U.S. patent application number 10/196703 was filed with the patent office on 2003-03-20 for genes and proteins predictive and therapeutic for stroke, hypertension, diabetes and obesity.
Invention is credited to Shimkets, Richard A..
Application Number | 20030055019 10/196703 |
Document ID | / |
Family ID | 22583461 |
Filed Date | 2003-03-20 |
United States Patent
Application |
20030055019 |
Kind Code |
A1 |
Shimkets, Richard A. |
March 20, 2003 |
Genes and proteins predictive and therapeutic for stroke,
hypertension, diabetes and obesity
Abstract
Common human diseases like diabetes and hypertension have been
demonstrated to possess an associated genetic component composed of
numerous underlying genetic defects. Traditional positional cloning
of genes which possess the mutations responsible for complex
disease has been hindered by both the low statistical power each
locus may afford, and by the technically-laborious nature of the
positional cloning methodologies. Disclosed herein is a methodology
for the rapid identification of the genes responsible for
quantitative trait loci (QTL) comprised of comprehensive gene
expression analysis in organs relevant to disease in combination
with the positional mapping of known QTL, so as to quantitatively
identify candidate genes. This aforementioned methodology was
applied to a total of five tissues/organs derived from the
spontaneously hypertensive rat (SHR), the stroke-prone variant of
the SHR (SHR-SP) and control Wistar Kyoto rats (WKY). Collectively
these animals vary genetically in their predisposition to stroke,
insulin sensitivity, blood pressure and body weight. These traits
segregate into more than a dozen identified. The present invention
discloses the differential-expression of sixty genes by
GeneCalling.RTM. within five different tissues/organs among these
animals. Additionally, five of the sixty genes were demonstrated to
be localized within chromosomal regions linked to these traits of
interest and possess amino acid substitutions which may contribute
to the phenotype.
Inventors: |
Shimkets, Richard A.; (West
Haven, CT) |
Correspondence
Address: |
MINTZ, LEVIN, COHN, FERRIS,
GLOVSKY AND POPEO, P.C.
One Financial Center
Boston
MA
02111
US
|
Family ID: |
22583461 |
Appl. No.: |
10/196703 |
Filed: |
July 15, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10196703 |
Jul 15, 2002 |
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09161939 |
Sep 28, 1998 |
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6486299 |
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Current U.S.
Class: |
514/44A ;
435/189; 435/193; 435/320.1; 435/325; 435/69.1; 530/388.26;
536/23.2 |
Current CPC
Class: |
A01K 67/0275 20130101;
A61P 3/04 20180101; C12N 9/1096 20130101; A61P 3/10 20180101; C07K
14/47 20130101; C07K 14/58 20130101; A61P 9/10 20180101; A01K
2217/05 20130101; A61K 38/00 20130101; Y10S 530/80 20130101; C07K
14/705 20130101; A61K 48/00 20130101; A61P 3/06 20180101; A61P 9/12
20180101; C07K 14/4716 20130101; A61P 43/00 20180101; C12N 9/88
20130101; C07K 14/70596 20130101 |
Class at
Publication: |
514/44 ;
435/69.1; 435/193; 435/189; 435/320.1; 435/325; 530/388.26;
536/23.2 |
International
Class: |
A61K 048/00; C07H
021/04; C12P 021/02; C12N 005/06; C07K 016/40 |
Claims
What is claimed is:
1. Isolated mutant proteins selected from a group consisting of:
SGLT2, kynurenine aminotransferase, FAT/CD36, aldolase A,
prepronatriodilatin, .alpha.-cardiac myosin and .alpha.-tubulin;
wherein the amino acid sequences of said mutant proteins possess
one or more amino acid substitutions in comparison to the amino
acid sequence of the corresponding native protein.
2. The isolated mutant proteins of claim 1, wherein said mutant
proteins are human proteins possessing the amino acid sequences
depicted in FIG. 4 consisting of: SGLT2 [SEQ ID NO:1]; kynurenine
aminotransferase [SEQ ID NO:2]; FAT/CD36 [SEQ ID NOS:3 AND 4];
aldolase A [SEQ ID NO:5]; prepronatriodilatin [SEQ ID NO:6];
.alpha.-cardiac myosin [SEQ ID NO:7] and .alpha.-tubulin [SEQ ID
NO:8].
3. The isolated mutant proteins of claim 1, wherein said mutant
proteins are substantially-purified proteins.
4. Antibodies which are specific for, and possess the ability to
bind to said mutant proteins of claim 1.
5. Isolated nucleic acid sequences encoding said mutant proteins of
claim 1.
6. Antibodies which are specific for, and possess the ability to
bind to said nucleic acid sequences of claim 5.
7. Isolated anti-sense nucleic acid derivatives of the nucleic acid
sequences encoding said mutant proteins of claim 1.
8. Antibodies which are specific for, and possess the ability to
bind to said anti-sense nucleic acid derivatives of claim 7.
9. The mutant proteins of claim 1, wherein said mutant proteins
possess a longer biological half life in vivo, relative to the
corresponding, native human proteins.
10. A method of treating or preventing hypertension comprising of
administering, to a subject in which such treatment or prevention
is desired, an amount of one or more of said mutant proteins of
claim 1, sufficient to treat or prevent hypertension.
11. A method of treating or preventing hypertension comprising of
administering, to a subject in which such treatment or prevention
is desired, an amount of one or more antibodies specific for, and
possessing the ability to bind to one or more of said mutant
proteins of claim 1 sufficient to treat or prevent
hypertension.
12. A method of treating or preventing hypertension comprising of
administering, to a subject in which such treatment or prevention
is desired, an amount of one or more of said nucleic acids of claim
5, sufficient to treat or prevent hypertension.
13. A method of treating or preventing hypertension comprising of
administering, to a subject in which such treatment or prevention
is desired, an amount of one or more of said anti-sense nucleic
acid derivatives of claim 7, sufficient to treat or prevent
hypertension.
14. A method of treating or preventing diabetes and insulin
resistivity comprising of administering, to a subject in which such
treatment or prevention is desired, an amount of one or more of
said mutant proteins of claim 1, sufficient to treat or prevent
diabetes and insulin resistivity.
15. A method of treating or preventing diabetes and insulin
resistivity comprising of administering, to a subject in which such
treatment or prevention is desired, an amount of one or more
antibodies specific for, and possessing the ability to bind to one
or more of said mutant proteins of claim 1 sufficient to treat or
prevent diabetes and insulin resistivity.
16. A method of treating or preventing diabetes and insulin
resistivity comprising of administering, to a subject in which such
treatment or prevention is desired, an amount of one or more of
said nucleic acids of claim 5, sufficient to treat or prevent
diabetes and insulin resistivity.
17. A method of treating or preventing diabetes and insulin
resistivity comprising of administering, to a subject in which such
treatment or prevention is desired, an amount of one or more of
said anti-sense nucleic acid derivatives of claim 7, sufficient to
treat or prevent diabetes and insulin resistivity.
18. A method of treating or preventing obesity and dyslipidemia
comprising of administering, to a subject in which such treatment
or prevention is desired, an amount of one or more of said mutant
proteins of claim 1, sufficient to treat or prevent obesity and
dyslipidemia.
19. A method of treating or preventing obesity and dyslipidemia
comprising of administering, to a subject in which such treatment
or prevention is desired, an amount of one or more antibodies
specific for, and possessing the ability to bind to one or more of
said mutant proteins of claim 1 sufficient to treat or prevent
obesity and dyslipidemia.
20. A method of treating or preventing obesity and dyslipidemia
comprising of administering, to a subject in which such treatment
or prevention is desired, an amount of one or more of said nucleic
acids of claim 5, sufficient to treat or prevent obesity and
dyslipidemia.
21. A method of treating or preventing obesity and dyslipidemia
comprising administering, to a subject in which such treatment or
prevention is desired, an amount of one or more of said anti-sense
nucleic acid derivatives of claim 7, sufficient to treat or prevent
obesity and dyslipidemia.
22. A method of treating or preventing or delaying stroke
comprising of administering, to a subject in which such treatment
or prevention or delay is desired, an amount of one or more of said
mutant proteins of claim 1, sufficient to treat or prevent
sufficient to treat or prevent or delay stroke.
23. A method of treating or preventing or delaying stroke
comprising of administering, to a subject in which such treatment
or prevention or delay is desired, an amount of one or more
antibodies specific for, and possessing the ability to bind to one
or more of said mutant proteins of claim 1 sufficient to treat or
prevent or delay stroke.
24. A method of treating or preventing or delaying stroke
comprising of administering, to a subject in which such treatment
or prevention or delay is desired, an amount of one or more of said
nucleic acids of claim 5, sufficient to treat or prevent sufficient
to treat or prevent or delay stroke.
25. A method of treating or preventing or delaying stroke
comprising of administering, to a subject in which such treatment
or prevention or delay is desired, an amount of one or more of said
anti-sense nucleic acid derivatives of claim 7, sufficient to treat
or prevent sufficient to treat or prevent or delay stroke.
26. The method of claim 22, wherein said mutant prepronatriodilatin
protein [SEQ ID NO:6] is administered to prevent stroke in a
subject possessing one or more risk factors for stroke.
27. The method of claim 22, wherein said stroke is ischemic
stroke.
28. The method of claim 22, wherein said mutant human
prepronatriodilatin [SEQ ID NO:6] increases latency to stroke in
stroke-prone rats fed a high salt diet.
29. A pharmaceutical composition comprising a therapeutically or
prophylactically effective amount of one or more of said mutant
proteins of claim 1 and a pharmaceutically acceptable carrier.
30. A kit, comprising in one or more containers, a therapeutically
or prophylactically effective amount of the pharmaceutical
composition of claim 29.
31. A pharmaceutical composition comprising a therapeutically or
prophylactically effective amount of one or more of said nucleic
acid of claim 5, and a pharmaceutically acceptable carrier.
32. The pharmaceutical composition of claim 31, wherein said
nucleic acid is a nucleic acid vector.
33. A kit, comprising in one or more containers, a therapeutically
or prophylactically effective amount of the pharmaceutical
composition of claim 31.
34. A pharmaceutical composition comprising a therapeutically or
prophylactically effective amount of one or more antibodies which
are specific for, and possess the ability to bind to, one or more
of said mutant proteins of claim 1, and a pharmaceutically
acceptable carrier.
35. A kit, comprising in one or more containers, a therapeutically
or prophylactically effective amount of the pharmaceutical
composition of claim 34.
36. A pharmaceutical composition comprising a therapeutically or
prophylactically effective amount of one or more of said anti-sense
nucleic acid derivatives of claim 7, and a pharmaceutically
acceptable carrier.
37. The pharmaceutical composition of claim 36, wherein said
anti-sense nucleic acid derivative is a nucleic acid vector.
38. A kit, comprising in one or more containers, a therapeutically
or prophylactically effective amount of the pharmaceutical
composition of claim 36.
39. A method for screening for an allele protective against
hypertension, diabetes, obesity and/or stroke in a subject
comprising detecting a mutant SGLT2 allele, the presence of said
mutant SGLT2 allele being indicative of an allele protective for
hypertension, diabetes, obesity and/or stroke.
40. The method of claim 39 in which said mutant SGLT2 allele
encodes a mutant SGLT2 having an amino acid substitution
corresponding to the amino acid sequence of human SGLT2 as depicted
in FIG. 4 (SEQ ID NO:1).
41. A method for screening for an allele protective against
hypertension, diabetes, obesity and/or stroke in a subject
comprising detecting a mutant kynurenine aminotransferase allele,
the presence of said mutant kynurenine aminotransferase allele
being indicative of an allele protective for hypertension,
diabetes, obesity and/or stroke.
42. The method of claim 41 in which said mutant kynurenine
aminotransferase allele encodes a mutant kynurenine
aminotransferase having an amino acid substitution corresponding to
the amino acid sequence of human kynurenine aminotransferase as
depicted in FIG. 4 (SEQ ID NO:2).
43. A method for screening for an allele protective against
hypertension, diabetes, obesity and/or stroke in a subject
comprising detecting a mutant FAT/CD36 allele, the presence of said
mutant FAT/CD36 allele being indicative of an allele protective for
hypertension, diabetes, obesity and/or stroke.
44. The method of claim 43 in which said mutant FAT/CD36 allele
encodes a mutant FAT/CD36 having an amino acid substitution
corresponding to the amino acid sequence of human FAT/CD36 as
depicted in FIG. 4 (SEQ ID NO:3 and NO:4).
45. A method for screening for an allele protective against
hypertension, diabetes, obesity and/or stroke in a subject
comprising detecting a mutant aldolase A allele, the presence of
said mutant aldolase A allele being indicative of an allele
protective for hypertension, diabetes, obesity and/or stroke.
46. The method of claim 45 in which said mutant aldolase A allele
encodes a mutant aldolase A having an amino acid substitution
corresponding to the amino acid sequence of human aldolase A as
depicted in FIG. 4 (SEQ ID NO:5).
47. A method for screening for an allele protective against
hypertension, diabetes, obesity and/or stroke in a subject
comprising detecting a mutant prepronatriodilatin allele, the
presence of said mutant prepronatriodilatin allele being indicative
of an allele protective for hypertension, diabetes, obesity and/or
stroke.
48. The method of claim 47 in which said mutant prepronatriodilatin
allele encodes a mutant prepronatriodilatin having an amino acid
substitution corresponding to the amino acid sequence of human
prepronatriodilatin as depicted in FIG. 4 (SEQ ID NO:6).
49. A method for screening for an allele protective against
hypertension, diabetes, obesity and/or stroke in a subject
comprising detecting a mutant .alpha.-cardiac myosin allele, the
presence of said mutant .alpha.-cardiac myosin allele being
indicative of an allele protective for hypertension, diabetes,
obesity and/or stroke.
50. The method of claim 49 in which said mutant .alpha.-cardiac
myosin allele encodes a mutant .alpha.-cardiac myosin having an
amino acid substitution corresponding to the amino acid sequence of
human .alpha.-cardiac myosin as depicted in FIG. 4 (SEQ ID
NO:7).
51. A method for screening for an allele protective against
hypertension, diabetes, obesity and/or stroke in a subject
comprising detecting a mutant .alpha.-tubulin allele, the presence
of said mutant .alpha.-tubulin allele being indicative of an allele
protective for hypertension, diabetes, obesity and/or stroke.
52. The method of claim 51 in which said mutant .alpha.-tubulin
allele encodes a mutant .alpha.-tubulin having an amino acid
substitution corresponding to the amino acid sequence of human
.alpha.-tubulin as depicted in FIG. 4 (SEQ ID NO:8).
53. A method for screening for a modulator of the activity or of
latency or predisposition to hypertension, diabetes, obesity and/or
stroke of said mutant SGLT2 protein of claim 1 comprising: (a)
administering a putative modulator of SGLT2 activity to a test
animal prone to hypertension, diabetes, obesity and/or stroke; and
(b) measuring one or more physiological parameters associated with
SGLT2 activity, in which a change in said one or more parameters
relative to an animal not administered the putative modulator
indicates that the putative modulator modulates SGLT2 activity or
latency or predisposition to hypertension, diabetes, obesity and/or
stroke.
54. The method of claim 53, wherein said test animal is a
recombinant test animal which expresses an SGLT2 transgene or
expresses SGLT2 under the control of a promoter which is not the
native SGLT2 gene promoter at an increased level relative to a
wild-type test animal.
55. A method for screening for a modulator of the activity or of
latency or predisposition to hypertension, diabetes, obesity and/or
stroke of said mutant kynurenine aminotransferase protein of claim
1 comprising: (a) administering a putative modulator of kynurenine
aminotransferase activity to a test animal prone to hypertension,
diabetes, obesity and/or stroke; and (b) measuring one or more
physiological parameters associated with kynurenine
aminotransferase activity, in which a change in said one or more
parameters relative to an animal not administered the putative
modulator indicates that the putative modulator modulates
kynurenine aminotransferase activity or latency or predisposition
to hypertension, diabetes, obesity and/or stroke.
56. The method of claim 55, wherein said test animal is a
recombinant test animal which expresses a kynurenine
aminotransferase transgene or expresses kynurenine aminotransferase
under the control of a promoter which is not the native kynurenine
aminotransferase gene promoter at an increased level relative to a
wild-type test animal.
57. A method for screening for a modulator of the activity or of
latency or predisposition to hypertension, diabetes, obesity and/or
stroke of said mutant FAT/CD36 protein of claim 1 comprising: (a)
administering a putative modulator of FAT/CD36 activity to a test
animal prone to hypertension, diabetes, obesity and/or stroke; and
(b) measuring one or more physiological parameters associated with
FAT/CD36 activity, in which a change in said one or more parameters
relative to an animal not administered the putative modulator
indicates that the putative modulator modulates FAT/CD36 activity
or latency or predisposition to hypertension, diabetes, obesity
and/or stroke.
58. The method of claim 57, wherein said test animal is a
recombinant test animal which expresses a FAT/CD36 transgene or
expresses FAT/CD36 under the control of a promoter which is not the
native FAT/CD36 gene promoter at an increased level relative to a
wild-type test animal.
59. A method for screening for a modulator of the activity or of
latency or predisposition to hypertension, diabetes, obesity and/or
stroke of said mutant aldolase A protein of claim 1 comprising: (a)
administering a putative modulator of aldolase A activity to a test
animal prone to hypertension, diabetes, obesity and/or stroke; and
(b) measuring one or more physiological parameters associated with
aldolase A activity, in which a change in said one or more
parameters relative to an animal not administered the putative
modulator indicates that the putative modulator modulates aldolase
A activity or latency or predisposition to hypertension, diabetes,
obesity and/or stroke.
60. The method of claim 59, wherein said test animal is a
recombinant test animal which expresses an aldolase A transgene or
expresses aldolase A under the control of a promoter which is not
the native aldolase A gene promoter at an increased level relative
to a wild-type test animal.
61. A method for screening for a modulator of the activity or of
latency or predisposition to hypertension, diabetes, obesity and/or
stroke of said mutant prepronatriodilatin protein of claim 1
comprising: (a) administering a putative modulator of
prepronatriodilatin activity to a test animal prone to
hypertension, diabetes, obesity and/or stroke; and (b) measuring
one or more physiological parameters associated with
prepronatriodilatin activity, in which a change in said one or more
parameters relative to an animal not administered the putative
modulator indicates that the putative modulator modulates
prepronatriodilatin activity or latency or predisposition to
hypertension, diabetes, obesity and/or stroke.
62. The method of claim 61, wherein said test animal is a
recombinant test animal which expresses a prepronatriodilatin
transgene or expresses prepronatriodilatin under the control of a
promoter which is not the native prepronatriodilatin gene promoter
at an increased level relative to a wild-type test animal.
63. A method for screening for a modulator of the activity or of
latency or predisposition to hypertension, diabetes, obesity and/or
stroke of said mutant .alpha.-cardiac myosin protein of claim 1
comprising: (a) administering a putative modulator of
.alpha.-cardiac myosin activity to a test animal prone to
hypertension, diabetes, obesity and/or stroke; and (b) measuring
one or more physiological parameters associated with
.alpha.-cardiac myosin activity, in which a change in said one or
more parameters relative to an animal not administered the putative
modulator indicates that the putative modulator modulates
.alpha.-cardiac myosin activity or latency or predisposition to
hypertension, diabetes, obesity and/or stroke.
64. The method of claim 63, wherein said test animal is a
recombinant test animal which expresses an .alpha.-cardiac myosin
transgene or expresses .alpha.-cardiac myosin under the control of
a promoter which is not the native .alpha.-cardiac myosin gene
promoter at an increased level relative to a wild-type test
animal.
65. A method for screening for a modulator of the activity or of
latency or predisposition to hypertension, diabetes, obesity and/or
stroke of said mutant .alpha.-tubulin protein of claim 1
comprising: (a) administering a putative modulator of
.alpha.-tubulin activity to a test animal prone to hypertension,
diabetes, obesity and/or stroke; and (b) measuring one or more
physiological parameters associated with .alpha.-tubulin activity,
in which a change in said one or more parameters relative to an
animal not administered the putative modulator indicates that the
putative modulator modulates .alpha.-tubulin activity or latency or
predisposition to hypertension, diabetes, obesity and/or
stroke.
66. The method of claim 65, wherein said test animal is a
recombinant test animal which expresses an .alpha.-tubulin
transgene or expresses .alpha.-tubulin under the control of a
promoter which is not the native .alpha.-tubulin gene promoter at
an increased level relative to a wild-type test animal.
67. A method for screening a SGLT2 mutant for its SGLT2 activity
comprising: (a) administering the SGLT2 mutant to a test animal
prone to hypertension, diabetes, obesity and/or stroke; and (b)
measuring stroke latency in the test animal, in which stroke
latency is indicative of SGLT2 activity.
68. The method of claim 67 in which the test animal is a
recombinant test animal which expresses an SGLT2 transgene or
expresses SGLT2 under the control of a promoter which is not the
native SGLT2 gene promoter at an increased level relative to a
wild-type test animal.
69. A method for screening a kynurenine aminotransferase mutant for
its kynurenine aminotransferase activity comprising: (a)
administering the kynurenine aminotransferase mutant to a test
animal prone to hypertension, diabetes, obesity and/or stroke; and
(b) measuring the latency of hypertension, diabetes, obesity and/or
stroke in the test animal, wherein such latency is indicative of
kynurenine aminotransferase activity.
70. The method of claim 69 in which the test animal is a
recombinant test animal which expresses a kynurenine
aminotransferase transgene or expresses kynurenine aminotransferase
under the control of a promoter which is not the native kynurenine
aminotransferase gene promoter at an increased level relative to a
wild-type test animal.
71. A method for screening a kynurenine aminotransferase mutant for
its kynurenine aminotransferase activity comprising: (a)
administering the kynurenine aminotransferase mutant to a test
animal prone to hypertension, diabetes, obesity and/or stroke; and
(b) measuring the latency of hypertension, diabetes, obesity and/or
stroke in the test animal, wherein such latency is indicative of
kynurenine aminotransferase activity.
72. The method of claim 71 in which the test animal is a
recombinant test animal which expresses an kynurenine
aminotransferase transgene or expresses kynurenine aminotransferase
under the control of a promoter which is not the native kynurenine
aminotransferase gene promoter at an increased level relative to a
wild-type test animal.
73. A method for screening a FAT/CD36 mutant for its kynurenine
aminotransferase activity comprising: (a) administering the
FAT/CD36 mutant to a test animal prone to hypertension, diabetes,
obesity and/or stroke; and (b) measuring the latency of
hypertension, diabetes, obesity and/or stroke in the test animal,
wherein such latency is indicative of FAT/CD36 activity.
74. The method of claim 73 in which the test animal is a
recombinant test animal which expresses a FAT/CD36 transgene or
expresses FAT/CD36 under the control of a promoter which is not the
native FAT/CD36 gene promoter at an increased level relative to a
wild-type test animal.
75. A method for screening a aldolase A mutant for its aldolase A
activity comprising: (a) administering the aldolase A mutant to a
test animal prone to hypertension, diabetes, obesity and/or stroke;
and (b) measuring the latency of hypertension, diabetes, obesity
and/or stroke in the test animal, wherein such latency is
indicative of aldolase A activity.
76. The method of claim 75 in which the test animal is a
recombinant test animal which expresses an aldolase A transgene or
expresses aldolase A under the control of a promoter which is not
the native aldolase A gene promoter at an increased level relative
to a wild-type test animal.
77. A method for screening a prepronatriodilatin mutant for its
prepronatriodilatin activity comprising: (a) administering the
prepronatriodilatin mutant to a test animal prone to hypertension,
diabetes, obesity and/or stroke; and (b) measuring the latency of
hypertension, diabetes, obesity and/or stroke in the test animal,
wherein such latency is indicative of prepronatriodilatin
activity.
78. The method of claim 77 in which the test animal is a
recombinant test animal which expresses a prepronatriodilatin
transgene or expresses prepronatriodilatin under the control of a
promoter which is not the native prepronatriodilatin gene promoter
at an increased level relative to a wild-type test animal.
79. A method for screening an .alpha.-cardiac myosin mutant for its
prepronatriodilatin activity comprising: (a) administering the
.alpha.-cardiac myosin mutant to a test animal prone to
hypertension, diabetes, obesity and/or stroke; and (b) measuring
the latency of hypertension, diabetes, obesity and/or stroke in the
test animal, wherein such latency is indicative of .alpha.-cardiac
myosin activity.
80. The method of claim 79 in which the test animal is a
recombinant test animal which expresses an .alpha.-cardiac myosin
transgene or expresses .alpha.-cardiac myosin under the control of
a promoter which is not the native .alpha.-cardiac myosin gene
promoter at an increased level relative to a wild-type test
animal.
81. A method for screening an .alpha.-tubulin mutant for its
prepronatriodilatin activity comprising: (a) administering the
.alpha.-tubulin mutant to a test animal prone to hypertension,
diabetes, obesity and/or stroke; and (b) measuring the latency of
hypertension, diabetes, obesity and/or stroke in the test animal,
wherein such latency is indicative of .alpha.-tubulin activity.
82. The method of claim 81 in which the test animal is a
recombinant test animal which expresses an .alpha.-tubulin
transgene or expresses .alpha.-tubulin under the control of a
promoter which is not the native .alpha.-tubulin gene promoter at
an increased level relative to a wild-type test animal.
83. A method for screening for a modulator of activity or of
latency or predisposition to hypertension, diabetes, obesity and/or
stroke comprising measuring such latency in a test animal, which is
prone or predisposed to the development of hypertension, diabetes,
obesity and/or stroke, and which recombinantly-expresses a putative
modulator of SGLT2 activity, wherein a change in the latency of
hypertension, diabetes, obesity and/or stroke within said test
animal relative to an analogous animal which is also prone or
predisposed to the development of hypertension, diabetes, obesity
and/or stroke but does not recombinantly-express the putative
modulator, is indicative of the putative modulator possessing the
ability to modulate SGLT2 activity or latency or predisposition to
hypertension, diabetes, obesity and/or stroke.
84. The method of claim 83 in which said SGLT2 mutant is screened
for an increase in the latency or a decrease in predisposition to
hypertension, diabetes, obesity and/or stroke.
85. A method for screening for a modulator of activity or of
latency or predisposition to hypertension, diabetes, obesity and/or
stroke comprising measuring such latency in a test animal, which is
prone or predisposed to the development of hypertension, diabetes,
obesity and/or stroke, and which recombinantly-expresses a putative
modulator of kynurenine aminotransferase activity, wherein a change
in the latency of hypertension, diabetes, obesity and/or stroke
within said test animal relative to an analogous animal which is
also prone or predisposed to the development of hypertension,
diabetes, obesity and/or stroke but does not recombinantly-express
the putative modulator, is indicative of the putative modulator
possessing the ability to modulate kynurenine aminotransferase
activity or latency or predisposition to hypertension, diabetes,
obesity and/or stroke.
86. The method of claim 85 in which said kynurenine
aminotransferase mutant is screened for an increase in the latency
or a decrease in predisposition to hypertension, diabetes, obesity
and/or stroke.
87. A method for screening for a modulator of activity or of
latency or predisposition to hypertension, diabetes, obesity and/or
stroke comprising measuring such latency in a test animal. which is
prone or predisposed to the development of hypertension, diabetes,
obesity and/or stroke, and which recombinantly-expresses a putative
modulator of FAT/CD36 activity, wherein a change in the latency of
hypertension, diabetes, obesity and/or stroke within said test
animal relative to an analogous animal which is also prone or
predisposed to the development of hypertension, diabetes, obesity
and/or stroke but does not recombinantly-express the putative
modulator, is indicative of the putative modulator possessing the
ability to modulate FAT/CD36 activity or latency or predisposition
to hypertension, diabetes, obesity and/or stroke.
88. The method of claim 87 in which said FAT/CD36 mutant is
screened for an increase in the latency or a decrease in
predisposition to hypertension, diabetes, obesity and/or
stroke.
89. A method for screening for a modulator of activity or of
latency or predisposition to hypertension, diabetes, obesity and/or
stroke comprising measuring such latency in a test animal. which is
prone or predisposed to the development of hypertension, diabetes,
obesity and/or stroke, and which recombinantly-expresses a putative
modulator of aldolase A activity, wherein a change in the latency
of hypertension, diabetes, obesity and/or stroke within said test
animal relative to an analogous animal which is also prone or
predisposed to the development of hypertension, diabetes, obesity
and/or stroke but does not recombinantly-express the putative
modulator, is indicative of the putative modulator possessing the
ability to modulate aldolase A activity or latency or
predisposition to hypertension, diabetes, obesity and/or
stroke.
90. The method of claim 89 in which said aldolase A mutant is
screened for an increase in the latency or a decrease in
predisposition to hypertension, diabetes, obesity and/or
stroke.
91. A method for screening for a modulator of activity or of
latency or predisposition to hypertension, diabetes, obesity and/or
stroke comprising measuring such latency in a test animal, which is
prone or predisposed to the development of hypertension, diabetes,
obesity and/or stroke, and which recombinantly-expresses a putative
modulator of prepronatriodilatin activity, wherein a change in the
latency of hypertension, diabetes, obesity and/or stroke within
said test animal relative to an analogous animal which is also
prone or predisposed to the development of hypertension, diabetes,
obesity and/or stroke but does not recombinantly-express the
putative modulator, is indicative of the putative modulator
possessing the ability to modulate prepronatriodilatin activity or
latency or predisposition to hypertension, diabetes, obesity and/or
stroke.
92. The method of claim 91 in which said prepronatriodilatin mutant
is screened for an increase in the latency or a decrease in
predisposition to hypertension, diabetes, obesity and/or
stroke.
93. A method for screening for a modulator of activity or of
latency or predisposition to hypertension, diabetes, obesity and/or
stroke comprising measuring such latency in a test animal, which is
prone or predisposed to the development of hypertension, diabetes,
obesity and/or stroke, and which recombinantly-expresses a putative
modulator of .alpha.-cardiac myosin activity, wherein a change in
the latency of hypertension, diabetes, obesity and/or stroke within
said test animal relative to an analogous animal which is also
prone or predisposed to the development of hypertension, diabetes,
obesity and/or stroke but does not recombinantly-express the
putative modulator, is indicative of the putative modulator
possessing the ability to modulate .alpha.-cardiac myosin activity
or latency or predisposition to hypertension, diabetes, obesity
and/or stroke.
94. The method of claim 93 in which said .alpha.-cardiac myosin
mutant is screened for an increase in the latency or a decrease in
predisposition to hypertension, diabetes, obesity and/or
stroke.
95. A method for screening for a modulator of activity or of
latency or predisposition to hypertension, diabetes, obesity and/or
stroke comprising measuring such latency in a test animal, which is
prone or predisposed to the development of hypertension, diabetes,
obesity and/or stroke, and which recombinantly-expresses a putative
modulator of .alpha.-tubulin activity, wherein a change in the
latency of hypertension, diabetes, obesity and/or stroke within
said test animal relative to an analogous animal which is also
prone or predisposed to the development of hypertension, diabetes,
obesity and/or stroke but does not recombinantly-express the
putative modulator, is indicative of the putative modulator
possessing the ability to modulate .alpha.-tubulin activity or
latency or predisposition to hypertension, diabetes, obesity and/or
stroke.
96. The method of claim 95 in which said .alpha.-tubulin mutant is
screened for an increase in the latency or a decrease in
predisposition to hypertension, diabetes, obesity and/or
stroke.
97. A recombinant, non-human animal possessing a mutant SGLT2 gene
which is under the control of a promoter which is not the native
SGLT2 gene promoter, wherein said mutant SGLT2 gene encodes a
mutant SGLT2 which either increases the latency or decreases the
predisposition to hypertension, diabetes, obesity and/or
stroke.
98. A recombinant, non-human animal which is the product of a
process comprising introducing a nucleic acid into said non-human
animal, or an ancestor thereof, wherein said nucleic acid comprises
a mutant SGLT2 gene sequence.
99. A recombinant, non-human animal possessing a mutant kynurenine
aminotransferase gene which is under the control of a promoter
which is not the native kynurenine aminotransferase gene promoter,
wherein said mutant kynurenine aminotransferase gene encodes a
mutant kynurenine aminotransferase which either increases the
latency or decreases the predisposition to hypertension, diabetes,
obesity and/or stroke.
100. A recombinant, non-human animal which is the product of a
process comprising introducing a nucleic acid into said non-human
animal, or an ancestor thereof, wherein said nucleic acid comprises
a mutant kynurenine aminotransferase gene sequence.
101. A recombinant, non-human animal possessing a mutant FAT/CD36
gene which is under the control of a promoter which is not the
native FAT/CD36 gene promoter, wherein said mutant FAT/CD36 gene
encodes a mutant FAT/CD36 which either increases the latency or
decreases the predisposition to hypertension, diabetes, obesity
and/or stroke.
102. A recombinant, non-human animal which is the product of a
process comprising introducing a nucleic acid into said non-human
animal, or an ancestor thereof, wherein said nucleic acid comprises
a mutant FAT/CD36 gene sequence.
103. A recombinant, non-human animal possessing a mutant aldolase A
gene which is under the control of a promoter which is not the
native aldolase A gene promoter, wherein said mutant aldolase A
gene encodes a mutant aldolase A which either increases the latency
or decreases the predisposition to hypertension, diabetes, obesity
and/or stroke.
104. A recombinant, non-human animal which is the product of a
process comprising introducing a nucleic acid into said non-human
animal, or an ancestor thereof, wherein said nucleic acid comprises
a mutant aldolase A gene sequence.
105. A recombinant, non-human animal possessing a mutant
prepronatriodilatin gene which is under the control of a promoter
which is not the native prepronatriodilatin A gene promoter,
wherein said mutant prepronatriodilatin A gene encodes a mutant
prepronatriodilatin which either increases the latency or decreases
the predisposition to hypertension, diabetes, obesity and/or
stroke.
106. A recombinant, non-human animal which is the product of a
process comprising introducing a nucleic acid into said non-human
animal, or an ancestor thereof, wherein said nucleic acid comprises
a mutant prepronatriodilatin gene sequence.
107. A recombinant, non-human animal possessing a mutant
.alpha.-cardiac myosin gene which is under the control of a
promoter which is not the native .alpha.-cardiac myosin gene
promoter, wherein said mutant .alpha.-cardiac myosin gene encodes a
mutant .alpha.-cardiac myosin which either increases the latency or
decreases the predisposition to hypertension, diabetes, obesity
and/or stroke.
108. A recombinant, non-human animal which is the product of a
process comprising introducing a nucleic acid into said non-human
animal, or an ancestor thereof, wherein said nucleic acid comprises
a mutant .alpha.-cardiac myosin gene sequence.
109. A recombinant, non-human animal possessing a mutant
.alpha.-tubulin gene which is under the control of a promoter which
is not the native .alpha.-tubulin gene promoter, wherein said
mutant .alpha.-tubulin gene encodes a mutant .alpha.-tubulin which
either increases the latency or decreases the predisposition to
hypertension, diabetes, obesity and/or stroke.
110. A recombinant, non-human animal which is the product of a
process comprising introducing a nucleic acid into said non-human
animal, or an ancestor thereof, wherein said nucleic acid comprises
a mutant .alpha.-tubulin gene sequence.
Description
FIELD OF THE INVENTION
[0001] The present invention discloses a set of genes which have
been demonstrated to be anomalously regulated (i.e., dysregulated)
in a model of combining a pathophysiological predisposition towards
stroke, hypertension, diabetes and obesity. The present invention
also relates to methods of treating and/or preventing stroke,
hypertension, diabetes or obesity by the administration of the
nucleic acids or protein products (and derivative and analogs
thereof) of the GENE SET which are defective and/or are of low
abundance in humans. The present invention further relates to
methodologies of diagnosis, prognosis and screening for alleles of
the GENE SET which may cause or predispose to the aforementioned
diseases.
BACKGROUND OF THE INVENTION
[0002] Prevalent human diseases such as hypertension, non-insulin
dependent diabetes (NIDDM), stroke, and obesity (dyslipidemia) have
been shown to possess a significant genetic component composed of
multiple, perhaps numerous, underlying genetic defects. Human
Metabolic Syndrome X, a relatively common but poorly understood
disorder, has been shown to possess a significant genetic component
which is comprise of an association of the pathophysiologies of
hypertension, insulin resistance, dyslipidemia and abdominal
obesity. See e.g., Ferrannini, et al., 1987. New Engi. J Med.
317:350-357; Kaplan, 1989. Arteriosclerosis 9:335-344. Given the
prevalence of this combination of diseases, many research groups
have focussed their efforts upon determining the etiology (i.e.,
the primary causative genetic defects) of Metabolic Syndrome X in
humans and closely-associated animal models.
[0003] The most successfully studied of these aforementioned
diseases to date is hypertension, with strong evidence nucleotide
sequence variants, with their associated amino acid residue
substitutions, within a total of 11 human genes affect blood
pressure. See e.g., Shimkets, et al., 1994. Cell 79(3):407-414;
Simon, et al., 1997. Nat. Genet. 17(2):171-178; Geller, et al,
1998. Nat. Genet. 19(3):279-281. While in all probability these
variants, in toto, account for only a fraction of the variation in
blood pressure within the general population, they, nonetheless,
serve to illustrate the potential that the etiology of many such
diseases may involve the interaction of a large number of genetic
components. As the majority of the genetic components of complex
diseases such as human Metabolic Syndrome X have yet to be
elucidated, a comprehensive analysis for the genetic defects
responsible for the phenotype was undertaken in the present
invention within a closely-associated animal model of this
syndrome.
[0004] The most widely-utilized and generally-accepted model of
human Metabolic Syndrome X is the spontaneously hypertensive rat
(SHR), which is characterized by the pathophysiologies of
salt-induced hypertension, insulin resistance and increased
abdominal fat. See e.g. Yamori, 1984. Experimental and Genetic
Models of Hypertension In: Handbook of Hypertension (Elsevier
Science Publishers, New York, N.Y.). While many genetic loci have
been linked to various aspects of the SHR phenotype relative to
those of the Wistar Kyoto (WKY) control strain, only a single gene
defect has been implicated as a causative factor in the phenotype.
See e.g., Aitman, et al., 1997. Nat. Genet. 16(2):197-201; Clark,
et al., 1996. Hypertension 28(5):898-906; Bottger, et al., 1996. J
Clin. Invest. 98(3):856-862. In addition, a spontaneous variant of
the SHR was found which, in addition to the features of human
Metabolic Syndrome X, undergoes severe hemmhoragic or ischemic
stroke. This strain was designated SHR stroke-prone (SHR-SP). See
e.g., Okamoto, et al, 1974. Circ. Res. 33/34:1-143-153; Rabattu, et
al., 1996. Nat. Genet. 16(4):364-367. Prior to the present
invention, the gene(s) influencing the manifestation and latency
period of stroke within SHR-SP animals have also not been
identified.
[0005] In order to identify the primary genetic defects leading to
the phenotype of the SHR and SHR-SP, the present invention has
included a comprehensive gene expression analysis utilizing the
GeneCalling.RTM. technology to identify the majority of
differentially-expressed genes between the strains of animals that
were used for genetic linkage analysis. GeneCalling.RTM. not only
identifies both known and novel differentially-expressed genes, but
also identifies sequence variations in complementary DNA (cDNA)
between the various strains being compared. These variations
detected by GeneCalling.RTM. can include, but are not limited to,
insertions, deletions and single base-pair changes.
[0006] In order to identify, in the most efficacious manner
possible, which of the differentially-expressed genes may
contribute directly to the phenotype, the genes were placed on the
physical map of the rat using a radiation hybrid panel. Given the
statistical improbability of each "event" occurring by chance,
genes which were found to be both differentially-expressed within
the different animal strains and tissues/organs and to map within
quantitative trait loci (QTL), were deemed to have a high
probability of possessing mutations which would affect the
phenotype.
[0007] Presented herein is the first comprehensive organ survey of
differences in gene expression in a genetic disease model coupled
with a comprehensive mapping and mutation detection strategy to
identify the gene(s) responsible for causing or predisposing to
these aforementioned disease traits.
[0008] It should be noted that the citation or discussion of a
reference herein shall not be construed as an admission that such
is prior art to the present invention.
SUMMARY OF THE INVENTION
[0009] The present invention discloses the use of genes within a
GENE SET, or mutations of the genes within the GENE SET, as
diagnostics and therapeutics for disease.
[0010] More specifically, the present invention discloses nucleic
acid sequences comprising the genes of a GENE SET, the proteins
encoded therefrom and derivatives, analogs and mutations thereof,
for use in the diagnosis, prognosis and screening, as well as the
treatment, both prophylactic and therapeutic, of diseases such as
hypertension, diabetes (insulin resistance), obesity/dyslipidemia
and stroke (ischemic disease).
[0011] Further disclosed herein are methodologies of diagnosis,
prognosis, and screening by detecting genes from the GENE SET.
Diagnostic, prognostic and screening kits are also provided.
[0012] Additionally, the present invention also discloses methods
of screening for modulators of GENE SET activity which affect
hypertension, diabetes, obesity and both the latency period and
severity of stroke.
BRIEF DESCRIPTION OF THE FIGURES
[0013] In order that the present invention disclosed herein is
better understood and appreciated, the following detailed
description is set forth.
[0014] FIG. 1: Illustrates multiple GeneCalling.RTM. fragments
derived from the same gene. Independent gene fragments from
3-.beta.-hydroxysteroid dehydrogenase/.DELTA.-5-.DELTA.-4 isomerase
were found to be differentially-expressed, and the region of the
cDNA from which they were derived is indicated. The red vertical
line indicates the peak of the expression difference. Fragment
length in nucleotides is indicated on the x-axis, relative peak
intensity is indicated on the y-axis. Each trace represents the
composite of multiple reactions from a single animal.
[0015] FIG. 2: Illustrates the differential-expression of selected
genes. Data indicating differential expression of selected genes
between the different genotypes is presented. In panel E, the
GeneCalling.RTM. reaction identified a two base-pair deletion in
the untranslated region of prepronatriodilatin which accounts for
the shift of the peak from 172 to 170. The red vertical line
indicates the peak of the expression difference. Fragment length in
nucleotides is indicated on the x-axis, relative peak intensity is
indicated on the y-axis. Each trace represents the composite of
multiple reactions from a single animal.
[0016] FIG. 3: Illustrates the mapping of differentially-expressed
candidate genes. Examples of mapping data of
differentially-expressed genes are shown and the LOD score is
indicated below each gene name. The vertical bar to the right of
the map indicates the 95% confidence interval in which the gene
lies.
[0017] FIG. 4: Illustrates the variation of amino acid residues
found between WKY, SHR and SHR-SP genes. Predicted amino acid
residue variation, based on nucleotide changes found in cDNAs
encoding the proteins shown, are indicated by underlining. The
first and last amino acid are numbered relative to the start of
translation, as indicated in the corresponding GenBank entry.
[0018] Also shown are the amino acid sequences of the mutant human
proteins consisting of: SGLT2 [SEQ ID NO:1]; kynurenine
arninotransferase [SEQ ID NO:2]; FAT/CD36 [SEQ ID NOS:3 AND 4];
aldolase A [SEQ ID NO:5]; prepronatriodilatin [SEQ ID NO:6];
.alpha.-cardiac myosin [SEQ ID NO:7] and .alpha.-tubulin [SEQ ID
NO:8].
[0019] Table 1: Total gene expression differences by genotype and
organ. For each set of organs from each animal genotype that was
compared, the number of gene fragments analyzed, the number of
differences found and the percentage differences are
illustrated.
[0020] Table 2: Differential gene expression between the SHR and
WKY rats. Differential gene expression across a total of five (5)
tissues is illustrated, where a "+" indicates increased mRNA
abundance in the SHR and a "-" indicates a decreased MRNA abundance
in the SHR. The chromosome to which the gene maps is provided and a
"*" denotes that the gene maps to a position within a known QTL. In
addition, amino acid residue substitutions which were identified by
the present invention are also provided, where applicable. Table 3:
Differential gene expression between the SHR-SP and SHR rats.
Differential gene expression across a total of five (5) tissues is
presented, where a "+" indicates increased mRNA abundance in the
SHR-SP and a "-" indicates a decreased mRNA abundance in the
SHR-SP. The chromosome to which the gene maps is provided and a "*"
denotes that the gene maps to a position within a known QTL. In
addition, amino acid residue substitutions which were identified by
the present invention are also provided, where applicable.
DETAILED DESCRIPTION OF THE INVENTION
[0021] The present invention discloses a GENE SET comprising a
total of 6 genes which were found to be both
differentially-expressed and mutated (i e., possessing amino acid
residue substitutions) between disease and control states in
genetic rat models of hypertension, obesity. diabetes and stroke.
The GENE SET (hereinafter "GENE SET") includes; (i) CD36 (also
known as fatty acid transport protein (FAT)); (ii) sodium dependent
glucose co-transporter (SGLT2): (iii) aldolase A; (iv) kynurenine
aminotransferase; (v) .alpha.-cardiac myosin and (vi)
.alpha.-tubulin; as well as the previously-described mutation in
the atrial natriuretic peptide (ANP).
[0022] Accordingly, the present invention relates to mutants of the
proteins which are encoded by aforementioned GENE SET (and
derivatives, fragments and homologs thereof) and the nucleic acids
which encode them (and derivatives, fragments and homologs
thereof), which function so as to increase predisposition to
stroke, hypertension, diabetes and obesity.
[0023] The present invention relates to methods of diagnosis,
prognosis and screening for stroke, hypertension, diabetes and
obesity. In one embodiment, subjects are screened for a mutant
allele of the GENE SET. In another embodiment, subjects are
screened to differentiate the expression of mRNAs derived from the
GENE SET, relative to their expression within controls.
[0024] The present invention also relates to methods of screening
members of the GENE SET for the ability to affect the onset of, or
predisposition to, hypertension, diabetes (insulin resistance) or
obesity (dyslipidemia) or stroke, and to methodologies of screening
for modulators (i.e., agonists, antagonists and inhibitors) of
these genes.
[0025] (1) Mutated GENE SET
[0026] Proteins produced from the GENE SET, and mutants of
derivatives, fragments, homologs and analogs of GENE SET proteins
and the nucleic acids encoding the mutants, protein derivatives and
protein analogs are disclosed by the present invention. The GENE
SET mutants can be proteins possessing substitutions, deletions or
insertions of one or more amino acid residues within the amino acid
sequence wild-type GENE SET protein. Preferably, the GENE SET
mutants are capable of binding to an anti-GENE SET antibody.
[0027] In another embodiment of the present invention, the GENE SET
mutant increases latency to hypertension or stroke in stroke-prone
rats (e.g., rats possessing the stroke-predisposing locus located
on chromosome 1) which are fed a high salt diet (for example, but
not limited to, a diet of 17.5% protein, 3.7 mg/g body weight
Na.sup.+, 6.3 mg/kg body weight K.sup.+, and 0.03 mg/g body weight
methionine and 1% NaCl drinking water).
[0028] Derivatives or analogs of GENE SET include, but are not
limited to, those molecules comprising regions which are
substantially homologous to the wild-type GENE SET or mutant GENE
SET, or fragments thereof. For example, in various embodiments, at
least 60-70% homology, preferably 70-80% homology, more preferably
90-95% homology and most preferably .gtoreq.95% homology over an
amino acid sequence of identical size or when compared to an
aligned sequence in which the alignment is performed by a computer
homology program known within the art, or whose encoding nucleic
acid is capable of hybridizing to a coding GENE SET sequence, under
stringent, moderately stringent, or non-stringent conditions.
[0029] The GENE SET, as well as fragments, derivatives, homologs
and analogs GENE SET. ot the present invention can be produced by
various methods known within the art. The manipulations which
result in their production can occur at the gene or protein level.
For example, the cloned GENE SET gene sequence can be modified by
any of numerous strategies known within the art. See e.g.,
Sambrook, et al., 1990. Molecular Cloning, A Laboratory Manual, 2d
ed (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
The sequence of interest may be cleaved at appropriate sites with a
restriction endonuclease (RE), followed by further enzymatic
modification (if necessary), isolated, and ligated in vitro. In the
production of the gene encoding a mutant, derivative or analog of
the GENE SET, care should be taken to ensure that the modified gene
remains within the same translational reading frame as the GENE SET
and is uninterrupted by translational stop signals within the
exonic region where the desired GENE SET activity is encoded.
[0030] The GENE SET-encoding nucleic acid sequence may be mutated
in vitro or in vivo, to make changes within the coding regions
(e.g., amino acid substitutions, additions or deletions) as well as
to create and/or destroy translation, initiation, and/or
termination sequences, or to form new restriction endonuclease
sites or destroy pre-existing ones, to facilitate further in vitro
modification. Any technique for mutagenesis known within the art
may be utilized including, but not limited to, chemical
mutagenesis; in vitro site-directed mutagenesis (see e.g.,
Hutchinson, et al, 1978. J Biol Chem. 253:6551); use of TAB7.RTM.
linkers (Pharmacia), and the like.
[0031] Manipulations of the GENE SET sequence may also be made at
the protein level. Included within the scope of the invention are
GENE SET protein fragments or other derivatives or analogs which
are differentially-modified during or after translation (e.g., by
glycosylation, acetylation, phosphorylation, amidation,
derivatization by known protectingiblocking groups, proteolytic
cleavage, linkage to an antibody molecule or other cellular ligand,
etc). Any of numerous chemical modifications may be performed by
known techniques, including but not limited to specific chemical
cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8
protease, NaBH.sub.4; acetylation, formylation, oxidation,
reduction; metabolic synthesis in the presence of tunicamycin; etc.
Particularly included within the scope of the present invention are
those modifications which reduce the level or activity of the GENE
SET.
[0032] In addition, mutant GENE SET proteins (or analogs and
derivatives thereof) which mediate the desired activity in vivo or
in vitro, may be synthesized by use of a peptide synthesizer.
Furthermore, if desired, non-classical amino acids or chemical
amino acid analogs may be introduced as a substitution or addition
into the GENE SET sequence. Non-classical amino acids include, but
are not limited to: the D-isomers of the common amino acids,
.alpha.-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino
butyric acid, .gamma.-Abu, .epsilon.-Ahx, 6-amino hexanoic acid,
Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine,
norleucine, norvaline, hydroxyproline, sarcosine, citrulline,
cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,
cyclohexylalanine, .beta.-alanine, fluoro-amino acids, designer
amino acids such as .beta.-methyl amino acids, C.alpha.-methyl
amino acids, N.alpha.-methyl amino acids, and amino acid analogs in
general. Furthermore, the amino acid may either be D (dextrorotary)
or L (levorotary) isomers.
[0033] In another embodiment of the present invention, the GENE SET
derivative is a chimeric, or fusion, protein comprising an GENE SET
protein or fragment thereof (preferably consisting of at least 10
amino acids of the GENE SET protein or a mutant GENE SET protein)
joined at its amino- or carboxyl-terminus via a peptide bond to an
amino acid sequence of a different protein. In one embodiment, such
a chimeric protein is produced by recombinant expression of a
nucleic acid encoding the protein (i.e., comprising a GENE
SET-coding sequence joined in-frame to a coding sequence for a
different protein). Such a chimeric product can be made by ligating
the appropriate nucleic acid sequences encoding the desired amino
acid sequences to each other by methods known in the art, in the
proper coding frame, and expressing the chimeric product by methods
commonly known in the art. Alternatively, such a chimeric product
may be made by protein synthetic techniques (e.g., by use of a
peptide synthesizer). Chimeric genes comprising portions of the
wild-type GENE SET or the mutant GENE SET fused to any heterologous
protein-encoding sequences may be constructed. A specific
embodiment of the present invention discloses a chimeric protein
comprising a fragment of GENE SET or mutant GENE SET of at least
six amino acids.
[0034] Additionally, due to the degeneracy of nucleotide-coding
sequences, other DNA sequences which encode substantially the same
amino acid sequence as the mutant GENE SET of the present invention
may be utilized in the practice of the present invention. The genes
indigenous to the mutant GENE SET may be obtained by alteration of
nucleotide sequences comprising all or portions of GENE SET gene by
the substitution of different codons which encode the desired amino
acid. For example, one or more amino acid residues within the
sequence may be substituted by another amino acid of a similar
polarity which acts as a functional equivalent, resulting in a
"silent alteration." Substitutes for an amino acid within the
sequence may be selected from other members of the class to which
the amino acid belongs. For example, the nonpolar (hydrophobic)
amino acids include, but are not limited to, alanine, leucine,
isoleucine, valine, proline, phenylalanine, tryptophan and
methionine. The polar neutral amino acids include, but are not
limited to, glycine, serine, threonine, cysteine, tyrosine,
asparagine, and glutamine. The positively charged (basic) amino
acids include, but are not limited to, arginine, lysine and
histidine. The negatively charged (acidic) amino acids include, but
are not limited to, aspartic acid and glutamic acid.
[0035] In addition, the present invention discloses mutant GENE SET
molecules, containing the aforementioned mutations for mutant GENE
SET molecules.
[0036] (2) Nucleic Acid Sequences and Proteins of the GENE SET
[0037] GENE SET proteins and nucleic acids can be obtained by any
methodology known within the art. The GENE SET amino acid and
nucleotide sequences for, inter alia, human, rat, hamster, dog,
mouse, bovine, porcine, equine, dogfish, Drosophila melanogaster
and Xenopus are available in the public databases (e.g.,
GenBank).
[0038] Any eukaryotic cell potentially can serve as the nucleic
acid source for the isolation of GENE SET nucleic acids. The
nucleic acid sequences of the GENE SET may be isolated from
vertebrate, mammalian, human, porcine, bovine, feline, avian,
equine, canine, primate, and like sources. The DNA may be obtained
by standard protocols known within the art from cloned DNA (e.g., a
DNA "library"), by chemical synthesis, by cDNA cloning, or by the
cloning of genomic DNA (or fragments thereof) purified from the
desired cell. See e.g., Sambrook, et al., 1990. Molecular Cloning,
A Laboratory Manual, 2d ed (Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y.); Glover, 1985. DNA Cloning: A Practical
Approach (MRL Press, Ltd., Oxford, U.K.). Clones which are derived
from genomic DNA may contain regulatory and non-coding, intron DNA
regions, in addition to coding, exonic regions; whereas clones
which are derived from cDNA will contain only coding, exon
sequences. Following isolation, the sequence of interest is then
molecularly-cloned into a suitable vector for propagation.
[0039] In the molecular cloning of the gene from complementary DNA
(cDNA), the cDNA is synthesized by reverse transcription from total
cellular RNA or poly(A).sup.+ mRNA by methods which are well-known
within the art. The gene(s) of interest may also be obtained from
genomic DNA, wherein random DNA fragments are generated (e.g., by
use of restriction endonucleases or by mechanical shearing), some
of which will encode the desired sequence(s). The linear DNA
fragments may then be separated as a function of their size by
standard techniques including, but not limited to, agarose and
polyacrylamide gel electrophoresis and column chromatography.
[0040] Once the DNA fragments have been generated, identification
of the specific DNA fragment containing all or a portion of the
GENE SET gene may be accomplished in a number of ways. In a
preferred embodiment of the present invention, a GENE SET gene is
isolated by use of the polymerase chain reaction (PCR), which can
be utilized to amplify the desired GENE SET sequence within a
genomic or cDNA library, or directly from genomic DNA or cDNA which
has not been incorporated into a library. Synthetic
oligonucleotides may then be utilized as primers in PCR-mediated
amplification of sequences from an RNA or DNA source, preferably
from a cDNA library, of potential interest. In addition, several
different degenerate primers may be synthesized for use in the PCR
amplification reactions. The PCR amplification reaction may be
performed, for example, by use of a Perkin-Elmer Cetus.RTM. Thermal
Cycler and Taq polymerase (Gene AmpJ).
[0041] It is also possible to vary the stringency of hybridization
conditions utilized during the priming of the PCR amplification
reactions, to allow for greater or lesser degrees of nucleotide
sequence similarity between the known GENE SET nucleotide sequence
and the nucleic acid of an GENE SET homolog being isolated. For
cross-species hybridization, low stringency conditions are
preferred. For same-species hybridization, moderately stringent
conditions are preferred. Following successful amplification of a
fragment or segment of a GENE SET homolog, that segment may be
molecularly-cloned, sequenced and utilized as a probe to isolate a
complete cDNA or genomic clone. This, in turn, will permit the
subsequent determination and isolation of the gene's complete
nucleotide sequence. Alternately, PCR amplification may also be
used to detect and quantitate GENE SET mRNA levels.
[0042] Additionally, a portion of a GENE SET gene or its associated
mRNA (or a fragment thereof) may be purified, or an oligonucleotide
synthesized, and the generated DNA fragments may be analyzed by
nucleic acid hybridization to the labeled probe. See e.g., Benton
& Davis. 1977. Science 196:180; Grunstein & Hogness, 1975.
Proc. Natl. Acad Sc. U.S.A. 72:3961. Those DNA fragments which
possess substantial homology to the labeled oligonucleotide probe
will hybridize. GENE SET nucleic acids may be also identified and
isolated by expression cloning using, for example, anti-GENE SET
antibodies for selection. Alternatives to obtaining the GENE SET
DNA by cloning or amplification include, but are not limited to,
chemically synthesizing the gene sequence itself from the known
GENE SET sequence or synthesizing a cDNA from the mRNA encoding the
GENE SET protein of interest. It should be noted that the use of
other methodologies is possible and within the scope of the present
invention.
[0043] Once a clone has been obtained, its identity may be
ascertained by nucleic acid sequencing and computer
database-mediated comparison to known GENE SET sequences. DNA
sequence analysis may be performed by any techniques known within
the art including, but not limited to: chemical-based sequencing
(see Maxam & Gilbert, 1980. Meth. Enzymol. 65:499-560);
enzymatic dideoxynucleotide chain termination sequencing (see
Sanger, et al., 1977. Proc. Natl. Acad. Sci. U.S.A. 74:5463); T7
DNA polymerase sequencing (see Tabor & Richardson, U.S. Pat.
No. 4,795,699); automated DNA sequenator (e.g., Applied Biosystems,
Foster City, Calif.) or the sequencing methodology described in PCT
Publication WO 97/15690.
[0044] Nucleic acids which are hybridizable to a GENE SET nucleic
acid (e.g., possessing a nucleotide sequence homologous or
complementary to SEQ. ID NO:3 or NO:6), or a derivative or analog
thereof, may be isolated by nucleic acid hybridization under
conditions of low, moderate or high stringency. By way of example
and not limitation, procedures using such conditions of low
stringency are as follows (see also e.g., Shilo & Weinberg,
1981. Proc. Natl. Acad. Sci. USA 78:6789-6792): filters containing
immobilized DNA were pre-hybridized for 6 hours at 40.degree. C. in
a solution containing 35% formamide, 5.times.SSC, 50 mM Tris-HCl
(pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 .mu./ml
denatured salmon sperm DNA. Hybridizations were performed in the
same solution with the following modifications: 0.02% PVP, 0.02%
Ficoll, 0.2% BSA, 100 .mu.g/ml salmon sperm DNA, 10% (wt/vol)
dextran sulfate and 5-20.times.10.sup.6 cpm .sup.32P-labeled probe
was utilized. The filters were incubated in hybridization mixture
for 18-20 hours at 40.degree. C., and then washed for 1.5 hour at
55.degree. C. in a solution containing 2.times.SSC, 25 mM Tris-HCl
(pH 7.4), 5 mM EDTA and 0.1% SDS. The wash solution was then
replaced with fresh solution and the filters were re-incubated for
an additional 1.5 hour at 60 C The filters were blotted dry and
autoradiographed. If necessary, filters were washed for a third
time at 65-68.degree. C. and re-exposed to X-ray film. Various
other conditions of low stringency hybridization which are
well-known within the art may be utilized for low stringency
hybridization protocols (e.g., as employed for cross-species
hybridizations).
[0045] By way of example, but not of limitation, procedures
utilizing conditions of moderate stringency hybridization are as
follows: filters containing immobilized DNA were pre-hybridized for
6 hours at 55.degree. C. in a solution containing 6.times.SSC,
5.times.Denhardt's solution, 0.5% SDS and 100 .mu.g/ml denatured
salmon sperm DNA. Hybridizations were performed in the same
solution and 5-20.times.10.sup.6 cpm .sup.32P-labeled probe was
utilized. The filters were incubated in hybridization mixture for
18-20 hours at 55.degree. C. and then washed twice for 30 minutes
at 60.degree. C. in a solution containing 1.times.SSC and 0.1% SDS.
Filters were then blotted dry and autoradiographed. Other
conditions of moderate stringency hybridizations which may be
utilized are well-known within the art.
[0046] Again, by way of example and not of limitation, procedures
utilizing conditions of high stringency hybridization were as
follows: pre-hybridization of filters containing immobilized DNA
was carried out for 8 hours to overnight at 65.degree. C. in buffer
composed of 6.times.SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02%
PVP, 0.02% Ficoll, 0.02% BSA, and 500 .mu.g/ml denatured salmon
sperm DNA. Filters were hybridized for 48 hours at 65.degree. C. in
pre-hybridization mixture containing 100 .mu.g/ml denatured salmon
sperm DNA and 5-20 .times.10.sup.6 cpm of .sup.32P-labeled probe.
Washing of filters was done at 37.degree. C. for 1 hour in a
solution containing 2.times.SSC, 0.01% PVP, 0.01% Ficoll and 0.01%
BSA. This was followed by a wash in 0.1.times.SSC at 50.degree. C.
for 45 minuets prior to autoradiography. Other conditions of high
stringency hybridization which may be used are well-known within
the art.
[0047] Nucleic acids encoding derivatives and analogs of GENE SET
proteins, GENE SET anti-sense nucleic acids and primers which can
be utilized to detect mutant GENE SET alleles and GENE SET gene
expression are disclosed by the present invention. As used herein,
a "nucleic acid encoding a fragment or portion of an GENE SET
protein" refers to a nucleic acid encoding only the recited
fragment or portion of the GENE SET protein, and not the other
contiguous portions of the GENE SET protein as a continuous
sequence.
[0048] GENE SET proteins (and derivatives, analogs and fragments
thereof) of GENE SET proteins may be obtained by any method known
within the art including, but not limited to, recombinant
expression methods, purification from natural sources, chemical
synthesis and the like. For example, GENE SET proteins may be
obtained by recombinant protein expression techniques, wherein the
GENE SET gene or portion thereof is inserted into an appropriate
cloning vector for expression within a particular host cell. A
large number of vector-host systems known within the art may be
used. Possible vectors include, but are not limited to,
bacteriophage (e.g., lambda derivatives); plasmids (e.g., pBR322,
pUC plasmid derivatives or the Bluescript vector (Stratagene)) or
other vector which are well-known within the art. The insertion of
the DNA fragment of interest into a cloning vector may, for
example, be accomplished by ligating the fragment into a cloning
vector which has complementary cohesive termini. However, if the
complementary restriction sites used to fragment the DNA are not
present in the cloning vector, the ends of the DNA molecules may be
enzymatically modified. Alternatively, any site desired may be
produced by ligating nucleotide sequences (e.g., linkers) onto the
DNA termini; these ligated linkers may comprise specific chemically
synthesized oligonucleotides encoding restriction endonuclease
recognition sequences. In an alternative methodology, the digested
vector and GENE SET gene may be modified by homopolymeric tailing.
The recombinant molecule may subsequently introduced into the host
cell via transformation, transfection, infection, electroporation,
and the like, to facilitate the generation of a plurality of copies
of the GENE SET gene sequence of interest.
[0049] In an alternative methodology, the desired gene may be
identified and isolated after insertion into a suitable cloning
vector in a "shot-gun" approach. Enrichment for the desired gene
by, for example, size fractionation, may be performed prior to its
insertion into the cloning vector.
[0050] In specific embodiments of the present invention,
transformation of host cells with recombinant DNA molecules which
incorporate the isolated GENE SET gene, cDNA or synthesized DNA
sequence, facilitates the generation of multiple copies of the
gene. Thus, the gene may be obtained in large quantities by growing
transformants, isolating the recombinant DNA molecules from the
transformants and, when necessary, retrieving the inserted gene
from the isolated recombinant DNA.
[0051] The nucleotide sequence encoding a GENE SET protein (or a
functionally-active analog, fragment or other derivative thereof),
may be inserted into an appropriate expression vector (i.e., a
vector which contains the necessary elements for the transcription
and translation of the inserted protein-coding sequence).
Alternately, the necessary transcriptional and translational
signals may also be supplied by the native GENE SET gene and/or its
flanking regions. A variety of host-vector systems may be utilized
to express the protein-coding sequence including, but are not
limited to, mammalian cell systems infected with virus (e.g.,
vaccinia virus, adenovirus, etc.); insect cell systems infected
with virus (e.g., baculovirus); microorganisms such as yeast
containing yeast vectors, or bacteria transformed with
bacteriophage, DNA, plasmid DNA, or cosmid DNA. The expression
elements of vectors vary in their strengths and specificities.
Depending on the host-vector system utilized, any one of a number
of suitable transcription and translation elements may be employed
in the practice of the present invention.
[0052] Any of the methodologies previously-described for the
insertion of DNA fragments into a vector may be used to construct
expression vectors containing a chimeric gene consisting of
appropriate transcriptional/translational control signals and the
protein coding sequences. These methods may include in vitro
recombinant DNA and synthetic techniques and in vivo recombinants
(i.e., genetic recombination). Expression of nucleic acid sequence
encoding a GENE SET protein or peptide fragment, may be regulated
by a second nucleic acid sequence so that the GENE SET protein or
peptide is expressed within a host cell which has been transformed
with the recombinant DNA molecule. For example, expression of a
GENE SET protein may be controlled by any promoter/enhancer element
known within the art including, but not limited to: (i) the SV40
early promoter region (see e.g., Bernoist & Chambon, 1981.
Nature 290:304-310); (ii) the promoter contained in the 3'-terminus
long terminal repeat (LTR) of Rous sarcoma virus (see e.g.,
Yamamoto, et al., 1980. Cell 22:787-797); (iii) the Herpesvirus
thymidine kinase promoter (see e.g., Wagner, et al., 1981. Proc.
Natl. Acad Sci. U.S.A. 78:1441-1445); (iv) the regulatory sequences
of the metallothionein gene (see e.g., Brinster, et al., 1982.
Nature 296:39-42); (v) prokaryotic expression vectors such as the
.beta.-lactamase promoter (see e.g., Villa-Kamaroff, et al, 1978.
Proc. Natl. Acad Sci. USA. 75:3727-3731) or the tac promoter (see
e.g., DeBoer, et al., 1983. Proc. Natl. Acad. Sci. U.S.A.
80:21-25). Additionally, the following animal transcriptional
control regions, exhibiting tissue specificity, have been utilized
in transgenic animals including: (i) the elastase I gene control
region which is active in pancreatic acinar cells (see e.g., Swift,
et al, 1984. Cell 38:639-646); (ii) the insulin gene-control region
which is active in pancreatic .beta.-cells (see e.g., Hanahan,
1985. Nature 315:115-122); (iii) the immunoglobulin gene control
region which is active in lymphoid cells (see e.g., Grosschedl, et
al., 1984. Cell 38:647-658; (iv) the .alpha.-1-antitrypsin gene
control region which is active in the liver (see e.g., Kelsey, et
al., 1987. Genes and Devel. 1:161-171) and the .beta.-globin gene
control region which is active in myeloid cells (see e.g., Mogram,
et al., 1985. Nature 315:338-340.
[0053] In a specific embodiment of the present invention, a vector
may be used which comprises a promoter operably-linked to a GENE
SET-encoding nucleic acid, one or more origins of replication and,
optionally, one or more selectable markers (e.g., an antibiotic
resistance gene). In another specific embodiment, an expression
construct is produced by sub-cloning a GENE SET coding sequence
into the EcoRI restriction site of each of the three pGEX vectors
(i.e., Glutathione S-Transferase expression vectors; Smith &
Johnson, 1988. Gene 7:3140), thus allowing the expression of the
GENE SET protein-product in the correct reading frame.
[0054] Expression vectors containing GENE SET gene inserts may be
identified by the use of three general approaches: (i) nucleic acid
hybridization; (ii) presence or absence of "marker" gene functions
and (iii) expression of inserted nucleotide sequences. In the first
approach, the presence of a GENE SET gene which has been inserted
into an expression vector is detected by nucleic acid hybridization
using oligonucleotide probes comprising sequences which are
complementary to the aforementioned inserted GENE SET gene. In the
second approach, the recombinant vector/host system is identified
and selected based upon the presence or absence of certain "marker"
gene functions (e.g., thymidine kinase activity, resistance to
antibiotics, transformation phenotype, occlusion body formation in
baculovirus, and the like) which is caused by the insertion of the
GENE SET gene of interest into the vector. Specifically, if the
GENE SET gene is inserted into the marker gene sequence of the
vector, the recombinant species possessing the GENE SET insert may
be identified by the absence of the marker gene function. In the
third approach, recombinant expression vectors are identified by
assaying the GENE SET protein product expressed by the recombinant.
Such assays may be based, for example, upon the physical or
functional properties of the GENE SET protein in various in vitro
assay systems (e.g., binding of an anti-GENE SET protein
antibody).
[0055] Following the isolation and identification of the
recombinant DNA molecule of interest, several methodologies
well-known within the art may be employed for its propagation. Once
a suitable host system and growth conditions have been established,
the recombinant expression vectors may be propagated and prepared
in quantity. As previously disclosed, expression vectors which may
be utilized include, but are not limited to the following vectors
or their derivatives: human or animal viruses (e.g., vaccinia virus
or adenovirus); insect viruses (e.g., baculovirus); yeast vectors;
bacteriophage vectors (e.g., lambda), and plasmid and cosmid DNA
vectors; and the like.
[0056] Similarly, a host cell strain may be chosen which modulates
the expression of the inserted sequences or, alternately, modifies
and processes the gene product in the specific fashion desired.
Expression from certain promoters may be elevated by the presence
of inducers; hence expression of the recombinant GENE SET protein
may be controlled. Moreover, different host cells possess
characteristic and/or specific mechanisms for the translational and
post-translational processing and modification (e.g.,
glycosylation, phosphorylation of proteins), thus appropriate cell
lines or host systems may be chosen to ensure the desired
modification and processing of the recombinant protein is
accomplished. For example, expression of the recombinant protein in
a bacterial system can be used to produce an non-glycosylated core
protein product; whereas expression in yeast will produce a
glycosylated protein product. Similarly, expression in mammalian
cells may be utilized to ensure "wild-type" glycosylation of a
heterologous protein.
[0057] In other specific embodiments of the present invention, the
GENE SET protein (or fragment, analog, or derivative) may be
expressed as a fusion, or chimeric protein product (comprising the
protein, fragment, analog, or derivative joined via a peptide bond
to a heterologous protein sequence of a different protein). These
chimeric products may be produced by the ligation of the
appropriate nucleic acid sequences encoding the desired amino acid
sequences to one another, in the proper reading flame, by methods
known within the art, in the proper coding frame, and expressing
the chimeric product by methods commonly known in the art.
Alternatively, such a chimeric product may be made by protein
synthetic techniques (e.g., by use of a peptide synthesizer). It
should be noted that the cloning and subsequent expression of both
cDNA and genomic DNA sequences are within the scope of the present
invention.
[0058] The recombinant GENE SET proteins of the present invention
may also be isolated and purified by standard methods including
chromatography (e.g., ion exchange, affinity, and partition column
chromatography); centrifugation; differential solubility or by any
other standard technique for the purification of proteins. In an
alternate embodiment, native GENE SET proteins may be purified from
natural sources utilizing standard methods such as those described
above (e.g., immunoaffinity purification). In another embodiment,
the GENE SET proteins may be synthesized by standard chemical
methods known within the art (see e.g., Hunkapiller, et al., 1984.
Nature 310:105-111). The functional properties of the GENE SET
proteins may be evaluated using any suitable assay.
[0059] (3) Methods of Treatment
[0060] The present invention discloses methodologies of treating
and preventing ischemic and metabolic diseases and disorders by
administration of a therapeutic compound (hereinafter designated
"Therapeutics"). In one embodiment, such "Therapeutics" include
GENE SET mutant proteins (and derivatives, fragments and analogs
thereof), as well as nucleic acids which encode the mutant GENE SET
proteins (and derivatives, fragments or analogs thereof).
[0061] In another embodiment, the protein product, which are not
produced as a direct result of the diminution of the activity of an
enzyme indigenous to the GENE SET, may also be utilized as a
Therapeutic of the present invention. As an example, but not a
limitation, the mutation (i.e., amino acid substitution) in the
kynurenine aminotransferase enzyme putatively blocks the enzyme's
ability to produce kynurenic acid, a small, aqueous-soluble
molecule which may function as an anti-hypertensive.
[0062] In another embodiment, the Therapeutic is a mutant GENE SET
protein possessing one or more substitutions of amino acid residues
relative to the "wild-type" GENE SET protein The subject to which
the Therapeutic is administered is preferably an animal including,
but not limited to, animals such as cows, pigs, horses, chickens,
cats, dogs, etc., and is preferably a mammal. In a preferred
embodiment, the subject is a human.
[0063] Generally, the administration of products of a species
origin or species reactivity (in the case of antibodies) which is
the same species as that of the subject is preferred. Thus, in a
preferred embodiment, a human mutant GENE SET protein or nucleic
acid (or derivative, fragment or analog thereof) is therapeutically
or prophylactically administered to a human patient.
[0064] Accordingly, in a specific embodiment of the invention, GENE
SET antagonists and inhibitors including, but not limited to,
anti-GENE SET antibodies and GENE SET anti-sense nucleic acids and
GENE SET derivatives (e.g., which function as competitive
inhibitors of GENE SET) are administered to treat or prevent stroke
or ischemic disease, hypertension, diabetes or obesity.
[0065] (a) Gene Therapy
[0066] In a specific embodiment of the present invention, nucleic
acids comprising a sequence encoding a GENE SET mutant protein (or
derivative thereof) or a GENE SET anti-sense nucleic acid, are
administered by way of gene therapy. Gene therapy refers to therapy
performed by the administration of a nucleic acid to a subject. In
this embodiment of the invention, the nucleic acid produces its
encoded protein or is an anti-sense nucleic acid which mediates a
therapeutic effect. Any of the methods for gene therapy which are
well-known within the art may be utilized in the practice of the
present invention. See e.g., Ausubel, et al, 1993. Current
Protocols in Molecular Biology (John Wiley & Sons, New York,
N.Y.); Kriegler, 1990. Gene Transfer and Expression: A Laboratory
Manual (Stockton Press, New York, N.Y.).
[0067] In a preferred embodiment, the Therapeutic comprises an GENE
SET nucleic acid that is part of an expression vector that
expresses an GENE SET protein or fragment or chimeric protein,
preferably a mutant GENE SET protein or fragment or chimeric
protein, or an GENE SET anti-sense nucleic acid thereof in a
suitable host. In a specific embodiment, the nucleic acid possesses
a promoter which is operably-linked to the mutant GENE SET coding
region or to a sequence encoding an GENE SET anti-sense nucleic
acid, wherein the promoter is inducible or constitutive and,
optionally, tissue-specific. In another specific embodiment, a
nucleic acid is used in which the mutant GENE SET coding sequences
and any other desired sequences are flanked by regions which
promote homologous recombination at a desired site in the genome,
thus providing for intra-chromosomal expression of the mutant GENE
SET nucleic acid. See e.g., Koller & Smithies, 1989. Proc. Natl
Acad. Sci. USA 86:8932-8935. Delivery of the nucleic acid into a
patient may be either direct, in which case the patient is directly
exposed to the nucleic acid or nucleic acid-carrying vector, or
indirect, in which case, cells are first transformed with the
nucleic acid in vitro, then transplanted into the patient. These
two approaches are known, respectively, as in vivo or ex vivo gene
therapy.
[0068] In a specific embodiment, the nucleic acid is directly
administered in vivo, where it is expressed to produce the encoded
product. This may be accomplished by any of numerous methods known
in the art including, but not limited to, constructing it as part
of an appropriate nucleic acid expression vector and administering
it so that it becomes intracellular by: (i) infection using a
defective or attenuated retroviral or other viral vector (see e.g.,
U.S. Pat. No. 4,980,286); (ii) direct injection of naked DNA; (iii)
use of microparticle bombardment; (iv) coating with lipids or
cell-surface receptors or transfecting agents, encapsulation in
liposomes, microparticles, or microcapsules; (v) by administering
it in linkage to a peptide which is known to enter the nucleus;
(vi) administering it in linkage to a ligand subject to
receptor-mediated endocytosis which can be used to target cell
types specifically-expressing the receptors and the like. In
another embodiment, a nucleic acid-ligand complex can be formed in
which the ligand comprises a fusogenic viral peptide to disrupt
endosomes, allowing the nucleic acid to avoid lysosomal
degradation. In yet another embodiment, the nucleic acid can be
targeted in vivo for cell specific uptake and expression, by
targeting a specific receptor (see e.g., PCT Publications WO
92/06180 and WO 93/20221. Alternatively, the nucleic acid may be
introduced intracellularly and incorporated within host cell DNA
for expression, by homologous recombination. See e.g., Zijlstra, et
al., 1989. Nature 342:435438.
[0069] In a specific embodiment, a viral vector that contains the
mutant GENE SET nucleic acid or codes for GENE SET anti-sense
nucleic acid is used. For example, a retroviral vector can be used.
See e.g., Miller, et al., 1993. Meth. Enzymol. 217:581-599. These
retroviral vectors have been modified to delete retroviral
sequences that are not necessary for packaging of the viral genome
and integration into host cell DNA. The GENE SET nucleic acid to be
used in gene therapy is cloned into the vector, which facilitates
delivery of the gene into a patient. See e.g, Clowes, et al, 1994.
J Clin. Invest. 93:644-651; Kiem, et al., 1994. Blood
83:1467-1473.
[0070] Adenoviruses are other viral vectors that can be used in
gene therapy. Adenoviruses are especially attractive vehicles for
delivering genes to respiratory epithelia. Adenoviruses naturally
infect respiratory epithelia where they cause a mild disease. Other
targets for adenovirus-based delivery systems are liver, the
central nervous system, endothelial cells, and muscle. Adenoviruses
have the advantage of being capable of infecting non-dividing
cells. See e.g., Rosenfeld, et al, 1991 Science 252:431.-434;
Mastrangeli, et al, 1993. J Clin. Invest. 91:225-234. In addition,
adeno-associated virus (AAV) has also been proposed for use in gene
therapy. See e.g., Walsh, et al., 1993. Proc. Soc. Exp. Biol. Med.
204:289-300 (1993).
[0071] Another approach to gene therapy involves transferring a
gene to cells in tissue culture by such methods as electroporation,
lipofection, calcium phosphate mediated transfection, or viral
infection. Usually, the method of transfer includes the transfer of
a selectable marker to the cells. The cells are then placed under
selection to isolate those cells that have taken up and are
expressing the transferred gene. Those cells are then delivered to
a patient. In this embodiment, the nucleic acid is introduced into
a cell prior to administration in vivo of the resulting recombinant
cell. Such introduction can be carried out by any method known in
the art, including but not limited to transfection,
electroporation, microinjection, infection with a viral or
bacteriophage vector containing the nucleic acid sequences, cell
fusion, chromosome-mediated gene transfer, microcell-mediated gene
transfer, spheroplast fusion, and the like. Numerous techniques are
well-known within the art for the introduction of foreign genes
into cells (see e.g., Loeffler & Behr, 1993. Meth Enzymol.
217:599-618) and may be used in accordance with the present
invention, provided that the necessary developmental and
physiological functions of the recipient cells are not disrupted.
The technique should provide for the stable transfer of the nucleic
acid to the cell, so that the nucleic acid is expressible by the
cell and preferably heritable and expressible by its cell
progeny.
[0072] The resulting recombinant cells can be delivered to a
patient by various methods known in the art. In a preferred
embodiment, epithelial cells are injected (e.g., subcutaneously).
In another embodiment, recombinant skin cells may be applied as a
skin graft onto the patient. Recombinant blood cells (e.g.,
hematopoietic stem or progenitor cells) are preferably administered
intravenously. The amount of cells envisioned for use depends on
the desired effect, patient state, etc., and can be determined by
one skilled in the art.
[0073] Cells into which a nucleic acid can be introduced for
purposes of gene therapy encompass any desired, available cell
type, and include but are not limited to epithelial cells,
endothelial cells, keratinocytes, fibroblasts, muscle cells,
hepatocytes; blood cells such as T-lymphocytes, B-lymphocytes,
monocytes, macrophages, neutrophils, eosinophils, megakaryocytes,
granulocytes; various stem or progenitor cells, in particular
hematopoietic stem or progenitor cells (e.g, as obtained from bone
marrow, umbilical cord blood, peripheral blood, fetal liver, and
the like). In a preferred embodiment of the present invention, the
cell used for gene therapy is autologous to the patient.
[0074] In an embodiment in which recombinant cells are used in gene
therapy, a mutant GENE SET nucleic acid or nucleic acid encoding a
GENE SET anti-sense nucleic acid is introduced into the cells such
that it is expressible by the cells or their progeny, and the
recombinant cells are then administered in vivo for therapeutic
effect. In a specific embodiment, stem or progenitor cells are
used. Any stem and/or progenitor cells which can be isolated and
maintained in vitro may potentially be used in accordance with this
embodiment of the present invention. Such stem cells include but
are not limited to hematopoietic stem cells (HSC), stem cells of
epithelial tissues such as the skin and the lining of the gut,
embryonic heart muscle cells, liver stem cells (see e.g., PCT
Publication WO 94/08598) and neural stem cells (see e.g., Stemple
& Anderson. 1992. Cell 71:973-985).
[0075] Epithelial stem cells (ESCs) or keratinocytes can be
obtained from tissues such as the skin and the lining of the gut by
known procedures. See e.g., Rheinwald, 1980. Meth Cell Bio 21A:229.
In stratified epithelial tissue such as the skin, renewal occurs by
mitosis of stem cells within the germinal layer, the layer closest
to the basal lamina ESCs or keratinocytes obtained from the skin or
lining of the gut of a patient or donor can be grown in tissue
culture. See e.g. Pittelkow & Scott, 1986. Mayo Clinic Proc.
61:771. If the ESCs are provided by a donor, a method for
suppression of host versus graft reactivity (e.g., irradiation,
drug or antibody administration to promote moderate
immunosuppression) may also be utilized.
[0076] With respect to hematopoietic stem cells (HSC), any
technique that provides for the isolation, propagation, and
maintenance in vitro of HSC can be used in this embodiment of the
invention. Techniques by which this may be accomplished include:
(i) the isolation and establishment of HSC cultures from bone
marrow cells isolated from the future host, or a donor or (ii) the
use of previously established long-term HSC cultures, which may be
allogeneic or xenogeneic. Non-autologous HSC are used preferably in
conjunction with a method of suppressing transplantation immune
reactions of the future host/patient. In a particular embodiment of
the present invention, human bone marrow cells can be obtained from
the posterior iliac crest by needle aspiration. See e.g, Kodo, et
al., 1984. J Clin. Invest. 73:1377-1384. In a preferred embodiment
of the present invention, the HSCs can be made highly enriched or
in substantially-pure form. This enrichment can be accomplished
before, during or after long-term culturing, and can be done by any
techniques known in the art. Long-term cultures of bone marrow
cells can be established and maintained by using, for example,
modified Dexter cell culture techniques (see Dexter, et al., 1977.
J Cell Physiol 91:335) or Witlock-Witte culture techniques (see
Witlock & Witte, 1982. Proc. Natl. Acad. Sci. USA
79:3608-3612).
[0077] In a specific embodiment, the nucleic acid to be introduced
for purposes of gene therapy comprises an inducible promoter
operably-linked to the coding region, such that expression of the
nucleic acid is controllable by controlling the presence or absence
of the appropriate inducer of transcription.
[0078] (b) Anti-GENE SET Antibodies
[0079] In one embodiment of the present invention, as previously
discussed hereinabove, antibodies which bind GENE SET proteins or
nucleic acids (or derivative, fragments or analogs thereof) are
used to treat or prevent hypertension, diabetes, obesity or
ischemic stroke. Anti-GENE SET antibodies may also be used in the
diagnostic, prognostic and screening methods the present invention.
Such antibodies include, but are not limited to, polyclonal,
monoclonal. chimeric, single chain, Fab fragments, and an Fab
expression library. In a specific embodiment. antibodies to a human
GENE SET protein are produced. In another specific embodiment,
antibodies which reduce or inhibit GENE SET activity in vitro
and/or in vivo, are provided.
[0080] Various procedures known in the art may be used for the
production of polyclonal antibodies to an GENE SET protein or
derivative or analog. In a particular embodiment, rabbit polyclonal
antibodies to an epitope of a GENE SET protein (e.g., the protein
of amino acid sequences SEQ ID NOS:1 and 4 or encoded by the
nucleotide sequences of SEQ ID NOS:3 and 6, or a subsequence
thereof) may be obtained. For the production of antibody, various
host animals can be immunized by injection with the native GENE SET
protein, a synthetic version, or a derivative or fragment thereof
including, but not limited to, rabbits, mice, rats, and the like.
Various adjuvants may be used to increase the immunological
response, depending on the host species (e.g., Freund's
adjuvant).
[0081] For preparation of monoclonal antibodies directed toward an
GENE SET protein sequence (or derivative or analog thereof) any
technique which provides for the production of antibody molecules
by continuous in vitro cell lines may be used including, but not
limited to: the hybridoma technique (see Kohler & Milstein,
1975. Nature 256:495-497); the trioma technique; the human B-cell
hybridoma technique (see Kozbor, 1983. et aL, Immunology Today
4:72) and the EBV-hybridoma technique to produce human monoclonal
antibodies (see Cole, et al, Monoclonal Antibodies and Cancer
Therapy (Alan R. Liss, Inc., New York, N.Y.). In an additional
embodiment of the invention, monoclonal antibodies can be produced
in germ-free animals utilizing recent technology (see e.g., PCT
Publication US90/02545). Human antibodies are within the scope of
the present invention and may be obtained by using human hybridomas
(see e.g., Cote, et al, 1983. Proc. Natil. Acad. Sci. U.S.A.
80:2026-2030) or by transforming human B-cells with Epstein-Barr
virus (EBV) in vitro (see e.g., Cole, et al, 1985. Monoclonal
Antibodies and Cancer Therapy (Alan R. Liss, New York, N.Y.).
Additionally within the scope of the present invention are the
production of "chimeric antibodies" (see e.g., Morrison, et al.,
1984. Proc. Natl. Acad Sci. U.S.A. 81:6851-6855) which may be
produced by splicing the genes from a mouse antibody molecule
specific for GENE SET together with genes from a human antibody
molecule of appropriate biological activity.
[0082] In one embodiment of the present invention, single chain
antibodies (see U.S. Pat. No. 4,946,778) may be adapted to produce
GENE SET-specific single chain antibodies. An additional embodiment
of the invention discloses the utilization of Fab expression
libraries (see e.g., Huse, et al., 1989. Science 246:1275-1281) so
as to allow rapid and easy identification of monoclonal Fab
fragments with the desired specificity for GENE SET proteins (or
derivatives or analogs thereof).
[0083] Antibody fragments which contain the idiotype of the
molecule can be generated by known techniques. For example, such
fragments include but are not limited to: the F(ab').sub.2 fragment
which can be produced by pepsin digestion of the antibody molecule;
the Fab' fragments which can be generated by reducing the disulfide
bridges of the F(ab').sub.2 fragment, the Fab fragments which can
be generated by treating the antibody molecule with papain and a
reducing agent, and F.sub.v fragments.
[0084] In the production of antibodies, screening for the desired
antibody may be accomplished by techniques known in the art (e.g.,
enzyme-linked immunosorbent assay(ELISA)). In a specific
embodiment, the selection of antibodies which recognize a specific
portion of an GENE SET protein may be accomplished by an assay
which utilize hybridomas specific for a product which binds to a
GENE SET fragment containing such portion. For selection of an
antibody which possesses the ability to reduce or inhibit GENE SET
activity, one may screen the antibody in any of the assays for GENE
SET activity described infra.
[0085] (c) Anti-Sense GENE SET Nucleic Acids
[0086] In a specific embodiment of the present invention, the
function of GENE SET protein(s) is reduced or inhibited by GENE SET
anti-sense nucleic acids, utilized to treat or prevent stroke,
hypertension, diabetes or obesity. The present invention provides
the therapeutic or prophylactic use of nucleic acids of at least
six nucleotides which are anti-sense to a gene or cDNA encoding
GENE SET or a portion thereof. An GENE SET "anti-sense" nucleic
acid, as utilized herein, refers to a nucleic acid species which is
capable of hybridizing to a portion of an GENE SET RNA (preferably
mRNA) by virtue of sequence complementarily. The anti-sense nucleic
acid may be complementary to a coding and/or noncoding region of an
GENE SET mRNA. Such anti-sense nucleic acids have utility as
Therapeutics that reduce or inhibit GENE SET function, and can be
used in the treatment or prevention of disorders as described,
supra.
[0087] The GENE SET anti-sense nucleic acids are of at least six
nucleotides and are preferably oligonucleotides (ranging from 6-150
nucleotides or, more preferably, 6-50 nucleotides). In specific
aspects, the oligonucleotide is at least 10 nucleotides, at least
15 nucleotides, at least 100 nucleotides, or at least 125
nucleotides. The oligonucleotides may be DNA, RNA or chimeric
mixtures (or derivatives or modified versions thereof) and may be
single-stranded or double-stranded. The oligonucleotide can be
modified at the base moiety, sugar moiety, or phosphate backbone.
The oligonucleotide may include other appending groups such as
peptides, or agents facilitating transport across the cell membrane
(see e.g., Letsinger, et al., 1989. Proc. Natl. Acad. Sci. U.S.A.
86:6553-6556) or blood-brain barrier (see e.g., PCT Publication No.
WO 89/10134); hybridization-triggered cleavage agents (see e.g.,
Krol, et al, 1988. BioTechniques 6:958-976) or intercalating agents
(see e.g., Zon, 1988. Pharm. Res. 5:539-549).
[0088] The GENE SET anti-sense nucleic acid of the present
invention is preferably an oligonucleotide and more preferably, a
single-stranded DNA. In a preferred embodiment, the oligonucleotide
comprises a sequence anti-sense to a portion of human GENE SET. The
oligonucleotide may be modified at any position on its structure
with substituents generally known within the art.
[0089] Oligonucleotides of the invention may be synthesized by
standard methods known within the art, for example, by use of an
automated DNA synthesizer (e.g., Biosearch, Applied Biosystems,
etc). As an example, but of limitation, phosphorothioate
oligonucleotides may be synthesized by the method of Stein, et al.,
(1988. Nucl. Acids Res. 16:3209); methylphosphonate
oligonucleotides may be prepared by use of controlled pore glass
polymer supports (see e.g., Sarin, et al., 1988. Proc. Nati. Acad
Sci. U.S.A. 85:7448-7451) and similar synthesis methodologies.
[0090] In a specific embodiment of the present invention, the GENE
SET anti-sense oligonucleotide comprises catalytic RNA or a
ribozyme (see e.g., PCT International Publication WO 90/11364;
Sarver, et al., 1990. Science 247:1222-1225). In another specific
embodiment, the oligonucleotide is a 2N-0-methylribonucleotide (see
e.g., Inoue, et al., 1987. Nuc. Acids Res. 15:6131-6148) or a
chimeric RN.DELTA.-DNA analogue (see e.g., Inoue, et al., 1987.
FEBS Lett. 215:327-330).
[0091] In another embodiment, the GENE SET anti-sense nucleic acid
of the present invention is produced intracellularly by in vivo
transcription from an exogenous sequence. For example, a vector may
be introduced in vivo such that the vector (or a portion thereof)
is transcribed, producing an anti-sense nucleic acid (RNA) of the
present invention. Such a vector would contain a sequence encoding
the GENE SET anti-sense nucleic acid, and can remain episomal or
become chromosomally-integrated, so long as it can be transcribed
to produce the desired anti-sense RNA. The aforementioned vectors
may be comprised of plasmid, viral, or others known in the art
which are utilized for replication and expression in mammalian
cells and may be constructed by recombinant DNA technology
methodologies standard within the art. Expression of the sequence
encoding the GENE SET anti-sense RNA may be by any promoter known
in the art to act in mammalian, preferably human, cells. Such
promoters can be inducible or constitutive and include, but are not
limited to: (i) the SV40 early promoter region (see e.g., Bernoist
& Chambon, 1981. Nature 290:304-310); (ii) the promoter
contained in the 3'-terminus long terminal repeat (LTR) of Rous
sarcoma virus (see e.g., Yamamoto, et al., 1980. Cell 22:787-797);
(iii) the Herpesvirus thymidine kinase promoter (see e.g., Wagner,
et al., 1981. Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445); (iv) the
regulatory sequences of the metallothionein gene (see e.g.,
Brinster, et al., 1982. Nature 296:39-42), and the like.
[0092] The anti-sense nucleic acids of the present invention
comprise a sequence complementary to at least a portion of an RNA
transcript of an GENE SET gene, preferably a human GENE-SET gene.
However, absolute complementarily, although preferred, is not a
requirement. A sequence "complementary to at least a portion of an
RNA," as utilized herein refers to a sequence possessing sufficient
complementarily to be able to hybridize with the RNA, forming a
stable duplex; in the case of double-stranded GENE SET anti-sense
nucleic acids. Similarly, a single strand of the duplex DNA or
triplex formation may be assayed in a similar manner. The ability
to hybridize will dependent upon both the degree of complementarily
and the length of the anti-sense nucleic acid. Generally, the
longer the hybridizing nucleic acid, the more base mismatches with
a an GENE SET RNA it may contain and still form a stable duplex (or
triplex, as the case may be). One skilled in the art may ascertain
a tolerable degree of mismatch by use of standard procedures to
determine the melting point of the hybridized complex.
[0093] The invention further provides pharmaceutical compositions
(i.e., "Therapeutics") comprising an effective amount of the GENE
SET anti-sense nucleic acids of the present invention within a
pharmaceutically acceptable carrier. In a specific embodiment,
pharmaceutical compositions comprising GENE SET anti-sense nucleic
acids may be administered via liposomes, microparticles, or
microcapsules. It may be useful to use such compositions to achieve
sustained release of the GENE SET anti-sense nucleic acids.
[0094] The amount of GENE SET anti-sense nucleic acid which will be
effective in the treatment or prevention of ischemic disease will
depend on the nature of the disease, and can be determined by
standard clinical techniques. Where possible, it is desirable to
determine the anti-sense cytotoxicity in cells in vitro, and then
in useful animal model systems prior to testing and use in
humans.
[0095] (4) Methods of Diagnosis, Prognosis and Screening
[0096] The present invention also discloses methodologies which
relate to the diagnosis, prognosis and screening of stroke,
hypertension, diabetes and/or obesity.
[0097] In one embodiment, anti-GENE SET-antibodies are used to
detect and quantitate mutant GENE SET levels in one or more tissues
(e.g., blood) of a subject by use of an immunoassay- based
methodology. Specifically, such an immunoassay is performed by
contacting a sample derived from a patient with an anti-GENE SET
antibody under conditions such that immunospecific-binding can
occur, and detecting or measuring the amount of any
immunospecific-binding by the antibody. It should be noted,
however, that the particular amino acid deletion, insertion or
substitution within the amino acid sequence of the mutant GENE SET
protein can change the epitope recognized by a specific
anti-(wild-type) GENE SET antibody, such that antibody may bind the
mutant GENE SET protein to a lesser extent, or not at all.
Additionally, antibodies may be generated against the mutant GENE
SET protein, or portion thereof, which bind specifically to the
particular mutant GENE SET, but not the wild-type GENE SET (as
determined by the in vitro immunoassay methodology described
infra). These specific anti-mutant GENE SET antibodies may be used
to detect the presence of GENE SET by measuring the
immunospecific-binding by the anti-mutant GENE SET antibodies and,
optionally, the lack of immunospecific-binding by the
anti-(wild-type) GENE SET antibodies Moreover, GENE SET proteins
possessing deletion or insertion mutations may be detected by
either an increase or decrease in protein size by methodologies
which include, but are not limited to, for example, but not limited
to, Western blot analysis using an anti-GENE SET antibody which
recognizes both the mutant and wild-type GENE SET.
[0098] Immunoassays which may be utilized in the practice of the
present invention include, but are not limited to, competitive and
non-competitive assay systems using techniques such as Western
blots, radioimmunoassays (RIAs), enzyme-linked immunosorbent
assays(ELISA), "sandwich" immunoassays, immunoprecipitation assays,
precipitation reactions, gel diffusion precipitin reactions,
immunodiffusion assays, agglutination assays, complement-fixation
assays, immunoradiometric assays, fluorescent immunoassays,
protein-A immunoassays, and the like.
[0099] In a specific embodiment of the present invention, methods
of diagnosis, prognosis and screening are disclosed and utilize the
detection of mutant GENE SET alleles in genomic DNA or mRNA (i.e.,
genetic screening). These aforementioned mutant GENE SET alleles
may be detected by any method known in the art for detecting
mutations in genomic DNA including, but not limited to: DNA
hybridization methods (e.g. Southern Blotting), RFLP mapping,
PCR-based amplification methodologies, and the like, may be used
with nucleic acid probes which are complementary to both the
mutation and the corresponding position within the wild-type GENE
SET sequence.
[0100] In a preferred embodiment, allele-specific PCR (ASP) may be
used to detect mutant GENE SET alleles. In the ASP methodology, a
target DNA is, preferentially, amplified only if it is completely
complementary to the 3'-terminus of a specific PCR amplification
primer. The 3'-terminus of the primer is designed so as to
terminate at, or within one or two nucleotides of a known mutation
site within the GENE SET gene (target DNA) to which it possesses a
complementary sequence. Under the appropriate reaction conditions,
the target DNA is not amplified if there is a single nucleotide
mismatch (e.g., a nucleotide substitution caused by a mutation) or
a small deletion or insertion, at the 3'-terminus of the primer.
See e.g., Okayama, et al, 1989. J Lab. Clin. Med 114:105-113;
Sommer, et al., 1992. BioTechniques 12:82-87). Accordingly, ASP may
be utilized to detect either the presence or absence of (at least)
a single nucleotide mismatch between the primer sequence (which is
complementary to the pre-selected GENE SET target sequence) and a
nucleic acid within the sample. Amplification of the GENE SET
sequence is indicative of a lack of even a single mismatched
nucleotide.
[0101] Additionally, where the mutant comprises a deletion or
insertion mutation, mutant GENE SET alleles may be detected by
screening for an increase or decrease in the length of the GENE SET
nucleic acid sequence, or portion thereof. The increase or decrease
in length may be detected by any method known within the art for
measuring the length of nucleic acids, including, but not limited
to, amplification of a specific fragment of the GENE SET sequence
from the subject to be diagnosed or screened and from a standard or
control sample and comparison of the length of the fragments by any
size fractionation method (e.g., denaturing polyacrylamide gel
electrophoresis.
[0102] Additionally, kits for diagnostic or screening use are also
disclosed herein which comprise, in one or more containers, an
anti-GENE SET antibody or anti-GENE SET mutant antibody and,
optionally, a labeled binding partner to the antibody.
Alternatively, the anti-GENE SET antibody or anti-GENE SET mutant
antibody may be detectably-labeled (e.g., with a chemiluminescent,
enzymatic, fluorescent, or radioactive moiety). In another
embodiment, a kit is provided which comprises, in one or more
containers, a nucleic acid probe which is capable of
specifically-hybridizing to GENE SET RNA or, preferably, to mutant
GENE SET RNA. In a specific embodiment of the present invention, a
kit is provided which comprises, in one or more containers, a pair
of primers (e.g., each in the size range of 6-30 nucleotides) which
are capable of priming amplification reactions, under appropriate
reaction conditions, of at least a portion of a GENE SET nucleic
acid. These amplification reactions include, but are not limited
to, polymerase chain reaction (PCR); ligase chain reaction; Q.beta.
replicase, cyclic probe reaction or other amplification methods
known within the art. A kit may, optionally, further comprise, in a
container, a predetermined concentration of a purified GENE SET
protein or nucleic acid, for use as a standard or control.
[0103] (5) Assays for Modulators of GENE SET Proteins and Nucleic
Acids
[0104] A variety of methodologies are available within the art for
assaying the activity of GENE SET proteins (and derivatives,
analogs, fragments and homologs of GENE SET proteins), as well as
for the nucleic acids encoding the GENE SET proteins (and
derivatives, analogs, fragments and homologs thereof). Methods are
also available for the screening of putative GENE SET modulators
(e.g., GENE SET agonists, antagonists and inhibitors). Such
modulators of GENE SET activity include, but are not limited to,
GENE SET anti-sense nucleic acids, anti-GENE SET antibodies, and
competitive inhibitors of GENE SET proteins for binding to the GENE
SET protein receptors.
[0105] The activity of the GENE SET proteins (and derivatives,
fragments, analogs and homologs of GENE SET proteins), the nucleic
acids encoding these GENE SET proteins (and derivatives, fragments,
analogs and homologs thereof) and putative modulators of GENE SET
protein activity may also be ascertained in vivo. For example,
infusion of GENE SET proteins in humans causes significant
increases in cGMP levels in plasma and urine. See e.g., Vesely, et
al., 1995. Am. J Med Sci. 310:143-149; Vesely, et al., 1996.
Metabolism: Clin & Exp. 45:315-319. Administration of GENE SET
proteins to humans also elicits significant diuresis and reduction
in blood pressure (see e.g., Vesely, et al., 1996. Life Sciences
59:243-254); similar effects have also been observed in rodents
(see e.g., Garcia, et al., 1989. Hypertension 13:567-574). In
accord, the mutant GENE SET proteins and nucleic acids (and
derivatives, analogs, fragments and homologs thereof) and putative
GENE SET modulators may be assayed by the administration of a "test
compound" to an animal, preferably a non-human test animal,
followed by the measurement of the one or more of the physiological
parameters described above (e.g., cGMP levels in urine and/or
plasma, diuretic effect, decrease in blood pressure, and the
like).
[0106] Another embodiment of the present invention discloses a
methodology for screening a GENE SET mutant for a change in
activity comprising (i) administering the GENE SET mutant to a test
animal prone to stroke, hypertension, diabetes or obesity and (ii)
measuring of stroke latency within the test animal in which stroke
latency is indicative of GENE SET activity. In a specific
embodiment, a recombinant test animal, which expresses a GENE SET
transgene or expresses a member of the GENE SET under the control
of a promoter which is not the native GENE SET gene promoter at an
increased level relative to a wild-type test animal, is used to
screen the GENE SET for a change in GENE SET activity.
[0107] In another embodiment of the present invention, a method for
screening for a modulator of GENE SET activity, or of latency or
predisposition to stroke, is provided which comprises measuring
stroke latency within a stroke-prone animal that recombinantly
expresses a putative modulator of GENE SET activity, in which a
change in stroke latency relative to an analogous stroke-prone
animal which does not recombinantly-express the putative modulator,
indicates that the putative modulator possesses the ability to
modulate GENE SET activity, or latency or predisposition to
stroke.
[0108] In yet another embodiment, a method is provided for
screening an GENE SET mutant for an effect on latency or
predisposition to stroke comprising measuring stroke latency within
a stroke-prone animal which recombinantly-expresses a GENE SET
mutant, in which a change in stroke latency relative to an
analogous stroke-prone animal which does not recombinantly express
the GENE SET mutant indicates that the GENE SET mutant has an
effect on latency or predisposition to stroke, hypertension,
diabetes or obesity. In a preferred embodiment, a GENE SET mutant
is screened for an increase in stroke latency or a decrease in
predisposition to stroke.
[0109] (6) Pharmaceutical Compositions and Therapeutics
[0110] The present invention discloses methods of treatment and
prophylaxis by administering to a subject of an effective amount of
a Therapeutic of the invention. In a preferred embodiment, the
Therapeutic is substantially-purified. The subject is preferably an
animal which, preferably, is a mammal and most preferably
human.
[0111] Formulations and methods of administration which may be
employed when the Therapeutic comprises a nucleic acid are
described in Sections 3(a) and 3(c), supra; whereas additional
appropriate formulations and routes of administration may be
selected from among those described infra.
[0112] Numerous types of pharmaceutical composition delivery
systems are well-known within the art and may be utilized to
administer a Therapeutic of present the invention. These
aforementioned delivery systems include, but are not limited to:
(i) encapsulation in liposomes, microparticles and microcapsules;
(ii) recombinant cells capable of expressing the Therapeutic; (iii)
receptor-mediated endocytosis (see e.g., Wu & Wu, 1987. J Biol.
Chem. 262:4429-4432); (iv) construction of a Therapeutic nucleic
acid as part of a retroviral or other vector, and the like. Methods
of administration/introduction include, but are not limited to,
intradermal, intramuscular, intraperitoneal, intravenous,
subcutaneous, intranasal, epidural, and oral routes. The
Therapeutic may be administered by any convenient route, for
example by infusion or bolus injection, by absorption through
epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and
intestinal mucosa, etc.) and may be administered together with
other biologically active agents. Administration can be systemic or
local. In addition, it may be desirable to introduce the
Therapeutic of the present invention into the central nervous
system by any suitable route (e.g., intraventricular and
intrathecal injection). Intraventricular injection may be
facilitated by the use of an intraventricular catheter, for
example, attached to a reservoir, such as an Ommaya reservoir
Pulmonary administration may also be employed (e.g., by use of an
inhaler or nebulizer) and formulation with an aerosolizing
agent.
[0113] In a specific embodiment of the present invention, it may be
desirable to administer the Therapeutic of the present invention
locally to the area in need of treatment; this may be achieved by,
for example, and not by way of limitation, local infusion during
surgery, topical application (e.g., in conjunction with a wound
dressing after surgery), by injection, by means of a catheter, by
means of a suppository, or by means of an implant, said implant
being of a porous, non-porous, or gelatinous material, including
membranes, such as sialastic membranes, or fibers.
[0114] In another specific embodiment of the present invention, the
Therapeutic may be delivered in a vesicle, in particular a
liposome. See e.g., Langer, 1990. Science 249:1527-1533. In yet
another specific embodiment, the Therapeutic may be delivered via a
controlled release system including, but not limited to: a pump
(see e.g., Sefton, 1987. CRC Crit. Ref Biomed. Eng. 14:201) and
polymeric materials (see e.g., Smolen & Ball, 1983. Controlled
Drug Bioavailability, Drug Product Design and Performance (Wiley,
New York, N.Y.). In addition, a controlled release system may be
placed in proximity of the therapeutic target (e.g., the brain),
thus requiring only a fraction of the total systemic dose. See
e.g., Goodson, 1984. Medical Applications of Controlled Release,
(Wiley, New York, N.Y.).
[0115] In a specific embodiment of the present invention where the
Therapeutic is a nucleic acid encoding a protein-based Therapeutic,
the nucleic acid may be administered in vivo to promote expression
of its encoded protein, by constructing of the aforementioned
protein as part of an appropriate nucleic acid expression vector,
and administering the construct so that it becomes intracellular by
methodologies which include, but are not limited to: (i) use of a
retroviral vector (see e.g., U.S. Pat. No. 4,980,286); (ii) use
direct injection; (iii) use of microparticle bombardment (e.g., a
gene gun; Biolistic, DuPont); (iv) coating with lipids or
cell-surface receptors or transfecting agents; (v) administering it
in linkage to a homeobox-like peptide which is known to enter the
nucleus (see e.g., Joliot, et al, 1991. Proc. Natl. Acad Sci. USA
88:1864-1868) and the like.
[0116] In an alternate embodiment of the present invention, a
nucleic acid-based Therapeutic may be introduced intracellularly
and incorporated by homologous recombination within host cell DNA
for expression.
[0117] The present invention also discloses pharmaceutical
compositions. Such compositions comprise a
therapeutically-effective amount of a Therapeutic within a
pharmaceutically-acceptable carrier. In a specific embodiment, the
term "pharmaceutically acceptable," as utilized herein, is defined
as the composition being approved by a regulatory agency of the
Federal or a state government or listed in the U.S. Pharmacopeia or
other generally recognized pharmacopera for use in animals and,
more particularly, in humans. The term "carrier," as utilized
herein refers to a diluent, adjuvant, excipient, or vehicle with
which the therapeutic is administered. Such pharmaceutical carriers
include, but are not limited to: sterile liquids (e.g., water,
physiological saline and the like) and oils (e.g., oils of
petroleum, animal, vegetable or synthetic origin, such as peanut
oil, soybean oil, mineral oil, sesame oil and the like). Water is a
preferred carrier when the pharmaceutical composition is
administered intravenously. Additionally, saline solutions and
aqueous dextrose and glycerol solutions may also be employed as
liquid carriers, particularly for injectable solutions. Suitable
pharmaceutical excipients include starch, glucose, lactose,
sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium
stearate, glycerol monostearate, talc, sodium chloride, dried skim
milk, glycerol, propylene, glycol, water, ethanol and the like. The
composition, if desired, can also contain minor amounts of wetting
or emulsifying agents, or pH buffering agents. These compositions
can take the form of solutions, suspensions, emulsion, tablets,
pills, capsules, powders, sustained-release formulations and the
like. The composition can be formulated as a suppository, with
traditional binders and carriers such as triglycerides. Oral
formulation can include standard carriers such as pharmaceutical
grades of mannitol, lactose, starch, magnesium stearate, sodium
saccharine, cellulose, magnesium carbonate, etc. Examples of
suitable pharmaceutical carriers are described in Martin, 1965.
Remington's Pharmaceutical Sciences. Such compositions will contain
a therapeutically-effective amount of the Therapeutic, preferably
in purified form and, most preferably, in a substantially-purified
form, together with a suitable amount of carrier so as to provide
the form for proper administration to the patient. The formulation
should be suited to the mode of administration.
[0118] In a preferred embodiment of the present invention, the
composition is formulated in accordance with routine procedures as
a pharmaceutical composition adapted for intravenous administration
to human beings. Typically, compositions for intravenous
administration are solutions in sterile isotonic aqueous buffer.
Where necessary, the composition may also include a solubilizing
agent and a local anesthetic such as lignocaine to ease pain at the
site of the injection. Generally, the ingredients are supplied
either separately or mixed together in unit dosage form, for
example, as a dry lyophilized powder or water-free concentrate in a
hermetically sealed container such as an ampoule or sachette
indicating the quantity of active agent. Where the composition is
to be administered by infusion, it can be dispensed with an
infusion bottle containing sterile pharmaceutical grade water or
saline. Where the composition is administered by injection, an
ampoule of sterile water for injection or saline can be provided so
that the ingredients may be mixed prior to administration.
[0119] The Therapeutics of the present invention may be formulated
with pharmaceutically-acceptable salts including those derived from
hydrochloric, phosphoric, acetic, etc., and those formed with free
carboxyl groups such as those derived from sodium, potassium,
calcium, ferric hydroxides, isopropylamine, triethylarnine,
2-ethylamino ethanol, histidine, procaine, etc.
[0120] The amount of the Therapeutic of the present invention which
will be effective in the treatment of a particular disorder or
condition will be dependent upon the exact nature of the disorder
or condition, and can be quantitatively-determined by standard
clinical techniques. In addition, in vitro assays may (optionally)
be employed to help identify optimal dosage ranges. The precise
dose to be employed in the formulation will also depend on the
route of administration, and the seriousness of the disease or
disorder, and should be decided according to the judgment of the
practitioner and each patient's circumstances. However, suitable
dosage ranges for intravenous administration are generally about
20-500 .mu.g of active compound per kilogram (kg) body weight.
Suitable dosage ranges for intranasal administration are generally
about 0.01 pg/kg body weight to 1 mg/kg body weight. Effective
doses may be extrapolated from dose-response curves derived from in
vitro or animal model test systems. Suppositories generally contain
active ingredient in the range of 0.5% to 10% by weight; oral
formulations preferably contain 10% to 95% active ingredient.
[0121] The present invention also provides a pharmaceutical pack or
kit comprising one or more containers filled with one or more of
the ingredients of the pharmaceutical compositions of the
invention. Optionally associated with such container(s), a notice
in the form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
which notice reflects approval by the agency of manufacture, use or
sale for human administration.
[0122] (7) Animal Models
[0123] The present invention discloses animal models. In one
embodiment, animal models for stroke, hypertension, diabetes or
obesity are provided. Transgenic animals may be bred or produced
through molecular-biological means, which over-express or
under-express one or more of the GENE SET genes (e.g., by
introducing a member or members of the GENE SET gene under the
control of a heterologous promoter or a promoter which facilitates
the expression of GENE SET proteins and/or nucleic acids in tissues
which do not normally express GENE SET components. Additionally,
"knockout" mice may be initially produced by promoting homologous
recombination between a GENE SET gene in its chromosome and an
exogenous GENE SET gene that has been rendered biologically
inactive, preferably by insertion of a heterologous sequence (e.g.,
an antibiotic resistance gene) or by non-homologous
recombination.
[0124] In a preferred embodiment of the present invention,
introduction of heterologous DNA is carried out by transforming
embryo-derived stem (ES) cells with a vector containing the
insertionally-inactivated GENE SET gene or a GENE SET gene which is
under the control of a heterologous promoter, followed by injecting
the ES cells into a blastocyst and implanting the blastocyst into a
"foster mother" animal. Accordingly, the resulting mice are
chimeric animals ("knockout animal" or "transgenic animal") in
which an GENE SET gene has been inactivated or overexpressed or
misexpressed (see e.g., Capecchi, 1989. Science 244:1288-1292). The
chimeric animal can then be bred to produce additional knockout or
transgenic animals. Such chimeric/transgenic animals include, but
are not limited to, mice, hamsters, sheep, pigs, cattle, etc., and
are, preferably, non-human mammals. Transgenic and knockout animals
can also be made in D. melanogaster, C. elegans, and the like, by
methods which are commonly-known within the art.
[0125] Another embodiment of the present invention provides a
recombinant non-human animal containing a mutant GENE SET gene,
under the control of a promoter which is not the native GENE SET
gene promoter, in which the mutant GENE SET gene encodes a mutant
GENE SET which increases latency to stroke. Yet another embodiment
discloses a recombinant non-human animal that is the product of a
process comprising introducing a nucleic acid into the non-human
animal, or an ancestor thereof, said nucleic acid comprising a
mutant GENE SET gene sequence.
[0126] (8) Specific Examples
[0127] Heart, brain, fat, liver and kidney tissue from
spontaneously hypertensive rats (SHR), stroke-prone SHR (SHR-SP)
and control Wistar Kyoto rats (WKY) were analyzed by the
GeneCalling.RTM. methodology (see PCT Publication WO 97/15690) to
facilitate the identification and characterization of genes which
are differentially-expressed in the SHR and SHR-SP rats, as
compared to the control WKY animals.
[0128] (A) Materials and Methodologies
[0129] (i) Isolation of Total Cellular RNA and Polv(A).sup.+
mRNA
[0130] SHR, SHR-SP and WKY rats were maintained on normal rat chow
(Purina) and water ad libitum. Thirteen week old rats were
sacrificed and the hearts, liver, fat, kidney and brain tissues
were removed and quick-frozen in liquid nitrogen immediately
following dissection. The whole organs were stored at -70.degree.
C. for subsequent processing.
[0131] Total cellular RNA was extracted from 5 mg of heart, liver,
fat, kidney, or brain tissue by initially grinding the tissue into
a fine powder in liquid nitrogen. The powdered tissue was then
transferred to a tube containing 500 .mu.l Triazol Reagent.RTM.
(Life Technologies; Gaithersburg, Md.) and was dispersed in the
Triazol Reagent.RTM. using a Polytron homogenizer (Brinkman
Instruments; Westbury, N.Y.). See e.g., Chomszynski, et al., 1987.
Annal. Biochem. 162 156-159; Chomszynski, et al., 1993.
BioTechniques 15:532-533, 536-537. The total cellular RNA fraction
was then extracted with 50 .mu.l BCP (1-bromo-3-chloropropane;
Molecular Research; Cincinnati, Ohio) to facilitate phase
separation. The extraction mixture was centrifuged for 15 minutes
at 4.degree. C. at 12,000.times.G, and the aqueous phase was
removed and transferred to a fresh tube. The RNA was then
precipitated with 0.5 volume of isopropanol per original volume of
Triazol Reagent.RTM. used, and the sample was re-centrifuged at
room temperature for 10 minutes at 12,000.times.G. The supernatant
was then discarded, the pellet washed with 70% ethanol and
re-centrifuged at room temperature for 5 minutes at 12,000.times.G.
Finally the 70% ethanol was removed and the centrifuge tube was
inverted and let stand to dry in this position. The resulting RNA
pellet was re-suspended in 100 .mu.l water (i.e., 1 .mu.l/mg of
original tissue weight) and heated to 55.degree. C. until
completely dissolved. The final concentration of total cellular RNA
was quantitated by fluorometry with OliGreen.RTM. (Molecular
Probes; Eugene, Oreg.). In addition, the quality of the total
cellular RNA was determined by both spectrophotometry and
formaldehyde agarose gel electrophoresis.
[0132] Poly(A).sup.+ RNA was prepared from 100 .mu.g of total
cellular RNA by use of affinity chromatography with oligo(dT)
magnetic beads (PerSeptive; Cambridge, Ma.) or with the Dynabeads
mRNA Direct Kit.RTM. (Dynal; Oslo, Norway) as directed by the
manufacturer. The Poly(A).sup.+ RNA was harvested in a small volume
of sterile water, and the final yield quantified by OD.sub.260
measurement and fluorometry with OliGreen.RTM. (Molecular Probes;
Eugene. OR). The Poly(A).sup.+ RNA was stored at -20.degree. C. for
subsequent utilization in cDNA synthesis and GeneCalling.RTM.
protocols.
[0133] (ii) cDNA Synthesis
[0134] Prior to cDNA synthesis, each of the Poly(A).sup.+ RNA
samples from the aforementioned tissues were treated with DNase to
remove endogenous, contaminating DNA. 28 .mu.l of 5> reverse
transcriptase buffer (Life Technologies; Gaithersburg, Md.), 10
.mu.l 0.1 M DTT, 5 units RNAguard.RTM. (Pharmacia Biotech, Upsala,
Sweden) per 100 mg tissue and 1 unit RNase-free DNase 1 (Pharmacia
Biotech) per 100 mg tissue, were added to the re-suspended RNA
samples. The reaction mixtures were then incubated at 37.degree. C.
for 20 minutes. The total RNA concentration was quantified by
measuring OD260 of a 100-fold dilution and the samples were stored
at -20.degree. C.
[0135] cDNA was synthesized from the Poly(A).sup.+ RNA as follows:
the Poly(A).sup.+ RNA isolated from each of the aforementioned
tissues was mixed with 50 ng random hexamer primers (50 ng/.mu.l)
in 10 .mu.l of water. The mixtures were heated to 70.degree. C. for
10 minutes, quick-chilled in an ice-water slurry, and kept on ice
for 1-2 min. The condensates were then collected by centrifugation
in a microfuge for approximately 10 seconds.
[0136] The first-strand synthesis was performed by adding to the
reaction mixtures: 4 .mu.l 5.times.first-strand buffer (from a BRL
cDNA Synthesis Kit; Grand Island, N.Y.), 2 .mu.l 100 mM DTT, 1
.mu.l 10 mM dNTP mix, and 2 .mu.l water to each of the
primer-annealed Poly(A).sup.+ RNA. Alternately, 200 pmols of
oligo(dT).sub.25V (V=A, C or G) was utilized as a primer in the
first-strand synthesis reactions. The reaction mixtures were then
incubated at 37.degree. C. for 2 minuets, followed by the addition
of 1 .mu.l of Superscript II.RTM. reverse transcriptase (BRL) and
the reactions were incubated at 37.degree. C. for 1 hour.
[0137] Second-strand cDNA synthesis was then performed. The samples
were placed on ice and to each of the first-strand reaction mixture
was added: 30 .mu.l of 5.times.second-strand buffer, 90 .mu.l of
cold water, 3 .mu.l of 10 mM DNTP, 1 .mu.L (10 units) of E. coli
DNA ligase (BRL), 4 .mu.l (40 units) of E. coli DNA polymerase I
(BRL), and 1 .mu.l (3.5 units) of E. coli RNaseH (BRL) and the
reaction mixtures were incubated for 2 hours at 16.degree. C. The
resulting double-stranded cDNA was then incubated with 2 .mu.l of
T.sub.4 DNA polymerase (5 units) at 16.degree. C. for 5
minutes.
[0138] The resulting cDNA was then dephosphorylated with Arctic
Shrimp Alkaline Phosphatase ("SAP"; USB; St. Louis, Mich.) by
adding to each reaction mixture: 20 .mu.l 10.times.SAP buffer, 25
.mu.l of water, and 5 .mu.l (5 units) of SAP. The reactions were
incubated at 37.degree. C. for 30 minutes.
[0139] The cDNA was extracted with phenol/chloroform (50:50 v/v),
chloroform/isoamyl alcohol (99:1 v/v) and precipitated from the
aqueous phase by the addition of NaOAc pH 5.0 to 0.3 M, 20 .mu.g
glycogen, and 2.5 volumes of ethanol followed by incubation at
-20.degree. C. for 10 minuets. The cDNA was collected by
centrifugation at 14,000.times.g for 10 minuets. The supernatant
was then aspirated and the resulting cDNA pellet was washed with
75% ethanol. resuspended in TE buffer (pH 7.0) and the yield of
cDNA was estimated using fluorometry with Picogreen.RTM. (Molecular
Probes; Eugene, Oreg.).
[0140] (iii) GeneCalling.RTM. Methodology
[0141] The GeneCalling.RTM. methodology is comprised of a 3-step
process which involves cDNA fragmentation, tagging and
amplification. Fragmentation was achieved by restriction enzyme
digestions in a 50 .mu.l reaction mix containing 5 units of each
restriction enzyme, 1 ng of double-stranded cDNA and 5 .mu.l of the
appropriate 1.times.restriction endonuclease buffer. Analysis of
all mRNAs was achieved by performing 80 separate sets of cDNA
fragmentation reactions, each with a different pair of restriction
enzymes. Tagging was achieved by ligation of amplification
cassettes with ends compatible to the 5'- and 3-termini of the cDNA
fragments. A FAM label was incorporated onto the 5'-terminus of one
of the PCR primers. Incubation of the ligation reaction was
performed at 16.degree. C. for 1 hour in 10 mM ATP, 2.5% PEG, 10
units T4 DNA ligase and 1.times.ligase buffer.
[0142] PCR amplification was performed by the addition of the
following reagents to each of the reaction tubes: 2 .mu.l 10 mM
dNTP, 5 .mu.l 10.times.TB buffer (500 mM Tris, 160 mM
(NH.sub.4).sub.2SO.sub.4, 20 mM MgCl.sub.2, pH 9.15), 0.25 .mu.l
KlenTaq.RTM. (Clontech Advantage):PFU.RTM. (Stratagene, La Jolla
Calif.) (16:1) and 32.75 .mu.l H.sub.2O. Twenty (20) cycles of
amplification (30 seconds at 96.degree. C., 1 minute at 57.degree.
C., 2 minutes at 72.degree. C.), followed by 10 minutes at
72.degree. C., were performed in a PTC-100 Thermal Cycler equipped
with a mechanized lid (MJ Research; Watertown, Ma.).
[0143] Post-PCR amplification product purification was performed
using streptavidin magnetic beads (MPG.RTM. Beads; CPG, Lincoln
Park, N.J.). After washing the beads twice with buffer 1 (3 M NaCl,
10 mM Tris-HCl, 1 mM EDTA, pH 7.5), 20 .mu.l of buffer 1 was mixed
with the PCR product for 10 minutes at room temperature, separated
with a magnet, and washed once with buffer 2 (10 mM Tris, 1 mM
EDTA, pH 8.0). The beads were then dried and resuspended in 3 .mu.l
of buffer 3 (80% (v/v) formamide, 4 mM EDTA, 5% TAMRA- or
ROX-tagged molecular size standard (PE-Applied Biosystems, Foster
City Calif.). Following denaturation at 96.degree. C. for 3
minutes, samples were then loaded onto 5% polyacrylamide, 6M urea,
0.5.times.TBE ultrathin gels and electrophoresed on a proprietary
Niagara.RTM. gel electrophoresis system. PCR products were
visualized by virtue of the fluorescent FAM label at the 5' end of
one of the PCR primers, which ensures that all detected fragments
have been digested by both enzymes.
[0144] The primary components of the Niagara.RTM. gel
electrophoresis system are an interchangeable horizontal ultrathin
gel cassette mounted in a platform employing stationary laser
excitation and a multi-color CCD imaging system. Each gel cassette
may be loaded with a total of 48 lanes (4 cycles of 12 wide)
directly from a 96-well plate using a Beckman Biomek 2000.RTM.
robotic arm (Beckman, Sunnyvale, Calif.). The Niagara
electrophoresis system has the advantage of high throughput, with
separation of fragments between 30 and 450 bases in 45 minutes.
[0145] (iv) Niagara.RTM. Gel Interpretation
[0146] The output from the Niagara.RTM. gel electrophoresis system
was processed using the Java-based, internet-ready Open Genome
Initiative (OGI) software suite. Gels images were initially
visually checked and tracked. Each lane contained the FAM-labeled
products of a single GeneCalling.RTM. reaction plus a molecular
weight "sizing-ladder" spanning the range from 50 to 500 bp. The
ladder peaks provided a correlation between camera frames
(collected at 1 Hz) and DNA fragment size in base pairs (bp).
Following tracking, the lanes were extracted and the peaks in the
sizing ladder were ascertained. Linear interpolation between the
ladder peaks was utilized to convert the fluorescence traces from
frames to base pairs. A final quality control (QC) step checked for
various anomalies (e g, low signal-to-noise ratio, poor peak
resolution, missing ladder peaks, and lane-to-lane sample
bleeding). Data which passed all of the aforementioned criteria
were submitted as point-by-point length versus amplitude addresses
to an Oracle 8 database for subsequent difference
identification.
[0147] (v) Difference Identification
[0148] For each restriction enzyme pair (subsequence) comprising
each sample set, a composite trace was calculated. This composite
trace calculation entailed compiling all of the individual sample
replicates, followed by application of a scaling algorithm for
best-fit to normalize the traces of the experimental set versus
that of the control set. The scaled traces were then compared on a
point-by-point basis to define areas of amplitude difference which
meet the minimum, pre-specified threshold for a
statistically-significant difference. Once a region of difference
was characterized, the local maximum for the corresponding traces
of each set was identified. All difference peaks were stored as
unique database addresses in the specified expression difference
analysis.
[0149] (vi) Northern Blot Analysis
[0150] 1 .mu.g of Poly (A).sup.+ RNA from 3 animals of each
genotype was electrophoresed in agarose/formaldehyde gels, blotted
to PVD-nylon filters and probed with fill-in oligonucleotides
labeled with .sup.32P-dCTP. The hybridization was quantitated with
a storage phosphor imaging plate using the Fuji BAS2000.RTM.. Blots
were stripped and re-probed for GAPDH to normalize gene expression
levels. Induction levels are expressed as the ratio of average
normalized hybridization signals.
[0151] (vii) Radiation Hybrid Mapping
[0152] A commercially-available, rat T55 radiation hybrid panel
(Research Genetics; Huntsville, Ala.), consisting of 106 individual
hybrids was utilized in this analysis. It was determined
empirically, that hybrid samples 1, 20, 35, 38, 60 and 90 had very
low retention when compared to the other samples in the panel. In
addition, a total of 94 rat hybrid (RH) samples was utilized, in
order to efficiently perform PCR in a 96-well format (94 RH
samples, plus negative and positive control). Accordingly, RH
samples 1-100 from the T55 radiation hybrid panel, excluding the 6
low retention RH samples, previously discussed.
[0153] MapPairs.RTM. simple sequence repeat (SSR) markers (Research
Genetics) were utilized. All markers were run in duplicate and
evaluated/scored as a pair. RH templates were distributed into 384
well PCR plates (MJ Research) at 20 ng/well using a Tecan Genesis
100.RTM. pipetting robot, and allowed to desiccate. PCR reactions
were performed in 5 .mu.l total volumes using the following
concentrations of reagents: 0.5 .mu.M each primer, 200 .mu.M dNTPs,
1.times.PC2 buffer (50 mM Tris-HCl pH 9.1, 16 mM ammonium sulfate,
3.5 mM MgCl.sub.2, 150 .mu.g/ml BSA, 1.times.Rediload (Research
Genetics) and 0.25U KlenTaq.RTM. (Clonetech Advantage). PCR
amplification reaction conditions (Thermocycle) were as follows:
initial 3 minute denaturation at 94.degree. C.; subsequent
denaturation at 94.degree. C. for 30 seconds; initial annealing
temperature of 65.degree. C. for 30 seconds; elongation at
68.degree. C. for 30 seconds; second cycle annealing temperature of
63.degree. C. for 30 seconds; all subsequent cycles, annealing
temperature of 60.degree. C. for a total of 35 cycles.
[0154] PCR products were then subjected to electrophoresis through
a 3% agarose (BRL) gel using 1.times.TBE buffer, for 30 min at
200V. Gel images were documented using an Alpha Innotech 950.RTM.
imaging system.
[0155] (B) Experimental Results
[0156] (i) Identification of Differentially-expressed Genes between
the SHR-SP, SHR and WKY by GeneCalling
[0157] GeneCalling.RTM. reactions were performed on whole organs
from adipose, kidney, heart, brain and liver as previously
described, and gene expression profiles were compared between the
SHR-SP and SHR and between the SHR and WKY animals.
[0158] An average of 20,000 GeneCalling.RTM. fragments were
measured for relative abundance, yielding between 0.3% and 1.4%
differences in gene expression. The results of this analysis are
shown in Table 1. The SHR vs. WKY comparisons gave about 3-fold
more differences than the SHR-SP vs. SHR comparisons, which is not
surprising since the SHR-SP diverged from the SHR more recently
than the SHR diverged from the WKY. By percentage, the most changes
in gene expression were seen in the liver of the SHR relative to
the WKY (1.4%), whereas the fewest changes were seen in the heart
of the SHR-SP relative to the SHR (0.3%).
[0159] On average, three GeneCalling.RTM. fragments per gene were
measured for relative abundance, allowing fault-tolerance in the
event of sequence variation between the strains being compared. The
relative abundance of each GeneCalling.RTM. fragment is illustrated
in FIG. 1. Modulation was reported in fold-increase or
fold-decrease in each tissue measured, with positive modulation
(fold-increase) indicating increased mRNA abundance in the SHR
(Table 2) or SHR-SP (Table 3) GeneCalling.RTM. samples.
[0160] Comparison of the SHR and WKY sanples, resulted in the
finding of a total of 48 differentially-expressed genes with
identity or significant similarity to sequences which had been
previously reported to GenBank. This figure accounted for
approximately one-half the total number of gene expression
differences (Table 2, FIG. 2). Interestingly, two genes were found
to be modulated within all 5 tissues studied, indicating
organism-wide alterations in gene expression which may link the
phenotypes seen in the different organs.
[0161] Comparison of the SHR-SP and SHR samples, resulted in the
finding of 14 differential-expressed genes with significant
similarity to sequences which had been previously reported to
GenBank (Table 3, FIG. 2), with an average of 2.7 fragments per
gene and one gene modulated across all 5 tissues.
[0162] (ii) Mapping of Differentially-Expressed Genes
[0163] Differentially-expressed and previously unmapped genes of
interest were placed on the physical map utilizing a radiation
hybrid panel. Upon genotyping, map positions were constructed using
RHMAPPER.RTM. computer program. Examples of mapped genes are shown
in FIG. 3, Table 2, column 7 and Table 3, column 4. The map
locations of the differentially-expressed genes were compared to
quantitative trait loci (QTL) previously-reported in the literature
and/or reported to GenBank. Of the 26 differentially-expressed
genes whose map locations have been determined, six of the
aforementioned genes mapped to within previously described QTL
(FIG. 3, Table 1, Table 2). These six genes, in toto, as utilized
within the scope of the present invention, were designated the GENE
SET genes.
[0164] Specifically, the six differentially-expressed genes were
mapped to the following chromosomal positions: (i) kynurenine
aminotransferase mapped to chromosome 3 between D3rat100 and
D3kyo2; (ii) CD36 mapped to chromosome 4 between D4rat5 and D4rat7;
(iii) SGLT2 maps near the SA locus on chromosome 1; (iv) aldolase A
maps near the SA locus on chromosome 1 and (v) both .alpha. cardiac
myosin and .alpha.-tubulin are part of the RT complex on chromosome
20. In addition, the atrial natriuretic peptide (ANP) was found to
map between Elaiil and D5mgh16.
[0165] (iii) Detection of Mutated Genes Within the QTL
[0166] Sequence variations between the SHR-SP, SHR and WKY rodent
strains were detected by direct sequencing of cDNAs which derived
from PCR-amplified cDNA initially synthesized from
oligo(dT)-selected, Poly (A).sup.+ RNA from the tissues analyzed
for gene expression.
[0167] Amino acid substitutions were found in five of the six genes
which were demonstrated to be differentially-expressed between the
disease animal strains (i.e., SHR and SHP-SP) and the control
strain (WKY) and which additionally mapped to QTL identified as
contributing to the traits of interest in cross between the disease
model and control.
[0168] Specifically, a conserved leucine (Leu) was found to be
changed to glutamine (Glu) in the low-affinity, Na.sup.+-dependent
glucose transporter SGLT2 (L638Q) which was found to map within a
region which has been previously shown to contain a gene(s)
contributing to blood pressure variation (FIG. 3). The amino acid
sequence of the region possessing the amino acid substitution of
the SGLT2 variant (L638Q) in SHR, WKY, human, rabbit and pig is
shown in FIG. 4 [SEQ ID NO:1].
[0169] Kynurenine aminotransferase was demonstrated to possess an
amino acid substitution which involves the changing of a specific
charge at amino acid residue position 27 (E27G). Previously, this
residue was found to be conserved across all known family members
to the C elegans kynurenine aminotransferase homolog, and the
mutated gene also mapped to a region shown to be involved in blood
pressure regulation. The amino acid sequence of the region
possessing the amino acid substitution of the kynurenine
aminotransferase variant (E27G) in SHR, WKY, human and nematode is
shown in FIG. 4 [SEQ ID NO:2].
[0170] Similarly, the fatty acid transport protein (FAT)/CD36
homolog was found to contain numerous amino acid substitutions and
mapped to a region which was shown to be defective in the SHR, with
regards to both glucose uptake and impaired fatty acid secretion.
The amino acid sequence of the region possessing the amino acid
substitution in amino acid residues 148-191 and 213-257 of the
FAT/CD36 variant in SHR-SP, SHR, WKY and human is shown in FIG. 4
[SEQ ID NO:3] and [SEQ ID NO:4], respectively.
[0171] Aldolase A was demonstrated to map to the stroke
predisposition locus on chromosome 1 and possessed a methionine
(Met) to valine (Val) amino acid substitution near the protein's
amino terminus. It should be noted that the amino acid sequence of
aldolase A is extremely conserved throughout numerous genus and
species. The amino acid sequence of the region possessing the amino
acid substitution of the aldolase A variant in SHR-SP, SHR, WKY,
mouse, human, seal, dog and rabbit is shown in FIG. 4 [SEQ ID
NO:5].
[0172] The prepronatriodilatin gene, which encodes the atrial
natriuretic peptide (ANP) was found to contain an altered glycine
(Gly) amino acid residue in the SHR which is conserved across
species, and mapped to a region which has been implicated in the
etiology of both stroke and hypertension. See e.g., Rubattu, et
al., 1996. Nat. Genet. 13(4):429-434. The amino acid sequence of
the region possessing the amino acid substitution of the
prepronatriodilatin variant in SHR-SP, SHR, WKY, human, pig and
horse is shown in FIG. 4 [SEQ ID NO:6].
[0173] Similarly, the amino acid sequence of the region possessing
the amino acid substitution of the .alpha.-cardiac myosin variant
in SHR-SP, SHR, WKY, mouse and human is shown in FIG. 4 [SEQ ID
NO:7] and the amino acid sequence of the region possessing the
amino acid substitution of the .alpha.-tubulin variant in SHR, WKY,
mouse, chicken, human and fluke is shown in FIG. 4 [SEQ ID
NO:8].
[0174] (C) Physiological and Biochemical Significance of the
Experimental Results
[0175] Presented herein is the first comprehensive organ survey of
differential gene expression in a genetic disease animal model,
coupled with a comprehensive mapping and mutation-detection
strategy to facilitate the identification of the specific gene(s)
involved directly and/or indirectly in complex disease phenotypes
The present invention, for the first time, implicates many new
genes and their respective pathways in the mechanisms of these
complex diseases, and highlights those genes whose variants may
directly cause the disease phenotypes. Using this strategy to
define the overlap between differential gene expression in a
disease versus normal model and the map location(s) of the disease
trait(s) is a powerful way of rapidly identifying genes that cause
disease.
[0176] A total of sixty known rat genes (or rat homologs of genes
known in other species) were found to be differentially-expressed
in the analysis of five tissues. Six of these genes mapped to
within chromosomal regions which have been implicated in various
disease traits, and a total of seven genes were shown to possess
amino acid substitutions which may influence these aforementioned
disease traits.
[0177] (i) Hypertension- and Obesity-Related Genes
[0178] The differential-expression and amino acid substitution of
the L638Q variant of the low affinity sodium-dependent glucose
transporter, SGLT2, suggests that this transporter may be directly
involved in the SHR phenotype. Activating mutations affecting
selectivity, stoichiometry or activation state may increase sodium
reabsorption, leading to plasma volume expansion and concomitant
hypertension. Similarly, increased glucose reabsorption through
this transporter enzyme, might lead to increased plasma glucose.
Accordingly, antagonists/blockers of sodium-glucose transporter
activity are currently being developed for utilization in
anti-diabetic applications. See e.g., Tsujihara, al, 1996. Chem.
Pharm. Bull. (Tokyo) 44(6):1174-1180.
[0179] The identification of kynurenine aminotransferase variant
E27G, may help explain the previous finding that the activity of
this enzyme in the SHR brain is greatly reduced relative to
activity in the WKY brain. See e.g., Kapoor, et al., 1994. Clin.
Exp. Pharmacol. Physiol. 21(11):891-896. Kynurenine
aminotransferase catalyzes the conversion of kynurenine to
kynurenic acid, an excitatory amino acid receptor antagonist. This
implicates the kynurenine pathway in blood pressure regulation and
suggests the possibility that kynurenic acid may have
anti-hypertensive applications. An associated increase in the
expression of the water channel protein, aquaporin 3, suggests that
increased water reabsorption, leading to plasma volume expansion,
may be linked to anomalies in the kynurenine pathway. While
aquaporin 2 is differentially-expressed in a congestive heart
failure model (see e.g., Nielson, 1997. Proc. Natl. Acad. Sci. USA
94(10):5450-5455), aquaporin 3 is a thirst-responsive channel with
a role in osmotically-driven water absorption across the collecting
duct epithelium, which has no previous association with a disease
state (see e.g., Ecelbarger, et al., 1995. Am. J Physiol.
269(5):F663-672.
[0180] Increased expression of 21-hydroxylase, the final enzyme in
aldosterone synthesis which is mutated in patients with adrenal
hyperplasia (see e.g., Jospe, et al., 1987. Biochem. Biophys. Res.
Commun. 142(3):798-804), putatively suggests a
transcriptionally-regulate- d mechanism of increased aldosterone
production. Interestingly, 21-hydroxylase alteration has also
recently been associated with obesity/dyslipidemia (see e.g.,
Cornean, et al., 1998. Arch. Dis. Child. 78(3):261-263), which may
help to link these two features (i.e., obesity and hypertension) of
human Metabolic Syndrome X.
[0181] (ii) Insulin Resistance-Related Genes
[0182] The tissue-wide decrease in the differential-expression of
.alpha.-SNAP suggests an additional mechanism of insulin resistance
in the SHR. Due to the fact that .alpha.-SNAP plays a key role in
the translocation of the insulin-responsive glucose transporter
GLUT4 to the cell surface (see e.g., Mastick, et al., 1997.
Endocrinology 138(6):2391-2397), a significant decrease in the
levels of the .alpha.-SNAP protein may lead to insulin resistance
through a defective protein-trafficking or translocation
mechanism.
[0183] The finding of numerous amino acid substitutions and
coincident map location of the rat homologs of the fatty acid
transport protein (FAT)/CD36, platelet glycoprotein IV and the
class B scavenger receptor to a major locus of impaired fatty acid
secretion, strongly suggests that this multi-functional transporter
may be directly involved in glucose uptake and fatty acid
secretion. Previously, this chromosome 4 locus was also
demonstrated to be the only genetic determinant of impaired
isoproterenol-mediated, non-esterified fatty acid secretion in the
SHR (see e.g., Aitman, et al., 1997. Nat. Genet. 16(2):856-862). In
addition, FAT/CD36 has been shown to bind and internalize oxidized,
low density lipoprotein, bind thrombospondin and collagen 1, and
when mutated in humans leads to a defect in platelet-collagen
adhesion (see e.g., Frieda, et al., 1995. J Biol. Chem.
270(7):2981-2986; Rigotti, et al., 1995. J Biol. Chem.
270(27):16221-16224; Ibrahimi, et al., 1996. Proc. Natl. Acad. Sci.
U.S.A 93(7):2646-2651). The association between the SHR CD36 allele
and insulin resistance suggests further functionality of this
receptor, and the finding of linkage of NIDDM to a potentially
syntenic region of human 7q suggests that CD36 may play a causal
role in insulin resistance in humans. Coincidentally, the SHR-SP
version of CD36 differs from the SHR version and maps near the
chromosome 4 stroke protective locus (see e.g., Rubattu, et al.,
1996. Nat. Genet. 13(4):429-434), suggesting the possibility of its
involvement in the stroke phenotype as well.
[0184] Increased expression of the cardiac isoform of fatty acid
binding protein (FABP) in the SHR adipocytes suggests a role for
this member the FABP family in insulin resistance. Variants in the
intestinal and adipocyte FABPs have been linked to obesity,
dyslipidemia and insulin resistance (see e.g., Hotamisligil, et
al., 1996. Science 274:1377-1379; Baier, et al., 1995. J Clin.
Invest. 95(3):1281-1287). While the cDNA sequence of this gene was
identical in the SHR and WKY rats, the differing levels of mRNA
detected may affect protein levels contributing to the
phenotype.
[0185] (iii) Stroke Predisposition-Related Genes
[0186] The discovery of the differential expression of a novel
variant of prepronatriodilatin, which was mapped to a region
associated with an increased stroke latency period (see e.g.,
Rubattu, et al, 1996. Nat. Genet. 13(4):429-434) and with increased
infarct volume (see e.g, Jeffs, et al., 1997. Nat. Genet.
16(4):364-367), suggests a novel, blood pressure-independent role
in stroke predisposition for the peptide hormones encoded by this
gene. The fact that the products of this gene circulate in the
bloodstream and may serve a protective role in stroke, serves to
facilitate the development of pharmaceutical compounds for
utilization in therapeutic intervention of stroke.
[0187] The finding of differential expression and amino acid
substitution in the extremely conserved aldolase A gene, as well as
its coincident location near a region of stroke predisposition,
suggests that this enzyme (whose activity has long been known to be
elevated in the sera of patients with cerebrovascular insult) may
play a direct role in the onset of the cerebrovascular event. The
amino acid residue which is substituted in the SHR-SP has been
demonstrated to be completely conserved in every organisms from
which it has been sequenced. including at least 12 mammals. While
aldolase A is a well-characterized, glucose-induced glycolytic
enzyme, it has been shown to bind .alpha.-tubulin, whose mRNA
accumulates after transient ischemic brain insult (see e.g., Volker
& Knull, 1997. Arch Biochem. Biophys. 338(2):237-243).
Breakdown of the cytoskeleton has been proposed to be a central
event in the evolution of ischemic brain damage. In addition, an
amino acid substitution (S340T) was found in .alpha.-tubulin in the
SHR and SHR-SP, relative to the amino acid sequence of the protein
in both WKY control rodents and literature-reported sequences. This
evidence suggests the possible involvement of aldolase A and
.alpha.-tubulin in predisposition to vascular injury.
[0188] In addition, the demonstration of increased expression of
cystatin C in the SHR-SP brain may play a significant role in the
stroke phenotype, as mutations of this cysteine protease inhibitor,
in humans, has been shown to cause cerebral hemmhorage (see e.g.,
Ghiso, et al., 1986. Proc. Natl. Acad. Sci. USA 83(9):2974-2978).
While the human mutation does not alter the ability of cystatin C
to inhibit cathepsin B, it does, nonetheless, permit dimerization
and aggregation which leads to its subsequent deposition as
amyloid. In the SHR-SP model, increased cystatin C synthesis may
have an equally physiologically-deleterious effect on the
cerebrovasculature by an accumulation of protein leading to
aggregation.
[0189] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and accompanying figures. Such modifications
are intended to fall within the scope of the appended claims.
[0190] Various publication are cited herein, the disclosures of
which are incorporated by reference in their entireties.
1TABLE 1 Differential Gene Expression Summary Between SHRSP, SHR
and WKY Rats Genotype Comparison Tissue # Fragments # Diff.
Expressed % Diff. Expressed SHR vs WKY Heart 11,600 108 0.9% SHR vs
WKY Brain 36,500 384 1.1% SHR vs WKY Kidney 15,200 162 1.1% SHR vs
WKY Adipose 28,300 207 0.7% SHR vs WKY Liver 27,100 382 1.4% SHR-SP
vs SHR Heart 14,500 44 0.3% SHR-SP vs SHR Brain 35,700 150 0.4%
SHR-SP vs SHR Kidney 7,500 39 0.5%
[0191]
2TABLE 2 Differential Gene Expression Between SHR and WKY Rats
.DELTA. in .DELTA. in .DELTA. in .DELTA. in amino Gene heart
.DELTA. in brain .DELTA. in kidney adipose liver Map acid y00979
Beta beta enolase +4.3 -6.3 -29.4 -4.1 -7.9 10 m15868
Prepronatriodilatin (ANF) +2.5 0 0 0 0 5* G99S x15939 Beta cardiac
myosin heavy +8.5 0 0 0 0 11 chain 106433 c-HA-ras proto-oncogene
+8.8 0 0 0 0 x90375 RT1.A1(N) alpha chain. +10.7 0 -6.7 0 0 20*
Collagen11A2-like 0 +14.6 0 0 0 Mitogen activated protein (MAP) 0
+12.9 0 0 0 kinase-like Lysophospholipase-like 0 -3 -3.5 -7.3 0
m95763 GABA transporter GAT-3 0 -2.4 0 0 0 X Alpha enolase-like 0
-20.6 0 0 0 5 x60352 Alpha B-crystallin 0 +2.6 -2.6 +2.8 0 20
m54919 S100 protein beta subunit 0 -22.2 0 -20.0 0 7 m38179
3-beta-hydroxysteroid 0 0 +85.2 0 +2.6 dehydrogenase/delta-5-delta-
-4 isomerase x67156 (S)-2-hydroxy acid oxidase 0 0 -53.5 0 0 3
x89968 Alpha-soluble NSF attachment -3.2 -3.8 -23.6 -17.3 -4.7
protein s74029 Kynurenine aminotransferase 0 0 +22.4 0 0 3* E27G
137333 Flucose-6-phosphatase 0 0 -20.5 0 0 (G6Pase) Saccharopine
dehydrogenase-like 0 -2.4 +15.9 0 0 Organic anion transport protein
NKT- 0 0 -14.7 0 0 like Gluthathione-S-transferase-like 0 0 +13.6 0
0 Hemoglobinase-like (cysteine protease) 0 0 -7 0 0 af003944
Ovalbumin upstream 0 0 -6.9 0 0 promotoer beta nuclear receptor
u29881 Na-dependent glucose 0 0 -6.7 0 0 1* L638Q transporter SGLT2
Dermatopontin-like 0 0 +6.2 +20.7 0 d17695 Water channel aquaporin
3 0 0 +5.4 0 0 (AQP3) 20 alpha-hydroxysteroid 0 0 +4.7 0 0
dehydrogenase-like Nicotinic receptor alpha 7 subunit-like 0 +5.5
+4.7 u56853 21-hydroxylase 0 0 +4.4 0 0 20 NAD(+)-dependent 15- 0 0
-4.1 hydroxyprostaglandin dehydrogenase- like 119658 Fatty acid
transporter -14 0 0 -20.5 0 4* See. FIG. 3 (FAT/CD36) z24721
Superoxide dismutase 0 0 0 +6.2 0 11 m64755 Cysteine sulfunic acid
0 0 0 +3.2 0 decarboxylase x71127 Complenent protein C1q beta 0 0 0
+2.4 0 5 chain. j02773 Low molecular weight fatty 0 0 0 +2.4 0 acid
binding protein z50144 Kynurenine/alpha- 0 0 0 -2.0 0 aminoadipate
u51017 Kallistatin 0 0 0 0 +23.9 6 d00752 Contraspin-like protease
0 0 0 0 -17.4 6 inhibitor related protein Nuclear hormone receptor
MB67-like +17.2 m18335 Cytochrome P450 0 0 0 0 -9.4 m22360 Plasma
proteinase inhibitor 0 0 0 0 -7.3 alpha-1-inhibitor III group 3
m27440 Apolipoprotein B 0 0 0 0 -4.8 6 m95591 Hepatic squalene
synthetase 0 0 0 0 -3.5 x52477 Pre-pro-complment C3 0 0 0 -2.2 -2.7
x05300 Ribophorin 1 0 0 0 0 -2.5 4 m17714 Insulin-like growth
factor-I 0 0 0 0 -1.9 7 d90055 Peroxisomal 3-ketoacyl-CoA 0 0 0 0
+1.7 8 thiolase v01227 Alpha-tubulin 0 +15.6 0 0 0 5 S340T s76489
Estrogen sulfotransferase 0 0 0 -1.8 -2.6 1
[0192]
3TABLE 3 Differential Gene Expression Between SHSRP and SHR Rats
Gene .DELTA. in heart .DELTA. in brain .DELTA. in kidney Map
.DELTA. in amino acid m15868 Prepronatriodilatin (ANF) +2.5 -1.5 0
5* G99S 103294 Lipoprotein lipase +3.3 0 0 16 Alpha 2 actinin-like
-3.7 0 0 Batroxostatin-like 0 +27.7 0 Sodium bicarbonate
cotransporter (HNBC1)-like 0 +13.6 0 x16957 Cystatin C 0 +3.9 0 3
y00979 Beta beta enolase +4.7 +3.7 0 10 m12919 Aldolase A -51.7
-7.7 -9.7 1* M22V elF-4AII-like ND -9.6 ND SON3-like ND -4.6 ND
x15938 Alpha cardiac myosin heavy chain +9.1 0 0 15/16 T10221
Immunoglobulin light chain-like +5.6 0 0 C. elegenas YK51G8.3-like
+5.2 0 0 Sodium bicarbonate cotransporter-like ND +13.6 ND
* * * * *