecting the presence of Tau proteins or MAPT mRNA. For example, the kit can comprise a labeled compound or agent (e. g., nucleic acid probe) capable of detecting protein or mRNA (or cDNA produced from the mRNA) in a biological sample. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect protein or nucleic acid.

1. 15 Utility of the diagnostic methods of the invention In preferred examples of the present invention, the neurodegenerative disorder is a dementing neurodegenerative disorder. Preferably, the neurodegenerative disorder is a FTDP-17 disorder or a tauopathy. Further preferably, the FTDP-17 disorder or tauopathy is frontotemporal dementia, pallido-ponto-nigral degeneration, cortico-basal degeneration, progressive supranuclear palsy, familial progressive subcortical gliosis, a familial multisystem tauopathy, or any other tauopathy. In another preferred example of the present invention, the neurodegenerative disease is an Alzheimer's Disease or a Parkinson's Disease.

Correlation between a particular phenotype, e. g. , Parkinson's Disease, and the presence or absence of a particular polymorphism in the MAPT gene regulatory region may be performed for a population of subjects who have been tested for the presence or absence of the phenotype. Correlation can be performed by standard statistical methods such as a Chi-squared test and statistically significant correlations between polymorphic form (s) and phenotypic characteristics are noted. For example, as

described herein, it has been found that the frequency of H1/H1 promoter genotype was 1. 2-fold higher in Parkinson's Disease patients compared with normal controls (p <0. 03, X2 test).

This correlation can be exploited in several ways. In the case of a strong correlation between a particular polymorphic form and a neurodegenerative disorder, e. g.

Parkinson's Disease, detection of the polymorphic form in an subject may justify immediate administration of treatment, or at least the institution of regular monitoring of the subject. Detection of a polymorphic form correlated with a disorder in a couple contemplating a family may also be valuable to the couple in their reproductive decisions. For example, the female partner might elect to undergo donor sperm in vitro fertilization to avoid the possibility of transmitting such a polymorphism from her husband to her offspring. In the case of a weaker, but still statistically significant correlation between a polymorphic form and a particular disorder, immediate therapeutic intervention or monitoring may not be justified. Nevertheless, the subject can be motivated to begin simple life-style changes (e. g. , therapy or counselling) that can be accomplished at little cost to the subject but confer potential benefits in reducing the risk of conditions to which the subject may have increased susceptibility by virtue of the particular allele. Furthermore, identification of a polymorphic form correlated with enhanced receptiveness to one of several treatment regimes for a disorder indicates that this treatment regime should be followed for the subject in question.

Furthermore, it may be possible to identify a physical linkage between a genetic locus associated with a trait of interest (e. g. , Parkinson's Disease) and polymorphic markers that are not associated with the trait, but are in physical proximity with the genetic locus responsible for the trait and co-segregate with it. Such analysis is useful for mapping a genetic locus associated with a phenotypic trait to a chromosomal position, and thereby cloning gene (s) responsible for the trait. See Lander et al., Proc. Natl.

Acad. Sci. USA, 1986,83, 7353-7357; Lander et al., Proc. Natl. Acad. Sci. USA, 1987, 84,2363-2367 ; Donis-Keller et al., Cell, 1987,51, 319-337; Lander et al., Genetics, 1989,121, 185-199). Genes localized by linkage can be cloned by a process known as positional cloning. See Wainwright, Med. J. Australia, 1993,159, 170-174; Collins, Nature Genetics, 1992,1, 3-6.

Linkage studies are typically performed on members of a family. Available members of the family are characterized for the presence or absence of a phenotypic trait and for

a set of polymorphic markers. The distribution of polymorphic markers in an informative meiosis is then analyzed to determine which polymorphic markers co- segregate with a phenotypic trait. See, e. g. , Kerem et al., Science, 1989,245, 1073- 1080; Monaco et al., Nature, 1985, 316, 842 ; Yamoka et al., Neurology, 1990,40, 222- 226; and Rossiter et al., FASEB Journal, 1991,5, 21-27.

Linkage is analyzed by calculation of lod (log of the odds) values. A lod value is the relative likelihood of obtaining observed segregation data for a marker and a genetic locus when the two are located at a recombination fraction n, versus the situation in which the two are not linked, and thus segregating independently (Thompson & Thompson, Genetics in Medicine (5th ed, W. B. Saunders Company, Philadelphia, 1991); Strachan, "Mapping the human genome"in The Human Genome (BIOS Scientific Publishers Ltd, Oxford), Chapter 4). A series of likelihood ratios are calculated at various recombination fractions (II), ranging from n=0. 0 (coincident loci) to Il=0. 50 (unlinked). Thus, the likelihood at a given value of II is: probability of data if loci linked at n to probability of data if loci unlinked. The computed likelihoods are usually expressed as the logs of this ratio (i. e. , a lod score). For example, a lod score of 3 indicates 1000: 1 odds against an apparent observed linkage being a coincidence. The use of logarithms allows data collected from different families to be combined by simple addition. Computer programs are available for the calculation of lod scores for differing values of fI (e. g. , LIPED, MLINK (Lathrop, Proc. Nat. Acad. Sci. (USA) 81,<BR> 3443-3446 (1984) ). For any particular lod score, a recombination fraction may be determined from mathematical tables. See Smith et al., Mathematical tables for research workers in human genetics (Churchill, London, 1961); Smith, Ann. Hum.

Genet. , 1968,32, 127-150. The value of fI at which the lod score is the highest is considered to be the best estimate of the recombination fraction.

Positive lod score values suggest that the two loci are linked, whereas negative values suggest that linkage is less likely (at that value of II) than the possibility that the two loci are unlinked. By convention, a combined lod score of +3 or greater (equivalent to greater than 1000: 1 odds in favor of linkage) is considered definitive evidence that two loci are linked. Similarly, by convention, a negative lod score of-2 or less is taken as definitive evidence against linkage of the two loci being compared. Negative linkage data are useful in excluding a chromosome or a segment thereof from consideration.

The search focuses on the remaining non-excluded chromosomal locations.

2. Methods of screening for compounds that reduce or inhibit expression of MAPT gene products 2. 1 Methods of screening for therapeutics based on expressio of MAPT gene products or a reporter gene The present invention also provides a method of screening for a therapeutic for a neurodegenerative disorder, the method comprising determining the level of expression of a MAPT gene product in the presence and absence of a candidate compound, wherein decreased expression of the MAPT gene product in the presence of the compound indicates that the compound is a candidate therapeutic for a neurodegenerative disorder.

It is preferred that the method of screening is performed under conditions of relatively high levels of expression of the MAPT gene products.

Accordingly, in one preferred example, the regulatory region of the MAPT gene has one or more of the following polymorphisms: (i) AATTT at a position equivalent to nucleotides 763-767 of SEQ ID NO: 1; (ii) TT at a position equivalent to nucleotides 793 and 794 of SEQ ID NO: 1; (iii) A at a position equivalent to nucleotide 817 of SEQ ID NO: 1; (iv) G at a position equivalent to nucleotide 1011 of SEQ ID NO: 1; (v) T at a position equivalent to nucleotide 1036 of SEQ ID NO: 1; (vi) C at a position equivalent to nucleotide 1159 of SEQ ID NO: 1; or (vii) A at a position equivalent to nucleotide 1340 of SEQ ID NO: 1.

In another example, the MAPT gene has at least one polymorphism located in exon 1.

Preferably, the polymorphism is located at positions equivalent to nucleotide 1492 or 1583 of SEQ ID NO: 1. Preferably, the polymorphisms are 1492 (C) and/or 1583 (C).

In another example, the regulatory region of the MAPT gene has the sequence as shown in SEQ ID NO: 1.

In another example, the method of screening involves exposing a translation system capable of expressing MAPT to a candidate compound and comparing the levels of expression of MAPT in the presence of the compound to the levels achieved under similar conditions but in the absence of the compound. The translation system may be

a cell-free translation system. Alternatively, the translation system may comprise eukaryotic or prokaryotic cells.

In one example the present invention provides a screening method for a candidate compound which inhibits expression of a MAPT gene product involving the following steps: (i) contacting a candidate compound with cells capable of expressing a MAPT gene product, (ii) measuring the amount of expression of the MAPT gene product in the cells brought into contact with the candidate compound and comparing this amount of expression with the amount of expression (control amount of expression) of the MAPT gene product in the corresponding control cells not brought into contact with the candidate compound, and (iii) selecting a candidate compound showing a reduced amount of expression of the MAPT gene product as compared with the amount of control expression on the basis of the result of the above step (ii).

It will be appreciated that levels of expression of MAPT gene products, such as MAPT mRNA (or cDNA derived therefrom) or Tau protein can be detected or measured in accordance with methods described herein.

The present invention also provides a method of screening for a therapeutic for a neurodegenerative disorder, the method comprising determining the transcriptional activity of the MAPT regulatory region in the presence and absence of a candidate compound, wherein decreased transcriptional activity in the presence of the compound indicates that the compound is a candidate therapeutic for a neurodegenerative disorder.

In one example the present invention provides a screening method for a candidate compound which inhibits reporter expression involving the following steps: (i) contacting a candidate compound with cells which express a reporter gene via a MAPT regulatory region, (ii) measuring the amount of expression of the reporter gene in the cells brought into contact with the candidate compound and comparing this amount of expression with the amount of expression (control amount of expression) of

the reporter gene in the corresponding control cells not brought into contact with the candidate compound, and (iii) selecting a candidate compound showing a reduced amount of expression of the reporter gene as compared with the amount of control expression on the basis of the result of the above step (ii).

In a preferred example of this method of screening, the MAPT regulatory region is operably linked to a reporter gene. Preferably, the reporter gene is chloramphenicol acetyltransferase, B-galactosidase, B-glucuronidase, luciferase, green fluorescent protein, red fluorescent protein, placental alkaline phosphatase, or secreted embryonic alkaline phosphatase.

In a preferred example of this screening method, the MAPT regulatory region has a sequence as shown in SEQ ID NO: 1. The cells may be human derived, or may derive from mammals other than humans such as mice, or from other organisms. Examples of suitable cells are SK-N-MC (ATCC HTB 10) and embryonic kidney 293 cells (ATCC CRL 1573).

The conditions for allowing the candidate compound to come into contact with the cells that can express MAPT gene products or the reporter gene are not limited, but it is preferable to select from among culture conditions (temperature, pH, culture composition, etc. ) which will not kill the applicable cells, and in which the MAPT or reporter gene can be expressed.

The term"reduced"refers not only the comparison with the control amount of expression, but also encompasses cases where no MAPT/reporter gene is expressed at all. Specifically, this includes circumstances wherein the amount of expression of MAPT/reporter gene product is substantially zero.

2.2 Screening protocol based on binding of transcription factors to the MAPT regulatory region In a further example the present invention provides a method of screening for a therapeutic for a neurodegenerative disorder, the method comprising determining the ability of a candidate compound to modulate the binding of a transcription factor to the regulatory region of the MAPT gene, wherein a decreased level of binding of the

transcription factor to the regulatory region in the presence of the compound indicates that the compound is a candidate therapeutic for a neurodegenerative disorder.

Preferably, the transcription factor is S8 or Myc.

In one embodiment, the selected and amplified binding site (SAAB) method may be used to identify compounds which inhibit binding of the transciption factors (Blackwell, et al., Science 250: 1104-1110 (1990) ). By using this method, fragments of the regulatory region may be incubated with purified transcription factors in the presence and absence of candidate compounds. In another embodiment, a method similar to SAAB, multiplex selection technique (MuST) as developed by Nullur et al., (Proc. Natl. Acad. Sci. USA, 1996,93 : 1184-1189) may be used.

2. 3 Screening of natural product libraries The screening methods of the present invention may involve screening small molecule chemodiversity represented within libraries of parent and fractionated natural product extracts, to detect bioactive compounds as potential candidates for further characterization.

Candidate therapeutic agents or compounds encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having molecular weight of more than 100 and less than about 2,500 daltons.

Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids and steroids.

It is generally possible to test 100,000-250, 000 samples during a primary screening phase. With hit rates frequently in the range of 0.1%-1%, the number of bioactive samples identified in a primary screen usually range from several hundred to several thousand. The process of primary screening may involve the use of specialised assay technologies, coupled with automated systems, which allow test sample throughputs of up to 50,000 per day. For example, the screening process may involve the use of a robotic screening system comprising a precise, six-axis robotic arm mounted on a linear track. Such a system links to all instrumentation and hardware, allowing microtitre plates to be transferred to any location on the system. Hardware includes plate carousels for storage and access of sample and assay plates, an automated system with

robotic arm for liquid handling, a platewise microplate pipetting system, a plate shaker and a plate washer. This system also has a plate reader capable of fluorometric, photometric and luminometric detection. In addition to the robotic screening system, a number of stand-alone instruments for rapid microplate pipetting and for detection of a variety of signal read-outs from assay plates may be employed.

The process of dereplication may be used to select a small sub-population of hits identified in a primary screen that are most likely to contain active compounds with the desired characteristics. Experience has shown that dereplication is an important success-determining and rate-limiting step in natural products drug discovery.

Dereplication may be performed as follows. All hits from the initial screen are subjected to a high-capacity fractionation procedure designed to generate information about the relative polarity of all active compounds present. Based on this information, all extracts displaying bioactivity in one or more of these initial fractions are progressed for HPLC, separations using short gradients tailored to provide high resolution over the appropriate polarity ranges. With coupled UV/visible detection of eluates, testing of fractions for bioactivity both in the primary screen and relevant secondary assays, and analysis of active fractions by LC-MS, a package of physicochemical and bioactivity data on pure or nearly pure active HPLC fractions from all screen hits is generated.

Prioritized screen hits emerging from the dereplication process may be progressed for isolation and full chemical characterization of the active compounds present. In the case of microbial extracts, scaled-up quantities of the appropriate extracts may be first prepared by re-fermenting the producing organisms. In the case of plant tissue extracts, there are sufficient stocks of most of the dried and ground plant tissue specimens to prepare further quantities of extract for chemical isolation work.

In a preferred embodiment, the chemical isolation program aims to purify enough of each active compound to conduct structure elucidation work and further profiling of biological activity (typically 2-20 mg). Structures may be determined primarily on the basis of mass spectrometry (MS) and nuclear magnetic resonance (NMR) data.

Preferred bioassays developed as primary screens are also backed up by several secondary assays designed to detect false hits (eg due to interference with the assay detection system), hits due to unrelated modes of action (eg cytotoxicity in functional

cell-based screens) or hits that fail to show the desired profile of biological specificity.

Most secondary assays are reserved for use at a late stage in the dereplication process when they can be applied to pure or nearly pure active fractions derived from the hit extracts identified in primary screens.

2. 4 Characterisation and production of the candidate compounds identified in the methods of screening The present invention extends to the use of any in silico analytical method and/or industrial process for carrying the screening methods described herein into a pilot scale production or industrial scale production of an inhibitory compound identified in such screens. This invention also provides for the provision of information for any such production.

Accordingly, a further example of the present invention provides a process for identifying or determining a candidate compound, said method comprising: (i) performing a screening method as described herein to thereby identify or determine a compound for the treatment of a neurodegenerative disease ; (ii) optionally, determining the structure of the compound; and (iii) providing the compound or the name or structure of the compound such as, for example, in a paper form, machine-readable form, or computer- readable form.

Naturally, for compounds that are known albeit not previously tested for their function using a screen provided by the present invention, determination of the structure of the compound is implicit in step (i). This is because the skilled artisan will be aware of the name and/or structure of the compound at the time of performing the screen.

As used herein, the term'providing the compound"shall be taken to include any chemical or recombinant synthetic means for producing said compound or, alternatively, the provision of a compound that has been previously synthesized by any person or means.

In a preferred embodiment, the compound or the name or structure of the compound is provided with an indication as to its use e. g. , as determined by a screen described herein.

The present invention also provides a process for producing a compound, said process comprising : (i) performing a screening method as described herein to thereby identify or determine a compound for the treatment of a neurodegenerative disease; (ii) optionally, determining the structure of the compound; (iii) optionally, providing the name or structure of the compound such as, for example, in a paper form, machine-readable form, or computer-readable form; and (iv) providing the compound.

Preferably, the synthesized compound or the name or structure of the compound is provided with an indication as to its use e. g. , as determined by a screen described herein.

The present invention also provides a method of manufacturing a compound for the treatment of a neurodegenerative disease comprising: (i) determining a candidate compound for the treatment of a neurodegenerative disease; and (ii) using the compound in the manufacture of a therapeutic or prophylactic for the treatment of a neurodegenerative disease.

Preferably, the method comprises the additional step of isolating the candidate compound. Alternatively, a compound is identified and is produced for use in the manufacture of a compound for the treatment of a neurodegenerative disease.

Formulation of a compound to be administered will depend upon the route of administration selected (e. g. solution, emulsion, capsule). An appropriate composition or medicament comprising the compound can be prepared in a physiological carrier or vehicle (see, generally Remington's Pharmaceutical Sciences, 17th Edition, Mack Publishing Co. , Pa. , 1985).

3. Method of treatment, modes of administration of agents and pharmaceutical compositions The present invention also provides a method of treatment for a neurodegenerative disorder comprising administering to a subject in need thereof an agent that reduces or inhibits levels of Tau in the subject. In one embodiment, the agent is identified by a screening method of the invention as described above. Examples of agents that reduce or inhibit levels of Tau are described below.

3. 1 Antisense compounds, Catalytic nucleic acids and RNA inhibitors The term"antisense compounds"encompasses DNA or RNA molecules that are complementary to at least a portion of a MAPT mRNA molecule and capable of interfering with a post-transcriptional event such as mRNA translation. Antisense oligomers complementary to at least about 15 contiguous nucleotides of MAPT- encoding mRNA are preferred, since they are easily synthesized and are less likely to cause problems than larger molecules when introduced into the target MAPT- producing cell. The use of antisense methods is well known in the art (Marcus-Sakura, Anal. Biochem. 172: 289, 1988). Preferred antisense nucleic acid will comprise a nucleotide sequence that is complementary to at least 15 contiguous nucleotides of a sequence encoding the Tau protein.

The term catalytic nucleic acid refers to a DNA molecule or DNA-containing molecule (also known in the art as a"DNAzyme") or an RNA or RNA-containing molecule (also known as a"ribozyme") which specifically recognizes a distinct substrate and catalyzes the chemical modification of this substrate. The nucleic acid bases in the catalytic nucleic acid can be bases A, C, G, T and U, as well as derivatives thereof. Derivatives of these bases are well known in the art.

Antisense nucleic acid, ribozymes (e. g. Cech et al., US 4,987, 071; Cech et al., US 5,116, 742; Bartel and Szostak, Science, 1993,261, 1411-1418), nucleic acid capable of forming a triple helix (e. g. Helene, Anticancer Drug Res. , 1991,6, 569-584), PNAs<BR> (Hyrup et al., Bioorganic & Med. Chem. , 1996,4, 5-23; O'Keefe et al., Proc. Natl Acad. Sci. USA, 1996,93, 14670-14675), interfering RNAs (Elbashir et al., Nature, 2001, 411, 494-498; Sharp, Genes Devel., 2001,15, 485-490; Lipardi et al., Cell, 2001, 107,297-307 ; Nishikura, Cell, 2001,107, 415-418) or small interfering RNAs (siRNA)

may be produced by standard techniques known to the person skilled in the art, based upon a sequence encoding the Tau protein.

Typically, the catalytic nucleic acid contains an antisense sequence for specific recognition of a target nucleic acid, and a nucleic acid cleaving enzymatic activity (also referred to herein as the"catalytic domain"). To achieve specificity, preferred ribozymes and DNAzymes will comprise a nucleotide sequence that is complementary to at least about 12-15 contiguous nucleotides of a sequence encoding the Tau protein.

The types of ribozymes that are particularly useful in this invention are the hammerhead ribozyme (Haseloff and Gerlach 1988, Perriman et al., 1992) and the hairpin ribozyme (Shippy et al., 1999).

The ribozymes of this invention and DNA encoding the ribozymes can be chemically synthesized using methods well known in the art. The ribozymes can also be prepared from a DNA molecule (that upon transcription, yields an RNA molecule) operably linked to an RNA polymerase promoter, e. g. , the promoter for T7 RNA polymerase or SP6 RNA polymerase. Accordingly, also provided by this invention is a nucleic acid molecule, i. e. , DNA or cDNA, coding for the ribozymes of this invention. When the vector also contains an RNA polymerase promoter operably linked to the DNA molecule, the ribozyme can be produced in vitro upon incubation with RNA polymerase and nucleotides. In a separate embodiment, the DNA can be inserted into an expression cassette or transcription cassette. After synthesis, the RNA molecule can be modified by ligation to a DNA molecule having the ability to stabilize the ribozyme and make it resistant to RNase. Alternatively, the ribozyme can be modified to the phosphothio analog for use in liposome delivery systems. This modification also renders the ribozyme resistant to endonuclease activity. dsRNA is particularly useful for specifically inhibiting the production of a particular protein. Although not wishing to be limited by theory, Dougherty and Parks (1995) have provided a model for the mechanism by which dsRNA can be used to reduce protein production. This model has recently been modified and expanded by Waterhouse et al. (1998). This technology relies on the presence of dsRNA molecules that contain a sequence that is essentially identical to the mRNA of the gene of interest, in this case an mRNA encoding the Tau protein. Conveniently, the dsRNA can be produced in a single open reading frame in a recombinant vector or host cell, where the

sense and anti-sense sequences are flanked by an unrelated sequence which enables the sense and anti-sense sequences to hybridize to form the dsRNA molecule with the unrelated sequence forming a loop structure. The design and production of suitable dsRNA molecules targeted against MAPT is well within the capacity of a person skilled in the art, particularly considering Dougherty and Parks (1995), Waterhouse et al. (1998), WO 99/32619, WO 99/53050, WO 99/49029, and WO 01/34815.

As used herein, the terms"small interfeing RNA", and"RNAi"refer to homologous double stranded RNA (dsRNA) that specifically targets a gene product, thereby resulting in a null or hypomorphic phenotype. Specifically, the dsRNA comprises two short nucleotide sequences derived from the target RNA encoding MAPT and having self-complementarity such that they can anneal, and interfere with expression of a target gene, presumably at the post-transcriptional level. RNAi molecules are described by Fire et al., Nature 391, 806-811, 1998, and reviewed by Sharp, Genes & Development, 1999,13, 139-141).

Other examples of agents that reduce or inhibit levels of Tau are proteins or small molecules that interfere with transcription and/or translation of the MAPT gene.

3.2 Pharmaceutical compositions and methods of administration In the case where the agent is in the form of a low molecular weight compound, a peptide or a protein, the substance can be formulated into the ordinary pharmaceutical compositions (pharmaceutical preparations) which are generally used for such forms, and such compositions can be administered orally or parenterally.

Generally speaking, the following dosage forms and methods of administration can be utilized: tablets, troches, pills, powders, fine powders, granules, and capsules, and liquid preparations, e. g. solutions, suspensions, emulsions, syrups, and elixirs. These forms can be classified by the route of administration into said oral dosage forms or various parenteral dosage forms such as transnasal preparations, transdermal preparations, rectal preparations (suppositories), sublingual preparations, vaginal preparations, injections (intravenous, intraarterial, intramuscular, subcutaneous, intradermal, in particular intracerebral injection, more particularly intracerebral injection into the substantia nigra) and drip injections. The oral preparations, for instance, may for example be tablets, troches, pills, powders, fine powders, granules,

capsules, solutions, suspensions, emulsions, syrups, etc. and the rectal and vaginal preparations include tablets, pills, and capsules, among others. The transdermal preparations may not only be liquid preparations, such as lotions, but also be semi-solid preparations, such as reams, ointments, and so forth.

The injections may be made available in such forms as solutions, suspensions and emulsions, and as vehicles, sterilized water, water-propylene glycol, buffer solutions, and saline of 0.4 % weight/volume concentration can be mentioned as examples. These injections, in such liquid forms, may be frozen or lyophilized. The latter products, obtained by lyophilization, are extemporaneously reconstituted with distilled water for injection or the like and administered. The above forms of pharmaceutical composition (pharmaceutical preparation) can be prepared by formulating the compound having MAPT inhibitory action and a pharmaceutically acceptable carrier in the manner established in the art. The pharmaceutically acceptable carrier includes various excipients, diluents, fillers, extenders, binders, disintegrators, wetting agents, lubricants, and dispersants, among others. Other additives which are commonly used in the art can also be formulated. Depending on the form of pharmaceutical composition to be produced, such additives can be judiciously selected from among various stabilizers, fungicides, buffers, thickeners, pH control agents, emulsifiers, suspending agents, antiseptics, flavours, colours, tonicity control or isotonizing agents, chelating agents and surfactants, among others.

The dosage and dosing schedule of such a pharmaceutical preparation vary with the dosage form, the disease or its symptoms, and the patient's age and body weight, among other factors, and cannot be stated in general terms. The usual dosage, in terms of the daily amount of the active ingredient for an adult human, may range from about 0.0001 mg to about 500 mg, preferably about 0.001 mg to about 100 mg, and this amount can be administered once a day or in a few divided doses daily.

When the substance having a Tau production diminution effect is in the form of a polynucleotide such as an antisense compound, the composition may be provided in the form of an agent for gene therapy or a prophylactic agent. Recent years have witnessed a number of reports on the use of various genes, and gene therapy is by now an established technique.

The agent for gene therapy can be prepared by introducing the polynucleotide of present interest of interest into a vector or transfecting appropriate cells with the vector.

The modality of administration to a patient is roughly divided into two modes, viz. the mode applicable to (1) the case in which a non-viral vector is used and the mode applicable to (2) the case in which a viral vector is used. Regarding the case in which a viral vector is used as the vector and the case in which a non-viral vector is used, respectively, both the method of preparing an agent for gene therapy and the method of administration are dealt with in detail in several books relating to experimental protocols [e. g."Bessatsu Jikken Igaku, Idenshi Chiryo-no-Kosogijutsu (Supplement to Experimental Medicine, Fundamental Techniques of Gene Therapy), Yodosha, 1996; Bessatsu Jikken Igaku: Idenshi Donyu & Hatsugen Kaiseki Jikken-ho (Supplement to Experimental Medicine : Experimental Protocols for Gene Transfer & Expression Analysis), Yodosha, 1997; Japanese Society for Gene Therapy (ed. ) : Idenshi Chiryo Kaihatsu Kenkyn Handbook (Research Handbook for Development of Gene Therapies), NTS, 1999, etc.].

When using a non-viral vector, any expression vector capable of expressing the anti- MAPT nucleic acid may be used. Suitable examples include pCAGGS [Gene 108, 193-200 (1991)], pBK-CMV, pcDNA 3.1, and pZeoSV (Invitrogen, Stratagene).

Transfer of a polynucleotide into the patient can be achieved by inserting the polynucleotide of present interest of interest into such a non-viral vector (expression vector) in the routine manner and administering the resulting recombinant expression vector. By so doing, the polynucleotide of present interest of interest can be introduced into the patient's cells or tissue.

More particularly, the method of introducing the polynucleotide into cells includes the calcium phosphate transfection (coprecipitation) technique and the DNA (polynucleotide) direct injection method using a glass microtube, among others.

The method of introducing a polynucleotide into a tissue includes the polynucleotide transfer technique using internal type liposomes or electrostatic type liposomes, the HVJ-liposome technique, the modified HVJ-liposome (HVJ-AVE liposome) technique, the receptor-mediated polynucleotide transfer technique, the biolistic technique which comprises transferring the polynucleotide along with a vehicle (metal particles) into

cells with a particle gun, the naked-DNA direct transfer technique, and the transfer technique using a positively charged polymer, among others.

Suitable viral vectors include vectors derived from recombinant adenoviruses and retroviruses. Examples include vectors derived from DNA or RNA viruses such as detoxicated retrovirus, adenovirus, adeno-associated virus, herpesvirus, vaccinia virus, poxvirus, poliovirus, sindbis virus, Sendai virus, SV40, human immunodeficiency virus (HIV) and so forth. The adenovirus vector, in particular, is known to be by far higher in infection efficiency than other viral vectors and, from this point of view, the adenovirus vector is preferably used.

Transfer of the polynucleotide into the patient can be achieved by introducing the polynucleotide of present interest into such a viral vector and infecting the desired cells with the recombinant virus obtained. In this manner, the polynucleotide of present interest can be introduced into the cells.

The method of administering the thus-prepared agent for gene therapy to the patient includes the in vivo technique for introducing the agent for gene therapy directly into the body and the ex vivo technique which comprises withdrawing certain cells from a human body, introducing the agent for gene therapy into the cells in vitro and returning the cells into the human body [Nikkei Science, April, 1994 issue, 20-45; Pharmaceuticals Monthly, 36 (1), 23-48,1994 ; Supplement to Experimental Medicine, 12 (15), 1994; Japanese Society for Gene Therapy (ed. ) : Research Handbook for Development of Gene Therapies, NTS, 19991]. For use in the prevention or treatment of an inflammatory disease to which the present invention is addressed, the agent is preferably introduced into the body by the in vivo technique.

When the in vivo method is used, the agent can be administered by a route suited to the particular neurodegenerative disorder. For example, it can be administered intravenously, intra-arterially, subcutaneously or intramuscularly, for instance, or may be directly administered topically into the affected tissue.

The agent for gene therapy can be provided in a variety of pharmaceutical forms according to said routes of administration. In the case of an injectable form, for instance, an injection can be prepared by an established procedure, for example by dissolving the active ingredient polynucleotide in a solvent, such as a buffer solution,

e. g. PBS, physiological saline, or sterile water, followed by sterilizing through a filter where necessary, and filling the solution into sterile vitals, Where necessary, this injection may be supplemented with the ordinary carrier or the like. In the case of liposomes such as HVJ-liposome, the agent can be provided in various liposome- entrapped preparations in such forms as suspensions, frozen preparations and centrifugally concentrated frozen preparations.

Furthermore, in order that the product of the gene therapy may be easily localized in the neighbourhood of the affected site, a sustained-release preparation (eg. a minipellet) may be prepared and implanted near the affected site or the agent may be administered continuously and gradually to the affected site by means of an osmotic pump or the like.

The polynucleotide content of the agent for gene therapy can be judiciously adjusted according to the disease to be treated, the patient's age and body weight, and other factors but the usual dosage in terms of each polynucleotide is about 0.0001 to about 100 mg. preferably about 0.001 to about 10 mg. This amount is preferably administered several days or a few months apart.

The present invention will now be illustrated by the following Examples, which are not intended to be limiting in any way. The teachings of all references cited herein are incorporated herein by reference.

Example 1: Assessing the effect of polymorphisms located upstream of the equivalent of nucleotide-226 in the regulatory region of the gene encoding the microtubule associated protein Tau and in exon 1 on gene expression The late-onset idiopathic PD patient group comprised 206 subjects recruited from movement disorders clinics throughout Queensland as described previously (Buchanan et al., Neurosci Lett 2002; 327: 91-94). Probable PD was diagnosed by a specialist neurologist in accordance to the criteria of Calne et al. (Neurol 1992; 32 (suppl): S125- S127). Patients had an average age of 71 years, with an average onset age of 62 years.

Unaffected control subjects were matched to PD cases for age, gender and area of residence (Buchanan et al., Neurosci Lett 2002,327 : 91-94). The early-onset familial cohort of PD patient group comprised 13 probands who fulfilled three of the following five criteria, bradykinesia, muscular rigidity, rest tremor, improvement >30% with L-

dopa and asymmetry of signs at onset (Litvan et al., Movement Disorders, 2003, 18 : 467-486). The average age of onset for these probands was 52 years.

Mutation screen ofMAPTgene All coding exons of MAPT were amplified by PCR from genomic DNA using primers derived from 5'and 3'ionic sequences as described (Stanford et al., Brain, 2000, 123: 880-893). Approximately 800bp region upstream of the transcription start site of MAPT was screened using two overlapping primer pairs: PromlF 5'- cgaccagcagaatgaggaccact-3' (SEQ ID NO: 4) , PromlR 5'-AGAAGTCCTGAGCGGCCT TCCAC-3' (SEQ ID NO: 5) which amplifies a 463bp product and Prom2F 5'-AAGGAA GCAGCCTGGGGGAAAGA-3' (SEQ ID NO: 6), Prom2R 5'-GCGCTTACCTGATAGTCGACAGA-3' (SEQ ID NO: 7) which amplifies a 690bp product. Reactions were carried out in 251l1 volume, using lpmol/, l primer, 1 X reaction buffer (PE Biosystems, Forster City, CA), 1. 5mM MgCl2, 0.2mM dNTPs, 5% dimethylsulfoxide, 1 unit of AmpliTaq Gold (PE Biosystems, Foster City, CA) and 100ng of genomic DNA as template. PCR conditions were as follows: denaturation at 94°C for 12 minutes, followed by 38 cycles of amplification (30sec at 94°C, 30sec at 56°C and 1 minute at 72°C). Each sequence was analysed using the Sequencing Analysis software (version 3.3) and analysed by the Autoassembler software (version 2.1) (Applied Biosystems, Foster City, CA).

Luciferase Reporter Gene Assay A 1039bp DNA fragment comprising approximately 808bp of the promoter and 231bp of the 5'untranslated region of the MAPT gene was subcloned into the pGL3-Basic Luciferase vector (Promega, Madison, WI). Each of the promoter haplotypes, HI, H1' and H2, were assayed for transcriptional efficiency. Each recombinant vector was transiently transfected into the neuroblastoma cell line, SK-N-MC (ATCC HTB 10) and embryonic kidney 293 cells (ATCC CRL 1573) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to manufacturers instructions. Following transfection, cells were left for 48 hours before being lysed with TBS (50mM Tris. HCl (pH 7.4), 150mM NaCI and 1X complete cocktail protease inhibitor (Roche, Mannheim, Germany) ). Cell lysates were assayed for luciferase activity using the Bright-Glo Luciferase assay system (Promega) according to the manufacturer's instructions.

Single Nucleotide Polymorphisms Analysis The Celera database (http : //www. celera. com) and the UCSC Genome Bioinformatics Site (http ://genome. ucsc. edu) were used to identify genes adjacent to MAPT. Each flanking gene was examined for known SNPs using the CHIP Bioinformatics Tool (http://bio. chip. org : 8080/bio/). For each gene, two verified SNPs were amplified by PCR as follows: the SNP rs701984 SNP (OSBPL7. SNP1-F 5'TGCGTTATTGGCTGTGCTCTC-3' (SEQ ID NO : 8), OSBPL7. SNP1-R 5'-GACATCTGAAATGGAATCCTCTGC-3' (SEQ ID NO : 9)) and SNP rs2017884 (OSBPL7. SNP2-F 5'-GTGGCAGAACAGGGACTTGAAC-3' (SEQ ID NO : 10), OSBPL7. SNP2-R 5'-AGGGAGGACAACCGTGTAAACC-3' (SEQ ID NO : 11)) from the OSBPL7 gene ; the SNP rsl71440 (CRHR1. SNP2-F 5'-GGTAGATGTGGTTTGATTGCAGAG-3' (SEQ ID NO : 12), CRHR1. SNP2-R 5'-CAAAGAAGACACAAGGGGAAGAAA-3' (SEQ ID NO : 13)) and SNP rsl396862 (CRHR1. SNP3-F 5'-TGAAACGGATTCTGGGGGTC-3' (SEQ ID NO : 14), CRHR1. SNP3-R 5'-TCTGGGTTGGTGGCAATGAG-3' (SEQ ID NO : 15)) from the CRHR1 gene ; the SNP HCV448079 (NSF. SNP1-F 5'-AAGTGAGGGGGAAATGAGGTCTC-3' (SEQ ID NO : 16), NSF. SNP1-R 5'-GACAGGGAAAAGGCAGAAGGTAG-3' (SEQ ID NO : 17)) and SNP HCV44076 (NSF. SNP2-F 5'-GTTGGGCTGTCTGTCCATTCTG-3' (SEQ ID NO : 18), NSF. SNP2-R 5'-TGCGGTGCTGTTGTGTATTGG-3' (SEQ ID NO : 19)) from the NSF gene ; and

the SNP HCV2261819 (WNT3. SNP1-F 5'CTTCAAGGAATAGGTGTGCTGAGG-3' (SEQ ID NO: 20), WNT3. SNP1-R 5'-TGGGAGACTGACTTAGGGTTTGG-3' (SEQ ID NO: 21) ) and SNP HCV11623713 (WNT3. SNP2-F 5'-GCTGAGGAGCCAAAGAACATAGTC- 3' (SEQ ID NO: 22), WNT3. SNP2-R 5'-AGCCTGAGTGACAGAGCAAGAATC-3' (SEQ ID NO: 23) ) from the WNT3 gene. PCR conditions were as described above.

Each SNP was detected by direct sequencing of PCR product as described above.

Results MAPTPromoter Haplotype consists of a series of Polymorphisms We examined the upstream region of MAPT for polymorphisms in the promoter region by direct DNA sequence analysis in a cohort of 12 normal subjects. The MAPT promoter sequence used for our analysis was taken from the most recent complete genomic contig sequence obtained from Genbank (Accession Number # NT_010748).

We detected nine biallelic variants within a 1039bp region comprising 808bp of the promoter and 231bp of the 5'untranslated region of the MAPT gene (Figure 1A). Five SNPs were detected as well as four microdeletions and insertions of 1 to 11 bases in size (Figure 1A). The G/C SNP at position-226 and A/G SNP at position 45 correspond to the previously described-221 and-44 SNPs (Ezquerra et al., Neurosci Lett, 1999,275 : 183-186; De Silva et al., Neurosci Lett, 2001,311 : 145-148). This is due to discrepancies between the nucleotide sequence used by de Silva et al. (Neurosci Lett, 2001,311 : 145-148) and NT 010748.

We found that the series of biallelic variants formed the two major promoter haplotypes, H1 and H2. These were contiguous with the previously reported H1 and H2 haplotypes in the coding region of MAPT (Baker et al., Hum Mol Genet, 1999,8 : 711- 715; Higgins et al., Neurology, 2000,55 : 1364-1367). An insertion of a'T'nucleotide at position-568 distinguished the H1 haplotype from its minor variant H1'haplotype (Figure 1A). We confirmed the phase of the promoter haplotypes by subcloning and sequencing entire PCR products that encompass all the promoter biallelic variants. We did not identify a single recombination event within our promoter haplotypes in any of our unrelated patient and control subjects (>200 chromosomes), suggesting that the polymorphisms are in strong linkage disequilibrium with each other. We also looked

for recombination events between the promoter haplotypes and two exon 9 polymorphisms (a novel G/A intronic polymorphism at position-25 and the previously reported Ala227Ala polymorphism (Baker et al., Hum Mol Genet, 1999) ) in our case- control cohorts. We detected only four recombination events in 154 chromosomes, a frequency of 0.03. Our data strongly suggests that the promoter haplotypes form part of an extended haplotype with the entire MAPT gene. This is consistent with a previous study that showed significant association between the-226 promoter SNP and the AO microsatellite marker allele in intron 9 of MAPT (Higgins et al., Neurology, 2000, 55: 1364-1367).

Frequency of the Tau promoter Haplotypes differs significantly in Case-Control Cohorts of late-onset PD patients.

The extended MAPT haplotype has been shown to be associated with late-onset PD in a several independent cohorts (Martin et al., J Am Med Assoc, 2001,286 : 2245-2250; Maraganore et al., Ann Neurol, 2001,50 : 658-661; Golbe et al., Movement Disorder 2001,16 : 442-447; Pastor et al., Ann Neurol, 2000,47 : 242-245). We examined whether the frequencies of the MAPT promoter haplotypes were altered in a cohort of late-onset PD patients. The frequency of H1/H1 promoter genotype was 1. 2-fold higher in PD patients compared with normal controls (p <0.03, X2 test). This difference in allele frequency between PD and normal control is comparable to values (1. 1- to 1.2- fold) seen in other studies (Martin et al., J Am Med Assoc, 2001,286 : 2245-2250; Maraganore et al., Ann Neurol, 2001,50 : 658-661; Golbe et al., Movement Disorder, 2001,16 : 442-447; Pastor et al., Ann Neurol, 2000,47 : 242-245). When allele frequencies were analysed, we found that the H1 haplotype was over-represented in the PD cohort (p = 0.08, % 2) compared with the normal controls. Conversely, the frequency of the H1'and H2 promoter haplotypes were 0. 8- to 0. 9- fold in the PD cohort compared with the normal controls (Table 1).

A cohort of 13 probands with early-onset PD was also examined for mutations in all coding exons of MAPT. We did not detect any nucleotide substitutions that would be predicted to lead to changes in amino acid sequence or exon skipping. Well- characterised polymorphisms in the MAPT gene (Baker et al., Hum Mol Genet, 1999, 8: 711-715), corresponding to the two ancestral haplotypes were detected in this early- onset PD cohort. The frequency of the H1/H1 haplotype in the early-onset cohort (0.68) was 1. 6- fold higher compared with controls (0.43).

MAPTHaplotype Does Not extend beyond the Gene The SNPs detected within the MAPT gene appear to be in strong linkage disequilibrium and form two distinct haplotypes which are contiguous for over 100kb (Baker et al., Hum Mol Genet, 1999,8 : 711-715; Higgins et al., Neurology, 2000, 55: 1364-1367). However, the extent of this block of linkage disequilibrium has not been determined. Likewise, it is unknown whether the flanking genes are in linkage disequilibrium with MAPT and may be the result of significant association with PD.

There are several genes in close proximity to MAPT (Figure 2A) which have been implicated in neurological diseases, including the N-ethylmaeimide-sensitive factor (NSF) gene (Mimics et al., Neuron, 2000,28 : 53-67) which has been implicated in schizophrenia and the corticotropin-releasing factor receptor (CRFR1) gene, which may be involved in increased susceptibility to alcoholism (Sillaber et al., Science, 2002,296 : 931-933). We constructed haplotypes using two SNPs from the two genes flanking either side of MAPT (Figure 2A). This was used to infer whether any ancestral recombinations have occurred between the MAPT promoter haplotype and SNPs within adjacent genes in our panel of 12 normal subjects. As shown in Figure 2B, the number of chromosomal haplotypes which are contiguous with the MAPT promoter rapidly decreases with increasing physical distance. We infer from this that linkage disequilibrium does not include any entire gene in addition to MAPT.

Functional Analysis of the MAPTPromoter Haplotypes We examined the sequence around the transcription start site of MAPT for possible binding sites of transcription factors using the MatInspector v2.2 software and the TRANSFAC 4.0 database (Quandt et al., Nucleic Acids Res, 1995,23 : 4878-48840).

Using a high stringency of selection (maximal'Core similarity'setting of 1 and'Matrix similarity'of 0.9), a series of binding sites were detected for common transcription factors including AP-1 and SP-1 (Figure 1A). Two of the MAPT promoter variants, the deletion of a five base pair sequence (AATTT) and the G/C SNP at position-373, were predicted to alter the binding sites of the S8 and myc transcription factors respectively (Figure 1A). As both of these variants lie on the H2 haplotype, we examined the ability of each promoter haplotype to drive the expression of a luciferase reporter gene.

Three fragments containing all of the biallelic variants of each promoter haplotype were analysed for transcriptional efficiency in two human cell lines SK-N-MC and 293 using the luciferase reporter gene assay. Luciferase activity associated with each promoter haplotype was determined 48 hours post transfection. As shown in Figure 1B, the H2 haplotype was associated with a significant 1. 2-fold reduction in transcriptional efficiency relative to the H1 haplotype in 293 (p < 0.02, student t test) and SK-N-MC (p < 0.005, student t test) cell lines. The H1'promoter haplotype, which differs from the H1 haplotype by a single T nucleotide insertion at position-623, also showed a 1.1- fold decrease in transcriptional efficiency relative to the H1 promoter in SK-N-MC cells (p < 0.05, student t test), but not in 293 cells. This difference in the transcriptional efficiency of H1'haplotype may reflect cell type specific differences between the 293 and SK-N-MC cell lines.

DISCUSSION Late-onset idiopathic PD is considered to be a complex disease, where the interaction between multiple genetic loci and environmental factors can lead to the disease phenotype (Shastry, Neurosci Res 2001,41 : 5-12). This is in contrast to early-onset PD, where autosomal dominantly inherited mutations in genes such as Parkin and a- synuclein are important (Lansbury, Curr Op Genet Devel, 2002,12 : 299-306). One genetic locus which may be important in idiopathic PD is the MAPT gene on chromosome 17q. This locus has been implicated in several independent studies, including a genome-wide scan of late-onset PD families which identified a chromosomal region on 17q which co-localised with MAPT (Scott et al., J Am Med Assoc, 2001,286 : 2239-2244) and a number of case-control studies which demonstrated significant association between MAPT polymorphisms and idiopathic PD patients (Martin et al., J Am Med Assoc, 2001,286 : 2245-2250; Maraganore et al., Ann Neurol, 2001,50 : 658-661 ; Golbe et al., Movement Disorder, 2001,16 : 442-447; Pastor et al., Ann Neurol, 2000,47 : 242-245). We identified a series of biallelic variants in the promoter region of the MAPT gene, which are in complete linkage disequilibrium with each other and form two major haplotypes H1 and H2. We also identified a minor variant of H1 termed H1'. We have demonstrated that the three promoter haplotypes drive the expression of a luciferase reporter gene with significantly different efficiencies. We showed that the H1 promoter haplotype, which is the strongest at initiating transcription, is significantly over-represented in idiopathic PD patients compared with normal controls. We also found that the H2 and H1'

haplotype, which have decreased transcriptional efficiencies, were present with lower frequencies in our PD cohort.

The role of MAPT in PD has been controversial. Dramatic changes to Tau structure and function, such as those associated with missense or splicing mutations effects, give rise to autosomal dominant early-onset phenotypes of frontotemporal dementias with parkinsonism (Hutton et al., Nature, 1998,393 : 702-705), CBD (Spillantini et al., Ann Neurol, 2000,48 : 939-943) and PSP (Stanford et al., Brain, 2000,123 : 880-893).

Mutation screening of a cohort of early-onset PD probands failed to reveal any MAPT mutations. However, susceptibility alleles for complex disorders such as late-onset idiopathic PD are likely to be of smaller effect and insufficient to cause PD directly.

Thus, although the differences in promoter strength between the three MAPT haplotypes are small (1. 1-to 1. 2-fold), this is expected for the cumulative or interactive small effects that may be seen for a complex disease gene variant. This study, in agreement with other groups, has shown only a small difference in frequency of the H1 haplotype (1. 1- to 1. 2-fold) between PD and control cohorts (Martin et al., J Am Med Assoc, 2001,286 : 2245-2250; Maraganore et al., Ann Neurol, 2001,50 : 658-661; Golbe et al., Movement Disorder, 2001,16 : 442-447; Pastor et al., Ann Neurol 2000, 47: 242-245). This is in contrast with the strong association between the H1 haplotype and the two tauopathies CBD and PSP, where several studies have shown a 1. 4- to 1.8- fold difference in frequencies between disease and control cohorts (Baker et al., Hum Mol Genet, 1999,8 : 711-715; De Maria et al., Ann Neurol, 2000,47 : 374-377; Higgins et al., Neurology, 2000,55 : 1364-1367; Ezquerra et al., Neurosci Lett, 1999,275 : 183- 186; Houlden et al., Neurology, 2001, 56: 1702-1706; De Silva et al., Neurosci Lett, 2001,311 : 145-148). This is not unexpected as CBD and PSP are characterised neuropathologically by intracellular Tau deposits (Spillantini and Geodert, Trends Neurosci, 1998,21 : 428-433). It is conceivable that MAPT plays a more direct biochemical role in these tauopathies whereby an increase in MAPT expression, due to the inheritance of the H1 promoter haplotype, directly results in the increased deposition of Tau protein. Conversely, other genes in addition to MAPT may be important in determining the PD phenotype.

The presence of Lewy Bodies is currently an important diagnostic feature of PD. The main constituent of Lewy bodies is a-synuclein (Baba et al., Am J Pathol, 1998,152 : 879-884) and the current consensus is that Tau is absent from these lesions (Pollanen and Bergeron, J Neuropathol Exp Neurol, 1993,52 : 183-191; Schwab et al., Neurobiol

Aging, 2000,21 : 503-510). However, Tau immunoreactivity has recently been detected in some brain stem Lewy bodies (Arima et al., Brain Res, 1999,843 : 53-61; Ishizawa et al., J Neuropathol Exp Neurol, 2003,62 : 389-97). It has been suggested that there is an interaction between the negatively charged carboxy-terminal of a- synuclein and the positively charged microtubule region of Tau in vitro (Jensen et al., J Biol Chem, 1999,274 : 25481-25489). Our study suggests that the putative increase in expression of MAPT, in subjects with the H1/H1 genotype, may indirectly give rise to Lewy bodies by increasing the propensity of a-synuclein to aggregate in PD brains. Our results show that Tau protein levels are an important factor in the aetiology of idiopathic PD.

Example 2. Level of Tau transcripts in brain RNAs.

Total RNAs were isolated from 38 brain samples (cerebellum) using either Trizol reagent (Sigma) or SV Total RNA Isolation kit (Promega). For each sample, approximately 1 slg of RNA was reverse transcribed using an oligo dT primer, followed by Dnase 1 digestion. 0. 1 gag of brain cDNA was then used to amplify Tau transcript using primers which flank the constitutively spliced exon 9 sequence (Tau-RT-F : 5'- CAGGTGAACTTTGAACCAGG-3' (SEQ ID NO: 24) and TAU-RT-R : 5'- GGAGGAGACATTGCTGAGAT-3' (SEQ ID NO: 25) ). The absolute amount of Tau transcripts in each sample was quantified by real-time PCR (SYBR-Green chemistry).

The difference in total RNA levels for each sample was normalised to each other by measuring their absolute levels of p-actin transcripts. The Tau promoter haplotype was determined by a MspI restriction digest polymorphism that detects the-373 T/C single nucleotide polymorphism within the haplotype (Kwok et al., 2004, Annals Neurol. , 55: 329-334).

We observed a 3-fold difference (p = 0. 06, Student's T test) in Tau RNA levels between the two homozygote genotypes H1/H1 and H2/H2 (Figure 3).

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.