This invention relates to the discovery and identification of novel perlecan domain I splice variants and their utilization for the production of specific and unique perlecan domain I variant nucleotides, peptides, antibodies, and molecular biology probes for the diagnosis and therapeutic intervention of Alzheimer""s disease and other amyloid diseases. In addition new animal models to effectively screen and identify potential therapeutic compounds for Alzheimer""s disease and each of the amyloidoses are described.
Alzheimer""s disease is one of the so-called xe2x80x9camyloid diseases. The literature suggests that an interaction between an amyloid protein and a heparan sulfate proteoglycan, known as perlecan, is important in the pathogenesis of Alzheimer""s and other amyloid diseases. A more detailed description of the amyloid diseases, Alzheimer""s disease, heparan sulfate proteoglycans, and perlecan is contained in the xe2x80x9cDetailed Description of the Inventionxe2x80x9d.
What is not known is whether perlecan (or related macromolecules) present in the tissues of Alzheimer""s disease and other amyloid disorders are altered, abnormal and/or different than normal. This is a very important and puzzling question which has, of yet, not been answered. Since perlecan is usually found throughout the body in various organs and tissues and is synthesized by a variety of different cells, is it possible that perlecan (or related macromolecules) may exist in different form(s) in tissues, amyloid deposits and/or neurofibrillary tangles of patients afflicted with Alzheimer""s disease? Is it also possible that perlecan (or related macromolecules) may also exist in different form(s) in tissues and/or amyloid deposits of patients afflicted with any of the other amyloid diseases?
The present invention provides some answers to these questions and relates to the novel and surprising discovery that perlecan exists in unique splice variant forms which are the result of xe2x80x9cdeletionxe2x80x9d or xe2x80x9cadditionxe2x80x9d splicing in domain I of perlecan (the region normally containing the 3 GAG chains of perlecan). The splicing of perlecan domain I results in the production of unique amino acid sequences which usually contain a consensus sequence for an additional GAG chain attachment site (ie. Ser-Gly or Ser-Gly-Asp) creating new perlecan molecules that are predicted to have four or more GAG chains, instead of the three GAG chains found in normal perlecan. Two different anti-peptide polyclonal antibodies produced against unique peptide regions which occurred as a result of perlecan domain I splicing demonstrate the immunolocalization of perlecan domain I variants to either the neurofibrillary tangles (i.e. perlecan domain I exon 5 deletion variant detected using the xe2x80x9cexon 5 deletionxe2x80x9d antibody) or amyloid plaques (i.e. perlecan domain I variant exon 4a and/or perlecan domain I variant exon 3a using the xe2x80x9cperlecan domain I insetxe2x80x9d antibody) in Alzheimer""s disease brain. Immunolocalization studies utilizing the polyclonal anti-peptide antibodies produced suggest that some of the perlecan domain I splice variant molecules discovered (ie. perlecan domain I exon 5 deletion) do not reside on basement membranes, as does normal perlecan, and therefore should be regarded as distinctly different from perlecan or xe2x80x9cbasement membrane heparan sulfate PGsxe2x80x9d. Therefore, the perlecan domain I splice variants identified in the invention are different than normal perlecan in that their are distinct differences in their 1) nucleotide sequence(s), 2) corresponding amino acid sequence(s), 3) number of predicted GAG chains, and 4) distribution in specific tissues. Since recent studies have suggested that heparan sulfate GAGs are primarily involved in amyloid fibril formation (Castillo et al, Soc. Neurosc. Abst. 22:1172, 1996 Abstract) and in the induction of neurofibrillary tangle formation (Goedert et al, Nature 383:550-553, 1996), the surprising discovery in this invention of unique perlecan domain I splice variants which may contain additional heparan sulfate GAG chains and which co-localize to amyloid plaques or neurofibrillary tangles, further implicate their importance in plaque and tangle pathogenesis in Alzheimer""s disease. In addition, the presence of perlecan domain I splice variants in tissues outside the central nervous system, as discovered in this invention, also implicates these splice variants in other amyloid diseases which affect systemic organs and tissues.
The present invention identifies perlecan domain I splice variants and provides specific and unique perlecan domain I variant nucleotides, peptides, antibodies, and molecular biology probes for the diagnosis and therapeutic intervention of Alzheimer""s disease and other amyloid diseases. In addition new animal models to effectively screen and identify potential therapeutic compounds for Alzheimer""s disease and each of the amyloidoses are described.
Perlecan is known to play important and pathogenetic roles in the amyloid diseases including contributing to the formation, deposition, accumulation and/or persistence of fibrillar amyloid deposits in each of the amyloid diseases. This invention relates to the identification and the utilization of newly identified splice variants of domain I of human perlecan. One or all of the splice variants described in the present invention are believed to play similar, if not more important pathogenetic roles in Alzheimer""s and other amyloid diseases. The invention described herein concerns these perlecan domain I splice variants and their use for the diagnosis and therapeutic intervention for Alzheimer""s disease and other amyloidoses.
A primary object of the present invention is to establish new diagnostic and therapeutic methods and applications for the amyloid diseases. The amyloid diseases include, but are not limited to, the amyloid associated with Alzheimer""s disease and Down""s syndrome (wherein the specific amyloid is referred to as beta-amyloid protein or Axcex2), the amyloid associated with chronic inflammation, various forms of malignancy and Familial Mediterranean Fever (wherein the specific amyloid is referred to as AA amyloid or inflammation-associated amyloidosis), the amyloid associated with multiple myeloma and other B-cell dyscrasias (wherein the specific amyloid is referred to as AL amyloid), the amyloid associated with type II diabetes (wherein the specific amyloid is referred to as amylin or islet amyloid), the amyloid associated with the prion diseases including Creutzfeldt-Jakob disease, Gerstmann-Straussler syndrome, kuru and animal scrapie (wherein the specific amyloid is referred to as PrP amyloid), the amyloid associated with long-term hemodialysis and carpal tunnel syndrome (wherein the specific amyloid is referred to as beta2-microglobulin amyloid), the amyloid associated with senile cardiac amyloid and Familial Amyloidotic Polyneuropathy (wherein the specific amyloid is referred to as transthyretin or prealbumin), and the amyloid associated with endocrine tumors such as medullary carcinoma of the thyroid (wherein the specific amyloid is referred to as variants of procalcitonin).
One object of the present invention is to utilize novel and specific primer sequences for the detection of perlecan domain I splice variants in human tissues using standard reverse transcription polymerase chain reaction (RT-PCR) methodology, knowledgeable to one skilled in the art.
Yet another object of the present invention is to use standard RT-PCR methodology, but utilizing the specific primers described herein, which will aid in the amplification of each of the perlecan domain I splice variants, for the ultimate detection of these splice variants in various human tissues, cells and/or cells in biological fluids. In addition, quantitative competitive RT-PCR techniques can be utilized (Maresh et al, J. Neurochem. 67:1132-1144, 1996) to determine quantitative differences in these specific variants in total RNA derived from human tissues, cells, white blood cells and/or cells in biological fluids. Changes in quantitative levels of these perlecan domain I splice variants will aid in the diagnosis and monitoring of prognosis of patients who demonstrate amyloid and concurrent perlecan domain I splice variant and/or perlecan accumulation in tissues as part of the pathological process observed in the amyloid diseases.
Another object of the invention is to provide polyclonal and/or monoclonal peptide antibodies which can be utilized in a number of in vitro assays to specifically detect the perlecan domain I splice variants in human tissues and/or biological fluids. Polyclonal or monoclonal antibodies made specifically against a peptide portion or fragment of any of the perlecan domain I splice variants described herein can be utilized to detect and quantify perlecan domain I splice variants in human tissues and/or biological fluids. A preferred embodiment are the polyclonal antibodies described herein (i.e. xe2x80x9cexon 5 deletionxe2x80x9d antibody and the xe2x80x9cperlecan domain I insertxe2x80x9d antibody) which are made against unique perlecan domain I sequences as a result of splicing. These antibodies can be made by administering the peptides in antigenic form to a suitable host. Polyclonal or monoclonal antibodies may be prepared by standard techniques by one skilled in the art.
Another object of the present invention is to use each of the perlecan domain I splice variant antibodies described herein for the detection and specific localization of each of the perlecan domain I splice variants in human tissues, cells, and/or cell culture using standard immunohistochemical techniques.
Yet another object of the present invention is to use the xe2x80x9cexon 5 deletionxe2x80x9d antibody to detect perlecan domain I variant exon 5 (PerDI-v5) as a specific indicator for the presence and extent of neurofibrillary tangles in brain by monitoring biological fluids including, but not limited to, cerebrospinal fluid, blood, serum, urine, saliva, sputum, and stool.
Yet another object of the present invention is to use the xe2x80x9cexon 5 deletionxe2x80x9d antibody to detect perlecan domain I variant exon 5 (PerDI-v5) as a specific indicator for the presence and progression of Alzheimer""s disease and/or other amyloid diseases by monitoring biological fluids including, but not limited to, cerebrospinal fluid, blood, serum, urine, saliva, sputum, and stool.
Yet another object of the present invention is to use the xe2x80x9cperlecan domain I insertxe2x80x9d antibody to detect perlecan domain I variant exon 4a and/or perlecan domain I variant exon 3a as a specific indicator for the presence and extent of amyloid plaques in brain by monitoring biological fluids including, but not limited to, cerebrospinal fluid, blood, serum, urine, saliva, sputum, and stool.
Yet another object of the present invention is to use the xe2x80x9cperlecan domain I insertxe2x80x9d antibody to detect perlecan domain I variant exon 4a and/or perlecan domain I variant exon 3a as a specific indicator for the presence and progression of Alzheimer""s disease and/or other amyloid diseases by monitoring biological fluids including, but not limited to, cerebrospinal fluid, blood, serum, urine, saliva, sputum, and stool.
Yet another object of the present invention is to provide a method which can evaluate a compound or potential therapeutic""s ability to alter (diminish or eliminate) the affinity of a given amyloid protein (as described herein) or amyloid precursor protein, to perlecan domain I splice variant protein or perlecan domain I splice variant GAGs. By providing a method of identifying compounds which affect the binding of amyloid proteins, or amyloid precursor proteins to such perlecan domain I splice variant protein or perlecan domain I splice variant derived-GAGs or fragments thereof, the present invention is also useful in identifying compounds which can prevent or impair such binding interaction. Thus, compounds can be identified which specifically affect an event linked with the amyloid formation, amyloid deposition, and/or amyloid persistence condition associated with Alzheimer""s disease and other amyloid diseases as described herein.
Yet another object of the present invention is to use antibodies generated which recognize each of the perlecan domain I splice variants for in vivo labelling; for example, with a radionucleotide, for radioimaging to be utilized for in vivo diagnosis, and/or for in vitro diagnosis. Preferred embodiments include, but are not limited to, the xe2x80x9cexon 5 deletionxe2x80x9d antibody and the xe2x80x9cperlecan domain I insertxe2x80x9d antibodyxe2x80x9d described herein.
Yet another object of the present invention is to make use of peptides or fragments thereof which are specific against new and unique sequences of the perlecan domain I splice variants. These peptides or fragments thereof can be used as potential blocking therapeutics for the interaction of the perlecan domain I splice variants in a number of biological processes and diseases (such as in the amyloid diseases described herein).
Another object of the present invention is to use peptides or fragments thereof, in conjunction with polyclonal and/or monoclonal antibodies generated against these peptide fragments, using in vitro assays to detect potential perlecan domain I splice variant autoantibodies in human biological fluids. Specific assay systems can be utilized to not only detect the presence of autoantibodies against perlecan domain I splice variants in biological fluids, but also to monitor the progression of disease by following elevation or diminution of perlecan domain I splice variant autoantibody levels.
Yet another object of the invention is to utilize perlecan domain I splice variant antibodies to isolate the perlecan domain I splice variant proteins from tissues using procedures known to those in the art such as affinity column chromatography and immunoprecipitation methodology. Isolation of perlecan domain I splice variants from tissues will also allow one to further structurally characterize the GAG chains associated with said perlecan domain I splice variants. Therefore, another object of the invention is to utilize perlecan domain I splice variant derived-GAGs as described herein for use in diagnostic assays, therapeutic intervention and research purposes.
Yet another object of the invention is to make oligonucleotides utilizing the nucleotide sequences described herein, to be utilized as new molecular biological probes to detect perlecan domain I splice variants in human tissues by standard in situ hybridization techniques.
Another object of the present invention is to provide new animal models for the production, deposition, accumulation and/or persistence of fibrillar Axcex2 amyloid in brain as observed in Alzheimer""s disease and Down""s syndrome. These new animal models can also be used to effectively screen and identify new therapeutic agents that target fibrillar Axcex2 amyloid formation, deposition, accumulation and/or persistence in brain.
Yet another object of the present invention is to provide new animal models for the production, deposition, accumulation and/or persistence of fibrillar amyloid as observed in each of the other amyloidoses. This includes, but is not limited to, the amyloid associated with chro nic infl ammation, various forms of malignancy and Fanilial Mediterranean Fever (where in the specific amyloid is referred to as AA amyloid or inflammation-associated amyloidosis), the amyloid associated with multiple myeloma and other B-cell dyscrasias (wherein the specific amyloid is referred to as AL amyloid), the amyloid associated with type II diabetes (wherein the specific amyloid is referred to as amylin or islet amyloid), the amyloid associated with the prion diseases including Creutzfeldt-Jakob disease, Gerstmann-Straussler syndrome, kuru and animal scrapie (wherein the specific amyloid is referred to as PrP amyloid), the amyloid associated with long-term hemodialysis and carpal tunnel syndrome (wherein the specific amyloid is referred to as beta2-rnicroglobulin amyloid), the amyloid associated with senile cardiac amyloid and Familial Amyloidotic Polyneuropathy (wherein the specific amyloid is referred to as transthyretin or prealbumin), and the amyloid associated with endocrine tumors such as medullary carcinoma of the thyroid (wherein the specific amyloid is referred to as variants of procalcitonin). These new animal models can also be used for the evaluation of candidate drugs and therapies for the prevention and treatment of the amyloidoses as referred to above.
Yet another object of the invention is to produce new transgenic animals that overexpress or knock-out a particular perlecan domain I splice variant in an effort to produce specific phenotypes associated with a number of diseases and/or pathological processes, including, but not limited to, Alzheimer""s disease and/or other amyloid diseases.
Yet another object of the invention is to utilize specific perlecan domain I variant antibodies and/or molecular biology probes, described herein, for the detection of these splice variants in human tissues in the amyloid diseases.
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USPTO Backgrounds