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 α 2 M RNA transcripts and proteins. Based on this finding, the invention provides for new therapeutic agents for AD, including molecules having Aβ 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.

What is claimed is:  
     
         1 . A therapeutic agent for combating Alzheimer's disease, wherein said agent can replace or supplement α 2 M function, or suppress expression of A2M-2.  
     
     
         2 . An anti-LRP-Aβ molecule comprising, an Aβ binding domain, and an LRP binding domain, or a pharmaceutically acceptable salt thereof.  
     
     
         3 . The anti-LRP-Aβ molecule of  claim 2 , wherein said molecule is a peptide, or a pharmaceutically acceptable salt thereof.  
     
     
         4 . An anti-LRP-Aβ peptide comprising: 
 (a) an Aβ 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-Aβ peptide comprising: 
 (a) an Aβ 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-Aβ peptide comprising: 
 (a) an Aβ 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-Aβ peptide of  claim 4 ,  5  or  6 , wherein said Aβ binding domain is connected to said LRP binding domain by a peptide bond.  
     
     
         8 . The anti-LRP-Aβ peptide of  claim 4 ,  5  or  6 , wherein said Aβ binding domain is connected to said LRP binding domain by a linker.  
     
     
         9 . The anti-LRP-Aβ peptide of  claim 8 , wherein said linker is selected from the group consisting of a peptide, or polyethylene glycol.  
     
     
         10 . The anti-LRP-Aβ peptide of  claim 9 , wherein said peptide comprises 1-20 glycine residues.  
     
     
         11 . A nucleic acid comprising a polynucleotide encoding the anti-LRP-Aβ peptide of  claim 4 ,  5 ,  6 ,  7 ,  8 ,  9  or  10 .  
     
     
         12 . An anti-LRP-Aβ peptide comprising a polypeptide having the sequence of SEQ ID NO:14, or a pharmaceutically acceptable salt thereof.  
     
     
         13 . An anti-LRP-Aβ peptide comprising: 
 (a) an Aβ 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 Aβ binding domain to said LRP binding domain.  
 
     
     
         14 . A nucleic acid molecule comprising a nucleotide encoding the anti-LRP-Aβ peptide of  claim 12  or  13 .  
     
     
         15 . A nucleic acid molecule encoding an anti-LRP-Aβ peptide comprising: 
 (a) a region encoding an Aβ 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-Aβ peptide comprising: 
 (a) a region encoding an Aβ 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-Aβ peptide comprising: 
 (a) a region encoding an Aβ 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 Aβ 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 Aβ 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° C. in a solution consisting of 50% formamide, 5× SSC, 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and a 20 μg/ml denatured, sheared salmon sperm DNA; and    (b) washing at 65° C. in a solution consisting of 0.14× 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° C. in a solution consisting of 50% formamide, 5× SSC, 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and a 20 μg/ml denatured, sheared salmon sperm DNA; and    (b) washing at 65° C. in a solution consisting of 0.1× SSC.    
     
     
         27 . A pharmaceutical composition comprising an anti-LRP-Aβ molecule, and one or more pharmaceutically acceptable carriers.  
     
     
         28 . A pharmaceutical composition comprising the anti-LRP-Aβ 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-Aβ 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-Aβ molecule.  
     
     
         31 . The method of  claim 30 , wherein said anti-LRP-Aβ molecule is a peptide.  
     
     
         32 . A method of combating Alzheimer's Disease in a subject comprising administering the anti-LRP-Aβ 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-Aβ 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 α 2 M, and a gene encoding an anti-LRP-Aβ peptide.  
     
     
         44 . The viral vector of  claim 43 , wherein said transgene is a gene encoding α 2 M.  
     
     
         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-Aβ 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 α 2 M with a test agent; and    (b) determining whether said α 2 M 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 α 2 M electrophoretic mobility assay.  
     
     
         67 . A method of screening for a therapeutic agent for Alzheimer's Disease comprising the steps of: 
 (a) incubating α 2 M with a test agent; and    (b) determining whether said α 2 M 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 α 2 M 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 α 2 M;    (c) incubating said well with anti-α 2 M 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 α 2 M;    (b) incubating said well with anti-α 2 M 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 α 2 M in a well coated with an anti-α 2 M IgG specific for activated α 2 M;    (b) incubating said well with said α 2 M;    (c) incubating said well with anti-α 2 M 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-α 2 M IgG are used to perform said immunoblotting.  
     
     
         75 . The method of  claim 67 , wherein said step (b) comprises determining the ability of said α 2 M to undergo LRP mediated endocytosis.  
     
     
         76 . The method of  claim 67 , wherein said step (b) comprises determining the ability of said α 2 M to undergo LRP mediated degradation.