Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2001-11-20
2004-11-23
Kemmerer, Elizabeth (Department: 1647)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S252300, C435S254110, C435S320100, C536S023100, C536S023500
Reexamination Certificate
active
06821750
ABSTRACT:
BACKGROUND OF THE INVENTION
In humans the intestine presents a barrier that prevents the absorption of plant sterols and partially blocks the absorption of cholesterol. This barrier is disrupted in the rare autosomal recessive disorder, sitosterolemia (Bhattacharyya, et al.,
J. Clin. Invest.
53:1033 (1974)). Sitosterolemic patients Hyperabsorb the plant sterols such as sitosterol, which provide the identifying feature of this disease (Bhattacharyya, et al.,
J. Clin. Invest.
53:1033 (1974); Bjorkhem and Boberg, in
The Metabolic and Molecular Bases of Inherited Disease
, Scriver, et al., Eds., pp. 2073 vol. 2, chap. 65 [seventh edition] (McGraw Hill, New York, 1995); Salen, et al.,
J. Lipid Res.
33:945 (1992)). These patients also hyperabsorb cholesterol and are usually hypercholesterolemic, resulting in the development of xanthomas (cholesterol deposits in skin and tendons) and premature coronary artery disease (Bjorkhem and Boberg, in
The Metabolic and Molecular Bases of Inherited Disease
, Scriver, et al., Eds., pp. 2073 vol. 2, chap. 65 [seventh edition] (McGraw Hill, New York, 1995); Salen, et al.,
J. Lipid Res.
33:945 (1992)). Unlike other forms of hyperlipidemia, sitosterolemic subjects respond to restriction in dietary cholesterol and to bile acid resin treatment with dramatic reductions in plasma cholesterol levels (Bjorkhem and Boberg, in
The Metabolic and Molecular Bases of Inherited Disease
, Scriver et al., Eds., pp. 2073 vol. 2, chap. 65 [seventh edition] (McGraw Hill, New York, 1995); Salen, et al.,
J. Lipid Res.
33:945 (1992); T. A. Miettinen,
Eur. J. Clin. Invest.
10:27 (1980); Morganroth, et al.,
J. Pediatr.
85:639 (1974)).
Patients with sitosterolemia have markedly elevated (>30-fold) plasma levels of plant sterols (sitosterol, stigmasterol and campesterol) as well as other neutral sterols with modified side chains (Bhattacharyya, et al.,
J. Clin. Invest.
53:1033 (1974); Salen, et al.,
J. Lipid Res.
26:203 (1985); Gregg, et al.,
J. Clin. Invest.
77:1864 (1986)). Normal humans absorb only ~5% of the 200 to 300 mg of plant sterols consumed each day (Gould, et al.,
Metabolism
18:652 (1969); Salen, et al.,
J. Clin. Invest.
49:952 (1970)). Almost all of the absorbed sitosterol is quickly secreted into the bile so that only trace amounts of sitosterol and other plant sterols remain in the blood (Gould, et al.,
Metabolism
18:652 (1969); Salen, et al.,
J. Clin. Invest.
49:952 (1970)). In contrast, subjects with sitosterolemia absorb between 15 and 60% of ingested sitosterol, and they excrete only a fraction into the bile (Bhattacharyya, et al.,
J. Clin. Invest.
53:1033 (1974); Bjorkhem and Boberg, in
The Metabolic and Molecular Bases of Inherited Disease
, Scriver, et al., Eds., pp. 2073 vol. 2, chap. 65 [seventh edition] (McGraw Hill, New York, 1995); Salen, et al.,
J. Lipid Res.
33:945 (1992); T. A. Miettinen,
Eur. J. Clin. Invest.
10:27 (1980)). The liver secretes sitosterol into the bloodstream where it is transported as a constituent of low density and high density lipoprotein particles (Bhattacharyya, et al.,
J. Clin. Invest.
53:1033 (1974)). With the exception of the brain, the relative proportion of sterol represented by sitosterol in tissues matches that in plasma (10-25%) (Salen, et al.,
J. Lipid Res.
26:1126 (1985)). Hyperabsorption and inefficient secretion is not limited to plant sterols. Sitosterolemic subjects absorb a higher fraction of dietary cholesterol than normal subjects, and they secrete less cholesterol into the bile (Bhattacharyya, et al.,
J. Clin. Invest.
53:1033 (1974); Bjorkhem and Boberg, in
The Metabolic and Molecular Bases of Inherited Disease
, Scriver, et al., Eds., pp. 2073 vol. 2, chap. 65 [seventh edition] (McGraw Hill, New York, 1995); Salen, et al.,
J. Lipid Res.
33:945 (1992); T. A. Miettinen,
Eur. J. Clin. Invest.
10, 27 (1980)). Taken together, the genetic and metabolic data indicate that sitosterolemic patients lack a gene product that normally limits the absorption and accelerates the biliary excretion of sterols (Bjorkhem and Boberg, in
The Metabolic and Molecular Bases of Inherited Disease
, Scriver et al., Eds., pp. 2073 vol. 2, chap. 65 [seventh edition] (McGraw Hill, New York, 1995); Salen, et al.,
J. Lipid Res.
33:945 (1992)).
The molecular mechanisms that limit sterol absorption are poorly understood, but clues have emerged recently from studies of the orphan nuclear hormone receptor LXR (Repa, et al.,
Science
289:1524 (2000)). Mice treated with an LXR agonist have a marked decrease in cholesterol absorption and a corresponding increase in the intestinal expression of mRNA encoding the ATP binding cassette protein (ABC) 1, a membrane-associated protein that has been implicated in the transport of cholesterol (Repa, et al.,
Science
289:1524 (2000); Lawn, et al.,
J. Clin. Invest.
104:25 (1999)).
Clearly, new approaches for reducing the absorption of dietary cholesterol, for maximizing the elimination of excess cholesterol from the liver, and for treating sterol-associated disorders such as sitosterolemia would have tremendous public health benefits. The present invention addresses these and other needs.
SUMMARY OF THE INVENTION
The present invention provides nucleic acids encoding a novel ABC family sterol transporter, called ABCG8. The herein-disclosed sequences can be used for any of a number of purposes, including for the diagnosis and treatment of sterol-associated disorders, including sitosterolemia, and for the identification of molecules that associate with and/or modulate the activity of ABCG8 and, in turn, modulate the absorption of dietary cholesterol.
In one aspect, the present invention provides an isolated nucleic acid encoding an ABCG8 polypeptide, the polypeptide comprising at least about 70% amino acid sequence identity to an amino acid sequence as set forth in SEQ ID NO:4 or 8.
In one embodiment, the polypeptide specifically binds to polyclonal antibodies generated against a polypeptide that comprises an amino acid sequence of SEQ ID NO:4 or 8. In another embodiment, the polypeptide comprises an amino acid sequence of SEQ ID NO:4 or 8. In another embodiment, the polypeptide forms a dimer with a second ABC polypeptide, wherein the dimer comprises sterol transport activity. In another embodiment, the dimer is a heterodimer. In another embodiment, the sterol is cholesterol. In another embodiment, the second ABC polypeptide is ABCG5. In another embodiment, the ABCG5 polypeptide (1) comprises an amino acid sequence that is at least about 70% identical to an amino acid sequence as set forth in SEQ ID NO:2 or 6; (2) selectively binds to polyclonal antibodies generated against a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO:2 or 6; (3) comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:6; (4) is encoded by a nucleic acid that hybridizes under moderately stringent (or stringent conditions) conditions to a nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO:1 or 5; (5) is encoded by a nucleic acid that comprises a nucleotide sequence that is at least about 70% identical to a sequence as set forth in SEQ ID NO:1 or 5; or (6) is encoded by a nucleic acid that comprises a nucleotide sequence of SEQ ID NO:1 or 5.
In another embodiment, the nucleic acid hybridizes under moderately stringent hybridization conditions to a nucleic acid comprising a nucleotide sequence of SEQ ID NO:3 or 7. In another embodiment, the nucleic acid hybridizes under stringent hybridization conditions to a nucleic acid comprising a nucleotide sequence of SEQ ID NO:3 or 7. In another embodiment, the nucleic acid comprises a nucleotide sequence that is at least about 70% identical to SEQ ID NO:3 or 7. In another embodiment, the nucleic acid comprises a nucleotide sequence of SEQ ID NO:3 or 7. In another embodiment, the nucleic acid is greater than 500, 1000, 1500, 2000, or more nucleotides in length. In another embodiment, the nucleic a
Barnes Robert
Hobbs Helen H.
Shan Bei
Tian Hui
Kemmerer Elizabeth
Nichols Christopher James
Townsend and Townsend / and Crew LLP
Tularik Inc.
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