Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2002-07-23
2004-04-06
Achutamurthy, P. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C536S023200, C435S252300, C435S320100
Reexamination Certificate
active
06716614
ABSTRACT:
1.0 INTRODUCTION
The present invention relates to the discovery, identification, and characterization of novel human polynucleotides encoding proteins that share sequence similarity with human calcium dependent proteases, specifically calpains. The invention encompasses the described polynucleotides, host cell expression systems, the encoded proteins, fusion proteins, polypeptides and peptides, antibodies to the encoded proteins and peptides, and genetically engineered animals that either lack or overexpress the disclosed polynucleotides, antagonists and agonists of the proteins, and other compounds that modulate the expression or activity of the proteins encoded by the disclosed polynucleotides, which can be used for diagnosis, drug screening, clinical trial monitoring, the treatment of diseases and disorders, such as a reduced white blood cell count, and cosmetic or nutriceutical applications.
2.0 BACKGROUND OF THE INVENTION
Proteases are enzymes that mediate the proteolytic cleavage of polypeptide sequences. In particular, calcium-dependent proteases, such as calpains, have been found in virtually every vertebrate cell that has been examined for their presence. The calpain system has at least three well-characterized protein members that are activated in response to changes in calcium concentration. These proteins include at least two calpains that are activated at different concentrations of calcium, and a calpastatin that specifically inhibits the two calpains. Various tissue/species specific cDNAs have been described that are homologous to the calpains. Given the near ubiquitous expression of calpains, they have been implicated in a wide variety of cellular functions including, but not limited to, cell proliferation and differentiation, signal transduction, processes involving interactions between the cell membrane and cytoskeleton, secretion, platelet aggregation, cytokinesis, and disease. Accordingly, calpains represent a key target for the regulation of a variety of biological pathways.
Reduced white blood cell count, or neutropenia, is a major complication that occurs during many forms of chemotherapy, particularly those regimens involving myelosuppressive anti-cancer drugs, and as a result of certain infectious diseases. Although treatments for neutropenia currently exist in the art, they are not ideal for use in all circumstances, and are actually contraindicated in certain patients. Therefore, new treatments for neutropenia would represent a significant advance in the art.
3.0 SUMMARY OF THE INVENTION
The present invention relates to the discovery, identification, and characterization of nucleotides that encode novel human proteins, and the corresponding amino acid sequences of these proteins. The novel human proteins (NHPs) described for the first time herein share structural similarity with animal calcium-activated proteases, or calpains. As such, the novel genes represent a new class of protease proteins with a range of homologues and orthologs that transcend phyla and a broad range of species.
The novel human nucleic acid sequences described herein, encode proteins/open reading frames (ORFs) of 739, 723, 702, and 686 amino acids in length (see SEQ ID NOS:2, 4, 6, and 8 respectively).
The invention also encompasses agonists and antagonists of the described NHPs, including small molecules, large molecules, mutant NHPs, or portions thereof, that compete with native NHPs, peptides, and antibodies, as well as nucleotide sequences that can be used to inhibit the expression of the described NHPs (e.g., antisense and ribozyme molecules, and open reading frame or regulatory sequence replacement constructs) or to enhance the expression of the described NHPS (e.g., expression constructs that place the described polynucleotide under the control of a strong promoter system), and transgenic animals that express a NHP sequence, or “knock-outs” (which can be conditional) that do not express a functional NHP. Knock-out mice can be produced in several ways, one of which involves the use of mouse embryonic stem cell (“ES cell”) lines that contain gene trap mutations in a murine homolog of at least one of the described NHPS. When the unique NHP sequences described in SEQ ID NOS:1-9 are “knocked-out” they provide a method of identifying phenotypic expression of the particular gene, as well as a method of assigning function to previously unknown genes. In addition, animals in which the unique NHP sequences described in SEQ ID NOS:1-9 are “knocked-out” provide an unique source in which to elicit antibodies to homologous and orthologous proteins, which would have been previously viewed by the immune system as “self” and therefore would have failed to elicit significant antibody responses.
To these ends, gene trapped knockout ES cells have been generated in murine homologs of the described NHPs. Characterization of mice in which both copies of a NHP have been disrupted (homozygotes) has allowed the identification of a novel role for this enzyme, and a model for the study of certain disorders. In particular, NHP knockout mice (that are homozygous for the mutated gene) display, intra alia, increased white blood cell counts. This suggests that these mice can be used as models for the study of the treatment of a variety of human conditions, including, but not limited to, neutropenia, as exemplified by neutropenia associated with the administration of myelosuppressive anti-cancer drugs.
In addition, the invention includes animals containing at least a single disrupted NHP allele (e.g., “knock-out” mice) that do not express normal levels of a NHP, humanized “knock-in” animals where the endogenous murine NHP gene has been replaced by one or more polynucleotides encoding at least a first human NHP protein, or animals harboring one or more NHP transgene (e.g., mice overexpressing a NHP). These animals may either transiently, inducibly, or constitutively express a NHP.
Additionally, the unique NHP sequences described in SEQ ID NOS:1-9 are useful for the identification of protein coding sequences, and mapping an unique gene to a particular chromosome. These sequences identify biologically verified exon splice junctions, as opposed to splice junctions that may have been bioinformatically predicted from genomic sequence alone. The sequences of the present invention are also useful as additional DNA markers for restriction fragment length polymorphism (RFLP) analysis, and in forensic biology, particularly given the presence of nucleotide polymorphisms within the described sequences.
Further, the present invention also relates to processes for identifying compounds that modulate, i.e., act as agonists or antagonists of, NHP expression and/or NHP activity that utilize purified preparations of the described NHPs and/or NHP products, or cells expressing the same. Such compounds can be used as therapeutic agents for the treatment of any of a wide variety of symptoms associated with biological disorders or imbalances, such as reduced white blood cell count.
The present invention also provides novel methods and compositions that can be used to facilitate drug discovery, drug development, and/or as treatments of conditions such as reduced white blood cell count, and the complications resulting therefrom. The present invention is based on the identification and novel functional characterization of the NHPs described herein.
The invention encompasses diagnostic assays that make use of the NHP polynucleotide sequences, or portions thereof, host cells expressing such nucleotides, and the expression products of such nucleotides, nucleotides that encode mammalian versions of the NHPs, including human NHPs, nucleotides that encode NHP mutants and the corresponding mutant NHP expression products, nucleotides that encode portions of a NHP that correspond to one or more of the NHP functional domains and the polypeptide products specified by such nucleotide sequences, and nucleotides that encode fusion proteins containing a NHP or one or more of its domains fused to another polypeptide.
The present invention also fe
Donoho Gregory
Friedrich Glenn
Nehls Michael C.
Sands Arthur T.
Turner, Jr. C. Alexander
Achutamurthy P.
Lexicon Genetics Incorporated
Pak Yong
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