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
1995-11-14
2001-07-31
Spector, Lorraine (Department: 1646)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C530S350000, C536S023100, C536S023500, C536S024100, C435S320100, C435S325000, C435S252300, C435S254110, C514S012200, C549S408000
Reexamination Certificate
active
06268170
ABSTRACT:
DESCRIPTION
1. Technical Field of the Invention
The field of this invention is &agr;-tocopherol transport protein. More particularly, the present invention pertains to human &agr;-tocopherol transport protein, polynucleotides encoding that &agr;-tocopherol transport protein, and methods of making and using those polynucleotides and proteins.
2. Background of the Invention
Familial isolated vitamin E deficiency, also called ataxia with vitamin E deficiency (AVED) is a neurodegenerative syndrome characterized by cerebellar ataxia and symptoms of central and peripheral axonopathy resembling Friedreich ataxia. It is transmitted as an autosomal recessive trait. The failure to incorporate &agr;-tocopherol in very low density lipoprotein (VLDL) in liver cells is thought to be the primary cause of the decrease in the serum level of vitamin E in these patients. The absorption and metabolism of chylomicrons and VLDL are normal in AVED patients. This differentiates AVED from most causes of secondary vitamin E deficiency in which the perturbation of lipoprotein absorption, metabolism or transport is the primary cause of vitamin E deficiency. &agr; Tocopherol transfer protein (&agr;TTP) from rat liver enhances the transfer of the &agr;-tocopherol form of vitamin E between membranes. In vivo, &agr;TTP incorporates &agr;-tocopherol into lipoprotein particles during their assembly in the liver cells. A recent report has identified three mutations in two exons of the gene for &agr;TTP in families with AVED.
BRIEF SUMMARY OF THE INVENTION
In one aspect, the present invention provides an isolated and purified polynucleotide comprising a nucleotide sequence consisting essentially of a nucleotide sequence selected from the group consisting of (a) nucleotide position 8 to nucleotide position 842 of the sequence of SEQ ID NO:1; (b) sequences that are complementary to the sequences of (a), and (c) sequences that, when expressed, encode a polypeptide encoded by a sequence of (a). A preferred polynucleotide is a DNA molecule. In another embodiment, the polynucleotide is an RNA molecule. A preferred polynucleotide is SEQ ID NO: 1, from nucleotide position 8 to nucleotide position 842.
In another embodiment, a DNA molecule of the present invention is contained in an expression vector. The expression vector preferably further comprises an enhancer-promoter operatively linked to the polynucleotide. In an especially preferred embodiment, the DNA molecule has the nucleotide sequence of SEQ ID NO:1.
In another aspect, the present invention provides an oligonucleotide of from about 15 to about 50 nucleotides containing a nucleotide sequence of at least 15 nucleotides that is identical or complementary to a contiguous sequence of a polynucleotide of this invention. A preferred oligonucleotide is an antisense oligonucleotide that is complementary to a portion of the polynucleotide of SEQ ID NO: 1.
The present invention also provides a pharmaceutical composition comprising an antisense oligonucleotide of this invention and a physiologically acceptable diluent.
In another aspect, the present invention provides an &agr;-tocopherol transport protein of human origin. That &agr;-tocopherol transport protein is an isolated and purified polypeptide of 278 amino acid residues and has the sequence shown in SEQ ID NO:2. Preferably, an &agr;-tocopherol transport protein of the present invention is a recombinant human &agr;-tocopherol transport protein.
In another aspect, the present invention provides a process of making &agr;-tocopherol transport protein comprising transforming a host cell with an expression vector that comprises a polynucleotide of the present invention, maintaining the transformed cell for a period of time sufficient for expression of the &agr;-tocopherol transport protein and recovering the &agr;-tocopherol transport protein. Preferably, the host cell is an eukaryotic host cell such as a human cell, or a bacterial cell. An especially preferred host cell is an
E. coli
. The present invention also provides an &agr;-tocopherol transport protein made by a process of this invention.
The present invention still further provides for a host cell transformed with a polynucleotide or expression vector of this invention. Preferably, the host cell is a bacterial cell such as an
E. coli.
In another aspect, the present invention provides a process of diagnosing ataxia with vitamin E deficiency. That process comprises detecting the presence of a mutation in the gene for &agr;-tocopherol transport protein, wherein the mutation is (a) a deletion of the thymine at position 485 of exon 3, (b) an insertion of two thymines at position 514 of exon 4 or (c) a substitution of a guanine for an adenine at position 574 of exon 4. In one embodiment of the diagnostic process, the mutation is two or more of (a), (b) and (c).
The detection of mutations can also be used to determine the susceptibility of a child to ataxia with vitamin E deficiency, In accordance with this embodiment, the presence of a mutation in the gene for &agr;-tocopherol transport protein is detected in one or both parents of the child, wherein the mutation is (a) a deletion of the thymine at position 485 of exon 3, (b) an insertion of two thymines at position 514 of exon 4 or (c) a substitution of a guanine for an adenine at position 574 of exon 4.
REFERENCES:
patent: 5541220 (1996-07-01), Ismail et al.
Arita et al., Human alpha-tocopherol transfer protein: cDNA cloning, expression and chromosomal localization, Biochem. J., 306(pt. 2): 437-443, Mar. 1995.*
Ouahchi et al., Ataxia with isolated vitamin E deficiency is caused by mutations in the alpha-tocopherol transfer protein, Nat. Genetics, 9: 141-145, Feb. 1995.*
Langer, R., New methods of drug delivery, Science, 249: 1527-1532, Sep. 1990.
Deng Han-Xiang
Hentati Afif
Siddique Teepu
Kaufman Claire M.
Northwestern University
Rockey, Milnamow & Katz
Spector Lorraine
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