U.S. patent application number 10/052817 was filed with the patent office on 2002-08-22 for alpha-2-macroglobulin therapies and drug screening methods for alzheimer's disease.
This patent application is currently assigned to The General Hospital Corporation. Invention is credited to Kovacs, Dora, Saunders, Aleister J., Tanzi, Rudolph E..
Application Number | 20020114792 10/052817 |
Document ID | / |
Family ID | 22911392 |
Filed Date | 2002-08-22 |
United States Patent
Application |
20020114792 |
Kind Code |
A1 |
Tanzi, Rudolph E. ; et
al. |
August 22, 2002 |
Alpha-2-macroglobulin therapies and drug screening methods for
Alzheimer's disease
Abstract
The disclosed invention relates to the finding that the A2M-2
deletion mutation, which is a predisposing factor for Alzheimer's
Disease, leads to the production of altered .alpha..sub.2M RNA
transcripts and proteins. Based on this finding, the invention
provides for new therapeutic agents for AD, including molecules
having A.beta. and low density lipoprotein receptor-related protein
(LRP) binding domains, peptides, nucleic acid molecules, antisense
oligonucleotides, and viral vectors for gene therapy. In addition,
the invention relates to pharmaceutical compositions containing
these therapeutic agents, methods of using these therapeutic agents
to combat Alzheimer's Disease, and methods of screening for
therapeutic agents that can combat Alzheimer's Disease.
Inventors: |
Tanzi, Rudolph E.; (Hull,
MA) ; Kovacs, Dora; (Boston, MA) ; Saunders,
Aleister J.; (Cambridge, MA) |
Correspondence
Address: |
STERNE, KESSLER, GOLDSTEIN & FOX PLLC
1100 NEW YORK AVENUE, N.W., SUITE 600
WASHINGTON
DC
20005-3934
US
|
Assignee: |
The General Hospital
Corporation
|
Family ID: |
22911392 |
Appl. No.: |
10/052817 |
Filed: |
January 23, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10052817 |
Jan 23, 2002 |
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09241606 |
Feb 2, 1999 |
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09241606 |
Feb 2, 1999 |
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09148503 |
Sep 4, 1998 |
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60057655 |
Sep 5, 1997 |
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60093297 |
Jul 17, 1998 |
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Current U.S.
Class: |
424/93.21 ;
435/226; 435/7.21; 435/7.9; 514/44R |
Current CPC
Class: |
C12Q 1/6883 20130101;
C12Q 2600/136 20130101; A61K 48/00 20130101; A61P 25/28 20180101;
G01N 33/6896 20130101; G01N 2333/4709 20130101; G01N 2800/2821
20130101; C12Q 2600/156 20130101; C07K 14/8107 20130101; C12N
2799/021 20130101; A61K 38/00 20130101 |
Class at
Publication: |
424/93.21 ;
514/44; 435/226; 435/7.9; 435/7.21 |
International
Class: |
A61K 048/00; C12N
009/64; G01N 033/567; G01N 033/53; G01N 033/542 |
Claims
What is claimed is:
1. A therapeutic agent for combating Alzheimer's disease, wherein
said agent can replace or supplement .alpha..sub.2M function, or
suppress expression of A2M-2.
2. An anti-LRP-A.beta. molecule comprising, an A.beta. binding
domain, and an LRP binding domain, or a pharmaceutically acceptable
salt thereof.
3. The anti-LRP-A.beta. molecule of claim 2, wherein said molecule
is a peptide, or a pharmaceutically acceptable salt thereof.
4. An anti-LRP-A.beta. peptide comprising: (a) an A.beta. binding
domain comprising 10-50 contiguous residues of SEQ ID NO:6; and (b)
an LRP binding domain comprising 10-50 contiguous residues of SEQ
ID NO:8, wherein said 10-50 contiguous residues of SEQ ID NO:8
encompass residues 1366-1392, or a pharmaceutically acceptable salt
thereof.
5. An anti-LRP-A.beta. peptide comprising: (a) an A.beta. binding
domain having an amino acid sequence selected from the group
consisting of SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:16, SEQ ID
NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:26;
and (b) an LRP binding domain having the amino acid sequence of SEQ
ID NO:10, or a pharmaceutically acceptable salt thereof.
6. An anti-LRP-A.beta. peptide comprising: (a) an A.beta. binding
domain having an amino acid sequence selected from the group
consisting of SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:18, SEQ ID
NO:20, SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:26; and (b) an LRP
binding domain comprising 10-50 contiguous residues of SEQ ID NO:8,
or a pharmaceutically acceptable salt thereof.
7. The anti-LRP-A.beta. peptide of claim 4, 5 or 6, wherein said
A.beta. binding domain is connected to said LRP binding domain by a
peptide bond.
8. The anti-LRP-A.beta. peptide of claim 4, 5 or 6, wherein said
A.beta. binding domain is connected to said LRP binding domain by a
linker.
9. The anti-LRP-A.beta. peptide of claim 8, wherein said linker is
selected from the group consisting of a peptide, or polyethylene
glycol.
10. The anti-LRP-A.beta. peptide of claim 9, wherein said peptide
comprises 1-20 glycine residues.
11. A nucleic acid comprising a polynucleotide encoding the
anti-LRP-A.beta. peptide of claim 4, 5, 6, 7, 8, 9 or 10.
12. An anti-LRP-A.beta. peptide comprising a polypeptide having the
sequence of SEQ ID NO:14, or a pharmaceutically acceptable salt
thereof.
13. An anti-LRP-A.beta. peptide comprising: (a) an A.beta. binding
domain having an amino acid sequence selected from the group
consisting of SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:16, SEQ ID
NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:26;
(b) an LRP binding domain having the amino acid sequence of SEQ ID
NO:10; and (c) a linker connecting said A.beta. binding domain to
said LRP binding domain.
14. A nucleic acid molecule comprising a nucleotide encoding the
anti-LRP-A.beta. peptide of claim 12 or 13.
15. A nucleic acid molecule encoding an anti-LRP-A.beta. peptide
comprising: (a) a region encoding an A.beta. binding domain,
comprising 30-150 contiguous nucleotides of SEQ ID NO:5; and (b) a
region encoding an LRP binding domain comprising 30-150 contiguous
nucleotides of SEQ ID NO:7.
16. A nucleic acid molecule encoding an anti-LRP-A.beta. peptide
comprising: (a) a region encoding an A.beta. binding domain having
a nucleotide sequence selected from the group consisting of SEQ ID
NO:5, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ
ID NO:21, SEQ ID NO:23, and SEQ ID NO:25; and (b) a region encoding
an LRP binding domain having the nucleotide sequence of SEQ ID
NO:9.
17. A nucleic acid molecule encoding an anti-LRP-A.beta. peptide
comprising: (a) a region encoding an A.beta. binding domain having
a nucleotide sequence selected from the group consisting of SEQ ID
NO:11, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ
ID NO:23, and SEQ ID NO:25; and (b) a region encoding an LRP
binding domain comprising 30-150 contiguous nucleotides of SEQ ID
NO:7.
18. The nucleic acid molecule of claim 15, 16, or 17, wherein said
region encoding said A.beta. binding domain is connected to said
region encoding said LRP binding domain by a phosphodiester
bond.
19. The nucleic acid molecule of claim 15, 16 or 17, wherein said
region encoding said A.beta. binding domain is connected to said
region encoding said LRP binding domain by a nucleotide encoding
1-20 glycine residues.
20. A nucleic acid molecule comprising, a polynucleotide having at
least 95% homology to the nucleic acid molecule of claim 15, 16,
17, 18 or 19.
21. A nucleic acid molecule comprising, a first polynucleotide that
hybridizes to a second polynucleotide, wherein said second
polynucleotide is complementary to the nucleic acid molecule of
claim 15, 16, 17, 18 or 19.
22. The nucleic acid molecule of claim 21, wherein said first
polynucleotide hybridizes to said second polynucleotide under
conditions comprising: (a) incubating overnight at 42.degree. C. in
a solution consisting of 50% formamide, 5.times. SSC, 50 mM sodium
phosphate (pH 7.6), 5.times. Denhardt's solution, 10% dextran
sulfate, and a 20 .mu.g/ml denatured, sheared salmon sperm DNA; and
(b) washing at 65.degree. C. in a solution consisting of
0.14.times. SSC.
23. A nucleic acid molecule comprising a polynucleotide having the
nucleotide sequence of SEQ ID NO:13.
24. A nucleic acid molecule comprising a polynucleotide having at
least 95% identity to the nucleotide sequence of SEQ ID NO:13.
25. A nucleic acid molecule comprising a first polynucleotide that
hybridizes to a second polynucleotide, wherein said second
polynucleotide is complementary to the nucleotide sequence of SEQ
ID NO:13.
26. The nucleic acid molecule of claim 25, wherein said first
polynucleotide hybridizes to said second polynucleotide under
conditions comprising: (a) incubating overnight at 42.degree. C. in
a solution consisting of 50% formamide, 5.times. SSC, 50 mM sodium
phosphate (pH 7.6), 5.times. Denhardt's solution, 10% dextran
sulfate, and a 20 .mu.g/ml denatured, sheared salmon sperm DNA; and
(b) washing at 65.degree. C. in a solution consisting of 0.1.times.
SSC.
27. A pharmaceutical composition comprising an anti-LRP-A.beta.
molecule, and one or more pharmaceutically acceptable carriers.
28. A pharmaceutical composition comprising the anti-LRP-A.beta.
peptide of claim 4, 5, 6, 7, 8, 9, 10 or 13, or a pharmaceutically
acceptable salt thereof, and one or more pharmaceutically
acceptable carriers.
29. A pharmaceutical composition comprising an anti-LRP-A.beta.
peptide having an amino acid sequence selected from the group
consisting of SEQ ID NO:4 or SEQ ID NO:14, or a pharmaceutically
acceptable salt thereof, and one or more pharmaceutically
acceptable carriers.
30. A method of combating Alzheimer's Disease in a subject
comprising administering an anti-LRP-A.beta. molecule.
31. The method of claim 30, wherein said anti-LRP-A.beta. molecule
is a peptide.
32. A method of combating Alzheimer's Disease in a subject
comprising administering the anti-LRP-A.beta. peptide of claim 4,
5, 6, 7, 8, 9, 10 or 13, or a pharmaceutically acceptable salt
thereof.
33. A method of combating Alzheimer's Disease in a subject
comprising administering an anti-LRP-A.beta. peptide having an
amino acid sequence selected from the group consisting of SEQ ID
NO:4 and SEQ ID NO:14, or a pharmaceutically acceptable salt
thereof.
34. An A2M-2 antisense oligonucleotide comprising a nucleotide
designed to target A2M-2 RNA.
35. The A2M-2 antisense oligonucleotide of claim 34, wherein said
RNA is hnRNA.
36. The A2M-2 antisense oligonucleotide of claim 34, wherein said
RNA is mRNA.
37. An A2M-2 antisense oligonucleotide comprising a nucleotide
having the sequence of SEQ ID NO:27.
38. An A2M-2 antisense oligonucleotide comprising a nucleotide
having the sequence of the last 15-30 contiguous nucleotides of SEQ
ID NO:27.
39. An A2M-2 antisense oligonucleotide comprising nucleotides 36-50
of SEQ ID NO:27.
40. An A2M-2 antisense oligonucleotide comprising nucleotides 20-50
of SEQ ID NO:27.
41. A pharmaceutical composition comprising the A2M-2 antisense
oligonucleotide of claim 34, 35, 36, 37, 38, 39 or 40, and one or
more pharmaceutically acceptable carriers.
42. A method of combating Alzheimer's Disease in a subject
comprising administering the A2M-2 antisense oligonucleotide of
claim 34, 35, 36, 37, 38, 39 or 40.
43. A vector for gene therapy of Alzheimer's Disease, comprising a
viral vector, wherein said viral vector carries a transgene
selected from the group consisting of a gene encoding
.alpha..sub.2M, and a gene encoding an anti-LRP-A.beta.
peptide.
44. The viral vector of claim 43, wherein said transgene is a gene
encoding .alpha..sub.2M.
45. The viral vector of claim 44, wherein said transgene has the
nucleotide sequence of nucleotides 44-4465 of SEQ ID NO:1.
46. The viral vector of claim 43, wherein said transgene is a gene
encoding an anti-LRP-A.beta. peptide.
47. The viral vector of claim 43, where in said transgene encodes
the anti-LRP-AB peptide of claim 4, 5, 6, 7, 8, 9, 10, 12 or
13.
48. The viral vector of claim 43, 44, 45, 46 or 47, wherein said
viral vector is an adeno-associated virus.
49. A pharmaceutical composition comprising the viral vector of
claim 43, 44, 45, 46, 47 or 48, and one or more pharmaceutically
acceptable carriers.
50. A method of combating Alzheimer's Disease in a subject by
administering the viral vector of claim 43, 44, 45, 46, 47 or
48.
51. A method of screening for a therapeutic agent for Alzheimer's
Disease, wherein said therapeutic agent is the agent of claim
1.
52. A method of screening for a therapeutic agent for Alzheimer's
Disease comprising the steps of: (a) incubating cells in the
presence of a test agent, wherein said cells are heterozygous or
homozygous for the A2M-2 allele, and wherein said cells express
A2M-2; and (b) determining whether the ratio of normal to aberrant
A2M mRNA has increased relative to the ratio of normal to aberrant
A2M mRNA found in cells untreated with test agent.
53. The method of claim 52, wherein said cells are glioma
cells.
54. The method of claim 52, wherein said cells are hepatoma
cells.
55. The method of claim 52, wherein said cells are heterozygous for
the A2M-2 allele.
56. The method of claim 52, wherein said cells are homozygous for
the A2M-2 allele.
57. The method of claim 52 wherein said step (b) comprises S1
nuclease analysis using a probe complementary to SEQ ID NO:1,
wherein said probe encompasses nucleotides 2057-2284 of SEQ ID
NO:1.
58. The method of claim 57, wherein said probe is 300 bp long.
59. The method of claim 52, wherein said step (b) comprises S1
nuclease analysis using a probe complementary to nucleotides
2024-2323 of SEQ ID NO:1.
60. The method of claim 52, wherein said step (b) comprises RT PCR
analysis.
61. The method of claim 60, wherein said step (b) comprises RT PCR
analysis using primers designed to amplify a region of A2M
encompassing exons 17-18.
62. The method of claim 61, wherein said region of A2M encompassing
exons 17-18 is 300 bp long.
63. The method of claim 61, wherein said primers are designed to
amplify nucleotides 2052-2289 of SEQ ID NO:1.
64. The method of claim 61, wherein said primers consist of a first
primer having a nucleotide sequence complementary to nucleotides
2024-2038 of SEQ ID NO:1, and a second primer having the nucleotide
sequence of nucleotides 2309-2323 of SEQ ID NO:1.
65. A method of screening for a therapeutic agent for Alzheimer's
Disease comprising the steps of: (a) incubating .alpha..sub.2M with
a test agent; and (b) determining whether said .alpha..sub.2M of
step (b) has undergone a conformational change; wherein said steps
are performed in sequential order.
66. The method of claim 65, wherein said step (b) comprises
performing an .alpha..sub.2M electrophoretic mobility assay.
67. A method of screening for a therapeutic agent for Alzheimer's
Disease comprising the steps of: (a) incubating .alpha..sub.2M with
a test agent; and (b) determining whether said .alpha..sub.2M of
step (b) can bind to LRP; wherein said steps are performed in
sequential order.
68. The method of claim 65, 66 or 67, wherein said .alpha..sub.2M
is tetrameric.
69. The method of claim 67, wherein said step (b) comprises
performing an ELISA.
70. The method of claim 69, wherein said ELISA comprises the steps
of: (a) incubating LRP in a well coated with anti-LRP IgG; (b)
incubating said well with said .alpha..sub.2M; (c) incubating said
well with anti-.alpha..sub.2M IgG conjugated to an enzyme; and (d)
incubating said well with a substrate for said enzyme; wherein said
steps are performed in sequential order.
71. The method of claim 69, wherein said ELISA comprises the steps
of: (a) incubating a well coated with LRP with said .alpha..sub.2M;
(b) incubating said well with anti-.alpha..sub.2M IgG conjugated to
an enzyme; and (c) incubating said well with the substrate for said
enzyme; wherein said steps are performed in sequential order.
72. The method of claim 69, wherein said ELISA comprises the steps
of: (a) incubating said .alpha..sub.2M in a well coated with an
anti-.alpha..sub.2M IgG specific for activated .alpha..sub.2M; (b)
incubating said well with said .alpha..sub.2M; (c) incubating said
well with anti-.alpha..sub.2M IgG conjugated to an enzyme; and (d)
incubating said well with a substrate for said enzyme; wherein said
steps are performed in sequential order.
73. The method of claim 67, wherein said step (b) comprises
immunoblotting.
74. The method of claim 73, wherein anti-LRP IgG and
anti-.alpha..sub.2M IgG are used to perform said
immunoblotting.
75. The method of claim 67, wherein said step (b) comprises
determining the ability of said .alpha..sub.2M to undergo LRP
mediated endocytosis.
76. The method of claim 67, wherein said step (b) comprises
determining the ability of said .alpha..sub.2M to undergo LRP
mediated degradation.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of application
Ser. No. 09/148,503, filed on Sep. 4,1998, which claims priority to
U.S. Provisional Application No. 60/057,655, filed on Sep. 5, 1997,
and U.S. Provisional Application No. 60/093,297, filed on Jul. 17,
1998, all of which are herein incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates to the field of medical genetics.
More specifically, the invention provides for therapeutic agents
for Alzheimer's Disease and methods of screening for therapeutic
agents for Alzheimer's disease that are based on affecting
alpha-2-macroglobulin function and expression.
[0004] 2. Related Art
[0005] Alzheimer's disease (AD) is a devastating neurodegenerative
disorder that affects more than 4 million people per year in the US
(Dobeli, H., Nat. Biotech. 15: 223-24 (1997)). It is the major form
of dementia occurring in mid to late life: approximately 10% of
individuals over 65 years of age, and approximately 40% of
individuals over 80 years of age, are symptomatic of AD (Price, D.
L., and Sisodia, S. S., Ann. Rev. Neurosci. 21:479-505 (1998)).
[0006] The first recognized clinical symptom of AD is usually the
loss of short-term memory (Schellenberg, G. D., Proc. Natl. Acad.
Sci. USA 92:8552-559 (1995)). Other common symptoms include
abnormal judgement and behavior, and difficulty with language,
orientation, problem solving, calculations, and visuospacial
perception (Price, D. L., and Sisodia, S. S., Ann. Rev. Neurosci.
21:479-505 (1998); Schellenberg, G. D., Proc. Natl. Acad. Sci. USA
92:8552-559 (1995)). These symptoms often worsen until cognitive
function is almost entirely lost, and the patient cannot function
independently (Schellenberg, G. D., Proc. Natl. Acad. Sci. USA
92:8552-559 (1995); Price, D. L., and Sisodia, S. S., Ann. Rev.
Neurosci. 21:479-505 (1998)). By late stages of the disease
patients typically lack verbal ability, cannot recognize people,
and are incontinent and bed-ridden (Price, D. L., and Sisodia, S.
S., Ann. Rev. Neurosci. 21:479-505 (1998); Sloane, P. D., Am.
Family Phys. 58: 1577-86 (1998)).
[0007] Known risk factors for AD include age, genetic
predisposition, abnormal protein (.beta.-amyloid) deposition in the
brain, and certain environmental factors such as head injury,
hypothyroidism, and a history of depression. The majority of AD
patients do not exhibit symptoms until their seventies (Price, D.
L., and Sisodia, S. S., Ann. Rev. Neurosci. 21:479-505 (1998)).
However, individuals who have inherited particular genetic defects
often exhibit symptoms in midlife (Price, D. L., and Sisodia, S.
S., Ann. Rev. Neurosci. 21:479-505 (1998)). This latter type of AD,
called early-onset familial AD (FAD), accounts for 5-10% of AD
cases, and has been linked to defects in three different genes,
APP, PSEN1, PSEN2 (Blacker, D. and Tanzi, R. E., Archives of
Neurology 55:294-296 (1998)). Mutations in these genes lead to
increased production of the amyloidogenic .beta.-amyloid peptide
(A.beta.) (Citron, M., et al., Nature Medicine 3:67-72 (1997);
Suzuki, N., et al., Science 264:1336-1340 (1994)).
[0008] The most prevalent form of AD, called late-onset AD (LOAD),
accounts for approximately 90% of AD cases, and has been
genetically linked to APOE and LRP (Kang, D. E., et al., Neurology
49:56-61 (1997); Kounnas, M. Z., et al., Cell 82:331-340 (1995)).
Recently, another gene, the alpha-2-macroglobulin gene (A2M), was
found to be linked to LOAD (Blacker, D., et al., Nature Genetics
19:357-360 (1998)). Carriers of a particular mutation in A2M were
discovered to be at increased risk of AD. This mutation is a
pentanucleotide deletion at the 5' splice site of the second exon
encoding the bait region of alpha-2-macroglobulin (.alpha..sub.2M),
and is referred to as the A2M-2 genotype. The A2M-2 genotype is
present in 30% of the population (Blacker, D., et al., Nature
Genetics 19:357-360 (1998)). The A2M-2 pentanucleotide deletion is
a predisposing factor for AD.
[0009] Presently, there is no cure for AD on the horizon and its
incidence is increasing as the population ages (Price, D. L., and
Sisodia, S. S., Ann. Rev. Neurosci. 21:479-505 (1998)). Due to the
lateness in life of the onset of AD symptoms, the ability to delay
onset by as little as 5 years could decrease the number of AD
patients by as much as 50% (Marx, J., Science 273:50-53 (1996)).
With the large number of people already affected, and projected to
be affected by AD, a drug that could merely delay the onset of AD
would be very valuable.
[0010] Therapeutic agents based on predisposing factors of AD might
be able to prevent, delay or slow progression of the disease.
However, presently, available treatments are primarily aimed at
treatment of the symptoms of the disease (Enz, A., "Classes of
drugs," in: Pharmacotherapy of Alzheimer's Disease, Gauthier, S.,
ed., Martin Dunitz, publ., Malden, M A (1998)). These AD drugs
offer only modest success, and at most, merely slow the progression
of the disease (Delagarza, V. W., Am. Family Phys.
58:1175-1182(1998); Enz, A., "Classes of drugs," in:
Pharmacotherapy of Alzheimer's Disease, Gauthier, S., ed., Martin
Dunitz, publ., Maiden, M A (1998)). Presently approved and
investigational drugs for treating AD can be characterized as those
whose actions enhance neurotransmitter effect, or those believed to
protect neurons (Delagarza, V., Am. Family Phys. 58:1175-1182
(1998)). The most well known drugs in the first category are the
cholinesterase inhibitors, such as tacrine (Cognex.TM.) and
doneprezil (Aricept.TM.), both of which have been approved by the
FDA (Delagarza, V., Am. Family Phys. 58:1175-1182 (1998); Sloan,
P., Am. Family Phys. 58:1577-1586 (1998)). Tacrine and doneprezil
are only modestly effective (Sloan, P., Am. Family Phys.
58:1577-1586 (1998)), and are associated with unpleasant side
effects including nausea and vomiting (Delagarza, V., Am. Family
Phys. 58:1175-1182 (1998)). Several neuro-protective drugs are
under investigation for the treatment of AD, including estrogen,
vitamin E, selegiline and non-steroidal anti-inflammatory drugs
(NSAIDs) (Sloan, P., Am. Family Phys. 58:1577-1586 (1998);
Delagarza, V., Am. Family Phys. 58:1175-1182 (1998)). None of these
drugs have been approved yet for the treatment of AD, and each has
significant drawbacks, including negative side-effects, or
association with increased risk of other diseases. (Sloan, P., Am.
Family Phys. 58:1577-1586 (1998); Delagarza, V., Am. Family Phys.
58:1175-1182 (1998); Enz, A., "Classes of drugs," in:
Pharmacotherapy of Alzheimer's Disease, Gauthier, S., ed., Martin
Dunitz, publ., Malden, M A (1998)).
[0011] Thus, there is a need for new AD therapeutic agents,
especially those based on predisposing factors of AD. In addition,
there is a need for drug screening systems to aid in developing
these therapeutic agents.
SUMMARY OF THE INVENTION
[0012] Based on the finding, described herein, that the A2M-2
deletion leads to the production of altered .alpha..sub.2M RNA
transcripts and proteins, strategies aimed at replacing or
supplementing normal .alpha..sub.2M function and activities, and/or
at suppressing defective .alpha..sub.2M function in the brain may
serve as a means for therapeutically preventing, treating, or even
reversing AD neuropathogenesis. In addition, these strategies may
be useful for treating other pathologies associated with defective
.alpha..sub.2M function. Moreover, methods described herein may be
used to screen for these therapeutic agents. Thus, the invention
provides for new therapeutic agents for AD, for pharmaceutical
compositions containing these therapeutic agents, for methods of
using these therapeutic agents, and for methods of screening for
these therapeutic agents.
[0013] The first aspect of the invention is to provide for a
therapeutic agent for Alzheimer's Disease, where the agent can
replace or supplement .alpha..sub.2M function, or can suppress the
expression of A2M-2. A molecule that can bind to A.beta. and to LRP
may be able to promote clearance of A.beta. through LRP mediated
endocytosis. Thus, one embodiment of the invention is an
anti-LRP-A.beta. molecule having an A.beta. binding domain, and an
LRP binding domain. In a preferred embodiment of the invention,
this molecule is a peptide.
[0014] In one embodiment of the invention the peptide is an
anti-LRP-A.beta. peptide having an A.beta. binding domain composed
of 10-50 contiguous residues of SEQ ID NO:6, and an LRP binding
domain comprising 10-50 contiguous residues of SEQ ID NO:8, which
encompass residues 1366-1392 of SEQ ID NO:8. In another embodiment
of the invention, the anti-LRP-A.beta. peptide has an A.beta.
binding domain with an amino acid sequence selected from the group
consisting of SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:16, SEQ ID
NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:26;
and an LRP binding domain composed of the amino acid sequence of
SEQ ID NO:10. In yet another embodiment of the invention, the
anti-LRP-A.beta. peptide has an A.beta. binding domain with an
amino acid sequence selected from the group consisting of SEQ ID
NO:12, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ
ID NO:24, and SEQ ID NO:26; and an LRP binding domain composed of
10-50 contiguous residues of SEQ ID NO:8.
[0015] The A.beta. binding domain may be connected to the LRP
binding domain of the anti-LRP-A.beta. molecule by a covalent bond,
linker molecule, or linkerless polyethylene glycol. In a preferred
embodiment, the A.beta. and LRP binding domains are connected by a
peptide bond. In another preferred embodiment of the invention, the
A.beta. and LRP binding domains are connected by a peptide composed
of 1-20 glycine residues.
[0016] In another embodiment, the anti-LRP-A.beta. peptide has the
amino acid sequence of SEQ ID NO:14. Alternatively, the
anti-LRP-A.beta. peptide has an A.beta. binding domain having an
amino acid sequence selected from the group consisting of SEQ ID
NO:6, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ
ID NO:22, SEQ ID NO:24, and SEQ ID NO:26; an LRP binding domain
having the amino acid sequence of SEQ ID NO:10; and a linker
connecting the A.beta. binding domain to the LRP binding
domain.
[0017] In addition, the invention provides for pharmaceutically
acceptable salts of the anti-LRP-A.beta. peptide and for nucleic
acid molecules encoding the anti-LRP-A.beta. peptide.
[0018] Another embodiment of the invention relates to a nucleic
acid molecule encoding an anti-LRP-.beta. peptide, where the
A.beta. binding domain is encoded by 30-150 contiguous nucleotides
of SEQ ID NO:5, and the LRP binding domain is encoded by 30-150
contiguous nucleotides of SEQ ID NO:7. In another embodiment of the
invention, the region of the nucleic acid molecule encoding the
A.beta. binding domain has a nucleotide sequence selected from the
group consisting of SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:15, SEQ ID
NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, and SEQ ID NO:25;
and the region encoding the LRP binding domain has the nucleotide
sequence of SEQ ID NO:9. In yet another embodiment of the
invention, the region of the nucleic acid molecule encoding the
A.beta. binding domain has a nucleotide sequence selected from the
group consisting of SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:17, SEQ
ID NO:19, SEQ ID NO:21, SEQ ID NO:23, and SEQ ID NO:25; and the
region encoding the LRP binding domain is encoded by 30-150
contiguous nucleotides of SEQ ID NO:7. In another embodiment of the
invention, the nucleic acid molecule has the nucleotide sequence of
SEQ ID NO:13.
[0019] The region encoding the A.beta. binding domain may be
connected to the region encoding the LRP binding domain of the
nucleic acid molecule by a phosphodiester bond. Alternatively,
these regions may be connected by a nucleotide encoding a linker
peptide. In a preferred embodiment of the invention, the connecting
nucleotide encodes 1-20 glycine residues.
[0020] In addition, the invention relates to nucleic acid molecules
having at least 95% homology to these nucleic acid molecules.
[0021] Another embodiment of the invention relates to a nucleic
acid molecule that is a first polynucleotide that hybridizes to a
second polynucleotide that is complementary to the nucleic acid
molecules described above. In another embodiment of the invention,
the nucleic acid molecule is a first polynucleotide that hybridizes
to a second polynucleotide that is complementary to the nucleotide
sequence of SEQ ID NO:13. In yet another embodiment of the
invention, the hybridizing conditions for the hybridization of the
first and second polynucleotides are as follows: (a) incubate
overnight at 42.degree. C. in a solution consisting of 50%
formamide, 5.times. SSC, 50 mM sodium phosphate (pH 7.6), 5.times.
Denhardt's solution, 10% dextran sulfate, and a 20 .mu.g/ml
denatured, sheared salmon sperm DNA; and (b) wash at 65.degree. C.
in a solution consisting of 0.1.times. SSC.
[0022] A related embodiment of the invention is a pharmaceutical
composition containing an anti-LRP-A.beta. molecule, and one or
more pharmaceutically acceptable carriers. In addition, the
invention provides for a pharmaceutical composition containing an
anti-LRP-A.beta. peptide, or a pharmaceutically acceptable salt
thereof. In a preferred embodiment, the pharmaceutical composition
contains an anti-LRP-A.beta. peptide having an amino acid sequence
selected from the group consisting of SEQ ID NO:4 or SEQ ID NO:14,
or a pharmaceutically acceptable salt thereof, and one or more
pharmaceutically acceptable carriers. The invention also relates to
a method of combating Alzheimer's Disease in a subject by
administering an anti-LRP-A.beta. molecule, or a pharmaceutically
acceptable salt thereof. In a preferred embodiment, the
anti-LRP-A.beta. molecule is a peptide. In another preferred
embodiment, the anti-LRP-A.beta. peptide is a polypeptide having an
amino acid sequence selected from the group consisting of SEQ ID
NO:4 or SEQ ID NO:14, or a pharmaceutically acceptable salt
thereof.
[0023] The invention also relates to an A2M-2 antisense
oligonucleotide designed to target A2M-2 RNA. In one preferred
embodiment of the invention, the A2M-2 antisense oligonucleotide is
designed to target A2M-2 heteronuclear RNA. In another preferred
embodiment, the A2M-2 antisense oligonucleotide is designed to
target A2M-2 mRNA. In one embodiment of the invention, the A2M-2
antisense oligonucleotide designed to target A2M hnRNA has the
nucleotide sequence of SEQ ID NO:27. The A2M-2 antisense
oligonucleotide is preferably from 8-50 nucleotides in length, and
more preferably is 15-30 nucleotides in length, and is most
preferably 15 nucleotides in length. Thus, in another preferred
embodiment of the invention an A2M-2 antisense oligonucleotide
designed to target A2M-2 hnRNA has the nucleotide sequence of the
last 15-30 contiguous nucleotides of SEQ ID NO:27. In another
embodiment of the invention the A2M-2 antisense oligonucleotide
designed to target A2M-2 has the sequence of nucleotides 36-50 of
SEQ ID NO:27 or of nucleotides 20-50 of SEQ ID NO:27. The invention
also relates to a pharmaceutical composition containing an A2M-2
antisense oligonucleotide, and one or more pharmaceutically
acceptable carriers. In addition, the invention relates to a method
of combating Alzheimer's Disease in a subject by administering the
A2M-2 antisense oligonucleotide.
[0024] The invention also provides for a viral vector carrying a
transgene encoding .alpha..sub.2M, or an anti-LRP-A.beta. peptide.
In a preferred embodiment of the invention, the viral vector
carries a gene encoding .alpha..sub.2M. In another preferred
embodiment of the invention, the gene encoding .alpha..sub.2M has
the nucleotide sequence of nucleotides 44-4465 of SEQ ID NO:1. The
invention also relates to a viral vector carrying a gene encoding
an anti-LRP-A.beta. peptide. In another preferred embodiment of the
invention, the viral vector is an adeno-associated virus. In
addition, the invention provides for a pharmaceutical composition
containing the viral vector, and one or more pharmaceutically
acceptable carriers, and for a method of combating Alzheimer's
Disease in a subject by administering the viral vector.
[0025] The second aspect of the invention is to provide for a
method of screening for therapeutic agents for Alzheimer's Disease
that can replace or supplement .alpha..sub.2M function, or can
suppress the expression of A2M-2. One embodiment of the invention
is a method of screening for a therapeutic agent for AD by
incubating a cell that is heterozygous or homozygous for the A2M-2
allele in the presence of a test agent, and then determining
whether the ratio of normal to aberrant A2M mRNA has increased
relative to the ratio of normal to aberrant A2M mRNA found in cells
untreated with the test agent. In one preferred embodiment of this
method, the cells are glioma cells. In another preferred
embodiment, the cells are hepatoma cells. In yet another preferred
embodiment of the invention, the cells are heterozygous for the
A2M-2 allele.
[0026] In a related embodiment of this method, S1 nuclease is used
to determine the ratio of normal to aberrant A2M mRNA, and the
probe used is complementary to a nucleotide encoding A2M (SEQ ID
NO:1). Thus, in one embodiment of the invention, S1 nuclease
analysis using a probe complementary to SEQ ID NO:1, where the
probe encompasses nucleotides 2057-2284 of SEQ ID NO:1, is used to
determine whether the ratio of normal to aberrant A2M mRNA has
increased. In a preferred method of the invention, the probe used
in the S1 nuclease analysis is 300 bp long. In another embodiment
of the invention, the probe used in the S1 nuclease analysis is
complementary to nucleotides 2024-2323 of SEQ ID NO:1.
[0027] Alternatively, RT PCR analysis is used to determine whether
the ratio of normal to aberrant A2M mRNA has increased. In a
preferred method of RT PCR analysis, the primers are designed to
amplify a region of A2M encompassing exons 17-18. In a more
preferred method of RT PCR analysis, the amplified region of A2M
encompassing exons 17-18 is 300 bp long. In another embodiment of
the invention, the primers used for the RT PCR analysis are
designed to amplify nucleotides 2052-2289 of SEQ ID NO:1. Another
embodiment of the invention relates to the use of a first primer
having a nucleotide sequence complementary to nucleotides 2024-2038
of SEQ ID NO:1, and a second primer having the nucleotide sequence
of nucleotides 2309-2323 of SEQ ID NO:1 for the RT PCR
analysis.
[0028] The invention also provides for a method of screening for a
therapeutic agent for Alzheimer's disease by incubating
.alpha..sub.2M with a test agent, and then determining whether the
treated .alpha..sub.2M has undergone a conformational change, or
determining whether the treated .alpha..sub.2M can bind to LRP. In
a preferred embodiment of the invention, the .alpha..sub.2M treated
with a test agent is tetrameric .alpha..sub.2M. In another
preferred embodiment of the invention, an .alpha..sub.2M
electrophoretic mobility assay is ued to determine whether the
treated .alpha..sub.2M has undergone a conformational change. In
another embodiment of the invention, an ELISA is used to determine
whether the treated .alpha..sub.2M can bind to LRP. In a related
embodiment of the invention, the ELISA includes the following steps
in sequential order: incubating LRP in a well coated with anti-LRP
IgG, incubating the well with treated .alpha..sub.2M, incubating
the well with anti-.alpha..sub.2M IgG conjugated to an enzyme, and
incubating the well with a substrate for the enzyme. In an
alternative embodiment, the ELISA includes the following steps in
sequential order: incubating a well coated with LRP with treated
.alpha..sub.2M, incubating the well with anti-.alpha..sub.2M IgG
conjugated to an enzyme, and incubating the well with the substrate
for the enzyme. In another embodiment, the ELISA includes the
following steps in sequential order: incubating treated
.alpha..sub.2M in a well coated with an anti-.alpha..sub.2M IgG
specific for activated .alpha..sub.2M, incubating the well with an
anti-.alpha..sub.2M IgG conjugated to an enzyme, and incubating the
well with a substrate for the enzyme. In another embodiment of the
invention, immunoblotting with anti-LRP IgG and anti-.alpha..sub.2M
IgG is used to determine whether the treated .alpha..sub.2M can
bind to LRP. In yet another embodiment of the invention, a test for
the ability of the treated .alpha..sub.2M to undergo LRP mediated
endocytosis is used to determine whether the treated .alpha..sub.2M
can bind to LRP. In another embodiment of the invention, a test for
the ability of the treated .alpha..sub.2M to undergo LRP mediated
degradation is used to determine whether the treated .alpha..sub.2M
can bind to LRP.
BRIEF DESCRIPTION OF THE FIGURES
[0029] FIG. 1. FIG. 1 is an autoradiograph depicting the results of
.sup.33P-labeled .alpha..sub.2M mRNA transcripts from A2M from
human glioma cell lines that express either wild-type A2M
((Blacker, D., et al., Nat. Genet. 19:357-360 (1998)) or are
heterozygous for the A2M-2 deletion allele obtained by RT-PCR, and
separated on a polyacrylamide gel. A2M-1/2 lines are indicated as
lanes marked "2", A2M-1/1 lines are indicated as lanes marked
"1."
[0030] FIG. 2. FIG. 2 is a schematic representation of four of the
altered A2M transcripts produced by human glioma cell lines
expressing the A2M-2 allele.
[0031] FIG. 3. FIG. 3 is a photograph of immunoblots of media and
extracts from CHO cells transfected with .alpha..sub.2M truncated
after exon 18 that were probed with an anti-.alpha..sub.2M
antibody. The anti-.alpha..sub.2M antibody detected truncated
.alpha..sub.2M in transfected CHO cells. Panel A: cell lysate;
Panel B: media; (-) indicates samples from untransfected cells;
(wt) indicates samples from cells transfected with full-length
.alpha..sub.2M construct; (.DELTA.) indicates samples from cells
transfected with the .alpha..sub.2M construct truncated after exon
18; m, d and t indicate monomer, dimer and trimer forms of the
truncated protein, respectively. These forms of wild type
.alpha..sub.2M are also visible but not marked.
[0032] FIG. 4. FIG. 4 is a photograph of an immunoblot from cell
lysates from wild-type cells (A2M-1) (lane labeled 1/1) and cells
heterozygous for the A2M-2 deletion (lanes labeled 1/2) probed with
an anti-.alpha..sub.2M antibody. The lane labeled (+) indicates
lysate from CHO cells transfected with full length .alpha..sub.2M,
and probed with an anti-.alpha..sub.2M antibody. The media (data
not shown) from A2M-1 and A2M-2 cells contained primarily
full-length .alpha..sub.2M monomers, but in the media from the
A2M-2 cells, small amounts of truncated species could also be
observed (data not shown).
[0033] FIG. 5. FIG. 5 depicts the .alpha..sub.2M conformational
change induced by protease (represented by the letter P in a
circle) cleavage. Note the exposure of the LRP binding domain
(represented by .quadrature.) after the conformational change.
[0034] FIG. 6. FIG. 6 depicts one possible amino acid sequence for
the anti-LRP-A.beta. polypeptide.
[0035] FIG. 7. FIG. 7 is a schematic of the yeast three-hybrid
system for detecting the anti-LRP-A.beta. peptide binding to
A.beta. and LRP.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0036] Definitions
[0037] In the description that follows, a number of terms used in
recombinant DNA technology, molecular and cell biology, and
pharmacology are extensively used. To provide a clearer and
consistent understanding of the specification and claims, including
the scope to be given such terms, the following definitions are
provided.
[0038] Nucleotide: "Nucleotide" refers to abase-sugar-phosphate
combination. Nucleotides are monomeric units of a nucleic acid
sequence (DNA and RNA). The term nucleotide includes
deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP,
dGTP, dTTP, or derivatives thereof. Such derivatives include, for
example, [.alpha.S]dATP, 7-deaza-dGTP and 7-deaza-dATP. The term
nucleotide as used herein also refers to dideoxyribonucleoside
triphosphates (ddNTPs) and their derivatives. Illustrated examples
of dideoxyribonucleoside triphosphates include, but are not limited
to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. According to the present
invention, a "nucleotide" may be unlabeled or detectably labeled by
well known techniques. Detectable labels include, for example,
radioactive isotopes, fluorescent labels, chemiluminescent labels,
bioluminescent labels and enzyme labels.
[0039] Polynucleotide: A "polynucleotide" is a linear polymer of
nucleotides linked by phosphodiester bonds between the 3' position
of one nucleotide and the 5' position of the adjacent
nucleotide.
[0040] Oligonucleotide: "Oligonucleotide" refers to an oligomer or
polymer of nucleotide or nucleoside monomers consisting of
naturally occurring bases, sugars and intersugar (backbone)
linkages. The term "oligonucleotide" also includes oligomers
comprising non-naturally occurring monomers, or portions thereof,
which function similarly. Such modified or substituted
oligonucleotides are often preferred over native forms in that they
may exhibit enhanced cellular uptake, increased stability in the
presence of nucleases, and other features which render them more
acceptable as therapeutic or diagnostic reagents.
[0041] Nucleic acid molecule: By "nucleic acid molecule" is meant a
polymeric molecule composed of nucleotides. Nucleic acid molecules
of the present invention may be in the form of RNA, such as mRNA,
or in the form of DNA, including, for instance, cDNA and genomic
DNA obtained by cloning or produced synthetically. The DNA may be
double-stranded or single-stranded. Single-stranded DNA or RNA may
be the coding strand, also known as the sense strand, or it may be
the non-coding strand, also referred to as the anti-sense
strand.
[0042] Complementary: As used herein, "complementary" refers to the
subunit sequence complementarity between two nucleic acids, for
example, two DNA molecules. When a nucleotide position in both of
the molecules is occupied by nucleotides normally capable of base
pairing with each other, then the nucleic acids are considered to
be complementary to each other at this position. Thus, two nucleic
acids are complementary to each other when a substantial number (at
least 60%) of corresponding positions in each of the molecules are
occupied by nucleotides which normally base pair with each other
(for example, A:T and G:C nucleotide pairs).
[0043] Hybridization: The terms "hybridization" and "specifically
hybridizes to" refer to the pairing of two complementary
single-stranded nucleic acid molecules (RNA and/or DNA) to give a
double-stranded molecule. These terms are used to indicate that the
nucleotides are sufficiently complementary such that stable and
specific binding occurs between the DNA or RNA target and the
oligonucleotide. It is understood that an oligonucleotide need not
be 100% complementary to its target nucleic acid sequence to be
specifically hybridizable. An oligonucleotide specifically
hybridizes to another when binding of the oligonucleotide to the
target interferes with the normal function of the target molecule
to cause a loss of utility, and there is a sufficient degree of
complementarity to avoid non-specific binding of the
oligonucleotide to non-target sequences under conditions in which
specific binding is desired, i.e., under physiological conditions
in the case of in vivo assays or therapeutic treatment, or, in the
case of in vitro assays, under conditions in which the assays are
conducted.
[0044] Primer: As used herein "primer" refers to a single-stranded
oligonucleotide that is extended by covalent bonding of nucleotide
monomers during amplification or polymerization of a DNA molecule.
Minisatellite primers used for the amplification of minisatellite
dimer, trimer, tetramer, etc., sequences are well-known in the
art.
[0045] Template: The term "template" as used herein refers to a
double-stranded or single-stranded nucleic acid molecule which is
to be amplified, synthesized or sequenced. In the case of a
double-stranded DNA molecule, denaturation of its strands to form a
first and a second strand is performed before these molecules may
be amplified, synthesized or sequenced. A primer, complementary to
a portion of a DNA template is hybridized under appropriate
conditions and the DNA polymerase of the invention may then
synthesize a DNA molecule complementary to the template or a
portion thereof. The newly synthesized DNA molecule, according to
the invention, may be equal or shorter in length than the original
DNA template. Mismatch incorporation or strand slippage during the
synthesis or extension of the newly synthesized DNA molecule may
result in one or a number of mismatched base pairs. Thus, the
synthesized DNA molecule need not be exactly complementary to the
DNA template.
[0046] Amplification: As used herein "amplification" refers to any
in vitro method for increasing the number of copies of a nucleotide
sequence with the use of a DNA polymerase. Nucleic acid
amplification results in the incorporation of nucleotides into a
DNA or molecule or primer thereby forming a new DNA molecule
complementary to a DNA template. The formed DNA molecule and its
template can be used as templates to synthesize additional DNA
molecules. As used herein, one amplification reaction may consist
of many rounds of DNA replication. DNA amplification reactions
include, for example, polymerase chain reactions (PCR). One PCR
reaction may consist of 5 to 100 "cycles" of denaturation and
synthesis of a DNA molecule.
[0047] 95%, 96%, 97%, 98% or 99% Homology: By a polynucleotide
having a nucleotide sequence at least, for example, 95% "identical"
to a reference nucleotide sequence is intended that the nucleotide
sequence of the polynucleotide is identical to the reference
sequence except that the polynucleotide sequence may include up to
five point mutations per each 100 nucleotides of the reference
nucleotide sequence. In other words, to obtain a polynucleotide
having a nucleotide sequence at least 95% identical to a reference
nucleotide sequence, up to 5% of the nucleotides in the reference
sequence may be deleted or substituted with another nucleotide, or
a number of nucleotides up to 5% of the total nucleotides in the
reference sequence may be inserted into the reference sequence.
These mutations of the reference sequence may occur at the 5' or 3'
terminal positions of the reference nucleotide sequence or anywhere
between those terminal positions, interspersed either individually
among nucleotides in the reference sequence or in one or more
contiguous groups within the reference sequence.
[0048] As a practical matter, whether any particular nucleic acid
molecule is at least 95%, 96%, 97%, 98% or 99% identical to, for
instance, the nucleotide sequence shown in SEQ ID NO:1 can be
determined conventionally using known computer programs such as the
Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for
Unix, Genetics Computer Group, University Research Park, 575
Science Drive, Madison, Wis. 53711. Bestfit uses the local homology
algorithm of Smith and Waterman, Advances in Applied Mathematics 2:
482-489 (1981), to find the best segment of homology between two
sequences. When using Bestfit or any other sequence alignment
program to determine whether a particular sequence is, for
instance, 95% identical to a reference sequence according to the
present invention, the parameters are set, of course, such that the
percentage of identity is calculated over the full length of the
reference nucleotide sequence and that gaps in homology of up to 5%
of the total number of nucleotides in the reference sequence are
allowed.
[0049] Polypeptide: A polypeptide is a polymer composed of amino
acid monomers joined by peptide bonds.
[0050] Peptide Bond: A peptide bond is a covalent bond between two
amino acids in which the alpha-amino group of one amino acid is
bonded to the alpha-carboxyl group of the other amino acid.
[0051] Isolated nucleic acid molecule or polypeptide: a nucleic
acid molecule, DNA or RNA, or a polypeptide, which has been removed
from its native environment. For example, recombinant DNA molecules
contained in a vector are considered isolated for the purposes of
the present invention. Further examples of isolated DNA molecules
include recombinant DNA molecules maintained in heterologous host
cells or purified (partially or substantially) DNA molecules in
solution. Isolated RNA molecules include in vivo or in vitro RNA
transcripts of the DNA molecules of the present invention. Isolated
nucleic acid molecules or polypeptides according to the present
invention further include such molecules produced
synthetically.
[0052] Linker: By "linker" is intended a molecule that connects the
LRP binding domain to the A.beta. binding domain of the
anti-LRP-A.beta. molecule. When referring to a linker composed of
amino acid residues, linker is used to refer to the amino acid
residues connecting the two domains. When referring to a nucleic
acid encoding a linker, linker refers to the nucleotide sequence
encoding the linking amino acid residues. Where the linker is
composed of amino acid residues, it will typically consist of one
or more glycine residues, or the nucleotide sequence encoding these
residues, however, proline may also be used.
[0053] Combating Alzheimer's Disease: The term "combating
Alzheimer's Disease" is intended to mean a slowing, delaying, or
even reversing the AD process. Thus, for example, the therapeutic
agents of the invention may be administered either therapeutically
in a patient where symptoms of AD are present, or prophylactically,
in a subject at risk of developing AD.
[0054] Pharmaceutically acceptable carrier: By pharmaceutically
acceptable carrier is meant anon-toxic solid, semisolid or liquid
filler, diluent, encapsulating material, or formulation auxiliary
of any type.
[0055] Performed in sequential order: By "performed in sequential
order" is intended that the steps described by this term are
performed in the order that the steps are recited, but that other
unrecited steps may be performed in between the recited steps.
[0056] Test agent: By "test agent" is meant any molecule that is of
interest for the treatment or prevention of AD, and is to be tested
using the screening methods of the invention.
[0057] Ranges: various ranges of numbers are described herein. When
a range is used, the range of numbers is meant to be inclusive of
the boundary numbers. For example, an oligonucleotide composed of
nucleotides 20-50 of SEQ ID NO:27, is meant to include nucleotides
20, and 50 and every nucleotide in between.
[0058] Other terms used in the fields of recombinant DNA
technology, molecular and cell biology, and pharmacology as used
herein will be generally understood by one of ordinary skill in the
applicable arts.
[0059] Alpha-2-macroglobulin Alpha-2-Macroglobulin (.alpha..sub.2M)
is a 718 kD glycoprotein found at high concentrations in the serum
(Borth, W., FASEB J. 6:3345-3353 (1992)). The structure of
.alpha..sub.2M consists of four identical 180 kD monomeric units,
of 1451 amino acids each (Sottrup-Jensen, L., et al., J. Biol.
Chem. 259:8318-8327 (1984)). Disulfide bonds link these monomers
into dimers, and noncovalent interactions between dimers lead to
formation of the functional homotetramer (Harpel, P. C., J. Exp.
Med. 138:508-521 (1973); Swenson, R. P. and Howard, J. B., J. Biol.
Chem. 254:4452-4456 (1979)). In addition to the ability to bind
A.beta., .alpha..sub.2M binds a variety of polypeptides proteases,
growth factors, and cytokines) and ions (Zn, Cu, Fe)(Borth, W.,
FASEB J. 6:3345-3353 (1992); James, K., Immunol. Today 11:163-166
(1990); Parisi, A. F. and Vallee, B. L., Biochem. 9:2421-2426
(1970)).
[0060] The best studied function of .alpha..sub.2M is its
pan-protease inhibitory activity (Barret, A. J. and Starkey, P. M.,
Biochem. J. 133:709-724 (1973)). A protease molecule binds the bait
region of a .alpha..sub.2M tetramer, amino acids 666-706, and
cleaves any of a number of susceptible peptide bonds in this region
((Harpel, P. C., J. Exp. Med. 138:508-521 (1973); Barret, A. J. and
Starkey, P. M., Biochem. J. 133:709-724 (1973); Sottrup-Jensen, L.,
et al., J. Biol. Chem. 264:15781-15789 (1989)). Protease binding
and cleavage triggers a large conformational change in the
.alpha..sub.2M/protease complex, referred to as activation, that
ultimately results in entrapment of the protease within the
tetramer (FIG. 5) (Borth, W., FASEB J. 6:3345-3353 (1992)). In each
monomer a unique .beta.-Cys-.gamma.-Glu thiol ester bond exists
between Cys-949 and Glu-952(Borth, W., FASEB J. 6:3345-3353
(1992)). Upon activation this thiol ester bond emerges from a
hydrophobic environment and can undergo nucleophilic attack, for
example, by lysine residues from the reacting proteases. The result
of this nucleophilic attack is a covalent bond between Glu-952 of
.alpha..sub.2M and surface lysine residues of the protease (FIG.
5). The protease is effectively trapped, unable to dissociate from
.alpha..sub.2M but still able to cleave small peptide substrates
(Qui, W. Q., et al., J. Biol. Chem. 271:8443-8451 (1996)).
Protease-mediated activation results in exposure of the
.alpha..sub.2M receptor/low density lipoprotein receptor-related
protein binding domain (FIG. 5) (Strickland, D., et al., J. Biol.
Chem. 265:17401-17404 (1990)). Low density lipoprotein
receptor-related protein (LRP) is a 600 kD endocytic membrane-bound
receptor belonging to the low-density lipoprotein receptor family
(Borth, W., FASEB J. 6:3345-3353 (1992)). LRP is a multifunctional
receptor, because it binds ligands from different classes (Kounnas,
M. Z., et al., Cell 82:331-340 (1995)). Exposure of this LRP
binding domain is a prerequisite for LRP mediated endocytosis of
.alpha..sub.2M/ligand complexes and targeted degradation (Borth,
W., FASEB J. 6:3345-3353 (1992)). In summary, .alpha..sub.2M serves
to bind a number of protein substrates, including A.beta., and
target them for internalization and degradation.
[0061] .alpha..sub.2M binds A.beta. specifically and tightly. The
A.beta. binding region of .alpha..sub.2M is located between
residues 1202-1312, approximately 600 residues C-terminal to the
bait region (Hughes, S. R., et al., Proc. Natl. Acad. Sci. USA
95:3275-3280 (1998)). Binding does not require .alpha..sub.2M
activation and binding stoichometry is approximately 1.1
A.beta./mol of .alpha..sub.2M (Du, Y., et al., J. Neurochem.
69:299-305 (1997)). The apparent dissociation constant (K.sub.D)
for the A.beta./.alpha..sub.2M complex has been reported as
3.8*10.sup.-10 M for .beta..sub.2M/.sup.125I-A.beta. (Du, Y., et
al., J. Neurochem. 69:299-305 (1997)) and 3.5*10.sup.-7 M for
biotinA.beta./(ruthenium (II) tris-bipyridine-n-hydroxysuccinimide
ester) modified-.alpha..sub.2M (Hughes, S. R., et al., Proc. Natl.
Acad. Sci. USA 95:3275-3280 (1998)). Despite this discrepancy in
K.sub.D values (which are most likely due to methodological
differences), a strong interaction between A.beta. and
.alpha..sub.2M exists. This interaction prevents A.beta. fibril
formation and fibril associated neurotoxicity ((Hughes, S. R., et
al., Proc. Natl. Acad. Sci. USA 95:3275-3280 (1998); Du, Y., et
al., J. Neurochem. 70:1182-1188 (1998)). Recently, it has been
demonstrated that a region of .alpha..sub.2M encompassing only the
A.beta. and LRP binding domains is sufficient for A.beta. binding
in vivo ((Hughes, S. R., et al., Proc. Natl. Acad. Sci. USA
95:3275-3280 (1998)). These data suggest that the A.beta. binding
domain is an independent structural unit and successful
.alpha..sub.2M/A.beta. interaction may only rely on a few key
interactions. Recent work by Soto and colleagues show that an
eleven residue peptide is capable of binding A.beta. and inhibiting
A.beta. fibril formation (Soto, C., et al., Nature Medicine
4:822-826 (1998)), supporting the idea that only a few key
interactions are needed to bind A.beta.. In summary, .alpha..sub.2M
can mediate the catabolism of A.beta. in a LRP dependent
process.
[0062] A2M-2 Genotype
[0063] The A2M-2 genotype, which is linked to late-onset AD, is
present in 30% of the population (Blacker, D., et al., Nature
Genetics 19:357-360 (1998)). This genotype has a pentanucleotide
deletion at the 5' splice site of the second exon encoding the bait
region of .alpha..sub.2M (exon 18) (Blacker, D., et al., Nature
Genetics 19:357-360 (1998)).
[0064] Low resolution X-ray data and biochemical data suggest that
the bait regions are located at the dimer interface and are crucial
for the formation of functional tetramers, and the mediation of the
conformational change that accompanies activation (Andersen, G. R.,
et al., J. Biol. Chem. 270:25133-25141 (1995); Bowen, M. E. and
Gettins, P. G. W., J. Biol. Chem. 273:1825-1831 (1998)). The
.alpha..sub.2M-2 deletion in the bait region could prevent A.beta.
clearance and degradation if (i) proteases can not cleave the
altered bait region, (ii) protease-induced activation cannot occur,
(iii) LRP binding is disrupted, and/or (iv) A.beta. binding is
disrupted.
[0065] Low Density Lipoprotein Receptor-Related Protein
[0066] LRP is a 600 kD endocytic membrane-bound receptor belonging
to the low-density lipoprotein receptor family (Borth, W., FASEB J.
6:3345-3353 (1992)). LRP is expressed in a variety of cell types
including: adipocytes, astrocytes, fibroblasts, hepatocytes,
macrophages, monocytes, and syncytiotrophoblasts. LRP is translated
as a 4525 residue single chain precursor (Nielsen, K. L., et al.,
J. Biol. Chem. 271:12909-12912 (1996)). It is then processed into a
515 kD A chain and an 85 kD .beta. chain. The .beta. chain
possesses a single transmembrane segment and a cytoplasmic tail
containing two copies of the NPXY endocytosis signal sequence
(Nielsen, K. L., et al., J. Biol. Chem. 271:12909-12912 (1996)).
The extracellularly located a chain contains four cysteine-rich LDL
receptor ligand-binding repeats flanked by epidermal growth factor
(EGF) repeats (Nielsen, K. L., et al., J. Biol. Chem.
271:12909-12912 (1996)). The noncovalent association of the a chain
with the extracellular portion of the .beta. chain forms a
functional LRP (Borth, W., FASEB J. 6:3345-3353 (1992)). LRP is a
multifunction receptor because it binds ligands from different
classes (Kounnas, M. Z., et al., Cell 82:331-340 (1995)). These
include .alpha..sub.2M-protease complexes, plasminogen activator
inhibitor-plasminogen activator complexes, lipoprotein lipase,
apoe, bovine pancreatic trypsin inhibitor, lactoferrin,
Pseudomonas, exotoxin A, nexin-1 complexes, and receptor associated
protein (RAP) (Kounnas, M. Z., et al., Cell 82:331-340 (1995)).
Most of these ligands do not compete for the same binding site.
RAP, however, inhibits the binding of all these ligands.
[0067] .alpha..sub.2M/LRP Association
[0068] The association of activated .alpha..sub.2M and LRP is
highly pH dependent, acidification to pH 6.8 or below abolishes
binding (Borth, W., FASEB J 6:3345-3353 (1992)). This suggests that
upon endocytosis .alpha..sub.2M dissociates from LRP. After
endocytosis .alpha..sub.2M and its associated ligands are degraded
in the lysosome and LRP is recycled to the membrane (Borth, W.,
FASEB J. 6:3345-3353 (1992)). The half-life for internalization and
degradation varies between 15 and 60 minutes (Borth, W., FASEB J.
6:3345-3353 (1992)).
[0069] The .alpha..sub.2M-protease binding site of LRP has been
mapped to residues 776-1399 of the .beta. chain (Nielsen, K. L., et
al., J. Biol. Chem. 271:12909-12912 (1996)). This region includes
EGF repeats 4-6 and LDL receptor ligand binding repeats 3-10. The
LRP binding domain of .alpha..sub.2M is located between residues
1312 and 1451, directly C-terminal to the A.beta. binding domain
(Hughes, S. R., et al., Proc. Natl. Acad. Sci. USA 95:3275-3280
(1998)). This domain is very flexible relative to the core of
.alpha..sub.2M (Andersen, G. R., et al., J. Biol. Chem.
270:25133-25141 (1995)). Low resolution crystal structures (10
.ANG.) indicate that activated .alpha..sub.2M is roughly the shape
of an H and the LRP binding domains are located at the tips of the
H (FIG. 5) (Andersen, G. R., et al., J. Biol. Chem. 270:25133-25141
(1995)). A LRP consensus binding sequence has been proposed based
on 31 LRP ligands from 7 different protein families (Nielsen, K.
L., et al., J. Biol. Chem. 271:12909-12912 (1996)). This 27 residue
consensus sequence is located between residues 1365 and 1393 of
human .alpha..sub.2M. Once again, experimental evidence suggests
that a few key interactions may be important in LRP/.alpha..sub.2M
binding. Mutations at positions 5 and 10 of the consensus sequence,
corresponding to Lys-1370 and Lys-1374 in the human .alpha..sub.2M,
abolish binding unlike mutations at other highly conserved
residues.
[0070] Implication of .alpha..sub.2M in Alzheimer's Disease
[0071] Cerebral deposition of amyloid is a central event in AD
(Soto, C., et al., Nat. Med. 4:822-826 (1998)). Genetic,
neuropathological, and biochemical evidence indicate that
inappropriate deposition of amyloid plays a fundamental role in the
pathogenesis of AD. The major component of AD amyloid plaques is
A.beta., a 39-43 amino acid peptide. A.beta. polymerizes as dense
(amyloid plaque) and diffuse extracellular deposits in the neuropil
(Masters, C. L., et al., Proc. Natl. Acad. Sci. USA 82:4245-4249
(1985)), and in cerebral blood vessels (congophilic angiopathy)
(Glenner, G. G. and Wong, C. W., Biochem. Biophys. Res. Comm.
120:885-890 (1984)) of both AD and Down syndrome (DS)patients.
Soluble A.beta. is found in the cerebrospinal fluid (CSF) and is
produced (Haass, C., et al., Nature 359:322-325 (1992); Seubert,
P., et al., Nature 359:325-327 (1992); Shoji, M., et al., Science
258:126-129 (1992)) by constitutive cleavage of its transmembrane
parent molecule, the amyloid protein precursor (APP) (Kang, J., et
al., Nature 325:733-736 (1987); Goldbarger, D., et al., Science
235:877-880 (1987); Robakis, N. K., et al., Proc. Natl. Acad. Sci.
USA 84:4190-4194 (1987); Tanzi, R. E., et al., Science 235:880-884
(1987)). APP is a family of alternatively-spliced proteins, of
unknown function, that are ubiquitously expressed (Tanzi, R. E., et
al., Nature 331:528-530 (1988)). Unknown proteases cleave APP to
produce a mixture of A.beta. peptides with carboxyl-terminal
heterogeneity. A.beta.1-40, the major soluble A.beta. species, is
found in the CSF at low nanomolar concentrations (Vigo-Pelfrey, C.,
et al., J. Neurochem. 61:1965-1968 (1993)). A.beta.1-42 is a minor
soluble A.beta. species, but is heavily enriched in amyloid plaques
(Masters, C. L., et al., Proc. Natl. Acad. Sci. USA 82:4245-4249
(1985); Kang, J., et al., Nature 325:733-736 (1987); Roher, A. E.,
et a., J. Biol. Chem. 268:3072-3083 (1993)).
[0072] The mechanism by which these amyloid deposits result in
dementia is unclear, but may be related to the neurotoxic effects
of A.beta. at micromolar concentrations (Pike, C. J., et al., Brain
Res. 563:311-314 (1991)). Insight into the mechanism of amyloid
deposit formation began with the discovery of pathogenic mutations
of APP close to, or within, the AB domain (van Broeckhoven, C., et
al., Science 248:1120-1122 (1990); Levy, E., et al., Science
248:1124-1126 (1990); Goate, A., et al., Nature 349:704-706 (1991);
Murrell, J., et al., Science 254:97-99 (1991); Mullan, M., et al.,
Nat. Genet. 1:345-347 (1992)). These studies indicated that the
metabolism of A.beta., and APP, is intimately involved with the
pathophysiology of AD. Increasing evidence suggests that increased
levels of A.beta.1-42 accelerates amyloid deposition in early-onset
familial AD (FAD). The FAD-linked APP670/671 mutation has been
shown to increase the secretion of A.beta. species several-fold
(Citron, M., et al., Nature 360:672-674 (1992)). While the APP717
mutation does not affect the quantity of A.beta. production (Cai,
X-D., et al., Science 259:514-516 (1993)), this mutation increases
the proportion of A.beta.1-42 produced (Suzuki, N., et al., Science
264:1336-1340 (1994)). Increased soluble A.beta.1-42 has also been
found in the brains of individuals affected by Down syndrome, a
condition complicated by premature AD (Teller, J. K., et al., Nat.
Med. 2:93-95 (1996)). Inheritance of the other FAD-linked mutations
of Presenilin-1 (PSEN1) or Presenilin-2 (PSEN2) (Sherrington, R.,
et al., Nature 375:754-760 (1995); Levy-Lahad, E., et al., Science
269:973-977 (1995)) correlates with increased cortical amyloid
burden. The emerging consensus is that the common effect of
FAD-linked presenilin mutations is to increase A.beta.1-42
production (Citron, M., et al., Nat. Med. 3:67-72 (1997); Xia, W.,
et al., J. Biol. Chem. 272:7977-7982 (1997)). Taken together these
studies suggest that mutations in the genes linked to FAD (APP,
PSEN1, PSEN2) can result in increased A.beta.1-42 production and
that this increase could cause FAD. In the vast majority of AD
patients, however, overproduction does not occur (Van Gool, W. A.,
et al., Ann. Neurol. 37:277-279 (1995)).
[0073] Ninety percent of AD patients suffer from late-onset AD
(LOAD). Three genes have been linked to this form of AD:APOE, LRP,
and A2M. Inheritance of the APOE-.epsilon.4 allele on chromosome 19
correlates with increased cortical amyloid burden (Rebeck, G. W.,
et al., Neuron. 11:575-580 (1993)). APOE promoter polymorphisms,
which upregulate transcription of APOE, have recently been shown to
be associated with AD (Bullido, M. J., et al, Nat. Genet. 18:69-71
(1998); Lambert, J. C., et al., Human Mol. Gen. 6:533-540 (1998)).
Higher expression of the APOE-.epsilon.4 allele, relative to
APOE-.epsilon.3, has been found in brains of APOE-.epsilon.4
positive AD patients, but not in age- and genotype-matched controls
(Lambert, J. C., et al., Human Mol. Gen. 6:2151-2154 (1997)). The
absence of apoE in transgenic mice expressing FAD mutant APP
attentuates A.beta. deposition (Bales, K. R., et al., Nature
Genetics 17:264 (1997)). The second gene linked to LOAD, the LRP
gene, encodes the low density lipoprotein receptor-related protein.
APP, apoE, and .beta..sub.2M are all ligands for this cell-surface
receptor (Blacker, D. and Tanzi, R. E., Archives of Neurology
55:294-296 (1998); Kang, D. E., et al., Neurology 49:56-61 (1997);
Blacker, D., et al., Neurology 48:139-147 (1997); Farrer, L. A., et
al., JAMA 278:1349-1356 (1997); Strittmatter, W. J., et al., Proc.
Natl. Acad. Sci. USA 90:1977-1981 (1993)). LRP internalizes ligands
via endocytosis, and targets them for lysosomal degradation (Borth,
W., FASEB J. 6:3345-3353 (1992)). Inheritance of a pentanucleotide
deletion in the third gene associated with LOAD, A2M (i.e,
inheritance of A2M-2), confers increased risk for AD and is present
in .about.30% of the population (Blacker, D., et al., Nat. Genet.
19:357-360 (1998)). The protein product of A2M, .alpha..sub.2M, is
an abundant pan-protease inhibitor found primarily in serum, but is
also present in brain and other organs (for example, liver).
.alpha..sub.2M binds A.beta. and can mediate its internalization
and degradation (Borth, W., FASEB J. 6:3345-3353 (1992); Narita,
M., et al., J. Neurochem. 69:1904-1911 (1997)).
[0074] .alpha..sub.2M has been implicated in the pathogenesis of AD
by both biological and genetic findings. .alpha..sub.2M-like
immunoreactivity was observed in AD cortical senile plaques (Bauer,
J., et al., FEBS Lett. 285:111-114 (1991)) and it was shown that
.alpha..sub.2M is upregulated in the AD brain where it localizes to
neuritic but not diffuse amyloid plaques (Strauss, S., et al., Lab.
Invest 66:223-230(1992); Van Gool, D., et al., Neurobiol. Aging
14:233-237 (1993)). In addition, A.beta. was found to bind to
.alpha..sub.2M with high affinity (Du, Y., et al., J. Neurochem.
69:299-305 (1997)), and binding prevented amyloid fibril formation
as well as neurotoxicity associated with aggregated A.beta. (Du,
Y., et al., J. Neurochem. 70:1182-1188 (1998); Hughes, S. R., et
al., Proc. Natl. Acad. Sci. USA 95:3275-3280 (1998)). Activated
.alpha..sub.2M-A.beta. complexes were recently shown to be
internalized and targeted for degradation by glioblastoma cells via
binding to LRP (Narita, M., et al., J. Neurochem. 69:1904-1911
(1997)). Moreover, LRP is especially abundant in brain regions
affected by AD such as the hippocampus (Rebeck, G. W., et al.,
Neuron 11:575-580 (1993); Tooyama, I., et al., Mol. Chem.
Neuropathol. 18:153-160 (1993)), and serves as a receptor for ApoE
(Rebeck, G. W., et al., Neuron 11:575-580 (1993)), a well
established genetic risk factor (Blacker, D., et al., Nature Gen.
19:357-360 (1998)).
[0075] The genetic linkage of APP, APOE, A2M, and their receptor
LRP to AD suggests that these proteins may participate in a common
neuropathogenic pathway leading to AD (Blacker, D., et al., Nat.
Genet. 19:357-360 (1998)). This pathway may be the .alpha..sub.2M
mediated clearance and degradation of A.beta. through
.alpha..sub.2M binding to LRP for endocytosis and lysosomal
degradation, and by serving as a direct mediator for A.beta.
degradation when .alpha..sub.2M is complexed with an unidentified
serine protease (Qiu, W. Q., et al., J. Biol. Chem. 271:8443-8451
(1996)). This hypothesis is supported, inter alia, by the fact that
apoE and .alpha..sub.2M are both ligands for LRP and, in addition,
that apoE has previously been reported to inhibit .alpha..sub.2M
mediated degradation of A.beta. (Rebeck, G. W., et al., Ann.
Neurol. 37:211-217 (1995); Zhang, Z., et al., Int. J. Exp. Clin.
Invest. 3:156-161 (1996)).
[0076] However, in its normal role, .alpha..sub.2M also binds a
host of cytokines, growth factors, and biologically active peptides
(Borth, W., FASEB J. 6:3345-3353 (1992)). It has also recently been
shown to activate the phosphatidylinositol 3-kinase suggesting a
role in signaling (Misra, U. K. and Pizzo, S. V., J. Biol. Chem.
273:13399-13402 (1998)). Thus, defective activity of .alpha..sub.2M
may lead to AD-related neurodegeneration by a variety of mechanisms
beyond possible effects on A.beta. accumulation and deposition.
[0077] A reduced steady-state level of secreted .alpha..sub.2M or
the presence of defective tetramers due to dominant negative
effects of A2M-2 could result in impaired .alpha..sub.2M function.
Partial or total deletion of the sequences coding for the bait
region in exons 17 and 18 are likely to modify protease binding,
activation, and internalization of potentially defective tetramers
containing mutant monomer(s). Therefore, the generation of very low
levels of mutant monomers may have an amplified effect as one
mutant monomer may potentially inhibit the function of three
wild-type monomers in the tetramer (dominant negative effect). Thus
a critical role for .alpha..sub.2M is indicated in AD
neuropathogenesis. The data described in Example 1 show that the
A2M-2 deletion leads to deleted/truncated forms of .alpha..sub.2M
RNA and protein that may have a dominant negative effect on normal
.alpha..sub.2M. Based on the finding, described herein, that the
A2M-2 deletion leads to the production of altered .alpha..sub.2M
transcripts and proteins, strategies aimed at replacing or
supplementing normal .alpha..sub.2M function and activities, and/or
at suppressing defective .alpha..sub.2M function in the brain may
effectively serve as a means for therapeutically preventing,
treating, or even reversing AD neuropathogenesis. In addition,
these strategies may be useful for treating other pathologies
associated with defective .alpha..sub.2M function. Moreover,
methods based on the results and experiments described herein may
be used to screen for these therapeutic agents.
[0078] The first aspect of present invention relates to therapeutic
agents for AD that can replace or supplement normal .alpha..sub.2M
function, and/or suppress expression of A2M-2.
[0079] In one embodiment of the invention, the therapeutic agent is
an anti-LRP-A.beta. molecule, which is a molecule containing LRP
and A.beta. binding domains. This molecule may be a peptide, or
other molecule, that is capable of binding to both AD and LRP. This
anti-LRP-A.beta. molecule may also contain other domains. An
anti-LRP-A.beta. molecule having A.beta. and LRP binding domains
could bind A.beta. and target it for LRP mediated endocytosis
followed by lysosomal degradation, and thus would be useful, inter
alia, as a therapeutic agent.
[0080] In one embodiment of the invention, the anti-LRP-A.beta.
molecule is a peptide, referred to herein as the anti-LRP-A.beta.
peptide. A 250-residue fragment of the .alpha..sub.2M monomer
contains both the A.beta. and LRP binding domains (Hughes, S. R.,
et al., Proc. Natl. Acad. Sci. U.S.A. 95:3275-3280 (1998)). Thus,
in one embodiment of the invention, the anti-LRP-A.beta. peptide
would be composed of the entire A.beta. and LRP binding domains of
.alpha..sub.2M (SEQ ID NO:4). Alternatively, the A.beta. and LRP
binding domains may be composed of portions of the A.beta. and LRP
binding domains of .alpha..sub.2M. The A.beta. binding domain of
.alpha..sub.2M is located between residues 1201 and 1313,
approximately 600 residues C-terminal to the bait region (Hughes,
S. R., et al., Proc. Natl. Acad. Sci. USA 95:3275-3280 (1998)).
Thus, in another embodiment of the invention, the A.beta. binding
domain of the anti-LRP-A.beta. peptide would consist of the full
A.beta. binding domain of .alpha..sub.2M (between residues
1201-1313, SEQ ID NO:6), but only a portion of the LRP binding
domain. In another embodiment of the invention, the A.beta. binding
domain would consist of at least 50 contiguous residues of the full
A.beta. binding domain of .alpha..sub.2M. In another embodiment of
the invention, the A.beta. binding domain would consist of 10-50
contiguous residues of the full A.beta. binding domain of
.alpha..sub.2M.
[0081] In addition, peptides that can bind A.beta. in vivo and
inhibit A.beta. fibril formation have been described by Soto et al
(Soto, C. et al., Nat. Med. 4:822-826 (1998); Soto, C., et al.,
Biochem. Biophys. Res. Comm. 226:672-680 (1996)). These peptides
(SEQ ID NOs:12, 16, 18, 20, 22, 24 and 26) have homology to A.beta.
and a similar degree of hydrophobicity, but have a low propensity
to adopt a .beta.-sheet conformation. In particular one 11 residue
A.beta. binding peptide, having the amino acid sequence of SEQ ID
NO:12, and encoded by the nucleic acid sequence of SEQ ID NO:11,
was particularly effective. Therefore, in a preferred embodiment of
the invention, the A.beta. domain of the anti-LRP-A.beta. peptide
would have the sequence of this 11-residue peptide. Thus, in a
preferred embodiment of the invention, the A.beta. domain of the
anti-LRP-A.beta. peptide has the amino acid sequence of SEQ ID
NO:12, and is encoded by the nucleic acid sequence of SEQ ID NO:11.
Two shorter derivatives of this 11 residue A.beta. binding peptide,
composed of a 5 residue peptide (SEQ ID NO:22) and a 7 residue
peptide (SEQ ID NO:18) also effectively bound A.beta. and inhibited
fibril formation (Soto, C. et al., Nat. Med. 4:822-826 (1998);
Soto, C., et al, Biochem. Biophys. Res. Comm. 226:672-680 (1996)).
Thus, in another preferred embodiment of the invention, the A.beta.
binding domain has the amino acid sequence of SEQ ID NO:22, and is
encoded by the nucleic acid sequence of SEQ ID NO:21, or has the
amino acid sequence of SEQ ID NO:18, and is encoded by the nucleic
acid sequence of SEQ ID NO:17. Alternatively, the A.beta. binding
domain may be composed of other derivatives of the 11 residue
A.beta. binding peptide having 3, 4 or 6 residues (SEQ ID NO:24,22
and 18 respectively). Thus in another embodiment of the invention,
the A.beta. binding domain has the amino acid sequence of SEQ ID
NO:24, 22 or 18, and is encoded by the nucleic acid sequence of SEQ
ID NO:23, 21 or 17, respectively.
[0082] The LRP binding domain of .alpha..sub.2M is located between
residues 1312 and 1451 of .alpha..sub.2M, directly C-terminal to
the A.beta. binding domain (Hughes, S. R., et al., Proc. Nat. Acad.
Sci. USA 95:3275-3280 (1998)). Thus, in one embodiment of the
invention, the LRP binding domain of the anti-LRP-A.beta. peptide
is composed of the full LRP binding domain of .alpha..sub.2M
(residues 1313-1451, SEQ ID NO:8). In another embodiment of the
invention, the LRP binding domain is composed of at least at least
50 contiguous residues of the full LRP binding domain of
.alpha..sub.2M. In yet another embodiment of the invention, the LRP
binding domain is composed of 10-50 contiguous residues of the full
LRP binding domain of .alpha..sub.2M. Within the LRP binding
domain, a 27 residue LRP binding consensus sequence exists at
residues 1366-1392 (Nielsen, K. L., et al., J. Biol. Chem.
271:12909-12912 (1996)). Thus, in a preferred embodiment of the
invention, the LRP binding domain of the anti-LRP-A.beta. peptide
is composed of residues 1366-1392 (SEQ ID NO:10) of .alpha..sub.2M.
Alternatively, the LRP binding domain may be composed of a
contiguous portion of residues 1313-1451 of .alpha..sub.2M that
includes residues 1366-1392. In another preferred embodiment, the
anti-LRP-A.beta. peptide is composed of the 11 residue A.alpha.
binding domain and the 27 residue consensus sequence of the
.alpha..sub.2M LRP binding domain (SEQ ID NO:14).
[0083] The A.beta. binding domain and the LRP binding domain of the
anti-LRP-A.beta. molecule may be connected to each other directly
by a covalent bond, or indirectly by another molecule, such as a
linker, or linkerless polyethylene glycol. Linker molecules include
polymers such as polyethylene glycol (PEG) and peptides or amino
acid residues. In addition, linkerless PEG modification
(PEGylation) may be used (Francis, G. E., et al., Int. J. Hematol.
68:1-18 (1998)). Various methods of connecting molecules using
linkers and other molecules are well known in the art, and may be
used to connect the A.beta. and LRP binding domains (See, for
example, Francis, G. E., et al., Int. J. Hematol. 68:1-18 (1998);
Raag, R. and Whitlow, M., FASEB J. 9:73-80 (1995); Deguchi, Y., et
al., Bioconjug. Chem. 10:32-37 (1999); Luo, D., et al., J.
Biotechnol. 65:225-228 (1998); Reiter, Y., and Pastan, I., Clin
Cancer Res. 2:245-52 (1996); DeNardo, G. L., et al., Clin. Canc.
Res. 4:2483-90 (1998); Taremi, S. S., Protein Sci. 7:2143-2149
(1998); Schaffer, D. V., and Lauffenburger, D. A., J. Biol. Chem.
273:28004-28009 (1998); Skordalakes, E., et al., Biochem.
37:14420-14427 (1998); Czerwinski, G., et al., Proc. Natl. Acad.
Sci. U.S.A. 95:11520-11525 (1998); Daffix, I., et al., J. Pept.
Res. 52:1-14 (1998); Liu, S. J., et al., Blood 92:2103-2112 (1998);
Chandler, L. A., et al, Int. J. Cancer 78:106-111 (1998); Park, C.
J., Appl. Microbiol. Biotechnol. 50:71-76 (1998); Suzuki, Y., et
al., J. Biomed. Mater. Res. 42:112-116(1998); Filikov, A. V., and
James, T. L., J. Comput. Aided Mol. Des. 12:229-240 (1998);
MacKenzie, R., and To, R., J. Immunol. Methods 220:39-49
(1998)).
[0084] In one preferred embodiment of the invention, the linker is
composed of amino acid residues, for example, glycine residues or
proline residues. Where the linker is composed of amino acid
residues, it may be from 1-20 residues, but will preferably be 5-10
residues, and more preferably will be 5 residues.
[0085] Where the anti-LRP-A.beta. molecule is apeptide, within the
peptide, the A.beta. binding domain may be C-terminal, or
N-terminal to the LRP binding domain. However, preferably, the
A.beta. binding domain will be N-terminal to the LRP binding
domain, which is the order of the A.beta. and LRP binding domains
in naturally occurring .alpha..sub.2M.
[0086] In addition, the invention provides for nucleic acid
molecules that encode an anti-LRP-A.beta. peptide. Thus, in another
embodiment of the invention, the nucleic acid molecules would
encode an anti-LRP-A.beta. peptide having the sequences described
above. The invention also relates to nucleic acids having at least
95% homology to these nucleic acids. In addition, the invention
relates to nucleic acids that hybridize to a nucleic acid that is
complementary to a nucleic acid encoding the anti-LRP-A.beta.
peptide. The conditions under which the first and second
polynucleotides hybridize are preferably as follows: (a) incubate
overnight at 42.degree. C. in a solution consisting of 50%
formamide, 5.times. SSC, 50 mM sodium phosphate (pH 7.6), 5.times.
Denhardt's solution, 10% dextran sulfate, and a 20 .mu.g/ml
denatured, sheared salmon sperm DNA; and (b) wash at 65.degree. C.
in a solution consisting of 0.1.times. SSC.
[0087] The anti-LRP-A.beta. peptide may be produced using standard
solid phase synthesis methods for protein synthesis, and purified
by high performance liquid chromatography (HPLC) which are well
known in the art (See "Preparation and Handling of Peptides," in:
Current Protocols in Protein Science, Coligan, J. E., et al., eds.,
John Wiley and Sons, Inc., pub., Vol. 2., Chapter 18 (Suppl. 14
1998)). Alternatively, the anti-LRP-A.beta. peptide may be produced
using standard recombinant DNA methods. For example, The DNA coding
for the desired sequence of the LRP binding domain (for example,
the 27 residue consensus sequence) may be obtained by PCR
amplification of the codons encoding the desired LRP binding domain
using primers designed to flank the desired codons. This DNA may
then be used as a template for PCR mediated integration of the
sequence coding for the desired A.beta. binding domain. For PCR
mediated insertion of the A.beta. domain, a nucleotide 5' PCR
primer can be designed having (1) a region homologous to the end of
the DNA sequence encoding the desired LRP binding domain that was
amplified as described immediately above, and (2) immediately 5' to
this region, a region encoding the desired A.beta. binding domain,
and (3) immediately 5' to this region a start codon. For the 3'
primer, the 3' flanking primer used to amplify the LRP binding
domain, which sequence is now being used as the template, may be
used. Alternatively, to produce an anti-LRP-A.beta. peptide having
the entire A.beta. and LRP binding domains of .alpha..sub.2M
(residues 1202-1451), primers may be designed to flank the coding
sequence for these domains, to amplify this region (nucleotides
3713-4465). A start codon may be then added by PCR mediated
insertion. To amplify a coding region that encodes less than the
entire AB and LRP binding domains, the primers may instead be
designed to flank this smaller region of .alpha..sub.2M. The
resulting nucleic acid molecule is DNA encoding a fusion protein
having LRP and A.beta. binding domains, and a start codon, such
that this molecule may be inserted into an expression vector to
produce the anti-LRP-A.beta. peptide.
[0088] Once DNA encoding the desired fusion protein is obtained,
PCR mediated insertion may be used to insert restriction enzyme
sites at the 5' and 3' ends of the fusion gene so that the fusion
protein gene may then be cleaved with these restriction enzymes for
insertion into an expression vector, and a vector for use in the
yeast three hybrid system (Tirode, F., et al., J. Biol. Chem.
272:22995-22999 (1997)). For example, an Xho I and Kpn I
restriction sites can be inserted at the 5' and 3' ends of the
fusion protein gene, respectively. Cleavage with these restriction
enzymes will then facilitate cloning of the fusion protein gene
into (i) the pBAD/His expression vector (Invitrogen), for arabinose
dependent expression of anti-LRP-A.beta. in E. coli, and (ii) the
pLex9-3H vector for use in the yeast three hybrid system (Tirode,
F., et al., J. Biol. Chem. 272:22995-22999 (1997)). The protein
product, named anti-LRP-A.beta. peptide, of the resulting gene
should have both A.beta. and LRP binding properties.
[0089] The ability of anti-LRP-A.beta. molecule to bind A.beta. and
LRP and to undergo LRP mediated endocytosis and degradation may be
tested using gel-filtration chromatography, immunoblotting and cell
culture techniques. If the anti-LRP-A.beta. molecule is a peptide,
a yeast-three-hybrid system may also be used to evaluate the
anti-LRP-A.beta. peptide (Tirode, F., et al., J. Biol. Chem.
272:22995-22999 (1997)). If necessary, the binding properties of an
anti-LRP-A.beta. peptide may be reoptimized using in vivo evolution
techniques (Buchholz, F., et al., Nat. Biotechnol. 16:657-662
(1998)).
[0090] Gel-filtration chromatograpy can be performed as described
by Narita et al. (Narita, M., et al., J. Neurochem. 69:1904-1911
(1997)) to test the ability of an anti-LRP-A.beta. molecule to bind
A.beta.. The anti-LRP-A.beta. molecule is incubated with
A.beta.1-42 that is radiolabeled with .sup.3H, .sup.14C or
.sup.121I. In the following discussion, .sup.125I-A.beta. is used
as an example of radiolabeled A.beta.. Methylamine or trypsin
activated .alpha..sub.2M, and .alpha..sub.2M, and unactivated
.alpha..sub.2M and .alpha..sub.2M-2, may be used as controls.
anti-LRP-A.beta./.sup.125I-A.beta.,
.alpha..sub.2M/.sup.125I-A.beta. and
.alpha..sub.2M-2/.sup.125I-A.beta. complexes are then separated
from unbound .sup.125I-A.beta. using a Superose 6 gel-filtration
column (0.7.times.20 cm) under the control of an FPLC (Pharmacia)
that has been standardized with molecular weight markers from 1000
kD-4 kD. If anti-LRP-A.beta. has bound .sup.125I-A.beta.,
.sup.125I-A.beta. should be detected by gamma counter at two peaks,
one corresponding to the molecular weight of the
anti-LRP-A.beta./.sup.125I-A.beta. complex (about 8-9 kD for a
complex containing an anti-LRP-A.beta. of about 40 residues), and
one corresponding to the molecular weight of .sup.125I-A.beta. (4.5
kD).
[0091] Alternatively, or in addition to gel-filtration
chromatography, immunoblotting methods (Narita, M., et al., J.
Neurochem. 69:1904-1911 (1997)) may be used to determine whether an
anti-LRP-A.beta. molecule can bind A.beta.. Unlabeled A.beta. is
incubated separately with anti-LRP-A.beta., unactivated
.alpha..sub.2M, unactivated .alpha..sub.2M-2, .alpha..sub.2M
activated by methylamine or trypsin, or .alpha..sub.2M-2 activated
by methylamine or trypsin. Samples are then electrophoresed on a 5%
SDS-PAGE, under non-reducing conditions, transferred to polyvinyl
difluoride nitrocellulose membrane, and probed with anti-A.beta.
IgG, or an antibody specific for the anti-LRP-A.beta. molecule.
Where one or more domains of the anti-LRP-A.beta. molecule are
derived from .alpha..sub.2M, an anti-.alpha..sub.2M IgG that
recognizes the domain derived from .alpha..sub.2M may be used, such
as anti-.alpha..sub.2M IgG raised against the LRP binding domain of
.alpha..sub.2M (for example, Marynen, P., et al., J. Immunol.
127:1782-1787 (1981)). If the anti-LRP-A.beta./A.beta. sample may
be detected by both the antibody against anti-LRP-A.beta., and
anti-A.beta. IgG it can be concluded that the anti-LRP-A.beta.
molecule can bind A.beta.. Where the A.beta. binding domain of the
anti-LRP-A.beta. molecule is derived from A.beta., the anti-A.beta.
antibody should be tested to ensure that it does not recognize the
anti-LRP-A.beta. molecule. Several antibodies against A.beta. are
available, including 6310, WO2, 4G8, G210 and G211. Antibody 4G8
may recognize an anti-LRP-A.beta. molecule for which A.beta.
binding domain is derived from A.beta.. In addition, some
anti-.alpha..sub.2M antibodies may not recognize an
anti-LRP-A.beta. molecule derived from .alpha..sub.2M, therefore,
they should be tested for the ability to recognize the peptide
prior to performing the immunoblotting, endocytosis, and
degradation protocols described herein. Marynen et al., (Marynen,
P., et al., J. Immunol. 127:1782-1787 (1981)) describe an
anti-.alpha..sub.2M antibody raised against the LRP binding domain
that may be able to recognize an anti-LRP-A.beta. peptide having an
LRP binding domain derived from .alpha..sub.2M. Other
anti-.alpha..sub.2M antibodies are available from Sigma and Cortex
Biochem (San Leandro, Calif., U.S.A.). .alpha..sub.2M can be
obtained from Sigma, or purified from human plasma and activated as
described in Warshawsky, I., et al., J. Clin. Invest. 92:937-944
(1993). Synthetic A.beta..sub.1-42 can be purchased from Bachem
(Torrance, Calif., U.S.A.).
[0092] Gel-filtration chromatography and immunoblotting as
described above may also be used to determine the ability of
anti-LRP-A.beta. to bind LRP, by using labeled soluble LRP (for
example, the extracellular region of LRP) in place of labeled
A.beta. for gel-filtration chromatography experiments, and anti-LRP
IgG in place of anti-A.beta. IgG for immunoblotting experiments.
Alternatively, for the immunoblotting protocol, the
anti-LRP-A.beta. molecule may be labeled with fluorescent or
radioactive label. For a labeled anti-LRP-A.beta. molecule, it can
be concluded that the anti-LRP-A.beta. molecule can bind A.beta. if
the labeled band corresponds to a band recognized by anti-A.beta.
antibody.
[0093] The ability of A.beta./anti-LRP-A.beta. complexes to undergo
LRP mediated endocytosis and subsequent degradation can be
determined using cell culture experiments using cells that express
LRP as described by Kounnas et al. (Kounnas, M. Z., et al., Cell
82:331-340 (1995); Kounnas, M. Z., et al., J. Biol. Chem.
270:9307-9312 (1995)). The amount of radioligand that is
internalized or degraded by cells has been described previously
(Kounnas, M. Z., et al., Cell 82:331-340 (1995); Kounnas, M. Z., et
al, J. Biol. Chem. 270:9307-9312 (1995)). Cells that express LRP
include, but are not limited to, adipocytes, astrocytes,
fibroblasts, hepatocytes, macrophages, monocytes, and
syncytiotrophoblasts. In one preferred embodiment of the invention,
mouse embryo fibroblasts are used for the cell culture
experiment.
[0094] Cells expressing LRP are incubated for 18 hours with
.sup.125I-A.beta. (alternatively, A.beta. may be labeled with
.sup.3H or .sup.14C) in the presence or absence of
anti-LRP-A.beta., unactivated .alpha..sub.2M, unactivated
.alpha..sub.2M-2, .alpha..sub.2M activated by methylamine or
trypsin, or .alpha..sub.2M-2 activated by methylamine or trypsin;
in the presence or absence of RAP (400 nM). RAP is an inhibitor of
LRP ligand binding, and is added to determine if endocytosis is LRP
mediated. RAP can be isolated and purified from a glutathione
S-transferase fusion protein expressed in E. coli as described in
Warshawsky, I., et al., J. Clin. Invest. 92:937-944 (1993b). To
assess endocytosis rather than degradation, chloroquine (0.1 mM) is
added at the same time as anti-LRP-A.beta./.sup.125I-A.beta. to
inhibit lysosomal degradation of .sup.125I-A.beta..
[0095] The amount of radioactive ligand released by treatment with
trypsin-EDTA, proteinase K solution defines the surface-bound
material, and the amount of radioactivity associated with the cell
pellet defines the amount or internalized ligand. Activated
.alpha..sub.2M/.sup.125I-A.b- eta. will serve as positive control.
Under the conditions described, more than 8 fmoles/10.sup.4 cells
of activated .alpha..sub.2M/.sup.125I-A.beta- . should be
internalized after 18 hours of incubation (Kounnas, M. Z., et al.,
Cell 82:331-340 (1995)). Unactivated
.alpha..sub.2M/.sup.125I-A.beta- . will serve as the negative
control for endocytosis, because .alpha..sub.2M must be activated
by trypsin or methylamine to be recognized by LRP. If the amount of
anti-LRP-A.beta./.sup.125I-A.beta. is greater than 4-8
fmoles/10.sup.4 cells, it can be concluded that
anti-LRP-A.beta./.sup.125I-A.beta. has the ability to undergo LRP
mediated endocytosis. Unactivated .alpha..sub.2M/.sup.125I-A.beta.,
and activated .alpha..sub.2M/.sup.125I-A.beta. in the presence of
RAP should not be internalized, therefore no more than 2-4
fmoles/10.sup.4 cells should be internalized (Kounnas, M. Z., et
al., Cell 82:331-340 (1995)). Internalization of the
anti-LRP-A.beta./.sup.125I-A.beta. complex will be deemed abolished
if anti-LRP-A.beta./.sup.125I-A.beta., in the presence and absence
of RAP, and unactivated .alpha..sub.2M/.sup.125I-A.beta. show the
same amount of radioactivity associated with the cell pellet.
[0096] To determine the ability of A.beta./anti-LRP-A.beta.
complexes to undergo degradation after endocytosis, this cell
culture protocol is repeated without chloroquine. The radioactivity
appearing in the cell culture medium that is soluble in 10%
trichloroacetic acid is taken to represent degraded
.sup.125I-A.beta. (Kounnas, M. Z., et al., Cell 82:331-340 (1995);
Narita, M., et al., J. Neurochem. 69:1904-1911 (1997)). Total
ligand degradation is corrected for the amount of degradation that
occurs in control wells lacking cells. Because free
.sup.125I-A.beta. can be degraded in an LRP independent manner,
degradation is measured for anti-LRP-A.beta. and .alpha..sub.2M
complexes with .sup.125I-A.beta., as well as for free
.sup.125I-A.beta., in the presence and absence of RAP. Using the
same positive and negative controls as above, if RAP does not
decrease the amount of TCA soluble radioactivity by at least 30%
for the anti-LRP-A.beta./.sup.125I-A.beta. complex, it can be
concluded that .sup.125I-A.beta. ligand of anti-LRP-A.beta. is not
degraded.
[0097] Another method of testing the ability of anti-LRP-A.beta.
molecule to bind A.beta. and LRP is the yeast three-hybrid system
described by Tirode et al. (Tirode, F., et al., J. Biol. Chem.
272:22995-22999 (1997)). This method may be used where the
anti-LRP-A.beta. molecule is a peptide. In this system, yeast
growth only occurs when the "bait" recognizes both the "hook" and
the "fish" (FIG. 7). In this instance, the "hook" is constructed of
the DNA coding for A.beta. (Bales, K. R., et al., Nat. Genet.
17:264 (1997)), fused to the coding sequence of the LexA DNA
binding protein in pLex9-3H, a TRP1 episomal vector (Tirode, F., et
al., J. Biol. Chem. 272:22995-22999 (1997)). The "fish" is
constructed of the coding sequence for the 515 kD extracellular
domain of LRP, fused to the B42 activation domain in pVP 16, a LEU2
episomal vector (Tirode, F., et al., J. Biol. Chem. 272:22995-22999
(1997)). The "bait" is the DNA coding for anti-LRP-A.beta. in the
pLex9-3H vector, expression of anti-LRP-A.beta. is repressed by
methionine. After transformation of yeast with these vectors,
transcription of the Leu 2 reporter gene will occur only when the
A.beta. fused DNA binding domain is brought into proximity to the
transcriptional activation domain fused to LRP. The A.beta./LRP
binding fusion peptide should promote reporter gene transcription.
The interaction between anti-LRP-A.beta. and A.beta. and LRP (515
kD) will be considered positive only if reporter gene expression
(yeast growth) occurs when A.beta.-LexA, LRP (515 kD)-B42, and
anti-LRP-A.beta. are expressed.
[0098] The anti-LRP-A.beta. molecule of the invention may be
administered per se, or in the form of a pharmaceutically
acceptable salt with any non-toxic, organic or inorganic acid.
Illustrative inorganic acids which form suitable salts include
hydrochloric, hydrobromic, sulfuric and phosphoric acid, and acid
metal salts such as sodium monohydrogen orthophosphate and
potassium hydrogen sulfate. Illustrative organic acids which form
suitable salts include the mono, di and tricarboxylic acids.
Illustrative of such acids are, for example, acetic, glycolic,
lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic,
tartaric, citric, ascorbic, maleic, hydroxymaleic, benzoic,
hydroxybenzoic, phenylacetic, cinnamic, salicylic, 2-phenoxybenzoic
and sulfonic acids such as methane sulfonic acid and
2-hydroxyethane sulfonic acid. Salts of the carboxy terminal amino
acid moiety include the non-toxic carboxylic acid salts formed with
any suitable inorganic or organic bases. Illustratively, these
salts include those of alkali metals, as for example, sodium and
potassium; alkaline earth metals, such as calcium and magnesium;
light metals of Group IIIA including aluminum; and organic primary,
secondary and tertiary amines, as for example, trialkylamines,
including triethylamine, procaine, dibenzylamine, 1-ethenamine,
N,N'-dibenzylethylenediamine, dihydroabietylamine,
N-(lower)alkylpiperidine, and any other suitable amine.
[0099] The amount of the anti-LRP-A.beta. molecule administered to
a subject will vary depending upon the age, weight, and condition
of the subject. The course of treatment may last from several days
to several months or until a cure is effected or a diminution of
disease state is achieved, or alternatively may continue for a
period of years, for example, when used prophylactically. Optimal
dosing schedules can be calculated from measurements of drug
accumulation in the body. Persons of ordinary skill can easily
determine optimum dosages, dosing methodologies and repetition
rates. However, the amount of anti-LRP-A.beta. molecule
administered to a subject is generally from 0.1 nanograms to 10
mg/kg/day, and is typically an amount ranging from 1 nanogram to 1
mg/kg/day.
[0100] The present invention also relates to antisense
oligonucleotides targeted to A2M-2 RNA, and to their use as
therapeutic agents for AD and for suppressing A2M-2 expression.
Partial or total deletion of the sequences coding for the bait
region in exons 17 and 18 of .alpha..sub.2M is likely to modify
protease binding, interfering with .alpha..sub.2M activation.
Incorporation of one or more mutant monomers into tetramers might
thereby result in defective tetramers that could not be activated
efficiently and, therefore, could not undergo subsequent
endocytosis via LRP. Thus, the generation of very low levels of
mutant monomers may have an amplified effect as one mutant monomer
may potentially inhibit the function of three wild-type monomers in
the tetramer (dominant negative effect). One way to counter this
dominant negative effect is to decrease the level of abnormal
.alpha..sub.2M by interfering with gene expression at the RNA
level. For this purpose, an antisense oligonucleotide specific for
A2M-2 RNA can be used. This oligonucleotide will be referred to
herein as A2M-2 antisense oligonucleotide. The A2M-2 antisense
oligonucleotide may be targeted to any A2M-2 RNA molecule, but in a
preferred embodiment of the invention, it is targeted to
heterologous nuclear (hnRNA).
[0101] The A2M-2 deletion is found in the splicing region of exon
18, therefore, in one embodiment of the invention, the A2M-2
antisense oligonucleotide is designed to target A2M-2 RNA
transcripts before splicing occurs, referred to as hnRNA. In
addition, in order to be specific for A2M-2 hnRNA the A2M-2
antisense oligonucleotide is designed to target the pentanucleotide
deletion found in A2M-2. In another embodiment of the invention,
the A2M-2 antisense oligonucleotide is designed to target A2M-2
mRNA. The A2M-2 deletion results in several variant mRNA
transcripts with varying sequences. The A2M-2 antisense
oligonucleotides of the invention can be designed to target
individual variants, or to target more than one of these variants.
In addition, A2M-2 antisense oligonucleotides targeting different
A2M-2 mRNA variants, or targeting A2M-2 hnRNA, may be used either
alone, or in conjunction with one another.
[0102] In addition, the A2M-2 antisense oligonucleotide must be
long enough so that it targets only A2M-2, but short enough to
optimize delivery. Thus, the antisense oligonucleotide of the
invention is preferably 8-50 nucleotides in length, and more
preferably 15-30 nucleotides in length. Therefore, in one
embodiment of the invention, the A2M-2 antisense oligonucleotide is
8-50 nucleotides and is complementary to the coding strand of the
region of A2M-2 containing the site of the pentanucleotide
deletion. In a preferred embodiment of the invention, the A2M-2
antisense oligonucleotide is composed of 15-30 contiguous
nucleotides of a region complementary to the site on the coding
strand of A2M-2 that contains the pentanucleotide deletion. In
another embodiment of the invention, the A2M-2 antisense
oligonucleotide is composed of the last 8-50 contiguous nucleotides
of SEQ ID NO:27. In a preferred embodiment of the invention, the
A2M-2 antisense oligonucleotide is composed of the last 15-30
contiguous nucleotides of SEQ ID NO:27. In yet another preferred
embodiment, the A2M-2 antisense oligonucleotide is composed of
nucleotides 36-50 of SEQ ID NO:27. In another preferred embodiment
of the invention, the A2M-2 antisense oligonucleotide is composed
of nucleotides 20-50 of SEQ ID NO:27.
[0103] The A2M-2 antisense oligonucleotide may be DNA or RNA, i.e.,
it may be composed of deoxyribonucleic acids or ribonucleic acids,
respectively. Alternatively, the oligonucleotide may be composed of
nucleotides with a phosphorothioate backbone to render the
oligonucleotide more resistant to enzymatic degradation (van der
Krol, A. R., et al., Biotechniques 6:958-976 (1988); Cazenave, C.
& & Hlne, C., "Antisense Oligonucleotides," in: Antisense
nucleic acids and proteins: Fundamental and applications, Mol, J.
N. M. & van der Krol, A. R., eds., M. Dekker, publ., New York,
pp. 1-6 (1991); Milligan, J. F., et al., J. Med. Chem. 36:1923-1937
(1993)). In a preferred embodiment of the invention the A2M-2
antisense oligonucleotide is DNA.
[0104] Other modifications which may be used to protect the
oligonucleotide include chemical changes to the 3' end of the
oligonucleotide (van der Krol, A. R., et al., Biotechniques
6:958-976 (1988); Khan, I. M. & Coulson, J. M., Nucleic Acids
Res. 21:2957-2958 (1993); Tang, J. Y., et al. Nucleic Acids Re.
21:2729-2735 (1993)) or biotynylation of the 3' end followed by
conjugation with avidin (Boado, R. J. & Pardridge, W. M.,
Bioconjugate Chem. 3:519-523 (1992)). Alternatively, lipofection
may be used to deliver the oligonucleotide, i.e., packaging the
oligonucleotide in lipid (McCarthy, M. M., et al., Endocrin.
133:433-439 (1993b); Ogawa, S., et al., J. Neurosci. 14:1766-1774
(1994)). Lipofection protects the oligonucleotide from nucleases
and may aid in delivery to the central nervous system.
[0105] The A2M-2 antisense oligonucleotide can be easily
synthesized by means of commercially-available automatic DNA
synthesizers such as a DNA synthesizer manufactured by Applied
Biosystems, or MilliGen, etc. In addition, methods of synthesizing
oligonucleotides are well known in the art and are described, for
example, in Oligonucleotides and Analogues a Practical Approach,
Eckstein, F., ed.,Oxford University Press, publ. New York, (1991),
and "Synthesis and Purification of Oligonucleotides" in: Current
Protocols in Molecular Biology, Ausubel, F. M., et al., eds., John
Wiley & Sons, Inc., publ., Vol. 1, .sctn..sctn. 2.11-2.12
(Suppl. 9 1993).
[0106] The invention also relates to pharmaceutical compositions
containing the A2M-2 antisense oligonucleotide, and one or more
pharmaceutically acceptable carriers. In addition, the invention
provides a method of treating AD and/or of suppressing A2M-2
expression by administering the A2M-2 antisense oligonucleotide to
a subject. Preferably, the A2M-2 antisense oligonucleotide is
delivered to a subject who has been determined to be heterozygous
or homozygous for the A2M-2 allele. Procedures for selecting and
assessing subjects who are heterozygous or homozygous for A2M-2 are
described in Tanzi et al., U.S. Ser. No. 09/148,503, PCT
Application No. PCT/US98/18535, and Blacker, D., et al., Nat.
Genet. 19:357-360 (August 1998). In another preferred embodiment of
the invention, treatment of a subject with the A2M-2 antisense
oligonucleotide is done in conjunction with a therapy designed to
replace or supplement .alpha..sub.2M function.
[0107] Antisense oligonucleotides have been safely administered to
humans and several clinical trials are presently underway. Based on
these clinical trials, oligonucleotides are understood to have
toxicities within acceptable limits at dosages required for
therapeutic efficacy. One such antisense oligonucleotide,
identified as ISIS 2105, is presently in clinical trials, and is
used as a therapeutic against papillomavirus. Another antisense
oligonucleotide, ISIS 2922, has been shown to have clinical
efficacy against cytomegalovirus-associated retinitis Antiviral
Agents Bulletin 5: 161-163 (1992); BioWorld Today, Dec. 20, 1993.
Therefore, it has been established that oligonucleotides are useful
therapeutic agents and that they can be used for treatment of
animals, especially humans.
[0108] The amount of the A2M-2 antisense oligonucleotide
administered to a subject will vary depending upon the age, weight,
and condition of the subject. The course of treatment may last from
several days to several months or until a cure is effected or a
diminution of disease state is achieved, or alternatively may
continue for a period of years, for example, when used
prophylactically. Optimal dosing schedules can be calculated from
measurements of drug accumulation in the body. Persons of ordinary
skill can easily determine optimum dosages, dosing methodologies
and repetition rates. Optimum dosages may vary depending on the
relative potency of individual oligonucleotides, and can generally
be estimated based on EC.sub.50's in in vitro and in vivo animal
studies. In general, dosage is from 0.01 mg to 100 g and may be
given once daily, weekly, monthly or yearly.
[0109] Another therapeutic method of the invention is gene therapy
to supplement .alpha..sub.2M function. Because the A2M-2 deletion
may result in impaired .alpha..sub.2M function, a strategy aimed at
supplementing normal .alpha..sub.2M, such as gene therapy, could
serve as a means for treating, preventing or reversing AD. One
embodiment of the invention is a viral vector carrying a transgene
encoding wild type .alpha..sub.2M, or an anti-LRP-A.beta. peptide.
Viral vectors suitable for use in the invention are those that are
capable of transfecting nondividing, post-mitotic cells, and have
low cytotoxicity. These vectors include, but are not limited to
adenovirus, lentivirus, and HSV-1, but are preferably
adeno-associated virus vector (AAV). AAV is a DNA virus that is not
directly associated with any human disease, and therefore should
present a lower risk of cytotoxicity (Freese, A. et al., Epilesia
38:759-766 (1997)). It can transfect nondividing, post-mitotic
cells, such as neurons and dormant glial cells. In addition, there
is some evidence that AAV may stably integrate into the host
chromosome (Freese, Z. et al., Mov. Disord. 11:469-488 (1996);
Kaplitt, M. G. et al., Natur. Genet. 8:148-154 (1994); Samulski, R.
J., et al., J. Virol 63:3822-3888 (1989); Kotin, R. M. et al.,
Proc. Natl. Acad. Sci. U.S.A. 87:2211-2215 (1990); Samulski, R. J.
et al., E.M.B.O. J. 10:3941-3950 (1991); Muzyczka, N., Curr.
Topics. Microbiol. Immunol. 158: 97-129 (1992)). Recently, AAV was
successfully used to deliver a reporter transgene to human
hippocampal tissue (Freese, A. et al., Epilesia 38:759-766
(1997)).
[0110] Transgenes to be used in the viral vector include the full
length cDNA encoding .alpha..sub.2M (SEQ ID NO:1), or the
anti-LRP-A.beta. peptide described above. The construction of
AAVlacZ is described by Kaplitt, et al., and Samulski et al.
(Kaplitt, M. G., et al., Nature Genet. 8:148-154 (1994); Samulski,
R. J., et al., J. Virol. 63:3822-3888 (1989)). To insert the
transgene into the viral vector, the viral vector is first cut with
restriction enzymes. PCR mediated integration is used to create
corresponding restriction sites at the 3' and 5' ends of the
transgene, and the transgene is ligated with AAV.
[0111] The invention also provides a method of combating AD by
administering the viral vector carrying an .alpha..sub.2M, or an
anti-LRP-A.beta. peptide transgene and pharmaceutical compositions
containing this viral vector.
[0112] The gene therapy of the invention can be administered using
in vivo or ex vivo strategies. The in vivo approach involves the
introduction of the viral vector directly into the tissue of the
subject. In vivo methods of administration include direct injection
into cerebrospinal fluid, or by stereotactic intracerebral
inoculation into the hippocampus. In addition, some viral vectors,
such as adenovirus, can be transported in a retrograde manner from
the point of injection (Ridoux, V., et al., Brain Res. 648:171-175
(1994); Kuo, H., et al., Brain Res. 24:31-38 (1995)). Other routes
of administration include nasal inhalation (Draghia, R., Gene Ther.
2:418-423 (1995)) and injection into the carotid artery after
disruption of the blood brain barrier (Doran, S. E., et al.,
Neurosurgery 36:965-970 (1995); Muldoon, L. L., Am. J. Pathol.
147:1840-1851 (1995)).
[0113] For the ex vivo approach, a suitable cell type, such as
fibroblasts myoblasts, or neural progenitor cells, is harvested
from a donor and grown in tissue culture. The cells are then
transfected, and the cells harvested and implanted in the subject.
Ex vivo methods are described, for example, at Raymon, H. K., et
al., Exper. Neurol. 144:82-91 (1997); Rosenberg, M. B., et al.,
Science 2442:1575-1578 (1988); Suhr, S. T., and Gage, F. H., Arch.
Neurol. 50:1252-1268 (1993); Tuszynski, M. H., et al.,Exp. Neurol.
126:1-14 (1994); Ridoux, V. et al., Neuroreport 5:801-804 (1994);
Buc-Caron, M. H., Neurobiol. Dis 2:37-47 (1995); Sabat, O., et al.,
Nat. Genet. 9:256-260 (1995).
[0114] The amount of viral vector carrying a transgene administered
to a subject will vary depending upon the age, weight, and
condition of the subject. The course of treatment may last from
several days to several months or until a cure is effected or a
diminution of disease state is achieved, or alternatively may
continue for a period of years, for example, when used
prophylactically. Optimal dosing schedules can be calculated from
measurements of drug accumulation in the body. Persons of ordinary
skill can easily determine optimum dosages, dosing methodologies
and repetition rates. In general, dosage is from 1.times.10.sup.4
to 1.times.10.sup.10 plaque forming units (pfu), but is preferably
1.times.10.sup.6 to 5.times.10.sup.7 pfu/kg and may be given once
daily, weekly, monthly or yearly.
[0115] The therapeutic agents of the invention can be administered
alone, or in concert with one another or with other therapeutic
agents. For example, a subject may be treated with both the
anti-LRP-A.beta. molecule and the antisense oligonucleotide of the
invention, to provide both a supplement of A2M function, and to
block defective A2M function at the same time.
[0116] Suitable subjects for carrying out the present invention are
typically male or female human subjects, and include both those
which have previously been determined to be at risk of developing
AD, and those who have been initially diagnosed with AD. The
present invention may be employed in combating both familial AD
(late onset and early onset) as well as sporadic AD. One preferable
group of subjects are those who have been determined to be
heterozygous or homozygous for the A2M-2 allele. Procedures for
selecting and assessing subjects who are heterozygous or homozygous
for A2M-2 are described in Tanzi et al., U.S. Ser. No. 09/148,503,
PCT Application No. PCT/US98/18535, and Blacker, D., et al., Nat.
Genet. 19:357-360 (August 1998), all of which are herein
incorporated by reference.
[0117] When the therapeutic agents as mentioned above are used as
preventive or therapeutic agents for Alzheimer's disease, they may
be made into preparations which satisfy the necessary requirements
of the particular administering route together with usual carriers.
For example, in the case of oral administration, preparations in
the form of tablets, capsules, granules, diluted powder, liquid,
etc. are prepared.
[0118] Pharmaceutical compositions containing the therapeutic
agents of the invention, may be prepared in either solid or liquid
form. To prepare the pharmaceutical compositions of this invention,
one or more of the therapeutic agents is intimately admixed with a
pharmaceutical carrier according to conventional pharmaceutical
compounding techniques, which carrier may take a wide variety of
forms depending on the form of preparation desired for
administration, for example, oral or parenteral. By
"pharmaceutically acceptable carrier" is meant a non-toxic solid,
semisolid or liquid filler, diluent, encapsulating material, or
formulation auxiliary of any type. In preparing the compositions in
oral dosage form, any of the usual pharmaceutical media may be
employed. Thus, for liquid oral preparations, such as for example,
suspensions, elixirs and solutions, suitable carriers and additives
include water, glycols, oils, alcohols, flavoring agents,
preservatives, coloring agents and the like; for solid oral
preparations such as, for example, powders, capsules and tablets,
suitable carriers and additives include starches, sugars, diluents,
granulating agents, lubricants, binders, disintegrating agents and
the like. In addition to such pharmaceutical carriers, cationic
lipids may be included in the formulation to facilitate
oligonucleotide uptake. One such composition shown to facilitate
uptake is LIPOFECTIN (GIBCO-BRL, Bethesda, Md.).
[0119] Because of their ease in administration, tablets and
capsules represent the most advantageous oral dosage unit form, in
which case solid pharmaceutical carriers are employed. If desired,
tablets may be sugar coated or enteric coated by standard
techniques. For parenterally injectable compositions, the carrier
will usually comprise sterile, pyrogen-free water, or sterile,
pyrogen-free physiological saline solution, though other
ingredients, for example, for purposes such as aiding solubility or
for preservatives, may be included. Parenterally injectable
suspensions (for example, for intravenous or intrathecal injection)
may also be prepared, in which case appropriate liquid carriers,
suspending agents and the like may be employed. See generally
Remington's Pharmaceutical Sciences (18th ed.) Mack Publishing Co.
(1990).
[0120] The pharmaceutical compositions of this invention may be
administered in a number of ways depending upon whether local or
systemic treatment is desired, and upon the area to be treated.
Administration may be topical (including ophthalmic, vaginal,
rectal, intranasal, transdermal), oral or parenteral, for example,
by intravenous drip, subcutaneous, intraperitoneal or intramuscular
injection or intrathecal or intraventricular administration.
Formulations for topical administration may include transdermal
patches, ointments, lotions, creams, gels, drops, suppositories,
sprays, liquids and powders. Compositions for oral administration
include powders or granules, suspensions or solutions in water or
non-aqueous media, capsules, sachets, or tablets. Thickeners,
flavorings, diluents, emulsifiers, dispersing aids or binders may
be desirable. Compositions for intrathecal or intraventricular
administration may include sterile aqueous solutions which may also
contain buffers, diluents and other suitable additives.
Formulations for parenteral administration may include sterile
aqueous solutions which may also contain buffers, diluents and
other suitable additives.
[0121] When necessary, the pharmaceutical composition may be
prepared so that the therapeutic agent passes through the
blood-brain barrier. One way to accomplish transport across the
blood-brain barrier is to couple or conjugate the therapeutic agent
to a secondary molecule (a "carrier"), which is either a peptide or
a non-proteinaceous moiety. The carrier is selected such that it is
able to penetrate the blood-brain barrier. Examples of suitable
carriers are pyridinium, fatty acids, inositol, cholesterol, and
glucose derivatives. Alternatively, the carrier can be a compound
which enters the brain through a specific transport system in brain
endothelial cells, such as transport systems for transferring
insulin, or insulin-like growth factors I and II. This combination
of therapeutic agent and carrier is called a prodrug. Upon entering
the central nervous system, the prodrug may remain intact or the
chemical linkage between the carrier and therapeutic agent may be
hydrolyzed, thereby separating the carrier from the therapeutic
agent. See generally U.S. Pat. No. 5,017,566 to Bodor.
[0122] An alternative method for transporting the therapeutic agent
across the blood-brain barrier is to encapsulate the carrier in a
lipid vesicle such as a microcrystal or liposome. Such lipid
vesicles may be single or multi-layered, and encapsulate the
therapeutic agent either in the center thereof or between the
layers thereof. Such preparations are well known. For example, PCT
Application WO 91/04014 of Collins et al. describes a liposome
delivery system in which the therapeutic agent is encapsulated
within the liposome, and the outside layer of the liposome has
added to it molecules that normally are transported across the
blood-brain barrier. Such liposomes can target endogenous brain
transport systems that transport specific ligands across the
blood-brain barrier, including but not limited to, transferring
insulin, and insulin-like growth factors I and II. Alternatively,
antibodies to brain endothelial cell receptors for such ligands can
be added to the outer liposome layer. U.S. Pat. No. 4,704,355 to
Bernstein describes methods for coupling antibodies to
liposomes.
[0123] Another method of formulating the therapeutic agent to pass
through the blood-brain barrier is to prepare a pharmaceutical
composition as described above, wherein the therapeutic agent is
encapsulated in cyclodextrin. Any suitable cyclodextrin which
passes through the blood-brain barrier may be employed, including
.beta.-cyclodextrin, .gamma.-cyclodextrin, and derivatives thereof.
See generally U.S. Pat. No. 5,017,566 to Bodor; U.S. Pat. No.
5,002,935 to Bodor; U.S. Pat. No. 4,983,586 to Bodor.
[0124] Another method of passing the therapeutic agent through the
blood-brain barrier is to prepare and administer a pharmaceutical
composition as described above, with the composition further
including a glycerol derivative as described in U.S. Pat. No.
5,153,179 to Eibl.
[0125] An alternative method of delivering the therapeutic agent to
the brain is to implant a polymeric device containing the agent,
which device is able to provide controlled release delivery of the
agent to the brain for an extended period after implantation.
Examples of such implantable polymeric devices are described in
U.S. Pat. No. 5,601,835 to Sabel, and in U.S. Pat. No. 5,846,565,
to Brem.
[0126] Another aspect of the invention relates to methods of
screening for therapeutic agents for AD that can replace or
supplement normal .alpha..sub.2M function and activities, and/or
suppress defective .alpha..sub.2M function.
[0127] The invention provides for a method of screening for
therapeutic agents for AD that can suppress the production of RNA
encoding .alpha..sub.2M-2 variants, and thereby suppress the
production of .alpha..sub.2M-2 variants. One embodiment of the
invention is a method for screening for therapeutic agents by
incubating cells that are heterozygous or homozygous for A2M-2, and
that express A2M-2, with a test agent, and determining whether the
agent increases the ratio of normal to aberrant A2M mRNA.
Preferably the cells used are heterozygous for the A2M-2 allele,
with the other allele being A2M-1 (2M-1/2 cells). Examples of cells
that may be used for this assay include, but are not limited to,
glioma cells, hepatocytes, and hepatoma cell lines. In addition,
cells used for the assay may be transformed or transfected to
enable them to grow indefinitely in culture. To screen for these
agents, the cells carrying are incubated with the test agent,
preferably, for a period ranging from 2 hours to 24 hours. The
incubation period may be longer or shorter depending on the agent,
and suitable incubation periods can be determined by one of
ordinary skill in the art. Cells homozygous for A2M-1 are used as a
control. Procedures for A2M-2 genotyping are described in Tanzi et
al., U.S. Ser. No. 09/148,503, PCT Application No.
PCT/US98/18535,and Blacker, D., et al., Nat. Genet. 19:357-360
(August 1998). After incubation, the ratio of normal to aberrant
.alpha..sub.2M mRNA transcripts is determined, and compared to the
ratio for cells (with the same genotype as the cells treated with
agent) untreated with agent, and for A2M-1/1 cells untreated with
agent. An increase in the ratio of normal to aberrant
.alpha..sub.2M mRNA transcripts relative to this ratio for cells
untreated with the agent would indicate an effective agent. This
ratio for A2M-1/2 cells untreated with an agent is typically from
5:1 to 20:1. If the ratio of normal to aberrant .alpha..sub.2M mRNA
transcripts approaches the ratio found in A2M-1/1 cells untreated
with agent, the agent will be considered effective. Thus, for
example, if the ratio in A2M-1/2 cells is 10:1, and the ratio in
A2M-1/1 cells is100:1, a test agent that results in the ratio to
20:1 would be considered effective.
[0128] The ratio of normal to aberrant transcripts may be
quantitated by S1 nuclease analysis, or by RT PCR on RNA isolated
from the glioma cells. Protocols for RNA isolation for cells in
culture, and for S1 nuclease analysis is described in "Preparation
and Analysis of RNA" in: Current Protocols in Molecular Biology,
Ausubel, F. M., et al., eds., John Wiley & Sons, Inc., publ.,
Vol. 1, .sctn. 4 (Suppl. 37 1997). S1 nuclease analysis is
performed using a single-stranded antisense probe encompassing at
least exons 17-18 (bp 2057-2284 of SEQ ID NO:1), synthesized from a
full length A 2M cDNA template (SEQ ID NO:1). Preferably, the probe
would encompass exons 17, 18 and part of exon 19. The length of the
probe is preferably from 250 bp to 500 bp long, and is more
preferably 300 bp long. The probe may be up to 4353 bp (the length
of the coding region), however, increasing the length of the probe
may decrease the accuracy of the assay. In a preferred embodiment
of the invention, the probe is complementary to nucleotides
2024-2323 of SEQ ID NO:1, in another preferred embodiment, the
probe is complementary to nucleotides 2057-2384 of SEQ ID NO:1.
After the RNA has been hybridized with the probe, and digested with
S1 nuclease, samples are run on a polyacrylamide gel with molecular
weight markers. Wild type mRNA transcript (A2M-1) should appear as
a band corresponding to the length of the probe, for example, 300
bp, A2M-2 variant transcripts should appear as smaller bands. Total
normal mRNA to total variant mRNA is compared and the ratio of
normal to aberrant determined.
[0129] Alternatively, RT PCR may be used to quantitate mRNA
transcripts. Protocols for RT PCR are described in "The Polymerase
Chain Reaction" in: Current Protocols in Molecular Biology,
Ausubel, F. M., et al, eds., John Wiley & Sons, Inc., publ.,
Vol. 2, .sctn. 15.4 (Suppl. 17 1992). RNA isolated from the treated
and control cells is amplified using RT PCR with labeled primers
designed to amplify a region including at least exons 17-18 (bp
2057-2284 of SEQ ID NO:1), and preferably exons 17, 18 and part of
exon 19. In addition, the primers may designed to target mRNA by
synthesizing them so that they bind to the junction of two exons.
For example, in a preferred pair of primers, the first primer would
hybridize to A2M cDNA encoding the end of exon 16 and beginning of
exon 17, and the second primer would hybridize to A2M cDNA encoding
the end of exon 18, and beginning of exon 19. The primers may be
from 8-50 nucleotides in length, but are preferably 15-30
nucleotides in length, and are more preferably 15 nucleotides in
length. The PCR product is then run on a polyacrylamide gel with
molecular weight markers. Bands corresponding to wild type mRNA
transcripts should correspond to the length of A2M-1 cDNA
corresponding to the far ends of the primers used. For example,
wild type mRNA amplified by primers designed to amplify the last 5
base pairs of exon 16 to the first 5 base pairs of exon 19 (bp
2052-2289 of SEQ ID NO:1), would be 238 nucleotides. If the primers
were designed to amplify a region starting at the beginning of exon
17, including exon 18, and ending after the first 100 nucleotides
of exon 19 (bp 2057-2456 of SEQ ID NO:1) the expected fragment
length would be 400 nucleotides for normal mRNA. Variant mRNA
transcripts will be shorter. Total normal mRNA to total variant
mRNA is compared and the ratio of normal to aberrant
determined.
[0130] Other methods of RNA quantitation that may be used in the
invention are well known in the art, and are described in, for
example, PCR Protocols, A Guide to Methods and Applications, Innis,
A., et al., eds., Academic Press, Inc., San Diego, Calif., pub.,
pp. 60-75 (1990).
[0131] Another embodiment of the invention is to screen for
nontoxic agents that can activate .alpha..sub.2M through mechanisms
other than cleavage of the bait domain. For .alpha..sub.2M
tetramers having one or more .alpha..sub.2M-2 monomers, protease
activation of the bait domain may be impaired. Because activation
is required to expose the LRP binding domain, impairment of
activation of one or more monomers of a tetramer would result in a
decreased ability to bind to LRP. Consequently, these tetramers
would be inefficient at clearing A.beta. through LRP mediated
endocytosis. However, .alpha..sub.2M may be activated through
mechanisms other than protease cleavage of the bait domain. For
example, agents other than proteases, such as methylamine, activate
.alpha..sub.2M through the thiolester site. These agents would be
able to activate defective .alpha..sub.2M monomers, exposing the
LRP binding domain (and other domains) and potentially allowing for
LRP mediated clearance of A.beta.. In addition, these agents could
be used to increase the amount of active wild type .alpha..sub.2M
tetramers, to compensate for defective .alpha..sub.2M tetramers.
Presently, effective nontoxic agents capable of activating
.alpha..sub.2M at sites other than the bait domain are unknown. The
invention provides for a method of screening for such agents.
[0132] To screen for these agents, .alpha..sub.2M is treated with a
test agent, and then tested to determine whether it has undergone a
conformational change, or for its ability to bind to LRP. The
.alpha..sub.2M used for the assay may be wild type .alpha..sub.2M,
.alpha..sub.2M-2, or .alpha..sub.2M mutants that are missing all,
or a portion of the bait domain. However, preferably, wild type
.alpha..sub.2M is used. In addition, .alpha..sub.2M used for the
assay may be in the form of dimers or tetramers, but is preferably
in the form of tetramers. For treatment of .alpha..sub.2M with the
test agent, the .alpha..sub.2M is preferably incubated with the
test agent for 2-24 hours. However, the incubation period may be
longer or shorter according to the agent, and suitable incubation
periods can be determined by one of ordinary skill in the art. To
determine whether treated .alpha..sub.2M has undergone a
conformational change, the .alpha..sub.2M electrophoretic-mobility
assay may be used. To determine the ability of treated
.alpha..sub.2M to bind to LRP, any method of measuring LRP binding
may be used, however, preferred methods include enzyme-linked
immunosorbent assays (ELISA), immunoblotting, LRP mediated
endocytosis, and LRP mediated degradation.
[0133] The .alpha..sub.2M electrophoretic mobility assay can also
be used to determine whether treated .alpha..sub.2M has been
activated, by determining whether treated .alpha..sub.2M has
undergone the conformational change expected for activated
.alpha..sub.2M. The .alpha..sub.2M electrophoretic-mobility assay
consists of analyzing the electrophoretic mobility of
.alpha..sub.2M under non-denaturing conditions after incubation
with the test agent, or as a control, a protease, or other reagent
capable of converting .alpha..sub.2M to the fast form (Barret, A.
J., et al., Biochem. J. 181: 401-418 (1979); Bowen, M. E., and
Gettins, P. W., J. Biol. Chem. 273:1825-1831 (1998)).
.alpha..sub.2M can exist in two forms easily distinguishable by
mobility in gel electrophoresis (Barret, A. J., et al., Biochem. J.
181: 401-418 (1979)). The difference in mobility is due to the
conformational change that .alpha..sub.2M undergoes after
activation with a protease or other agent, such as methylamine.
This conformational change results in an increase in
electrophoretic mobility on poly-acrylamide gels run under
non-denaturing conditions (this form is referred to as the "fast
form" of .alpha..sub.2M) (Barret, A. J., et al., Biochem. J. 181:
401-418 (1979); Bowen, M. E., and Gettins, P. W., J. Biol. Chem.
273:1825-1831 (1998)). This "slow to fast" conversion is used as
the basis for an assay for this conformational change, and the two
different .alpha..sub.2M conformations are referred to as the slow
and fast forms (Bowen, M. E., and Gettins, P. W., J. Biol. Chem,
273:1825-1831 (1998)). Conversion from the slow to fast form for
.alpha..sub.2M treated with a test agent would indicate that the
agent had activated .alpha..sub.2M. Where this assay is used to
determine the effectiveness of a test agent, the .alpha..sub.2M
treated with the agent would preferably be tetrameric.
[0134] The .alpha..sub.2M electrophoretic mobility assay and
methods of purifying .alpha..sub.2M from serum are described by
Barret et al. in Barret, A. J., et al., Biochem. J. 181: 401-418
(1979), and by Bowen et al. in Bowen, M. E., et al., Arch. Biochem.
Biophys. 337:191-201 (1997), and in Bowen, M. E., and Gettins, P.
W., J. Biol. Chem. 273:1825-1831 (1998). After incubation with the
test agent, the .alpha..sub.2M sample may be run on polyacrylamide
gel under nondenaturing conditions, such as those described in
Bowen, M. E., et al., Arch. Biochem. Biophys. 337:191-201 (1997).
The .alpha..sub.2M sample may be detected by methods well known in
the art such as by radiolabelling the protease used, or by Western
Blot using anti-.alpha..sub.2M antibodies. Activated and
unactivated .alpha..sub.2M may be used as controls for comparison
of electrophoretic mobility with the sample being analyzed.
[0135] In one embodiment of the invention, ELISA is used to
determine the ability of treated .alpha..sub.2M to bind to LRP.
ELISA protocols are described in "Immunology" in: Current Protocols
in Molecular Biology, Ausubel, F. M., et al., eds., John Wiley
& Sons, Inc., publ., Vol. 2, .sctn. 11.2 (Suppl. 15 1991). In
this assay, microtiter plate wells coated with an
anti-.alpha..sub.2M IgG that recognizes only activated
.alpha..sub.2M, such as the antibody described by Marynen et al.,
(Marynen, P., et al., J. Immunol. 127: 1782-1786 (1981)), are
incubated with the treated .alpha..sub.2M, or control molecule. The
wells are then incubated with an enzyme-conjugated
anti-.alpha..sub.2M IgG and rinsed. Next, the wells are incubated
with the substrate for the enzyme conjugate, rinsed, and the amount
of .alpha..sub.2M sample bound in the well is determined.
Alternatively, microtiter plate wells are coated with anti-LRP IgG
and rinsed. The wells are then incubated with LRP and rinsed. This
LRP is preferably soluble LRP. Then the wells are incubated with
.alpha..sub.2M treated with the test agent, untreated
.alpha..sub.2M, or activated .alpha..sub.2M, and rinsed. Next the
wells are incubated with enzyme-conjugated anti-.alpha..sub.2M IgG,
rinsed again, and then incubated with the substrate for the enzyme
that is conjugated to the anti-.alpha..sub.2M IgG. The amount of
.alpha..sub.2M sample bound in the well is then determined. In
another embodiment, wells coated with LRP are incubated with
.alpha..sub.2M treated with the test agent, untreated unactivated
.alpha..sub.2M, or untreated activated .alpha..sub.2M, and rinsed.
The wells are then incubated with enzyme-conjugated
anti-.alpha..sub.2M IgG, rinsed, and then treated with the enzyme
substrate, and the amount of .alpha..sub.2M sample bound is
determined. The anti-.alpha..sub.2M IgG may be conjugated with, for
example, horseradish peroxidase, urease or alkaline phosphatase,
but is preferably labeled with a fluorescent label, such as
4-methylumbelliferyl phosphate (MUP). The appropriate substrate is
added to the wells, the wells are washed, and then quantitated with
a microtitre plate reader.
[0136] Alternatively, the ability of .alpha..sub.2M treated with
the test agent to bind to LRP may be determined by immunoblotting
methods. Unlabeled soluble LRP is incubated separately with
.alpha..sub.2M treated with the test agent, untreated unactivated
.alpha..sub.2M, and untreated .alpha..sub.2M activated by
methylamine or trypsin. Samples are then electrophoresed on a 5%
SDS-PAGE, under non-reducing conditions, transferred to polyvinyl
difluoride nitrocellulose membrane, and probed with anti-2M IgG and
anti-LRP IgG. If the .alpha..sub.2M treated with the test agent may
be detected by both anti-.alpha..sub.2M IgG and anti-LRP IgG it can
be concluded that the treated .alpha..sub.2M can bind A.beta.. In
another method of immunoblotting, an antibody specific for the LRP
binding domain of .alpha..sub.2M, such as that described by
Marynen, et al., (Marynen, P., et al., J. Immunol. 127: 1782-1786
(1981)), is used as the anti-.alpha..sub.2M IgG, and the samples
are not incubated with LRP. Recognition of the treated
.alpha..sub.2M by this antibody indicates that .alpha..sub.2M has
been activated.
[0137] In addition, the ability of .alpha..sub.2M treated with a
test agent to bind to LRP can be determined by its ability to
undergo LRP mediated endocytosis using cell culture experiments as
described by Kounnas et al. (Kounnas, M. Z., et al., Cell
82:331-340 (1995); Kounnas, M. Z., et al., J. Biol. Chem.
270:9307-9312 (1995)). Cells expressing LRP, mouse embryo
fibroblasts, are incubated for 18 hours with .sup.125I-A.beta.
(alternatively, A.beta. may be labeled with .sup.3H or .sup.14C) in
the presence or absence of with .alpha..sub.2M treated with the
test agent, untreated unactivated .alpha..sub.2M, and untreated
.alpha..sub.2M activated by methylamine or trypsin, in the presence
or absence of RAP (400 nM). RAP is an inhibitor of LRP ligand
binding, and is added to determine if endocytosis is LRP mediated.
In addition, chloroquine (0.1 mM) is added to inhibit lysosomal
degradation of .sup.125I-A.beta..
[0138] The amount of radioactive ligand released by treatment with
trypsin-EDTA, proteinase K solution defines the surface-bound
material, and the amount of radioactivity associated with the cell
pellet defines the amount of internalized ligand. Activated
.alpha..sub.2M/.sup.125I-A.b- eta. will serve as positive control.
Under the conditions described, more than 4-8 fmoles/10.sup.4 cells
of activated .alpha..sub.2M/.sup.125I-A.be- ta. should be
internalized after 18 hours of incubation (Kounnas, M. Z., et al.,
Cell 82:331-340 (1995)). Unactivated .alpha..sub.2M/.sup.125I-A.b-
eta. and activated .alpha..sub.2M/.sup.125I-A.beta. in the presence
of RAP should not be internalized, therefore, no more than 2-4
fmoles/10.sup.4 cells should be internalized. If the amount of test
agent treated .alpha..sub.2M/.sup.125I-A.beta. is greater than 4-8
fmoles/10.sup.4 cells, it can be concluded that
.alpha..sub.2M/.sup.125I-A.beta. has the ability to undergo LRP
mediated endocytosis. In addition, unactivated
.alpha..sub.2M/.sup.125I-A.beta., and activated
.alpha..sub.2M/.sup.125I-- A.beta. in the presence of RAP should
not be internalized, therefore no more than 2-4 fmoles/10.sup.4
cells should be internalized (Kounnas, M. Z., et al., Cell
82:331-340(1995)). Internalization of the treated
.alpha..sub.2M/.sup.125I-A.beta. complex will be deemed abolished
if treated .alpha..sub.2M/.sup.125I-A.beta., in the presence and
absence of RAP, and unactivated .alpha..sub.2M/.sup.125I-A.beta.
show the same amount of radioactivity associated with the cell
pellet.
[0139] To determine the ability of treated .alpha..sub.2M/A.beta.
complexes to undergo degradation after endocytosis, this cell
culture protocol is repeated without chloroquine. The radioactivity
appearing in the cell culture medium that is soluble in 10%
trichloroacetic acid is taken to represent degraded
.sup.125I-A.beta. (Kounnas, M. Z., et al., Cell 82:331-340 (1995);
Narita, M., et al., J. Neurochem. 69:1904-1911 (1997)). Total
ligand degradation is corrected for the amount of degradation that
occurs in control wells lacking cells. Because free
.sup.125I-A.beta. can be degraded in an LRP independent manner,
degradation is measured for treated .alpha..sub.2M, and untreated
.alpha..sub.2M complexes with .sup.125I-A.beta., as well as for
free .sup.125I-A.beta., in the presence and absence of RAP. Using
the same positive and negative controls as above, if RAP does not
decrease the amount of TCA soluble radioactivity by at least 30%
for the treated .alpha..sub.2M/.sup.125I-A.beta. complex, it can be
concluded that .sup.125I-A.beta. ligand of treated .alpha..sub.2M
is not degraded.
[0140] It will be readily apparent to those skilled in the relevant
arts that other suitable modifications and adaptations to the
methods and applications described herein are obvious and may be
made without departing from the scope of the invention or any
embodiment thereof. Having now described the present invention in
detail, the same will be more clearly understood by reference to
the following examples, which are included herewith for purposes of
illustration only and are not intended to be limiting of the
invention.
EXAMPLE 1
[0141] In view of the link between the inheritance of A2M-2, and
the role of .alpha..sub.2M in brain, the potential effects of the
A2M-2 deletion polymorphism on A2M mRNA and on the .alpha..sub.2M
protein were investigated. These studies were complicated by the
fact the polymorphism does not directly alter the coding sequence
of .alpha..sub.2M, but consists of an intronic deletion just before
the exon 18 splice acceptor site (Matthijs, G., et al., Nucleic
Acids Res. 19:5102 (1991)). If exon 18 were to be deleted as a
result of the A2M-2 polymorphism, this deletion would result in the
loss of half of the active center or "bait" region of
.alpha..sub.2M (specifically, deletion of the last 20 amino acids
out of the 39 amino acids forming the bait region), with likely
adverse functional consequences for .alpha..sub.2M activity. With
specific regard to A.beta., the peptide does not directly bind to
the bait region. However, recognition and cleavage of the bait
domain by target proteases is a necessary prerequisite in vivo for
activation of .alpha..sub.2M via a conformational change in the
.alpha..sub.2M tetramer. Activation of .alpha..sub.2M then results
in the presentation of the LRP-binding domains which is essential
for binding to LRP (Borth, W., FASEB J. 6:3345-3353 (1992)). Thus,
clearance of .alpha..sub.2M ligands (for example, cytokines, growth
factors, A.beta.), would be hampered by deletion of the bait domain
(exon 18).
[0142] A specific deletion of exon 18 due to the A2M-2 deletion
would also result in a frame-shift in the coding region in exon 19,
resulting in the synthesis of a truncated .alpha..sub.2M monomer.
Therefore, one likely consequence of a modification of the bait
region is the formation of a defective .alpha..sub.2M tetramer
(insertion of defective monomer) which could not be activated and
undergo subsequent endocytosis via LRP. Experiments with an exon 18
deleted .alpha..sub.2M construct expressed in cells indicate that a
truncated .alpha..sub.2M protein at the bait region can still be
secreted and form tetramers with itself. In addition, only human
glioma cell lines positive for the A2M-2 allele produced altered
A2M message and corresponding truncated .alpha..sub.2M monomers
consistent with a deletion of exon 18 followed by termination of
the amino acid sequence in exon 19.
[0143] Methods and Results
[0144] First, the effect of the A2M-2 deletion on RNA splicing and
on .alpha..sub.2M complex formation and secretion were
investigated. To study the biological effects of the A2M-2
polymorphism in an endogenous system, 15 human glioblastoma cell
lines expressing high levels of .sub.2M were genotyped (Blacker,
D., et al., Nature Genetics 19:357-360 (1998)). While the highest
levels of .alpha..sub.2M would be expected from hepatoma cell
lines, glioblastomas were chosen because of their CNS origin. Ten
primary glioblastoma cell lines (all derived from different
patients) were homozygotes for the A2M-1 (no deletion) allele,
while 3 cell lines were A2M-1/2 heterozygotes for the deletion. Two
cell lines did not qualify for either of these alleles and were
excluded from further studies. At the molecular level, the A2M-2
allele consists of a deletion of 5 bp (ACCAT) in the consensus
polypyrimidine tract immediately prior to the consensus 3' AG at
the splice acceptor site of exon 18 (Matthijs, G., et al., Nucleic
Acids Res. 19:5102 (1991)). Given the position of the polymorphism,
aberrant A2M RNA splicing might be expected to lead to a deletion
at exon 18 since the consensus polypyrimidine tract would be
reduced by 3 pyrimidines (to a minimal consensus configuration for
exon splicing). Deletion of exon 18 would, in turn, result in
termination of the protein due to a stop codon in exon 19. Reverse
transcription-PCR (RT-PCR) was employed in attempts to identify
aberrant splice products in the vicinity of exon 18 of the A2M
gene. An expected 399 bp fragment encompassing exons 17, 18, and 19
was amplified by RT-PCR of RNA isolated from the 13 human glioma
cell lines. Agarose gel/ethidium bromide staining was not sensitive
enough to reveal aberrant A2M transcripts in any of the cell lines
containing the A2M-2 allele. However, using polyacrylamide gels,
various .sup.33P-labeled PCR products ranging in size between
250-290 bp were detected. These products were found exclusively in
the A2M-1/2 cell lines (FIG. 1).
[0145] Next, these products were cloned into the vector pCR 2.1.
Four different clones representing aberrant mRNA transcripts have
been identified using this approach (FIG. 2). Sequencing of these
clones revealed aberrant splicing events around exon 18 leading to
the production of variably sized RNAs in which exon 17 and/or 19
may also be shortened. Clone 1 has a 208 bp deletion (2126-2334)
including exon 18 and, interestingly, also 42 and 50 bp of exons 17
and 19, respectively. The protein product resulting from such a
deletion would still be in frame with 69 amino acids missing,
including most of the bait region. Clones 2, 3, and 4 contain
unidentified DNA fragments which continue within exon 19 to bp
2355, 2320, and 2297 respectively. The unknown sequences are most
likely intronic sequences that are not accessible in DNA databases.
Therefore, aberrant splicing events around exon 18 do not appear to
simply result in the precise deletion of exon 18. Rather, they lead
to the production of variably sized RNAs in which exons 17 and/or
19 may also be partly deleted.
[0146] Next, experiments designed to detect mutant forms of
.alpha..sub.2M protein containing large deletions or truncations
were performed. Based on the low level of aberrant mRNA
transcripts, the expected amount of mutant proteins could be below
detection or not recognized by the antibody used, since the
antibody was raised against the holoprotein. Finally, a truncated
or grossly altered protein may be targeted by the quality control
system in the ER for degradation prior to secretion. These concerns
were addressed by producing an A2M cDNA construct in which a stop
codon is inserted in the middle of exon 18 and transfecting this
construct into chinese hamster ovary (CHO) cells, which do not
produce .alpha..sub.2M endogenously. As seen in FIG. 3, both media
and extracts from the transfected cells contained truncated and the
control full-length .alpha..sub.2M protein products. The gels shown
were run under denaturing but non-reducing conditions. Under these
conditions, monomers of the truncated protein and monomers and
dimers of the full-length protein were detected in the cell lysate.
In the media, however, almost all of the truncated protein formed
tetramers, and dimers were barely detectable. Wild-type full-length
.alpha..sub.2M was also present in the media mainly in the form of
tetramers and dimers. Besides demonstrating that the antibody used
is able to recognize the N-terminal half of .alpha..sub.2M and that
a truncated .alpha..sub.2M protein can be synthesized and secreted
by CHO cells, the results of this experiment (FIG. 3) also provided
preliminary data indicating that secreted .alpha..sub.2M levels may
dramatically decrease as a result of the truncation.
[0147] Next, the effects of the A2M-2 polymorphism on secretion and
tetramer formation of endogenous .alpha..sub.2M were examined. For
this purpose, endogenous secreted .alpha..sub.2M was analyzed by
Western blot analysis. Glioblastoma cells were cultured overnight
in OptiMem (Gibco) serum-free media (as bovine serum contains high
levels of .alpha..sub.2M), and secreted .alpha..sub.2M was
immunoprecipitated with a polyclonal .alpha..sub.2M antibody
obtained from Sigma. When the immunoprecipitate was resolved by SDS
PAGE, the expected 180 kD monomer was detected in all lines tested,
however, smaller aberrant forms of .alpha..sub.2M were detected
only in the A2M-2 positive cells. FIG. 4 shows cell lysates from
wild-type and A2M-2 deletion-bearing cells. The data revealed
protein bands consistent with truncated forms of .alpha..sub.2M
exclusively in the A2M-2 deletion-containing cells. The media (data
not shown) from A2M-1 and A2M-2 cells contained primarily
full-length .alpha..sub.2M monomers, but in the media from the
A2M-2 cells small amounts of truncated species could also be
observed.
[0148] Discussion
[0149] A reduced steady-state level of secreted .alpha..sub.2M, or
the presence of defective tetramers due to dominant negative
effects of A2M-2, could result in impaired .alpha..sub.2M function.
Partial or total deletion of the sequences coding for the bait
region in exons 17 and 18 are likely to modify protease binding,
activation, and internalization of potentially defective tetramers
containing mutant monomer(s). Therefore, the generation of very low
levels of mutant monomers may have an amplified effect as one
mutant monomer may potentially inhibit the function of three
wild-type monomers in the tetramer (dominant negative effect).
Based on these and the linkage between the A2M-2 deletion and AD
(Blacker, D., et al., Nat. Genet. 19:357-360 (1998)), a critical
role for .alpha..sub.2M is indicated in AD neuropathogenesis. The
data described herein show that the A2M-2 deletion leads to
deleted/truncated forms of .alpha..sub.2M RNA and protein that may
have a dominant negative effect on normal .alpha..sub.2M.
EXAMPLE 2
[0150] To test the A2M-2 antisense oligonucleotides of the
invention, and the S1 nuclease assay, A2M-2 antisense
oligonucleotides having the nucleotide sequences of nucleotides
35-50, and 20-50 of SEQ ID NO:27 are synthesized using an automatic
DNA synthesizer (MilliGen). The oligonucleotides recovered from 20%
acrylamide-urea gel, and purified by means of an ethanol
precipitating method, and the precipitate is dissolved in water at
a concentration of 1 .mu.mol. A2M-2 sense oligonucleotides
complementary to each of the antisense nucleotides are used as a
positive control. Each of the antisense or sense oligonucleotides
(1 .mu.mol) is added to 1 ml cell culture medium. Each 1 ml sample
is then incubated with glioma cells heterozygous for the A2M-2
allele, or homozygous for wild type A2M (A2M-1) at 37.degree. C.
for 24 hours. The cells are washed with phosphate buffered saline,
and homogenized in a denaturing solution containing 4 M guanidine
thiocyanate. RNA is extracted using phenol/chloroform extraction
and ultracentrifugation. The RNA pellet is then rinsed with 1 ml
75% ethanol/25% 0.1 M sodium acetate, and resuspended in 100 .mu.l
water. RNA from each sample is then probed using a 300 bp antisense
DNA probe encompassing exons 17 and 18 (nucleotides 2057-2356 of
the full length cDNA for .alpha..sup.2M (SEQ ID NO:1)) end labeled
with 32P. The probe is hybrized with 15 .mu.g RNA from each sample.
The RNA is then precipitated, washed and resuspended with S1
hybridization solution. The samples are then denatured for 10
minutes at 65.degree. C., and hybridized overnight at 30.degree. C.
300 U S1 nuclease buffer in 150 .mu.l S1 nuclease buffer with
single-stranded calf thymus DNA is then added to each sample and
incubated for 60 minutes at 30.degree. C. The reaction is stopped,
the RNA precipitated, washed, and resuspended, and the samples are
run on a polyacrylamide gel with molecular weight markers. Wild
type transcripts (A2M-1) should appear as 300 bp bands, A2M-2
variant transcripts should appear as smaller bands. Without A2M-2
antisense oligonucleotide treatment, this ratio is expected to be
approximately 10:1 wild type to variant mRNA. The ratio of wild
type to variant transcripts is determined and compared to the ratio
found for A2M mRNA from A2M-1/1 cells.
EXAMPLE 3
[0151] To screen for therapeutic agents capable of activating
.alpha..sub.2M through a site other than the bait domain,
unactivated tetrameric .alpha..sub.2M (Sigma) (about 1 mg/ml) is
incubated with 5, 20, 50 or 100 .mu.g of test agent in Tris/HCl or
sodium phospate buffer at 37.degree. C. for 2 hours. Untreated
unactivated .alpha..sub.2M, and untreated .alpha..sub.2M activated
with methylamine or trypsin are used as controls.
[0152] Microtiter plates are incubated for 2 h at 37.degree. with
50 .mu.l of LRP (10 .mu.g)/well, and then rinsed with deionized
water. The plates are then filled with blocking buffer and rinsed.
50 .mu.l of treated .alpha..sub.2M, untreated unactivated
.alpha..sub.2M, or untreated .alpha..sub.2M activated with
methylamine or trypsin is added to each well and incubated for 2 h
at room temperature. After rinsing, 50 .mu.l anti-.alpha..sub.2M
IgG conjugated with MUP in blocking buffer is added to the wells
and incubated for 2 h at room temperature. After rinsing, MUP
substrate is added to the wells, and incubated for 1 h at room
temperature. The amount of .alpha..sub.2M bound is quantitated with
a spectrofluorometer with a 365-nm excitation filter and 450 .mu.m
emission filter.
EXAMPLE 4
[0153] Given the evidence that only a few key interactions are
required for .alpha..sub.2M binding to LRP and A.beta. (as
discussed above), a small peptide containing LRP and A.beta.
binding domains could promote A.beta. binding, LRP mediated
endocytosis, and finally A.beta. degradation. Such a peptide could
serve as a substitute for .alpha..sub.2M-2 if it is not able to
promote A.beta. clearance and degradation.
[0154] Protein-protein interactions are usually mediated by a few
key interactions (Wells, J. A., Proc. Natl. Acad. Sci. U.S.A.
93:1-6 (1996)). The A.beta. clearance properties of .alpha..sub.2M
do not require all the domains of an intact 5804 residue
.alpha..sub.2M tetramer. A 250-residue fragment of the
.alpha..sub.2M monomer contains both the A.beta. and LRP binding
domains (Hughes, S. R., et al., Proc. Natl. Acad. Sci. U.S.A.
95:3275-3280 (1998)). An 11-residue peptide can bind A.beta. in
vivo and a 27 residue LRP binding consensus sequence exists (Soto,
C., et al., Nat. Med. 4:822-826 (1998); Nielsen, K. L., et al., J.
Biol. Chem. 271:12909-12912 (1996); Soto, C., et al., Biochem.
Biophys. Res. Commun. 226:672-680 (1996)). A peptide containing an
A.beta. and an LRP binding domain could bind A.beta. and target it
for LRP mediated endocytosis followed by lysosomal degradation. To
achieve this goal, first, a peptide consisting of an 11-residue
A.beta. binding peptide and a 27 residue LRP binding domain is
produced and tested for A.beta. binding and clearance properties.
If necessary, the binding properties of this anti-LRP-A.beta.
peptide can be reoptimized using in vivo evolution techniques
(Buchholz, F., et al., Nat. Biotechnol. 16:657-662 (1998)).
[0155] Methods
[0156] FIG. 6 shows the sequence of one possible anti-LRP-A.beta.
peptide. Using standard solid phase synthesis methods this peptide
is synthesized in quantities sufficient to carry out tests to
determine function in A.beta. clearance. (See "Preparation and
Handling of Peptides," in: Current Protocols in Protein Science,
Coligan, J. E., et al., eds., John Wiley and Sons, Inc., pub., Vol.
2., Chapter 18 (Suppl. 14 1998)). DNA encoding the fusion peptide
is then synthesized. The DNA coding for the 27 residue LRP binding
peptide is obtained by PCR amplification of codons 1366 to 1392 of
the A2M gene (Nielsen, K. L., et al., J. Biol. Chem.
271:12909-12912 (1996)). To integrate the 11 residue A.beta.
binding sequence into the LRP binding sequence PCR mediated
insertion is used. A 55 nucleotide 5' PCR primer is designed that
has 25 nucleotides of homology to the LRP binding sequence and 36
nucleotides corresponding to the 11 residues of the A.beta. binding
peptide and a start codon. PCR mediated insertion is also used to
insert an Xho I and Kpn I restriction enzyme sites at the 5' and 3'
ends of the fusion gene, respectively. Cleavage with these enzymes
will facilitate cloning of the fusion protein gene into (i) the
pBAD/His expression vector (Invitrogen), for arabinose dependent
expression of anti-LRP-A.beta. in E. coli, and (ii) the pLex9-3H
vector for use in the yeast three hybrid system (Tirode, F., et
al., J. Biol. Chem. 272:22995-22999 (1997)). The protein product,
named anti-LRP-A.beta., of the resulting gene should have both
A.beta. and LRP binding properties.
[0157] A.beta. Binding. The ability of anti-LRP-A.beta. to bind
A.beta. is first determined by gel-filtration chromatography and
immunoblotting. Both of these methods have been used successfully
by other investigators to investigate A.beta. binding to wild type
and variant .alpha..sub.2M (Narita, M., et al., J. Neurochem.
69:1904-1911 (1997); Du, Y., et al., J. Neurochem. 69:299-305
(1997)). A.beta.1-42 is iodinated with .sup.125I, following the
procedure of Narita et al. (Narita, M., et al., J. Neurochem.
69:1904-1911 (1997)). .sup.125I-A.beta. (5 nmol) is incubated
separately with anti-LRP-A.beta., unactivated .alpha..sub.2M,
unactivated .alpha..sub.2M-2, .alpha..sub.2M activated by
methylamine or trypsin, or .alpha..sub.2M-2 activated by
methylamine or trypsin. A ten fold molar excess of A.beta. is used
and the samples are incubated in 25 mM Tris-HCl, 150 mM NaCl, pH
7.4 for two hours at 37.degree. C. Controls containing only
.sup.125I-A.beta. are also incubated. The
anti-LRP-A.beta./.sup.125I-A.beta.,
.alpha..sub.2M/.sup.125I-A.beta., and
.alpha..sub.2M-2/.sup.125I-A.beta. complexes are separated from
unbound .sup.125I-A.beta. using a Superose 6 gel-filtration column
(0.7.times.20 cm) under the control of an FPLC (Pharmacia). 25 MM
Tris-HCl, 150 mM NaCl, pH 7.4 are used to equilibrate the column
and elute the samples. Using a flow rate of 0.05 ml/minute, 200
.mu.L fractions are collected. Having standardized the column with
molecular weight markers ranging from 1000 kD to 4 kD,
anti-LRP-A.beta./.sup.125I-A.beta.,
.alpha..sub.2M/.sup.125I-A.beta., and
.alpha..sub.2M-2/.sup.125I-A.beta. fractions are counted in a
.gamma. counter to determine the elution profile of
.sup.125I-A.beta.. If anti-LRP-A.beta. has bound .sup.125I-A.beta.,
.sup.125I-A.beta. should be detected by gamma counter at two peaks,
one corresponding to the molecular weight of the
anti-LRP-A.beta./.sup.125I-A.beta. complex (about 8-9 kD for this
anti-LRP-A.beta. peptide), and one corresponding to the molecular
weight of .sup.125I-A.beta. (4.5 kD).
[0158] It is unlikely, but possible, that iodinated A.beta. may
lead to a false positive or negative binding. Therefore,
immunoblotting experiments are undertaken to confirm the results of
the gel-filtration chromatography experiment (Narita, M., et al.,
J. Neurochem. 69:1904-1911 (1997); Du, Y., et al., J. Neurochem.
69:299-305 (1997)). Unlabeled A.beta. is incubated separately with
anti-LRP-A.beta., unactivated .alpha..sub.2M, unactivated
.alpha..sub.2M-2, .alpha..sub.2M activated by methylamine or
trypsin, or .alpha..sub.2M-2 activated by methylamine or trypsin,
under the same conditions described above. Samples are
electrophoresed on a 5% SDS-PAGE, under non-reducing conditions,
and transferred to polyvinyl difluoride nitrocellulose membrane
(Immobilon-P). These membranes are probed with polyclonal
anti-.alpha..sub.2M IgG or monoclonal anti-A.beta. IgG.
Immunoreactive proteins are visualized using ECL and peroxidase
conjugated anti-rabbit IgG. Molecular mass markers are used to
determine if the immunoreactive proteins from the
anti-.alpha..sub.2M and anti-A.beta. blots for corresponding lanes
display the same mobility. If the immunoreactive proteins display
the same mobility then it will be concluded that A.beta. binds
anti-LRP-A.beta..
[0159] Endocytosis. The ability of anti-LRP-A.beta./A.beta.
complexes to undergo LRP mediated endocytosis and subsequent
degradation is determined in cell culture experiments. The amount
of radioligand that is internalized or degraded by cells has been
described previously (Kounnas, M. Z., et al., Cell 82:331-340
(1995); Kounnas, M. Z., et al., J. Biol. Chem. 270:9307-9312
(1995)). Mouse embryo fibroblasts, which are cells that express
LRP, are plated in 12 well plates to a density of 2.times.10.sup.5
cells per well, and grown for 18 hours at 37.degree. C. in 5%
CO.sub.2. Cells are incubated in 1% Nutridoma (Boehringer
Mannheim), penicillin/streptomycin, 1.5% bovine serum albumin for
one hour prior to addition of .sup.125I-A.beta. in the presence or
absence of anti-LRP-A.beta., unactivated .alpha..sub.2M,
unactivated .alpha..sub.2M-2, .alpha..sub.2M activated by
methylamine or trypsin, or .alpha..sub.2M-2 activated by
methylamine or trypsin, in the presence or absence of RAP (400 nM).
To assess anti-LRP-A.beta./.sup.125I-A.beta. endocytosis by LRP,
chloroquine (0.1 mM) is added at the same time as
anti-LRP-A.beta./.sup.125I-A.beta. (4 nM) to inhibit lysosomal
degradation of .sup.125I-A.beta. (Kounnas, M. Z., et al., Cell
82:331-340 (1995)).
[0160] Following 18 hours of incubation with the
anti-LRP-A.beta./.sup.125- I-A.beta., cells are washed with
phosphate-buffered saline and treated with a trypsin-EDTA,
proteinase K solution. Surface-bound material is defined as the
amount of radioactive ligand released by this treatment, and the
amount of internalized ligand is defined as the amount of
radioactivity which remains associated with the cell pellet
following the treatment.
[0161] Activated .alpha..sub.2M/.sup.125I-A.beta. will serve as
positive control. Under the conditions described, more than 4-8
fmoles/10.sup.4 cells of activated .alpha..sub.2M/.sup.125I-A.beta.
should be internalized after 18 hours of incubation (Kounnas, M.
Z., et al., Cell 82:331-340 (1995)). Unactivated
.alpha..sub.2M/.sup.125I-A.beta. will serve as the negative
control, because .alpha..sub.2M must be activated by trypsin or
methylamine to be recognized by LRP. If the amount of
anti-LRP-A.beta./.sup.125I-A.beta. is greater than 2-4
fmoles/10.sup.4 cells, it can be concluded that
anti-LRP-A.beta./.sup.125I-A.beta. has the ability to undergo LRP
mediated endocytosis. Unactivated .alpha..sub.2M/.sup.125I-A.beta.,
and activated .alpha..sub.2M/.sup.125I-- A.beta. in the presence of
RAP should not be internalized, therefore no more than 2-4
fmoles/10.sup.4 cells should be internalized (Kounnas, M. Z., et
al., Cell 82:331-340 (1995)). Internalization of the
anti-LRP-A.beta./.sup.125I-A.beta. complex will be deemed abolished
if anti-LRP-A.beta./.sup.125I-A.beta., in the presence and absence
of RAP, and unactivated .alpha..sub.2M/.sup.125I-A.beta. show the
same amount of radioactivity associated with the cell pellet.
[0162] Degradation.
[0163] The experiment above to test endocytosis is repeated without
chloroquine. The radioactivity appearing in the cell culture medium
that is soluble in 10% trichloroacetic acid is taken to represent
degraded .sup.125I-A.beta. (Kounnas, M. Z., et al., Cell 82:331-340
(1995); Narita, M., et al., J. Neurochem. 69:1904-1911 (1997)).
Total ligand degradation is corrected for the amount of degradation
that occurs in control wells lacking cells. Because free
.sup.125I-A.beta. can be degraded in an LRP independent manner,
degradation is measured for anti-LRP-A.beta. and .alpha..sub.2M
complexes with .sup.125I-A.beta. as well as for free
.sup.125I-A.beta. in the presence and absence of RAP. Using the
same positive and negative controls as above, if RAP does not
decrease the amount of TCA soluble radioactivity by at least 30%
for the anti-LRP-A.beta./.sup.125I-A.beta. complex it can be
concluded that .sup.125I-A.beta. ligand of anti-LRP-A.beta. is not
degraded.
[0164] The anti-LRP-A.beta. peptide may not promote A.beta. binding
and degradation because of steric constrains. If the
anti-LRP-A.beta. polypeptide does not promote A.beta. binding and
degradation another peptide is synthesized with a penta-glycine
linker between the A.beta. and LRP binding regions to provide the
flexibility needed to bind both targets simultaneously. This
anti-LRP-A.beta. with linker is tested for A.beta. binding, and LRP
mediated endocytosis and degradation as described above. If this
anti-LRP-A.beta. does not provide for A.beta. and LRP binding, the
three hybrid system is used to reoptimize binding, and to screen
for anti-LRP-A.beta. with the ability to bind both A.beta. and
LRP.
[0165] The use of peptides in therapy is associated with two
problems, transport across the blood-brain barrier, and the
generation of an immune response. These problems can be minimized
by shortening the peptide length. Thus when optimizing the
anti-LRP-A.beta. peptide, shorter binding domains may be preferred
over longer domains, where binding capabilities are equally
effective.
[0166] Yeast Three Hybrid System.
[0167] The yeast three hybrid system is a genetic method to detect
ternary protein complex formation (FIG. 7) (Tirode, F., et al., J.
Biol. Chem. 272:22995-22999 (1997); Osborne, M. A., et al.,
Biotechnology 13:1474-1478 (1995); Zhang, J. and Lautar, S., Anal.
Biochem. 242:68-72 (1996); Licitra, E. J. and Liu, J. O., Proc.
Natl. Acad. Sci. U.S.A. 93:12817-12821 (1996)). In the system,
yeast growth only occurs when the "bait" recognizes both the "hook"
and the "fish" (FIG. 7). In this instance, the "hook" is
constructed of the DNA coding for A.beta. (Bales, K. R., et al.,
Nat. Genet. 17:264 (1997)), fused to the coding sequence of the
LexA DNA binding protein in pLex9-3H, a TRP1 episomal vector
(Tirode, F., et al., J. Biol. Chem. 272:22995-22999 (1997)). The
"fish" is constructed of the coding sequence for the 515 kD
extracellular domain of LRP, fused to the B42 activation domain in
pVP 16, a LEU2 episomal vector (Tirode, F., et al., J. Biol. Chem.
272:22995-22999 (1997)). The "bait" is the DNA coding for
anti-LRP-A.beta. in the pLex9-3H vector, expression of
anti-LRP-A.beta. is repressed by methionine. These vectors are
transformed into the L40 yeast strain. Transcription of the Leu 2
reporter gene occurs only when the A.beta. fused DNA binding domain
is brought into proximity to the transcriptional activation domain
fused to LRP.
[0168] The A.beta./LRP binding fusion peptide should promote
reporter gene transcription. The interaction between
anti-LRP-A.beta. and A.beta. and LRP (515 kD) will be considered
positive only if reporter gene expression (yeast growth) occurs
when A.beta.-LexA, LRP(515 kD)-B42, and anti-LRP-A.beta. are
expressed. It is not likely that expression of A.beta.-LexA will
cause activation of the reporter transcription since this construct
has been used successfully in the past. It is also unlikely that
LRP(515 kD)-B42 expression alone will cause reporter transcription,
LRP(515 kD) is not known to bind DNA. The interaction of
A.beta.-LexA and LRP (515 kD)-B42 would cause reporter
transcription and the A.beta. parent protein APP is known to
interact with LRP. However, the interaction between LRP and APP
occurs via the Kunitz protease inhibitory domain far removed from
the location of A.beta. in APP (Kounnas, M. Z., et al., Cell
82:331-340 (1995)). In addition biochemical evidence suggests that
LRP does not recognize A.beta. (Narita, M., et al., J. Neurochem.
69:1904-1911 (1997)). Transformation of the A.beta.-LexA and
LRP(515 kD)-B42 containing plasmids into EGY48 and monitoring the
growth on media lacking leucine is carried out to insure that
A.beta.-LexA and LRP(515 kD)-B42 do not interact. As positive
controls the DNA sequence encoding the entire .alpha..sub.2M
monomer and the sequence encoding residues 1202-1451 of
.alpha..sub.2M are cloned separately into pLex9-3H, in place of
anti-LRP-A.beta.. The C-terminal fragment of .alpha..sub.2M
contains the full length A.beta. and LRP binding domains (residues
1202-1451 of .alpha..sub.2M) and it, along with the monomer, should
give rise to reporter gene transcription.
[0169] If expression of anti-LRP-A.beta., A.beta.-LexA, and LRP(515
kD)-B42 does not activate reporter transcription then each of the
binary interactions of anti-LRP-A.beta. are tested in a traditional
two hybrid screen. That is, concomitant expression of
anti-LRP-A.beta.-B42 and A.beta.-LexA, as well as
anti-LRP-A.beta.-B-42 and LRP(515 kD)-LexA, is used to assess the
ability of anti-LRP-A.beta. to interact with A.beta.-LexA and
LRP(515 kD)-LexA individually. If anti-LRP-A.beta. interacts
individually with both targets then one or all of the following is
carried out: (i) a 5 residue glycine linker is added between the
A.beta. binding domain and the LRP binding to allow flexibility
between the two binding domains, (ii) the A.beta.-LexA and LRP(515
kD)-B42 fusion partners are switched to become LRP(515 kD)-LexA and
A.beta.-B42, and (iii) the polarity of the anti-LRP-A.beta. is
switched so that the LRP binding domain is N-terminal to the
A.beta. binding domain. If anti-LRP-A.beta. interacts with one or
neither of the targets, binding is reoptimized using random
mutagenesis and selection by three hybrid screen for binding to
both targets. The non-binding region of anti-LRP-A.beta. is
subjected to protein evolution techniques, error prone PCR and DNA
shuffling (Buchholz, F., et al., Nat. Biotechnol. 16:657-662
(1998)), followed by selection of constructs that bind target
proteins. This is repeated until target binding is achieved.
[0170] Modifications of the above-described modes for carrying out
the invention that are obvious to persons of skill in medicine,
genetics, molecular biology, biochemistry, pharmacology and/or
related fields are intended to be within the scope of the following
claims.
[0171] All publications and patents mentioned in this specification
are indicative of the level of skill of those skilled in the art to
which this invention pertains. All publications and patents
mentioned are herein incorporated by reference to the same extent
as if each individual publication or patent application were
specifically and individually indicated to be incorporated by
reference.
[0172] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications can be practiced within the scope of the appended
claims.
Sequence CWU 0
0
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