Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1999-09-14
2001-10-30
Allen, Marianne P. (Department: 1631)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C435S006120, C435S069100, C435S069700
Reexamination Certificate
active
06310182
ABSTRACT:
1. INTRODUCTION
The present invention relates to the identification of novel nucleic acid molecules and proteins encoded by such nucleic acid molecules or degenerate, especially naturally occurring, variants thereof, that, when mutated, lead to disorders involving abnormal intracellular vesicles, especially abnormal lysosomes, melanosomes, platelet dense granules and cytolytic granules, including Chediak-Higashi syndrome (CHS). The nucleic acid molecules of the present invention represent the genes corresponding to the mammalian bg gene, including the human bg gene, which are involved in the normal differentiation and/or function of such intracellular vesicles. Nucleic acid molecules representing loss-of-function alleles of the human bg gene bring about Chediak-Higashi syndrome (CHS), in individuals homozygous for such alleles.
In particular, the compositions of the present invention include nucleic acid molecules (e.g., bg gene), including recombinant DNA molecules, cloned genes or degenerate, especially naturally occurring, variants thereof, which encode novel bg gene products, and antibodies directed against such bg gene products or conserved variants or fragments thereof. The compositions of the present invention additionally include cloning vectors, including expression vectors, containing the nucleic acid molecules of the invention and hosts which have been transformed with such nucleic acid molecules.
In addition, this invention presents methods for the diagnostic evaluation and prognosis of disorders involving abnormal intracellular vesicles, especially abnormal lysosomes, melanosomes, platelet dense granules and cytolytic granules, including CHS, and for the identification of subjects having a predisposition to such conditions. For example, nucleic acid molecules of the invention can be used as diagnostic hybridization probes or as primers for diagnostic PCR analysis for the identification of bg gene mutations, allelic variations and regulatory defects in the bg gene.
Further, methods and compositions are presented for the treatment of disorders involving abnormal intracellular vesicles, especially abnormal lysosomes, melanosomes, platelet dense granules and cytolytic granules, including CHS. Such methods and compositions are capable of modulating the level of bg gene expression and/or the level of bg gene product activity.
Still further, the present invention relates to methods for the use of the bg gene, bg gene products and/or cells expressing wild type or mutant bg gene sequences for the identification of compounds which modulate bg gene expression and/or the activity of bg gene products. Such compounds can be used as agents to control disorders involving abnormal intracellular vesicles, especially abnormal lysosomes, melanosomes, platelet dense granules and cytolytic granules, in particular, therapeutic agents in the treatment of CHS.
2. BACKGROUND OF THE INVENTION
Chediak-Higashi syndrome (CHS) is a lethal autosomal recessive disorder of humans mapping to 1q43. The clinical manifestations of this disorder include hypopigmentation, defective immune cell function, including severely impaired natural killer cell activity, and defective antibody-dependent, lymphocyte-mediated cytolysis against tumor cell targets. Further, neural degeneration is observed and, finally, the occurrence of a mononuclear cell lymphoma develops, which causes the death of afflicted individuals.
As mentioned above, the disease is accompanied by a marked susceptibility to infections. Young children have repeated infections, usually with gram-positive organisms of the staphylococcal and streptococcal type. Further, during the course of the disease, children may develop a progressive peripheral neuropathy. Children surviving the early infectious episodes (8-18 years of age), most frequently develop terminal lymphoreticular malignancy. Few patients survive beyond twenty years.
Pathological manifestation of the syndrome includes enlarged vesicles affecting lysosomes, melanosomes, platelet dense granules, cytolytic granules and Schwann cell granules. The abnormal size of these vesicles is thought to result from a malregulation of vesicle fusion or fission. Abnormal membrane-bound lysosomal-like organelles have been found in cells of the buccal mucosa, Schwann cells, pancreas, liver, gastric and duodenal mucosa, adrenal, pituitary, spleen, kidney, bone marrow, hair skin, iris and conjunctiva. The giant granules observed resemble the normal granules of the specific cell type in both fine structure and cytochemic reactions and result from the fusion of small primary granules.
Similar phenotypes are found in other species, most notably the beige mouse and the Aleutian mink, but are also found in such species as the Persian cat, cattle and even the killer whale. Somatic cell fusion studies have suggested that mutations within the same gene in mouse, mink, and man were responsible for the CHS-like phenotype in each of these species. In mice, the gene responsible for such a phenotype is the beige (bg) gene. Such studies, however, were not able to elucidate either the function or the identity of the bg gene product.
Over the past thirty years numerous theories have been evoked to explain the nature of these disorders. For example, it has been suggested that the defect might be caused by alterations in membrane fluidity, defects in microtubules or microtubule associated proteins, or changes in cyclic nucleotides levels. Upon further examination, though, each of these theories has been found to be inadequate, thus highlighting the fact that a great need remains for the discovery of the causative agent of the lethal Chediak-Higashi syndrome genetic disorder.
3. SUMMARY OF THE INVENTION
The present invention relates to the identification of novel nucleic acid molecules and proteins encoded by such nucleic acid molecules or degenerate, especially naturally occurring, variants thereof, that, when mutated, lead to disorders involving abnormal intracellular vesicles, especially abnormal lysosomes, melanosomes, platelet dense granules and cytolytic granules, including Chediak-Higashi syndrome (CHS). The nucleic acid molecules of the present invention represent the genes corresponding to the mammalian bg gene, including the human bg gene, which are involved in the normal differentiation and/or function of such intracellular vesicles. Nucleic acid molecules representing loss-of-function alleles of the human bg gene bring about Chediak-Higashi syndrome (CHS), in individuals homozygous for such alleles.
In particular, the compositions of the present invention include nucleic acid molecules (e.g., bg gene), including recombinant DNA molecules, cloned genes or degenerate, especially naturally occurring, variants thereof, which encode novel bg gene products, and antibodies directed against such bg gene products or conserved variants or fragments thereof. The compositions of the present invention additionally include cloning vectors, including expression vectors, containing the nucleic acid molecules of the invention and hosts which have been transformed with such nucleic acid molecules.
Nucleic acid sequences of wild type and mutant forms of the murine bg gene are provided. Wild type murine bg gene produces a transcript of approximately 12-14 kb. The amino acid sequence of the predicted bg gene product indicates that the protein is novel.
Nucleic acid sequences of wild type forms of the human bg gene are also provided. The human bg gene produces alternatively spliced transcripts. The long, putatively full length bg transcript encodes a bg protein of 3801 amino acid residues, as shown in FIG.
7
A-
7
I
1
. A short form, alternatively spliced, human bg transcript encodes a bg protein of 3672 amino acid residues, as shown in FIG.
8
A-
8
H
1
. The amino acid sequence of the predicted human bg gene products indicates that the proteins are novel.
In addition, this invention presents methods for the diagnostic evaluation and prognosis of disorders involving abnormal intracellular vesicles, especially abnormal lysosomes, melan
Kaplan Jerry
Moore Karen J.
Perou Charles M.
Allen Marianne P.
Millennium Pharmaceuticals Inc.
Pennie & Edmonds LLP
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