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US5679635A
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Aspartoacylase gene, protein, and methods of screening for mutatons associated with canavan disease
Assignee: Miami Children's Hospital Research Institute Inc
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Filing: 1994-09-09
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Granted: 1997-10-21
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Expires: 2014-09-09
Abstract
Abstract Canavan disease, an autosomal recessive leukodystrophy, is caused by deficiency of aspartoacylase and accumulation of N-acetylaspartic acid in brain. Human aspartoacylase (ASP) cDNA spanning 1,435 bp has been cloned and expressed in E. coli. A base change, a854>c, has been found in 85% of the 34 Canavan alleles tested so far, which results in a missense glu285>ala mutation that is predicted to be part of the catalytic domain of aspartoacylase. Several additional mutations have also been identified. The invention therefore provides nucleic acid sequences, genes, polypeptides, antibodies, vectors containing the gene, host cells transformed with vectors containing the gene, animal models for the disease, methods for expressing the polypeptide, genetic screening methods and kits, diagnostic methods and kits, methods of treating Canavan disease and methods of genetic therapy for the disease.
Inventors
- 👤 Reuben Matalon
- 👤 Rajinder Kaul
- 👤 Guang Ping Gao
- 👤 Kuppareddi Balamurugan
- 👤 Kimberlee Michals-Matalon
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-07 14:48
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/68; G01N33/6893; G01N33/6896; C; C12; C12N; C12N9/00; C12N9/14; C12N9/78; C12N9/80; C12Q; C12Q1/00; C12Q1/68; C12Q1/6876; C12Q1/6883; G01N33/53; G01N33/573; A; A01; A01K; A01K2217/00; A01K2217/05; C12Q2600/00; C12Q2600/156; C12Q2600/166
Claims Analysis
Total Claims: 45
Claim 1
Independent Claim
54 words
An isolated nucleic acid molecule comprising: (a) a nucleic acid sequence encoding a human aspartoacylase polypeptide; (b) a nucleic acid sequence fully complementary to nucleic acid sequence (a); or (c) a nucleic acid sequence at least 16 nucleotides in length capable of hybridizing specifically with one of said nucleic acid molecules (a) or (b).
Claim 2
Dependent Claim
54 words
An isolated nucleic acid molecule comprising: (a) a nucleic acid sequence encoding a human aspartoacylase polypeptide; (b) a nucleic acid sequence fully complementary to nucleic acid sequence (a); or (c) a nucleic acid sequence at least 16 nucleotides in length capable of hybridizing specifically with one of said nucleic acid molecules (a) or (b).
Claim 3
Dependent Claim
118 words
A nucleic acid molecule of claim 1(a), comprising: (i) a DNA molecule having the DNA sequence of FIG. 1 from DNA position +1 to +891; (ii) a DNA molecule encoding a wild-type human aspartoacylase polypeptide having the amino acid sequence of FIG. 1 from amino acid position +1 to +313; (iii) a DNA molecule having a sequence of a fragment of the DNA sequence of FIG. 1, or a sequence fully complementary thereto, and including at least 16 sequential nucleotides; or (iv) a DNA molecule having a sequence of a fragment of the DNA sequence of FIG. 1 and including at least 16 sequential nucleotides, and which encodes a fragment of the amino acid sequence of FIG. 1.
Claim 4
Dependent Claim
154 words
A nucleic acid molecule of claim 1(a), comprising (i') a DNA molecule having the DNA sequence of FIG. 1 from DNA position +1 to +891; (ii') a DNA molecule encoding a human aspartoacylase polypeptide having the amino acid sequence of FIG. 1 from amino acid position +1 to +313; (iii') a DNA molecule having a sequence of a fragment of the DNA sequence of FIG. 1, or a sequence fully complementary thereto, and including at least 16 sequential nucleotides; or (iv') a DNA molecule having a sequence of a fragment of the DNA sequence of FIG. 1 and including at least 16 sequential nucleotides, and which encodes a fragment of the amino acid sequence of FIG. 1; wherein each of said DNA molecules (i'), (ii'), (iii') or (iv') differs by at least one nucleotide from the nucleotide sequence of FIG. 1, and is a naturally-occurring allele of human aspartoacylase having an altered biological activity.
Claim 5
Dependent Claim
16 words
An isolated DNA molecule of claim 1, comprising a DNA sequence encoding a human aspartoacylase polypeptide.
Claim 6
Dependent Claim
17 words
An isolated DNA molecule of claim 4, wherein the DNA codes for a wild-type human aspartoacylase polypeptide.
Claim 7
Dependent Claim
34 words
An isolated DNA molecule of claim 4, wherein the DNA codes for a naturally-occurring allele or a mutant of human aspartoacylase polypeptide having an altered biological activity as compared with a wild-type aspartoacylase polypeptide.
Claim 8
Dependent Claim
35 words
An isolated DNA molecule of claim 6, wherein the DNA codes for an aspartoacylase polypeptide which, if expressed in cells of the human body, is associated with altered cellular function which correlates with Canavan disease.
Claim 9
Dependent Claim
31 words
An isolated nucleic acid primer or probe molecule comprising a DNA sequence, or its RNA equivalent, of the sequence (iii) or (iv) of claim 2, or a sequence fully complementary thereto.
Claim 10
Dependent Claim
30 words
An isolated nucleic acid primer or probe molecule comprising a DNA sequence, or its RNA equivalent, of the sequence (iii') or (iv') of claim 3, or a sequence complementary thereto.
Claim 11
Dependent Claim
21 words
A nucleic acid molecule of claim 8, having the sequence 5'-CTTCTGAATTGCAGAAATCA-3' (HASP9) (SEQ ID NO:20) or 5'-GTAAGACACCGTGTAAGATG-3' (HASPC7) (SEQ ID NO:21).
Claim 12
Dependent Claim
40 words
A nucleic acid molecule of claim 9, having the sequence 5'-F→CCGGGATGAAAATGGAGAA-3' (HASP14F) (SEQ ID NO:6) or 5'-R→ACCGTGTAAGATGTAAGC-3' (HASPC7R) (SEQ ID NO:7), wherein F and R are M13 universal and/or reverse primer tags linked or ligated to said nucleic acid molecule.
Claim 13
Dependent Claim
19 words
A DNA molecule of claim 5, having the DNA sequence of FIG. 1 from DNA position +1 to +891.
Claim 14
Dependent Claim
32 words
A DNA molecule of claim 7, having the DNA sequence of FIG. 1 from DNA position +1 to +891, wherein the adenine nucleotide at position 854 is replaced by a cytosine nucleotide.
Claim 15
Dependent Claim
10 words
A recombinant vector comprising a DNA molecule of claim 1.
Claim 16
Dependent Claim
28 words
A vector of claim 14, wherein said DNA molecule is operably linked to an expression control sequence suitable for expression of said DNA sequence in a host cell.
Claim 17
Dependent Claim
10 words
A host cell transformed with a vector of claim 15.
Claim 18
Dependent Claim
39 words
A host cell of claim 16, selected from a strain of E. coli, Pseudomonas, Bacillus subtilis, Bacillus stearothermophilus, or other bacilli; other bacteria; yeast; other fungi; insect cells; plant cells; or murine, bovine, porcine, human or other mammalian cells.
Claim 19
Dependent Claim
51 words
A method of producing a wild-type aspartoacylase polypeptide, comprising (a) culturing a host cell transformed with a vector of claim 15 containing a DNA coding for a wild-type aspartoacylase polypeptide in a cell culture medium under conditions whereby the aspartoacylase polypeptide is expressed, and (b) isolating the thus-produced wild-type aspartoacylase polypeptide.
Claim 20
Dependent Claim
24 words
An isolated aspartoacylase gene, comprising a DNA sequence of claim 1 operably linked to a DNA sequence encoding a human aspartoacylase expression control sequence.
Claim 21
Dependent Claim
15 words
An isolated gene of claim 19, encoding and capable of expressing a wild-type aspartoacylase polypeptide.
Claim 22
Dependent Claim
16 words
An isolated gene of claim 19, which is not capable of expressing a wild-type aspartoacylase polypeptide.
Claim 23
Dependent Claim
37 words
An isolated gene of claim 21, encoding and capable of expressing a mutant aspartoacylase polypeptide having an altered ability of hydrolyze N-acetyl-aspartic acid to aspartate and acetate or incapable of hydrolyzing N-acetyl-aspartic acid to aspartate and acetate.
Claim 24
Dependent Claim
26 words
An isolated gene of claim 22, which, if expressed in cells of the human body, is associated with altered cellular function which correlates with Canavan disease.
Claim 25
Dependent Claim
21 words
An isolated gene of claim 21, wherein said expression control sequence is mutated and is not capable of expression of aspartoacylase.
Claim 26
Dependent Claim
91 words
A method of screening a subject to determine if said subject is a Canavan carrier or a Canavan patient, comprising (a) providing a biological sample of the subject to be screened; and (b) submitting the sample to an assay for detecting in the biological sample the presence of a wild-type aspartoacylase gene, a mutant aspartoacylase gene or a mixture thereof, wherein said gene has a DNA sequence of claim 1, and wherein detection of a mutant as aspartoacylase gene indicates that the subject is a Canavan carrier or a Canavan patient.
Claim 27
Dependent Claim
26 words
A method of claim 25, wherein the biological sample includes at least part of the genome of the subject and the assay comprises a hybridization assay.
Claim 28
Dependent Claim
15 words
A method of claim 25, wherein the assay comprises at least one labeled nucleotide probe.
Claim 29
Dependent Claim
42 words
A method of claim 27, wherein the assay comprises a labeled nucleotide probe comprising a DNA molecule having a sequence of a fragment of the DNA sequence of FIG. 1, or a sequence complementary thereto, and including at least 16 sequential nucleotides.
Claim 30
Dependent Claim
53 words
A method of claim 27, wherein the probe is selected from the sequences 5'-CTTCTGAATTGCAGAAATCA-3' (HASP9) (SEQ ID NO:20), 5'-GTAAGACACCGTGTAAGATG-3' (HASPC7) (SEQ ID NO:21), 5'-F→CCGGGATGAAAATGGAGAA-3' (HASP14F) (SEQ ID NO:6) or 5'-R→ACCGTGTAAGATGTAAGC-3' (HASPC7R) (SEQ ID NO:7), wherein F and R are M13 universal and/or reverse primer tags linked or ligated to said nucleic acid molecule.
Claim 31
Dependent Claim
94 words
A process for screening a potential Canavan disease carrier or patient for the presence of an identified mutation in an aspartoacylase gene, comprising (a) isolating genomic DNA from said potential Canavan disease carrier or patient, (b) hybridizing a DNA probe of claim 9 with said isolated genomic DNA, said DNA probe spanning said mutation in said aspartoacylase gene, wherein said DNA probe is capable of detecting said mutation, (c) determining the presence or absence of said DNA probe hybridized with said genomic DNA, said hybridization indicating the presence or absence of said aspartoacylase mutation.
Claim 32
Dependent Claim
73 words
A process for screening a potential Canavan disease carrier or patient for the presence of an identified mutation in an aspartoacylase gene having a nucleic acid sequence of claim 1a, comprising (a) isolating genomic DNA from said potential Canavan disease carrier or patient, (b) determining the presence or absence of a restriction endonuclease site in the gene, the presence or absence of which thereby indicates the presence or absence of said aspartoacylase mutation.
Claim 33
Dependent Claim
114 words
A process for screening a potential Caravan disease carrier or patient for the presence of an identified mutation in an aspartoacylase gene, comprising (a) isolating genomic DNA from said potential Canavan disease carrier or patient, (b) performing PCR on said genomic DNA, using a primer of claim 8, (c) adding PCR products produced from wild-type genomic DNA using the same primer, (d) forming heteroduplexes between said PCR products, and (e) determining the mobility of said heteroduplex PCR products in polyacrylamide gels as compared with the mobility of PCR products produced from wild-type genomic DNA using the same primer, wherein an altered mobility of said heteroduplex PCR products indicates the presence of said aspartoacylase mutation.
Claim 34
Dependent Claim
63 words
A kit for assaying for the presence of an aspartoacylase gene by hybridization assay, comprising: (a) an oligonucleotide probe of claim 8 which specifically hybridizes to the aspartoacylase gene; and (b) a reagent means for detecting of the hybridization of the oligonucleotide probe to the gene; wherein the probe and reagent means are each present in amounts effective to perform the hybridization assay.
Claim 35
Dependent Claim
14 words
A kit of claim 33, wherein said aspartoacylase gene is a wild-type aspartoacylase gene.
Claim 36
Dependent Claim
14 words
A kit of claim 33, wherein said aspartoacylase gene is a mutant aspartoacylase gene.
Claim 37
Dependent Claim
56 words
A kit of claim 33, further comprising an oligonucleotide probe which specifically hybridizes one or more additional mutant or wild-type genes, and, optionally, a corresponding additional reagent means for detecting hybridization of said oligonucleotide probe which specifically hybridizes to said additional gene, wherein said additional gene codes for a protein associated with an additional genetic disease.
Claim 38
Dependent Claim
16 words
A kit of claim 36, wherein the additional genetic disease is Tay-Sachs disease and/or Goucher disease.
Claim 39
Dependent Claim
23 words
A nucleic acid molecule of claim 1, selected from the sequences SEQ ID NO:28-SEQ ID NO:62 and nucleotides 119-139 of SEQ ID NO:1.
Claim 40
Dependent Claim
63 words
A method of producing a mutant aspartoacylase polypeptide having an altered biological activity as compared with a wild-type aspartoacylase polypeptide, comprising (a) culturing a host cell transformed with a vector of claim 15 containing a DNA coding for a mutant aspartoacylase polypeptide in a cell culture medium under conditions whereby the aspartoacylase polypeptide is expressed, and (b) isolating the thus-produced mutant aspartoacylase polypeptide.
Claim 41
Dependent Claim
84 words
A process for screening a potential Canavan disease carrier or patient for the presence of an identified mutation in an aspartoacylase gene, comprising (a) isolating genomic DNA from said potential Canavan disease carrier or patient, (b) hybridizing a DNA probe of claim 8 with said isolated genomic DNA, said DNA probe spanning said mutation in said aspartoacylase gene, (c) determining the presence or absence of said DNA probe hybridized with said genomic DNA, said hybridization indicating the presence or absence of said aspartoacylase mutation.
Claim 42
Dependent Claim
103 words
A process for screening a potential Canavan disease carrier or patient for the presence of an identified mutation in an aspartoacylase gene, comprising (a) isolating genomic DNA from said potential Canavan disease carrier or patient, (b) performing PCR on said genomic DNA, using a primer of claim 9, (c) optionally, adding PCR products produced from wild-type genomic DNA, and (d) determining the mobility of heteroduplex PCR products in polyacrylamide gels as compared with the mobility of PCR products produced from wild-type genomic DNA using the same primer or primers, the mobility of which thereby indicates the presence or absence of said aspartoacylase mutation.
Claim 43
Dependent Claim
62 words
A kit for assaying for the presence of an aspartoacylase gene by hybridization assay, comprising: (a) an oligonucleotide probe of claim 9 which specifically hybridizes to the aspartoacylase gene; and (b) a reagent means for detecting the hybridization of the oligonucleotide probe to the gene; wherein the probe and reagent means are each present in amounts effective to perform the hybridization assay.
Claim 44
Dependent Claim
58 words
A kit of claim 42, further comprising an additional oligonucleotide probe which specifically hybridizes to one or more additional mutant or wild-type genes, and, optionally, a corresponding additional reagent means for detecting hybridization of said oligonucleotide probe which specifically hybridizes to said additional gene, wherein said additional gene codes for a protein associated with an additional genetic disease.
Claim 45
Dependent Claim
16 words
A kit of claim 42, wherein the additional genetic disease is Tay-Sachs disease and/or Goucher disease.
References Cited (Backward Citations)
No citation records indexed.
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 2000-10-20 | 2001-04-21 | 2001-10-21 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2004-10-20 | 2005-04-21 | 2005-10-21 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2008-10-20 | 2009-04-21 | 2009-10-21 | $7700.00 | Window Closed / Lapsed |