Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-08-26
2002-08-13
Myers, Carla J. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S023500, C536S024310, C536S024330
Reexamination Certificate
active
06432636
ABSTRACT:
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.
BACKGROUND OF THE INVENTION
Alzheimer's disease is one of the so-called “amyloid 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 “Detailed Description of the Invention”.
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?
SUMMARY OF THE INVENTION
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 “deletion” or “addition” 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 “exon 5 deletion” antibody) or amyloid plaques (i.e. perlecan domain I variant exon 4a and/or perlecan domain I variant exon 3a using the “perlecan domain I inset” 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 “basement membrane heparan sulfate PGs”. 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.
FEATURES OF THE INVENTION
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 A&bgr;), 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 beta
2
-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 t
Maresh Grace A.
Snow Alan D.
Dwyer Patrick M.
Myers Carla J.
University of Washington
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