Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1999-04-22
2001-07-03
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S183000, C435S252300, C435S320100
Reexamination Certificate
active
06255096
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to mammalian &agr;-N-acetylglucosaminidase and to genetic sequences encoding same and to the use of these in the investigation, diagnosis and treatment of subjects suspected of or suffering from &agr;-N-acetylglucosaminidase deficiency.
Bibliographic details of the publications referred to by author in this specification are collected at the end of the description. Sequence Identity Numbers (SEQ ID NOs.) for the nucleotide and amino acid sequences referred to in the specification are defined following the bibliography.
Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
BACKGROUND TO THE INVENTION
The increasing sophistication of recombinant DNA technology is greatly facilitating the efficacy of many commercially important industries including areas of medical and pharmaceutical research and development. The ability to purify native proteins and subsequently clone genetic sequences encoding these proteins is an important first step in the development of a range of therapeutic and diagnostic procedures. However, practitioners have faced many difficulties in purifying target molecules to an extent sufficient to determine amino acid sequences to permit the development of oligonucleotide probes to assist in the cloning of genetic sequences encoding the target molecules.
Such difficulties have been particularly faced in the research and development of lysosomal enzymes. An important lysosomal enzyme is &agr;-N-acetylglucosaminidase (EC 2.1.50). This enzyme acts as a exoglycosidase in lysosomes to hydrolyse the terminal &agr;-N-acetylglucosamine residues present at the non-reducing terminus of fragments of heparan sulphate and heparin (Hopwood, 1989). A deficiency in this lysosomal hydrolase is responsible for the pathogenesis of Sanfilippo B (Mucopolysaccharidosis type IIIB [MPS-IIIB]) syndrome (von-Figura and Kresse, 1972; O’Brien, 1972). This is an autosomal recessive disorder of glycosaminoglycan catabolism leading to storage and excretion of excessive amounts of heparan sulphate and a variety of clinical phenotypes, but classically presenting with progressive mental retardation in conjunction with skeletal deformities (McKusick and Neufeld, 1983).
There is a need, therefore, to purify &agr;-N-acetylglucosaminidase and to clone genetic sequences encoding same to permit development of a range of therapeutic and diagnostic procedures to assist in the diagnosis and treatment of disease conditions arising from &agr;-N-acetylglucosaminidase deficiency.
SUMMARY OF THE INVENTION
One aspect of the invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides which encodes or is complementary to a sequence which encodes a mammalian &agr;-N-acetylglucosaminidase or fragment or derivative thereof.
A second aspect of the invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides which is capable of hybridising under at least low stringency conditions to a nucleotide sequence set forth in SEQ ID NO:1 SEQ ID NO:3 or a complementary strand or a homologue, analogue or derivative thereof.
Another aspect of the invention is directed an isolated nucleic acid molecule which is at least 40% identical to the nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:3 or to a complementary strand thereof or a homologue, analogue or derivative thereof.
A further aspect of the present invention provides a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a polypeptide capable of hydrolysing the terminal &agr;-N-acetylglucosamine residues present at the non-reducing terminus of figments of heparan sulphate and heparin residues and wherein said nucleotide sequence is capable of hybridising under low stringency conditions to the nucleotide sequence set forth in SEQ ID NO:1.
A further aspect of the invention is directed to a genetic construct comprising a sense molecule, for the expression or over-expression of &agr;-N-acetylglucosaminidase in prokaryotic or eukaryotic cells.
A further aspect of the present invention is directed to synthetic &agr;-N-acetylglucosaminidase or like molecule.
A further aspect of the invention contemplates antibodies to &agr;-N-acetylglucosaminidase and preferably synthetic &agr;-N-acetylglucosaminidase or a like molecule.
In still yet another aspect of the present invention there is contemplated a method of diagnosing a mutation or other abberations in the &agr;-N-acetylglucosaminidase gene in a human or animal patient.
Another aspect contemplates a method of treating patients suffering from &agr;-N-acetylglucosaminidase deficiency, such as in MPS-IIIB, said method comprising administering to said patient an effective amount of &agr;-N-acetylglucosaminidase or active like form thereof.
Another aspect of the present invention is directed to a pharmaceutical composition comprising a recombinant mammalian &agr;-N-acetylglucosaminidase or an active fragment or derivative thereof and one or more pharmaceutically acceptable carriers and/or diluents.
REFERENCES:
Sasaki et al, 1991, J Biochem, 110:842-6.
Von Figura et al, 1975, Z Klin Chem Klin Biochem, 13:285-9.
Von Figura et al, 1976, Eur J Biochem, 61:581-8.
Zhao et al, 1994, Am J Gen, 55: A 252 (Abstract 1473).
Zhao et al, 1995, Am J Gen, 57: A 185 (Abstract 1059).
Zhao et al, 1996, Proc Nat Acad Sci, 93:6101-6105.
Weber et al, 1996, Hum Mol Gen,5: 771-777.
Anson Donald Stewart
Blanch Lianne
Hopwood John Joseph
Scott Hamish Steele
Weber Birgit
Pokalsky Ann R.
Prouty Rebecca E.
Rao Manjunath
Women's and Children's Hospital
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