Patent 6,781,029

A transgenic animal, preferably a mouse, that expresses human antichymotrypsin (ACT) in brain tissues is provided, together with animal tissue-derived cell lines and progeny animals of said transgenic animal. Progeny are obtained by mating the transgeny animal with select animal strains used as models of Alzheimer's disease, related neurological disorders, or amyloidogenic diseases. Methods utilizing the parent and progeny animals and cells derived therefrom are disclosed for testing compounds for use as anti-inflammatory drugs, inhibitors of amyloidogenesis, and/or inhibitors of tau protein pathology associated with Alzheimer's disease, in the treatment of a variety of neurological diseases.

SUMMARY OF THE INVENTION

It is therefore a feature and advantage of the present invention to provide a transgenic mammal, preferably a mouse, which serves as a model for Alzheimer's disease and related neurological disorders having a pathology comprising amyloid plaque formation. This transgenic animal carries an exogenous polynucleotide which has a coding sequence functionally equivalent of the DNA sequence of a protease inhibitor such as, for example, human antichymotrypsin (ACT), together with DNA sequences directing its expression, preferably a glial fibrillary acidic protein (GFAP) promoter and 5'0 UTR, most preferably modified to remove ATG start codons from its sequence. The mammalian polynucleotide can be in the form of DNA, genomic DNA, cDNA, mRNA and various fragments and portions of the gene sequence encoding ACT. Accordingly, the transgenic mouse exhibits one or more of the symptoms of cognitive memory loss and/or behavioral disturbances, amyloid accumulation, neuronal cell death or synapse loss, formation or aggregation of abnormal protein filaments, or phosphorylation of one or more proteins related to Alzheimer's disease such as tau. In addition or alternatively, the symptoms can appear as another cellular tissue disorder such as in mouse liver, kidney, spleen, bone marrow or other organs in which the human ACT gene product is expressed.

According to the second embodiment of the invention, animals, primary cell cultures, and cell lines are provided which derive from the parent transgenic animal carrying ACT gene. Accordingly these descendant animals, primary cell cultures and cell lines, which are defined hereinafter as a "progeny", are either homozygous or heterozygous for ACT allele.

Accordingly, in another embodiment, the transgenic ACT animal expresses one or more additional transgenes of proteins whose expression may be associated with Alzheimer's disease or related neurological disorders, where the second transgene encodes a normal, mutant, or altered gene encoding, for example, tau-1, apolipoprotein E, APP, presenilin 1, presenilin 2, IL-1 alpha, or IL-1 beta.

As another embodiment of the invention a screening method is provided wherein various test compounds are screened using transgenic animals and/or progeny of the invention. Compounds that are found to have an effect on ACT expression, or to promote or inhibit any of the diverse biochemical effects of ACT expression, are then further tested and used in treatment of Alzheimer's disease and/or related neurological disorders. In accordance with another aspect of the invention, progeny of the invention can be used as starting points for rational drug design to provide ligands, therapeutic drugs or other types of small chemical molecules. Alternatively, small molecules or other compounds identified by the above-described screening assays can serve as "lead compounds" in rational drug design.

There has thus been outlined, rather broadly, the more important features of the invention in order that the detailed description thereof that follows may be better understood, and in order that the present contribution to the art may be better appreciated. There are, of course, additional features of the invention that will be described below and which will form the subject matter of the claims appended hereto.

In this respect, before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and to the arrangements of the components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced and carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein, as well as the abstract included below, are for the purpose of description and should not be regarded as limiting.

As such, those skilled in the art will appreciate that the conception upon which this disclosure is based may readily be utilized as a basis for the designing of other structures, methods and systems for carrying out the several purposes of the present invention. It is important, therefore, that the claims be regarded as including such equivalent constructions insofar as they do not depart from the spirit and scope of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The following definitions may be useful in comprehending the disclosure of the present invention.

Nucleic acid: refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, would encompass analogs of natural nucleotide that can function in a similar manner as naturally occurring nucleotide. Nucleic acids may be cloned or synthesized using any technique known in the art. They may also include non-naturally occurring nucleotide analogs, such as those which are modified to improve hybridization and peptide nucleic acids.

Transgenic: DNA can be introduced into a murine embryo during embryo genesis by injection into the nucleus of a fertilized egg using an injection needle. In this case, the gene of interest may be directly injected into the nucleus (particularly, pronucleus) of a fertilized egg, or alternatively transgenes carrying sequences such as suitable transcriptional control region (containing, for example, enhancer, GFAP promoter, silencer and the like) and poly A addition signal, the sequences being necessary for proper expression of the genes of interest on the murine genome, may be prepared and then injected into the nucleus of a fertilized egg. Injection can be performed using a microinjector, micromanipulator, or the like, preferably microinjector due to good efficiency of gene transfer and good operability. The genes of interest can be introduced into a fertilized egg separately or concurrently, preferably concurrently because time needed for introduction becomes shorter due to only one manipulation of introduction, and because the fertilized egg has less damage.

Transgene: as used herein refers to a construct for introducing into the murine genome to prepare a transgenic mouse, the construct comprising a DNA sequence of a gene of interest to be introduced. The transgene according to the present invention may be linear or circular, preferably linear in view of the efficiency of integration in the chromosome of a mouse.

Transgenic animal: is any animal, preferably a non-human mammal in which one or more of the cells of the animal contain heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art. The nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. The term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule. This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA. In the typical transgenic animals described herein, the transgene causes cells to express a recombinant form of the subject ACT protein. Preferred non-human animals are selected from the rodent family including rat and mouse, most preferably mouse.

Promoter: as used herein refers to DNA capable of acting as a promoter (i.e., having promoter function). The term "promoter" as used herein refers to a specific nucleotide sequence on DNA, which initiates the synthesis of mRNA (i.e., transcription) with the DNA as a template. The promoter has a common sequence of nucleotides, and RNA polymerase recognizes the nucleotide sequence and synthesizes mRNA. The term "promoter function" as used herein refers to a function that RNA polymerase binds to a specific region on DNA and initiates the transcription.

Tau proteins: without being limited by theory, it is believed now that, in Alzheimer's disease, the amyloid beta-protein is accumulated in the neurons and that, as a result of its correlation with the formation of PHF, death of the neurons results. It has been known that the tau proteins, e.g., tau-i is usually a series of related proteins forming several bands at the molecular weights of 48-65 kd on SDS polyacrylamide gel electrophoresis and that it promotes the formation of microtubules. It is known that the tau which is incorporated in the PHF of the brain of Alzheimer's disease is hyperphosphorylated compared to normal tau.

Normal, mutant, or altered gene: It is also possible to modify the structure of the subject protease inhibitor protein for such purposes as enhancing or decreasing biological or pathological activities, or to facilitate screening methods for compounds that modulate their activity. Such modified polypeptides, expressed from a mutant or altered gene when designed to retain at least one activity of the naturally-occurring form of the protein, are considered functional equivalents of the protease inhibitor proteins described in more detail herein. Such modified polypeptides can be produced, for instance, by amino acid substitution, deletion, or addition, for which corresponding base substitutions or deletions within the normal, mutant, or altered gene are required. It is reasonable to expect, for example, that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid (i.e., conservative mutations) will not have a major effect on the biological activity of the resulting molecule. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are can be divided into four families: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine, histidine; (3) nonpolar=alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. In similar fashion, the amino acid repertoire can be grouped as (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine histidine, (3) aliphatic=glycine, alanine, valine, leucine, isoleucine, serine, threonine, with serine and threonine optionally be grouped separately as aliphatic-hydroxyl; (4) aromatic=phenylalanine, tyrosine, tryptophan; (5) amide=asparagine, glutamine; and (6) sulfur-containing=cysteine and methionine. (see, for example, Biochemistry, 2 nd ed, Ed. by L. Stryer, W H Freeman and Co.: 1981). Whether a change in the amino acid sequence of a peptide results in a functional homolog can be readily determined by assessing the ability of the variant peptide to produce a response in cells in a fashion similar to the wild-type protein. For instance, such variant forms of ACT can be assessed for their ability to induce the pathological effects described herein. Peptides in which more than one replacement has taken place can readily be tested in the same manner.

Amyloidosis: is art recognized and is intended to include amyloid deposition related symptoms, such as progressive and undesirable memory impairment, loss of language and visuospatial skills, and behavior deficits. These changes in cognitive function are the result of degeneration of neurons in the cerebral cortex, hippocampus, basal forebrain, and other regions of the brain. The presence of large numbers of neurofibrillary tangles in degenerated neurons, neuritic plaques in the extracellular space and in the walls of the cerebral microvasculature are a result of amyloid deposition. For example, neuritic plaques which consist of deposits of proteinaceous material surrounding an amyloid core.

A person "suffering from AD" is a person who has been diagnosed as having AD, by a practitioner of at least ordinary skill in the art of clinically diagnosing (e.g., diagnosing in patients antemortem) AD, using methods and routines, such as those described above, that are standard in the art of such clinical diagnoses. The invention entails treating AD in persons suffering therefrom and preventing AD in persons susceptible thereto.

By "treating AD" is meant slowing or preventing the progression or worsening of the disease that is now known to occur when the disease is untreated.

Many proteins have been found that change their phosphorylation state (i.e. become phosphorylated or dephosphorylated) as a result of Alzheimer's disease. Such proteins include, but are not limited to, APP, cdc-2/cyclin B, cdk5, p53, cdc47, MAD, cyclin D, or cyclin E.

Many proteins have been found that either make onset of Alzheimer's disease more likely or faster. Such proteins are of great interest and are known to those of skill in the art. These proteins include, but are not limited to tau-I, apolipoprotein E, APP, presenilin 1, presenilin 2, IL-1 alpha, or IL-1 beta.

The preferred promoter of the present invention is GFAP, but it should be noted that other promoters that are capable of directing protein expression in the brain and optionally in other tissues from an operably attached transgene are suitable for use in the present invention. Most preferably, the GFAP promoter is modified to remove undesired ATG start codons located upstream from the ATG start codon of the desired transgene within the construct.

By "monitoring a pathological marker" is meant any measurement that is responsive to any of the known pathological markers recognized in the art as consistent with Alzheimer's disease, including, but not limited to, plaques, tangles, PFT, synapse loss, neuronal death, elevated or decreased levels of diagnostic proteins including A-beta peptides, and the like. For example, neuronal cell death or synapse loss may conveniently be measured by TUNEL staining, neurofilament antibody staining, or synaptophysin antibody staining.

One puzzle relating to Alzheimer's disease is that the major amyloid component--the A.beta. peptide--is expressed throughout the body and the brain in both normal and Alzheimer individuals, and yet deposits as mature amyloid only in specific regions of the Alzheimer brain. The identification of ACT and apoE4 as amyloid-associated proteins and as amyloid-promoting factors, provides a mechanism for the region-specific and disease-specific deposition of amyloid. The present inventors have discovered that (1) ACT is overexpressed only in areas of Alzheimer-affected brain showing mature amyloid neuropathology; (2) that ACT is expressed in astrocytes in response to IL-1; and (3) that IL-1-expressing microglia cells are only present in Alzheimer's brain in areas showing neuropathology and ACT overexpression, suggest that the reason that Alzheimer amyloid neuropathology develops only in certain brain regions, may, in part, be due to the region-specific inflammatory cascade that leads to expression of the amyloid promoting factor ACT as an early step in the Alzheimer pathogenic pathway (Das and Potter, 1995).

The present invention derives from an unexpected appreciation that the mRNA start site in GFAP is more upstream than previously thought, and the consequent removal by site-directed mutagenesis of several potentially confounding ATG codons in the 5'UTR of GFAP greatly increases the levels of ACT mRNA and protein expression in transfected glioblastoma cells. Based on this insight, the present inventors have generated a transgenic mouse line containing a cDNA fusion-construct with a 6 kbp mouse GFAP promoter and 200 bp of the 5'-end of the GFAP attached to the human ACT cDNA clone. In addition, several ATG start codons in the GFAP part of the transcript that previously interfered with ACT expression have been deleted and the human ACT gene placed downstream of the GFAP transcription start site. The non-coding 3'UTR of the mRNA is derived from the rat preproinsulin II gene, which provides a 3' intronic region and a polyadenylation (polya) site.

As a first test of function, the GFAP-ACT construct is assayed for its ability to support ACT mRNA and protein expression after transient transfection into C6 glioma cells. This cell-line is selected because of its rat origin and which allows the human ACT mRNA and protein to be easily distinguished from any rat species. The summary of results is shown in FIG. 1.

Transgenic mice (FVB/N strain) are then generated using the GFAP/ACT expression plasmid and conventional oocyte injection. PCR is used to confirm the presence of the complete transgene in three founder animals and to show that the transgene is passed intact to half of the progeny of these founders mated with wild type mice. Select animals of the heterozygous offspring are then inbred to generate homozygous transgenic animals. The successful expression of human ACT in the brains of several heterozygous transgenic ACT mice, but not in wild type mice, is demonstrated using non-radioactive Immunoprecipitation/Western blots (see FIG. 2). The major band comigrates with ACT purified from human serum, indicating that the mice not only express human ACT, but also correctly glycosylate it.

The ACT mice and/or the progeny of their crossing with other mice develop Alzheimer-like pathology such as amyloid deposits, neurofibrillary tangles, synapse loss, and neuronal degeneration and do develop behavioral and memory deficits. For example, the human ACT transgenic mice are also mated with transgenic mouse strains that express an Alzheimer's disease mutated form of the human APP gene (PDGF-APP), and which as a result produce numerous congophilic plagues (Congo stain positive) in the hippocampus and cortex. It is known that brain tissue from Alzheimer's disease (AD) patients contained amyloid plaques which are stainable with Congo Red. The additional presence of an expressed ACT gene in the progeny of this cross increases the rate or extent of amyloid formation and of the development of other Alzheimer-like pathology.

Recent results of mating the PDGF-APP mice to apoE knockout mice have indicated that apoE is essential for amyloid formation (Bales et al., 1997). These APP/apoE KO mice are mated to the ACT transgenics to determine whether and how ACT and apoE interact to promote amyloid formation. For example, in the APP+/+ apoE-/- mice, no amyloid develops up to two years of age. When ACT expression is introduced into this background, amyloid deposition occurs. One or two copies of apoE contribute a dose-dependent optimal amyloid promoting effect. The various strains are also useful for studies of relative behavior changes.

It is thus clear that this invention is not limited to ACT mice only but also to any progeny of mating the ACT mice to other mice such that the progeny express human ACT in the brain. The resulting mice are equally important for studies of Alzheimer and other related Amyloidogenic Diseases.

In addition to Alzheimer's Disease (AD), a large number of related "amyloidogenic diseases" are contemplated, including but not limited to scrapie, transmissible spongioform encephalopathies (TSE's), hereditary cerebral hemorrhage with amyloidosis Icelandic-type (HCHWA-I), hereditary cerebral hemorrhage with amyloidosis Dutch-type (HCHWA-D), Familial Mediterranean Fever, Familial amyloid nephropathy with urticaria and deafness (Muckle-Wells syndrome), myeloma or macroglobulinernia-associated idopathy associated with amyloid, Familial amyloid polyneuropathy (Portuguese), Familial amyloid cardiomyopathy (Danish), Systemic senile amyloidosis, Familial amyloid polyneuropathy (Iowa), Familial amyloidosis (Finnish), Gerstmann-Staussler-Scheinker syndrome, Medullary carcinoma of thyroid, Isolated atrial amyloid, Islets of Langerhans, Diabetes type II, and Insulinoma. Many of these conditions are associated with deposition of amyloid plaques.

In a preferred form of the invention, the method of using instant mice is contemplated which is eventually useful for treating, preventing and/or inhibiting conditions associating with plaques occurring in a tissue of the central nervous system of said animals. In another form, the method useful against a disease of the internal organs related to amyloid plaque formation, including plaques in the heart, liver, spleen, kidney, pancreas, brain, lungs and muscles.

In a preferred embodiment, the present invention provides assays for identifying small molecules or other compounds which are capable of inducing or inhibiting the expression of ACT other ACT-related genes and proteins. The assays can be performed in vitro using non-transformed cells, immortalized cell lines, or recombinant cell lines expressing ACT. Specifically the assays are designed in a such a way as to detect the presence of increased or decreased expression of ACT or other inflammatory genes or proteins acting in concert with ACT. These comprise assays to measure increased or decreased mRNA expression (using, e.g., the nucleic acid probes), increased or decreased levels of ACT or other ACT-related protein products (using, e.g., the anti-ACT antibodies produced by art-known methods), or increased or decreased levels of expression of a reporter gene (e.g., beta-galactosidase or luciferase) operatively joined to ACT 5' regulatory region in a recombinant construct.

Thus, for example, one can culture cells known to express the ACT and add to the culture medium one or more test compounds. After allowing a sufficient period of time (e.g., 6-72 hours) for the compound to induce or inhibit the expression of the ACT, any change in levels of expression from an established baseline is detected using any of the techniques described above and well known in the art. In particularly preferred embodiments, the cells are from an immortalized cell line such as a human glioblastoma cell line or an astrocyte cell line. Using the nucleic acid probes and/or antibodies disclosed and herein, detection of changes in the expression of ACT, and thus identification of the compound as an inducer or repressor of ACT expression, requires only routine experimentation.

In particularly preferred embodiments, a recombinant assay is employed in which a reporter gene such as .beta.-galactosidase or luciferase is operably joined to the 5' regulatory regions of ACT gene. Such regulatory regions are easily isolated and cloned by one of ordinary skill in the art in light of the present disclosure of the coding regions of these genes. The reporter gene and regulatory regions are joined so that transcription and translation of the reporter gene may proceed under the control of the ACT regulatory elements. The recombinant construct is then introduced into any appropriate cell type although mammalian cells are preferred. The transformed cells are grown in culture and, after establishing the baseline level of expression of the reporter gene, test compounds are added to the medium. The ease of detection of the expression of the reporter gene provides for a rapid, high throughput assay for the identification of inducers and repressors of ACT gene.

Compounds identified by this method will have the utility in modifying the expression of ACT and other inflammatory genes in vivo. These compounds are then further tested in the animal models disclosed and enabled herein to identify those compounds having the most potent in vivo effects. In addition, as described above with respect to small molecules having ACT-binding activity, these molecules can serve as "lead compounds" for the further development of pharmaceuticals by, for example, subjecting the compounds to sequential modifications, molecular modeling, and other routine procedures employed in rational drug design.

Thus, the present invention also contemplates therapeutic compounds preferably in a pharmaceutically acceptable carrier or diluent.

With respect to in vivo applications, the compounds identified by above screening methods can be administered to a patient in a variety of ways including, for example, parenterally, orally or intraperitoneally. Parenteral administration includes administration by the following routes: intravenous, intramuscular, interstitial, intraarterial, subcutaneous, intraocular, intrasynovial, transepithelial, including transdermal, pulmonary via inhalation, opthalmic, sublingual and buccal, topical, including ophthalmic, dermal, ocular, rectal, and nasal inhalation via insufflation or nebulization.

The compounds are preferably orally administered, for example, with an inert diluent or with an assimilable edible carrier, they can be enclosed in hard or soft shell gelatin capsules, or they can be compressed into tablets. For oral therapeutic administration, the active compounds can be incorporated with an excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, sachets, lozenges, elixirs, suspensions, syrups, wafers, and the like. The pharmaceutical composition comprising the active compounds can be in the form of a powder or granule, a solution or suspension in an aqueous liquid or non-aqueous liquid, or in an oil-in-water or water-in-oil emulsion.

The tablets, troches, pills, capsules and the like can also contain, for example, a binder, such as gum tragacanth, acacia, corn starch or gelating, excipients, such as dicalcium phosphate, a disintegrating agent, such as corn starch, potato starch, alginic acid and the like, a lubricant, such as magnesium stearate, and a sweetening agent, such as sucrose, lactose or saccharin, or a flavoring agent. When the dosage unit form is a capsule, it can contain, in addition to materials of the above type, a liquid carrier. Various other materials can be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules can be coated with shellac, sugar or both. A syrup or elixir can contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring. Any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic. In addition, the active compound can be incorporated into sustained-release preparations and formulations.

The active compounds can be administered parenterally or intraperitoneally. Solutions of the compound as a free base or a pharmaceutically acceptable salt can be prepared in water mixed with a suitable surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations can contain a preservative and/or antioxidants to prevent the growth of microorganisms or chemical degeneration.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It can be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size (in the case of a dispersion) and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.

Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and any of the other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying technique.

Pharmaceutical compositions which are suitable for administration to the nose or buccal cavity include powder, self-propelling and spray formulations, such as aerosols, atomizers and nebulizers.

The therapeutic compounds of this invention can be administered to a mammal alone or in combination with pharmaceutically acceptable carriers or as pharmaceutically acceptable salts, the proportion of which is determined by the solubility and chemical nature of the compound, chosen route of administration and standard pharmaceutical practice.

The compositions can also contain other therapeutically active compounds which are usually applied in the treatment of the diseases and disorders discussed herein. Treatments using the present compounds and other therapeutically active compounds can be simultaneous or in intervals.

It is also contemplated that ACT and ACT-related genes and gene products, as well as other products derived therefrom (e.g., probes, antibodies), will be useful in the diagnosis of Alzheimer's disease, presenile and senile dementias, psychiatric diseases such as schizophrenia, depression, etc., and neurologic diseases such as stroke and cerebral hemorrhage all of which are seen to a greater or lesser extent in subjects bearing mutations in the ACT gene. Diagnosis of inherited cases of these diseases can be accomplished by methods based upon the nucleic acids (including genomic and mRNA/cDNA sequences), proteins, and/or antibodies disclosed and enabled herein. Preferably, the methods and products are based upon the human ACT nucleic acids, proteins or antibodies disclosed herein. As will be obvious to one of ordinary skill in the art as to how exploit the present invention in the best possible mode. Thus, for brevity of exposition, but without limiting the scope of the invention, the following description will focus upon uses of the human homologues of ACT. It will be understood, however, that homologous sequences from other species, including those disclosed herein, will be equivalent for many purposes.

As will be appreciated by one of ordinary skill in the art, the choice of diagnostic methods of the present invention will be influenced by the nature of the available biological samples to be tested and the nature of the information required. ACT, for example, is highly expressed in brain tissue but brain biopsies are invasive and expensive procedures, particularly for routine screening. Other tissues which express ACT at significant levels, however, may demonstrate alternative splicing (e.g., (white blood cells do not express ACT) liver cells) and, therefore, ACT mRNA or protein from such cells may be less informative. Thus, assays based upon a subject's genomic DNA may be the preferred methods for diagnostics as no information will be lost due to alternative splicing and because essentially any nucleate cells may provide a usable sample. Diagnostics based upon other ACT-related proteins are subject to similar considerations: availability of tissues, levels of expression in various tissues, and alternative translation products resulting from alternative mRNA splicing.

When a diagnostic assay is to be based upon ACT-related proteins, a variety of approaches are possible. For example, diagnosis can be achieved by monitoring differences in the electrophoretic mobility of normal and mutant proteins. Such an approach will be particularly useful in identifying mutants in which charge substitutions are present, or in which insertions, deletions or substitutions have resulted in a significant change in the molecular mass of the resultant protein. Alternatively, diagnosis may be based upon differences in the proteolytic cleavage patterns of normal and mutant proteins, differences in molar ratios of the various amino acid residues, or by functional assays demonstrating altered function of the gene products. In some preferred embodiments, protein-based diagnostics will employ differences in the ability of antibodies to bind to normal and mutant ACT-related proteins. Such diagnostic tests can employ antibodies which bind to the normal proteins but not to mutant proteins, or vice versa. In particular, an assay in which a plurality of monoclonal antibodies, each capable of binding to a mutant epitope, can be employed. The levels of anti-mutant antibody binding in a sample obtained from a test subject (visualized by, for example, radiolabelling, ELISA or chemiluminescence) can be easily compared to the levels of binding to a control sample. Such antibody diagnostics can be used for in situ immunohistochemistry using samples of CNS tissues obtained antemortem or postmortem, including neuropathological structures associated with these diseases such as neurofibrillary tangles and amyloid plaques, or may be used with fluid samples such a cerebrospinal fluid or with peripheral tissues such as serum, plasma, or blood.

When the diagnostic assay is to be based upon nucleic acids from a sample, either mRNA or genomic DNA can be used. When mRNA is used from a sample, many of the same considerations apply with respect to source tissues and the possibility of alternative splicing. That is, there may be little or no expression of transcripts unless appropriate tissue sources are chosen or available, and alternative splicing may result in the loss of some information. With either mRNA or DNA, standard methods well known in the art may be used to detect the presence of a particular sequence either in situ or in vitro (see, e.g. Sambrook et al., eds. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.).

For in situ detection of ACT other ACT-related nucleic acid sequence, a sample of tissue will be prepared by standard techniques and then contacted with a probe, preferably one which is labeled to facilitate detection, and an assay for nucleic acid hybridization is conducted under optimal stringency conditions which permit hybridization only between the probe and highly or perfectly complementary sequences. As an example only, the following procedure can be employed on a subject: A mouse is anesthetized and transcardially perfused with cold PBS, followed by perfusion with a formaldehyde solution. The brain or other tissue of interest is then removed, frozen in liquid nitrogen, and cut into thin micron sections. The sections are placed on slides and incubated in proteinase K. Following rinsing in DEP, water and ethanol, the slides are placed in prehybridization buffer. A radioactive probe corresponding to the selected oligonucleotide corresponding to ACT sequence is incubated with the sectioned brain tissue. After incubation and air drying, the labeled areas are visualized by autoradiography or fluorescence. Positive spots on the tissue sample indicate hybridization of the probe with brain mRNA, demonstrating expression of the nucleic acid sequence.

A significant advantage of the use of either DNA or mRNA is the ability to amplify the amount of genetic material using the polymerase chain reaction (PCR), either alone (with genomic DNA) or in combination with reverse transcription (with mRNA to produce cDNA). PCR-based genetic methods may be preferred commercial embodiments for diagnostic screenings and the technical details as to how run PCR analysis are well know to those of ordinary skill in the art.

Claim 1 of 11 Claims

What is claimed is:

1. A method for testing a compound suspected of promoting or inhibiting phosphorylation of one or more proteins related to Alzheimer's disease, said method comprising: providing a mammalian cell; administering to said cell antichymotrypsin and said compound; and monitoring the phosphorylation state of said one or more proteins.

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