Synchronized cell cycle gene expression tests on alzheimer disease and related therapeutic methods

Abstract

To provide synchronized cell cycle gene expression tests on Alzheimer disease and related therapeutic methods.SOLUTION: The invention provides a gene-based accurate method for determining whether a human subject is afflicted with Alzheimer disease (AD) or with non-Alzheimer type dementia (ADD). The subject method is based on, at least in part, a surprising finding that by synchronizing an appropriate cell population of the patient, then measuring the expression level of a gene expressed differentially between AD cells and non-ADD cells, it is possible to accurately diagnose whether the patient is afflicted with AD or with non-ADD. The invention also provides a method for treating AD by using an agent that affects the expression of a certain gene.SELECTED DRAWING: Figure 8

Description

[Technical Field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 596,588, filed December 8, 2017, the contents of which are incorporated herein by reference.

[0002] Throughout this application, various publications are cited. The disclosures of these publications are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains. [Background technology]

[0003] Background of the Invention Alzheimer's disease ("AD") has long been the subject of considerable effort to develop accurate diagnostic and therapeutic methods. Despite these efforts, there remains an unmet need for methods to accurately diagnose AD and distinguish it from non-Alzheimer's dementia ("non-ADD"). There also remains an unmet need for effective methods to treat AD. Summary of the Invention [Means for solving the problem]

[0004] Summary of the Invention The present invention provides a method for determining whether a human subject is suffering from AD or a non-ADD when the subject is suspected of having AD or a non-ADD, comprising: (a) synchronizing a population of suitable cells from the subject; and (b) measuring in the resulting synchronized cell population the expression levels of genes known to be differentially expressed between corresponding synchronized cells derived from AD patients and corresponding synchronized cells derived from non-ADD patients; whereby (i) if the expression level measured in step (b) matches the expression level of the gene in corresponding synchronized cells derived from an AD patient, the subject is afflicted with AD, and (ii) if the expression level measured in step (b) matches the expression level of the gene in corresponding synchronized cells derived from a non-ADD patient, the subject is afflicted with a non-ADD.

[0005] The present invention also provides a method for determining whether a human subject is afflicted with AD or a non-ADD when the subject is suspected of being afflicted with AD or a non-ADD, comprising: (a) synchronizing a population of fibroblasts of cultured skin cells derived from the subject, wherein the synchronizing step comprises culturing the fibroblasts to overconfluence and then starving the resulting overconfluent fibroblasts; and (b) measuring the expression level of each of the following genes in the resulting synchronized fibroblast population: AC004057.1, AC092651.1, ACP6, ADAM20, ASXL2, C2CD5, CARNS1, FAM149B1, GLIS3-AS1, IL18R1, LINC01393, LZIC, MAP1LC3B2, NHLH1, NORAD, NPPA-AS1_3, OSMR-AS1, PAN3, PHBP8, PSMB9, RAB3IP, RDH16, RFESDP1, RPL5, SCG2, SDHD, SHISA5, SLC45A3, SNHG14, TTC26, URB2, USMG5, WASF2, ZCWPW2, ZNF444, and ZNF70, wherein measuring the expression level of each gene comprises measuring the number of its RNA transcripts per number of total transcripts; whereby (i) if the expression level measured in step (b) is consistent with the expression level of the gene in corresponding synchronized cells derived from an AD patient, the subject is afflicted with AD, and (ii) if the expression level measured in step (b) is consistent with the expression level of the gene in corresponding synchronized cells derived from a non-ADD patient, the subject is afflicted with a non-ADD.

[0006] The present invention provides a method for determining whether a human subject is suffering from AD or a non-ADD when the subject is suspected of having AD or a non-ADD, comprising: (a) synchronizing a population of cultured immortalized B lymphocytes derived from the subject, wherein the synchronizing step comprises culturing the lymphocytes to overconfluence and then starving the resulting overconfluent lymphocytes; and (b) measuring the expression level of each of the following genes in the resulting synchronized lymphocyte population: AC004057.1, AC092651.1, ACP6, ADAM20, ASXL2, C2CD5, CARNS1, FAM149B1, GLIS3-AS1, IL18R1, LINC01393, LZIC, MAP1LC3B2, NHLH1, NORAD, NPPA-AS1_3, OSMR-AS1, PAN3, PHBP8, PSMB9, RAB3IP, RDH16, RFESDP1, RPL5, SCG2, SDHD, SHISA5, SLC45A3, SNHG14, TTC26, URB2, USMG5, WASF2, ZCWPW2, ZNF444, and ZNF70, wherein measuring the expression level of each gene comprises measuring the number of its RNA transcripts per number of total transcripts; whereby (i) if the expression level measured in step (b) is consistent with the expression level of the gene in corresponding synchronized cells derived from an AD patient, the subject is afflicted with AD, and (ii) if the expression level measured in step (b) is consistent with the expression level of the gene in corresponding synchronized cells derived from a non-ADD patient, the subject is afflicted with a non-ADD.

[0007] The present invention further provides a method for treating a human subject suffering from Alzheimer's disease, comprising administering to the subject a therapeutically effective amount of an agent known to favorably affect the expression level of one or more genes whose expression levels correlate with Alzheimer's disease.

[0008] Finally, the present invention provides a method for treating a human subject suffering from Alzheimer's disease, comprising administering to the subject a therapeutically effective amount of carfilzomib, bortezomib, bumetanide, furosemide, or torasemide. In certain embodiments, for example, the following are provided: (Item 1) 1. A method for determining whether a human subject is afflicted with Alzheimer's disease ("AD") or non-Alzheimer's dementia (non-ADD) when the subject is suspected of having AD or non-ADD, comprising: (a) synchronizing a population of suitable cells from the subject; and (b) measuring in the resulting synchronized cell population the expression levels of genes known to be differentially expressed between corresponding synchronized cells derived from AD patients and corresponding synchronized cells derived from non-ADD patients; whereby (i) if the expression level measured in step (b) is consistent with the expression level of the gene in corresponding synchronized cells derived from an AD patient, the subject is afflicted with AD, and (ii) if the expression level measured in step (b) is consistent with the expression level of the gene in corresponding synchronized cells derived from a non-ADD patient, the subject is afflicted with a non-ADD. method. (Item 2) 2. The method of claim 1, wherein the suitable cells derived from the subject are cultured skin cell fibroblasts. (Item 3) 2. The method of claim 1, wherein the suitable cells derived from the subject are cultured B lymphocytes. (Item 4) 4. The method of claim 3, wherein the B lymphocytes are immortalized. (Item 5) 2. The method of claim 1, wherein the step of synchronizing the population of suitable cells comprises culturing the cells until they become overconfluent and then starving the resulting overconfluent cells. (Item 6) 2. The method of claim 1, wherein the gene is known to be differentially expressed by at least 50% between matched synchronized cells derived from AD patients and matched synchronized cells derived from non-ADD patients. (Item 7) 7. The method of item 6, wherein the gene is known to be at least 100% differentially expressed between matched synchronized cells derived from AD patients and matched synchronized cells derived from non-ADD patients. (Item 8) 2. The method of item 1, wherein the gene is selected from the group consisting of AC004057.1, AC092651.1, ACP6, ADAM20, ASXL2, C2CD5, CARNS1, FAM149B1, GLIS3-AS1, IL18R1, LINC01393, LZIC, MAP1LC3B2, NHLH1, NORAD, NPPA-AS1_3, OSMR-AS1, PAN3, PHBP8, PSMB9, RAB3IP, RDH16, RFESDP1, RPL5, SCG2, SDHD, SHISA5, SLC45A3, SNHG14, TTC26, URB2, USMG5, WASF2, ZCWPW2, ZNF444, and ZNF70. (Item 9) 2. The method of claim 1, wherein step (b) comprises measuring the expression levels of a plurality of genes, each gene known to be differentially expressed between matched synchronized cells derived from an AD patient and matched synchronized cells derived from a non-ADD patient. (Item 10) 10. The method of claim 9, wherein the plurality of genes is selected from the group consisting of at least 2 genes, at least 5 genes, at least 20 genes, at least 100 genes, and at least 1,000 genes. (Item 11) 10. The method of item 9, wherein each gene of the plurality of genes is known to be differentially expressed by at least 50% between corresponding synchronized cells derived from an AD patient and corresponding synchronized cells derived from a non-ADD patient. (Item 12) 12. The method of claim 11, wherein each gene of the plurality of genes is known to be differentially expressed by at least 100% between corresponding synchronized cells derived from an AD patient and corresponding synchronized cells derived from a non-ADD patient. (Item 13) 10. The method of item 9, wherein the plurality of genes comprises two or more genes selected from the group consisting of AC004057.1, AC092651.1, ACP6, ADAM20, ASXL2, C2CD5, CARNS1, FAM149B1, GLIS3-AS1, IL18R1, LINC01393, LZIC, MAP1LC3B2, NHLH1, NORAD, NPPA-AS1_3, OSMR-AS1, PAN3, PHBP8, PSMB9, RAB3IP, RDH16, RFESDP1, RPL5, SCG2, SDHD, SHISA5, SLC45A3, SNHG14, TTC26, URB2, USMG5, WASF2, ZCWPW2, ZNF444, and ZNF70. (Item 14) 2. The method of claim 1, wherein the step of measuring the expression level of a gene comprises measuring the number of RNA transcripts of that gene per number of total transcripts. (Item 15) 1. A method for determining whether a human subject is afflicted with Alzheimer's disease ("AD") or non-Alzheimer's dementia ("non-ADD") when the subject is suspected of having AD or non-ADD, comprising: (a) synchronizing a population of fibroblasts of cultured skin cells derived from the subject, wherein said synchronizing comprises culturing said fibroblasts to overconfluence and then starving the resulting overconfluent fibroblasts; and (b) measuring the expression level of each of the following genes in the resulting synchronized fibroblast population: AC004057.1, AC092651.1, ACP6, ADAM20, ASXL2, C2CD5, CARNS1, FAM149B1, GLIS3-AS1, IL18R1, LINC01393, LZIC, MAP1LC3B2, NHLH1, NORAD, NPPA-AS1_3, OSMR-AS1, PAN3, PHBP8, PSMB9, RAB3IP, RDH16, RFESDP1, RPL5, SCG2, SDHD, SHISA5, SLC45A3, SNHG14, TTC26, URB2, USMG5, WASF2, ZCWPW2, ZNF444, and ZNF70, wherein measuring the expression level of each gene comprises measuring the number of its RNA transcripts per number of total transcripts; whereby (i) if the expression level measured in step (b) is consistent with the expression level of said gene in corresponding synchronized cells derived from an AD patient, said subject is afflicted with AD, and (ii) if the expression level measured in step (b) is consistent with the expression level of said gene in corresponding synchronized cells derived from a non-ADD patient, said subject is afflicted with a non-ADD. method. (Item 16) 1. A method for determining whether a human subject is afflicted with Alzheimer's disease ("AD") or non-Alzheimer's dementia ("non-ADD") when the subject is suspected of having AD or non-ADD, comprising: (a) synchronizing a population of cultured immortalized B lymphocytes derived from the subject, wherein said synchronizing comprises culturing the lymphocytes to overconfluence and then starving the resulting overconfluent lymphocytes; and (b) measuring the expression level of each of the following genes in the resulting synchronized lymphocyte population: AC004057.1, AC092651.1, ACP6, ADAM20, ASXL2, C2CD5, CARNS1, FAM149B1, GLIS3-AS1, IL18R1, LINC01393, LZIC, MAP1LC3B2, NHLH1, NORAD, NPPA-AS1_3, OSMR-AS1, PAN3, PHBP8, PSMB9, RAB3IP, RDH16, RFESDP1, RPL5, SCG2, SDHD, SHISA5, SLC45A3, SNHG14, TTC26, URB2, USMG5, WASF2, ZCWPW2, ZNF444, and ZNF70, wherein measuring the expression level of each gene comprises measuring the number of its RNA transcripts per number of total transcripts; whereby (i) if the expression level measured in step (b) is consistent with the expression level of the gene in corresponding synchronized cells derived from an AD patient, the subject is afflicted with AD, and (ii) if the expression level measured in step (b) is consistent with the expression level of the gene in corresponding synchronized cells derived from a non-ADD patient, the subject is afflicted with a non-ADD. method. (Item 17) A method for treating a human subject suffering from Alzheimer's disease, comprising administering to the subject a therapeutically effective amount of an agent known to favorably affect the expression level of one or more genes whose expression levels correlate with Alzheimer's disease. (Item 18) 18. The method of claim 17, wherein the gene is selected from the group consisting of AC004057.1, AC092651.1, ACP6, ADAM20, ASXL2, C2CD5, CARNS1, FAM149B1, GLIS3-AS1, IL18R1, LINC01393, LZIC, MAP1LC3B2, NHLH1, NORAD, NPPA-AS1_3, OSMR-AS1, PAN3, PHBP8, PSMB9, RAB3IP, RDH16, RFESDP1, RPL5, SCG2, SDHD, SHISA5, SLC45A3, SNHG14, TTC26, URB2, USMG5, WASF2, ZCWPW2, ZNF444, and ZNF70. (Item 19) 1. A method for treating a human subject suffering from Alzheimer's disease, comprising administering to the subject a therapeutically effective amount of carfilzomib. (Item 20) 1. A method for treating a human subject suffering from Alzheimer's disease, comprising administering to said subject a therapeutically effective amount of bortezomib. (Item 21) 1. A method for treating a human subject suffering from Alzheimer's disease, comprising administering to the subject a therapeutically effective amount of bumetanide. (Item 22) 1. A method for treating a human subject suffering from Alzheimer's disease, comprising administering to said subject a therapeutically effective amount of furosemide. (Item 23) 1. A method for treating a human subject suffering from Alzheimer's disease, comprising administering to said subject a therapeutically effective amount of torasemide. [Brief explanation of the drawings]

[0009] [Figure 1]This figure shows statistically significant genes when comparing AD and non-ADD groups based on the first study ("Study 1"). Study 1 revealed that there were 2103 statistically significant genes for a P value of less than 0.1; 1099 statistically significant genes for a P value of less than 0.05; 285 statistically significant genes for a P value of less than 0.01; and 6 statistically significant genes for a P value of less than or equal to 0.001.

[0010] [Figure 2] This figure shows the top six statistically significant genes (P≦0.001) for six AD cases and two non-ADD cases based on Study 1. Squares represent the AD population, while circles represent the non-ADD population.

[0011] [Figure 3] This figure shows examples of the top 10 statistically significant genes (P≦0.01) based on Study 1. (A) Raw TPM data showing the AD population as boxes and the non-ADD population as circles. (B) Average TPM data showing the AD population as boxes and the non-ADD population as circles. Error bars are standard deviations. (C) Percent change in gene expression (%Ch) when comparing AD to controls (non-ADD), i.e., 100×(AD−non-ADD) / non-ADD.

[0012] [Figure 4] This figure shows genes ranked 11-20 at a 1% probability of overlap (P ≤ 0.01) based on Study 1. (A) Raw TPM data showing the AD population as squares and the non-ADD population as circles. (B) Average TPM data showing the AD population as squares and the non-ADD population as circles. Error bars represent standard deviation. (C) Percent change in gene expression (%Ch) when comparing AD to controls (non-ADD), i.e., 100 × (AD - non-ADD) / non-ADD.

[0013] [Figure 5]This figure shows genes ranked 21-30 at a 1% probability of overlap (P ≤ 0.01) based on Study 1. (A) Raw TPM data showing the AD population as squares and the non-ADD population as circles. (B) Average TPM data showing the AD population as squares and the non-ADD population as circles. Error bars represent standard deviation. (C) Percent change in gene expression (%Ch) when comparing AD to controls (non-ADD), i.e., 100 × (AD - non-ADD) / non-ADD.

[0014] [Figure 6] This figure shows genes ranked 31-40 at a 1% probability of overlap (P ≤ 0.01) based on Study 1. (A) Raw TPM data showing the AD population as squares and the non-ADD population as circles. (B) Average TPM data showing the AD population as squares and the non-ADD population as circles. Error bars represent standard deviation. (C) Percent change in gene expression (%Ch) when comparing AD to controls (non-ADD), i.e., 100 × (AD - non-ADD) / non-ADD.

[0015] [Figure 7] This figure shows the percent change in gene expression (%Ch) for the top 40 genes based on Study 1.

[0016] [Figure 8] This figure shows the number of statistically significant differentially expressed genes for the training set (first column—lighter shading) versus the number of statistically significant differentially expressed genes for the validation set (second column—darker shading) for various levels of statistical significance P<0.001, 0.01, 0.05, and 0.10 based on the second study ("Study 2").

[0017] [Figure 9]This figure shows the gene networks for (A) PAN3, (B) PSMB9, (C) TTC26, (D) ZNF444, (E) NHLH1, (F) URB2, and (G) ADAM20 based on Study 2.

[0018] [Figure 10] This figure shows network measures for cross-validated genes based on Study 2: (A) number of edges; (B) average node degree; and (C) average local clustering coefficient.

[0019] [Figure 11] This figure compares non-ADD (n=3) with non-demented controls (NDC; n=5) based on Study 2 and shows the number of differentially expressed genes in the non-ADD population compared to the NDC population. DETAILED DESCRIPTION OF THE INVENTION

[0020] Detailed Description of the Invention definition In this application, certain terms are used which shall have the meanings indicated as follows.

[0021] As used herein, "administer," in reference to an agent, means delivering the agent to the body of a subject via any known method. Specific modes of administration include, but are not limited to, intravenous, oral, sublingual, transdermal, subcutaneous, intraperitoneal, and intrathecal administration.

[0022] Furthermore, in the present invention, the various drugs can be formulated using one or more commonly used pharmaceutically acceptable carriers. Such carriers are well known to those skilled in the art. For example, oral delivery systems include tablets and capsules. These may contain additives such as binders (e.g., hydroxypropylmethylcellulose, polyvinylpyrrolidone, other cellulose substances and starches), excipients (e.g., lactose and other sugars, starch, dicalcium phosphate and cellulose substances), disintegrants (e.g., starch polymers and cellulose substances), and lubricants (e.g., stearates and talc). Injectable drug delivery systems include, for example, solutions, suspensions, gels, microspheres, and polymer injections, and may contain additives such as solubility-altering agents (e.g., ethanol, propylene glycol, and sucrose) and polymers (e.g., polycaprylactone and PLGA). Implantable systems include rods and discs, and may contain additives such as PLGA and polycaprolactone.

[0023] As used herein, "Alzheimer's disease" refers to the simultaneous occurrence of the following three conditions: (i) dementia; (ii) amyloid plaques; and (iii) neurofibrillary tangles. Dementia can be diagnosed during one's lifetime. Cerebral amyloid plaques and neurofibrillary tangles can be diagnosed, for example, during autopsy. This definition of Alzheimer's disease is provided by the National Institute of Neurological Disorders and Stroke (NINDS) of the National Institutes of Health (NIH) and is known as the "gold standard." All subjects from whom samples were collected and studied and whose data are presented herein are autopsy-confirmed AD and non-ADD patients.

[0024] As used herein, when the expression level of a gene is the same as or close to the expression level of the corresponding synchronized cell derived from AD patient, it is considered to be consistent with its expression level.For example, suppose that the TPM scale of gene X in the synchronized cell derived from AD patient is 10, and its TPM scale is 100 in the same type of cell derived from non-ADD patient synchronized in the same way.The gene X expression level of subject should be consistent with the AD expression level of gene X, for example, if its expression level is less than 50, less than 40, less than 30, less than 20, or ideally, 10 or less.

[0025] As used herein, "culturing" lymphocytes can be accomplished, for example, by culturing them in a temperature and growth factor environment that is permissive for cell growth. In another embodiment, "culturing" lymphocytes is performed under conditions that preserve lymphocyte viability (e.g., conditions described herein for proliferation). In one embodiment, the temperature, salt, and protein environment that is permissive for cell growth is RPMI 1640 medium at 37°C with 10% fetal bovine serum ("FBS") and 1% penicillin ("PS"). In one embodiment of the present invention, the step of culturing the lymphocytes is performed for more than 3 hours. Preferably, the step of culturing the lymphocytes is performed for more than 6 hours (e.g., overnight). B lymphocytes can be cultured until overconfluent, i.e., at a high density / μl. This high density is determined as the plateau in the growth curve, typically greater than 90%. The lymphocytes are then starved overnight.

[0026] Methods for obtaining lymphocytes from a subject's blood are known and include, for example, flow cytometry, Ficoll (a hydrophilic polysaccharide that separates the layers of blood), and gradient centrifugation. Furthermore, in the subject methods, the lymphocytes (e.g., B lymphocytes) can be used in an immortalized or primary (i.e., non-immortalized) form. Methods for immortalizing lymphocytes (e.g., B lymphocytes) are known and include, for example, treating the lymphocytes with Epstein-Barr virus ("EBV").

[0027] As used herein, "culturing" skin fibroblasts is accomplished, for example, by culturing them in a temperature and growth factor environment that permits cell growth. In another embodiment, "culturing" skin fibroblasts is performed under conditions that maintain skin fibroblast viability (e.g., conditions described herein for proliferation). In one embodiment, the temperature, humidity, and protein environment that permits cell growth is 37°C, DMEM medium containing 10% fetal bovine serum ("FBS") and 1% penicillin ("PS"). In one embodiment of the present invention, the step of culturing the skin fibroblasts is performed for more than 3 hours. Preferably, the step of culturing the skin fibroblasts is performed for more than 6 hours (e.g., overnight).

[0028] Methods for obtaining skin fibroblasts from the blood of a subject are known, including, for example, skin punch biopsy and growing cells from explants. When cell confluence reaches 100%, the cells are passaged. Typically, after two passages, fibroblasts are purified to a rate of more than 95%.

[0029] As used herein, cells "derived from" a subject are cells that arise through culturing and / or other physical manipulations performed on cells removed directly from the subject. For example, cultured skin fibroblasts derived from a subject are those skin fibroblasts that arise through culturing a sample of skin cells removed directly from the subject (e.g., contained in a punch biopsy).

[0030] As used herein, "diagnosing Alzheimer's disease" means, with respect to a symptomatic human subject, determining that there is a greater than 50% likelihood that the subject has Alzheimer's disease. Preferably, "diagnosing Alzheimer's disease" means determining that there is a greater than 60%, greater than 70%, greater than 80%, or greater than 90% likelihood that the subject has Alzheimer's disease. As used herein, the phrase "determining whether a subject has Alzheimer's disease" is synonymous with the phrase "diagnosing Alzheimer's disease."

[0031] As used herein, "diagnosing non-ADD" means, with respect to a symptomatic human subject, determining that the subject has a greater than 50% likelihood of having non-ADD. Preferably, "diagnosing non-ADD" means determining that the subject has a greater than 60%, greater than 70%, greater than 80%, or greater than 90% likelihood of having non-ADD. As used herein, the phrase "determining whether a subject has non-ADD" is synonymous with the phrase "diagnosing non-ADD."

[0032] As used herein, a gene is "differentially expressed between the synchronized cells of AD patients and the synchronized cells of non-ADD patients" when, for example, the TPM scale of the gene in the synchronized cells of AD patients is different from that in the cells of the same type that are synchronized in the same way that are non-ADD patients.For example, if the TPM scale of gene in the synchronized cells of AD patients is 10, and the TPM scale of gene X in the cells of the same type that are synchronized in the same way that are non-ADD patients, then gene X should be differentially expressed between the synchronized cells of AD patients and the synchronized cells of non-ADD patients.

[0033] As used herein, a drug "favorably" affects the expression level of a gene, and its expression level is correlated with AD if the drug either decreases or increases its expression toward a level correlated with a non-AD state.For example, if the expression level of gene X is lower in AD patients than in unaffected patients, a drug that favorably affects the expression level of that gene should increase its expression level.Similarly, if the expression level of gene X is higher in AD patients than in unaffected patients, a drug that favorably affects the expression level of that gene should decrease its expression level.

[0034] As used herein, "measuring" the expression level of a gene means quantitatively determining its expression level via any means for doing so (e.g., total RNA sequencing (20 million reads, 2x75bp PE)). Preferably, measuring the expression level of a gene is accomplished by measuring the number of RNA transcripts of that gene per million total RNA transcripts (i.e., "TPM" via FastQ data and FPKM prediction per sample) present in the cell-derived RNA population being studied. For example, measuring the expression level of gene X in a synchronized cell population may result in 50 TPM.

[0035] As used herein, a subject suffering from "non-Alzheimer's dementia" means a subject exhibiting dementia such as, for example, the dementias that characterize Parkinson's disease, Huntington's disease, and frontotemporal dementia.

[0036] As used herein, a "population" of cells includes any number of cells that allows for the manipulation and study required to assess gene expression. In one embodiment, the population of cells includes at least 1,000,000 cells. In another embodiment, the population of cells includes between 100,000 and 1,000,000 cells, between 10,000 and 100,000 cells, between 1,000 and 10,000 cells, between 100 and 1,000 cells, between 10 and 100 cells, and less than 10 cells (e.g., 1 cell).

[0037] As used herein, the term "subject" includes, but is not limited to, mammals (e.g., humans, non-human primates, dogs, cats, horses, sheep, goats, cows, rabbits, pigs, rats, and mice). When the subject is a human, the subject can be of any age. For example, the subject can be 50 or older, 55 or older, 60 or older, 65 or older, 70 or older, 75 or older, 80 or older, 85 or older, or 90 or older. The methods of the present invention are contemplated for all subjects, preferably humans (and preferably symptomatic).

[0038] As used herein, a human subject "suspected of having AD or non-ADD" is a subject who exhibits at least one symptom consistent with both AD and non-ADD (e.g., dementia).

[0039] As used herein, "synchronizing" a population of cells means placing at least a majority of the cells in the population in the same cell cycle stage (i.e., in G1, S, G2, or M phase, and preferably in G1, S, or G2 phase). In one embodiment, synchronizing a population of cells means placing at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or preferably at least 99% of the cells in the population in the same cell cycle stage. In another embodiment, synchronizing a population of cells means placing the cells in the population in the same cell cycle stage they would be in if cultured to overconfluence and then starved. Bringing the cells to confluence and then serum-starving typically arrests the cells in G0 / G1 phase [1-3].

[0040] Dose, i.e., "therapeutically effective amount," as used in connection with the present invention, includes, for example, a single administration and two or more administrations (i.e., divided). In one embodiment, a therapeutically effective amount of a drug approved for a non-Alzheimer's indication is the dose and administration regimen approved for that non-Alzheimer's indication.

[0041] As used herein, "treating" a subject afflicted with a disorder includes, but is not limited to: (i) slowing, halting, or reversing the progression of the disorder, (ii) slowing, halting, or reversing the progression of symptoms of the disorder, (iii) reducing the likelihood of recurrence of the disorder, and / or (iv) reducing the likelihood of future recurrence of symptoms of the disorder. In preferred embodiments, treating a subject afflicted with a disorder means (i) reversing the progression of the disorder, ideally to the point of eliminating the disorder, and / or (ii) reversing the progression of symptoms of the disorder, ideally to the point of eliminating the symptoms.

[0042] Treatment of AD can be measured according to a number of clinical endpoints, including, but not limited to, (a) reducing, stabilizing, or slowing the progression of (i) dementia, (ii) synapse loss, (iii) amyloid plaques, and / or (iv) neurofibrillary tangles, and / or (b) favorably affecting the expression levels of genes whose expression levels correlate with AD.

[0043] Embodiments of the present invention The present invention provides an accurate gene-based method for determining whether a human subject is suffering from AD or non-ADD when the subject is suspected of having AD or non-ADD.The subject method is based, at least in part, on the surprising discovery that synchronizing the appropriate cell population of a patient and then measuring the expression levels of genes that are differentially expressed between AD cells and non-ADD cells allows the patient to be accurately diagnosed as having either AD or non-ADD.The present invention also provides a method for treating AD using agents that alter the expression of certain genes.

[0044] Specifically, the present invention provides a method for determining whether a human subject is afflicted with AD or a non-ADD when the subject is suspected of having AD or a non-ADD, the method comprising: (a) synchronizing a population of appropriate cells from the subject; and (b) measuring in the resulting synchronized cell population the expression levels of genes known to be differentially expressed between corresponding synchronized cells derived from AD patients and corresponding synchronized cells derived from non-ADD patients; whereby (i) if the expression level measured in step (b) matches the expression level of the gene in corresponding synchronized cells derived from an AD patient, the subject is afflicted with AD, and (ii) if the expression level measured in step (b) matches the expression level of the gene in corresponding synchronized cells derived from a non-ADD patient, the subject is afflicted with a non-ADD.

[0045] In one embodiment of the subject method, the suitable cells derived from the subject are cultured skin cell fibroblasts, hi another embodiment, the suitable cells derived from the subject are cultured B lymphocytes (preferably immortalized B lymphocytes).

[0046] Methods for synchronizing cell populations are known in the art. In one embodiment of the subject method, synchronizing a population of suitable cells comprises culturing the cells to overconfluence and then starving the resulting overconfluent cells.

[0047] Ideally, in the subject method, the gene is known to be differentially expressed by a significant margin. In one embodiment, the gene is known to be at least 50% differentially expressed between matched synchronized cells from an AD patient and matched synchronized cells from a non-ADD patient. Preferably, the gene is known to be at least 100% differentially expressed between matched synchronized cells from an AD patient and matched synchronized cells from a non-ADD patient. Another way to express the degree of differential expression is "% change" or "% Ch," which is the percentage of the difference between the AD and non-ADD patients. 発現 -Non-ADD 発現 / non-ADD 発現 ] is equivalent to

[0048] In another preferred embodiment of the subject method, the gene is selected from the group consisting of CFAP97, LINC01393, ZNF623, HAUS2, PAN3, PSMB9, ZFP28, TTC26, RFESDP1, ZNF444, WASF2, NHLH1, NPPA-AS1_3, NORAD, URB2, ADAM20, ZCWPW2, AC004057.1, AC092651.1, ACP6, ACP2, C2CD5, CARNS1, FAM149B1, GLIS3-AS1, ASXL2, and IL18R1.

[0049] In one embodiment, the gene expression levels shown in Table 9, individually or collectively (e.g., one, two or more, three or more, four or more, etc.), are indicative of AD. In a preferred embodiment, the gene expression levels shown in Table 10, individually or collectively (e.g., one, two or more, three or more, four or more, etc.), are indicative of AD. For example, as shown in Table 10, a PSMB9 expression level of greater than 18 TPM is indicative of AD. In yet another embodiment, the expression levels indicative of AD for each other gene disclosed herein are readily determined based on the data shown.

[0050] In a further preferred embodiment of the subject method, step (b) comprises measuring the expression levels of a plurality of genes, each gene known to be differentially expressed between corresponding synchronized cells derived from an AD patient and corresponding synchronized cells derived from a non-ADD patient. The plurality of genes (e.g., at least 2 genes, at least 5 genes, at least 20 genes, at least 100 genes, and at least 1,000 genes) can be of any suitable size. Preferably, each gene of the plurality of genes is known to be differentially expressed by at least 50% (and more preferably at least 100%) between corresponding synchronized cells derived from an AD patient and corresponding synchronized cells derived from a non-ADD patient. In yet another preferred embodiment of the subject method, the plurality of genes comprises two or more genes selected from the group consisting of AC004057.1, AC092651.1, ACP6, ADAM20, ASXL2, C2CD5, CARNS1, FAM149B1, GLIS3-AS1, IL18R1, LINC01393, LZIC, MAP1LC3B2, NHLH1, NORAD, NPPA-AS1_3, OSMR-AS1, PAN3, PHBP8, PSMB9, RAB3IP, RDH16, RFESDP1, RPL5, SCG2, SDHD, SHISA5, SLC45A3, SNHG14, TTC26, URB2, USMG5, WASF2, ZCWPW2, ZNF444, and ZNF70.

[0051] In the subject methods in which the expression levels of multiple genes are measured, the expression levels measured in step (b) are "consistent" with the expression levels in corresponding synchronized cells derived from an AD patient if, for example, for at least a majority of the gene expression levels measured, each such level independently matches the expression level of that gene in corresponding synchronized cells derived from the AD patient.

[0052] In the subject method, measuring the expression level of a gene can be achieved by any suitable method known in the art. In a preferred embodiment, measuring the expression level of a gene comprises measuring the number of RNA transcripts of that gene per total number of transcripts.

[0053] In a preferred embodiment, the subject invention provides a method for determining whether a human subject is afflicted with AD or non-ADD, when the subject is suspected of being afflicted with AD or non-ADD, comprising: (a) synchronizing a population of fibroblasts from cultured skin cells derived from the subject, wherein the synchronizing step comprises culturing the fibroblasts to overconfluence and then starving the resulting overconfluent fibroblasts; and (b) measuring the expression level of each of the following genes in the resulting synchronized fibroblast population: AC004057.1, AC092651.1, ACP6, ADAM20, ASXL2, C2CD5, CARNS1, FAM149B1, GLIS3-AS1, IL18R1, LINC01393, LZIC, MAP1LC3B2, NHLH1, NORAD, NPPA-AS1_3, OSMR-AS1, PAN3, PHBP8, PSMB9, RAB3IP, RDH16, RFESDP1, RPL5, SCG2, SDHD, SHISA5, SLC45A3, SNHG14, TTC26, URB2, USMG5, WASF2, ZCWPW2, ZNF444, and ZNF70, wherein measuring the expression level of each gene comprises measuring the number of its RNA transcripts per number of total transcripts; whereby (i) if the expression level measured in step (b) matches the expression level of the gene in corresponding synchronized cells derived from an AD patient, the subject is afflicted with AD, and (ii) if the expression level measured in step (b) matches the expression level of the gene in corresponding synchronized cells derived from a non-ADD patient, the subject is afflicted with a non-ADD.

[0054] In another preferred embodiment, the subject invention provides a method for determining whether a human subject is afflicted with AD or a non-ADD when the subject is suspected of being afflicted with AD or a non-ADD, comprising: (a) synchronizing a population of cultured immortalized B lymphocytes derived from the subject, wherein the synchronizing step comprises culturing the lymphocytes to overconfluence and then starving the resulting overconfluent lymphocytes; and (b) measuring the expression level of each of the following genes in the resulting synchronized lymphocyte population: AC004057.1, AC092651.1, ACP6, ADAM20, ASXL2, C2CD5, CARNS1, FAM149B1, GLIS3-AS1, IL18R1, LINC01393, LZIC, MAP1LC3B2, NHLH1, NORAD, NPPA-AS1_3, OSMR-AS1, PAN3, PHBP8, PSMB9, RAB3IP, RDH16, RFESDP1, RPL5, SCG2, SDHD, SHISA5, SLC45A3, SNHG14, TTC26, URB2, USMG5, WASF2, ZCWPW2, ZNF444, and ZNF70, wherein measuring the expression level of each gene comprises measuring the number of its RNA transcripts per number of total transcripts; whereby (i) if the expression level measured in step (b) matches the expression level of the gene in corresponding synchronized cells derived from an AD patient, the subject is afflicted with AD, and (ii) if the expression level measured in step (b) matches the expression level of the gene in corresponding synchronized cells derived from a non-ADD patient, the subject is afflicted with a non-ADD.

[0055] The present invention further provides a method for determining whether a human subject suspected of having AD or non-ADD has AD, non-ADD, or neither disorder ("NDS"), comprising: (a) synchronizing a population of appropriate cells from the subject; and (b) measuring in the resulting synchronized cell population the expression levels of genes known to be differentially expressed between matched synchronized cells from AD patients, matched synchronized cells from non-ADD patients, and matched synchronized cells from NDS subjects; (i) if the expression level measured in step (b) is consistent with the expression level of the gene in corresponding synchronized cells derived from an AD patient, the subject is diagnosed with AD, (ii) if the expression level measured in step (b) is consistent with the expression level of the gene in corresponding synchronized cells derived from a non-ADD patient, the subject is diagnosed with a non-ADD, and (iii) if the expression level measured in step (b) is consistent with the expression level of the gene in corresponding synchronized cells derived from an NDS subject, the subject is diagnosed with neither AD nor a non-ADD. The various embodiments of the above-described diagnostic methods for determining whether a human subject is diagnosed with AD or a non-ADD apply mutatis mutandis to this method.

[0056] The present invention further provides a method for treating a human subject suffering from Alzheimer's disease, comprising administering to the subject a therapeutically effective amount of an agent known to favorably affect the expression level of one or more genes whose expression levels correlate with Alzheimer's disease. Preferably, the gene is selected from the group consisting of AC004057.1, AC092651.1, ACP6, ADAM20, ASXL2, C2CD5, CARNS1, FAM149B1, GLIS3-AS1, IL18R1, LINC01393, LZIC, MAP1LC3B2, NHLH1, NORAD, NPPA-AS1_3, OSMR-AS1, PAN3, PHBP8, PSMB9, RAB3IP, RDH16, RFESDP1, RPL5, SCG2, SDHD, SHISA5, SLC45A3, SNHG14, TTC26, URB2, USMG5, WASF2, ZCWPW2, ZNF444, and ZNF70. In one embodiment, the gene is selected from the group consisting of IL18R1, PSMB9, TTC26, WASF2, ACP6, CARNS1, NPPA-AS1_3, SCG2, and SDHD. In another embodiment, the gene is IL18R1, PSMB9, TTC26, WASF2, ACP6, CARNS1, NPPA-AS1_3, SCG2, or SDHD.

[0057] The present invention further provides a method for treating a human subject suffering from Alzheimer's disease, the method comprising administering to the subject a therapeutically effective amount of an agent selected from the group consisting of: carfilzomib (Kyprolis) (登録商標) , Onyx Pharmaceuticals), bortezomib (Velcade (登録商標) , Takeda Oncology), bumetanide (Bumex (登録商標) , Hoffman-La Roche), furosemide (Lasix (登録商標) ), torasemide (Demadex (登録商標)), flavin mononucleotide, phosphate, riboflavin, gamma-aminobutyric acid, adenosine monophosphate, histidine, L-arginine, cisplatin, clozapine, cyclosporine A, dexamethasone, etanercept, ethanol, filgrastim, glucose, haloperidol, heparin, infliximab, leflunomide, nitric oxide, oxygen, polyethylene glycol, prednisolone, progesterone, tacrolimus, thalidomide, zinc, calcitriol, calcium, serine, acetylcholine, capsaicin, dopamine, histamine, lithium, norepinephrine, succinic acid, formic acid, tromethamine, citric acid, 10Z-hymenialdisine (Tocris), JIB 04 (Tocris), CRT 0066101 (Tocris), celastrol, dihydroeponemycin, noradrenaline bitartrate (Tocris), or any other drug listed in Table 7. In a preferred embodiment, the agent is carfilzomib, which in one embodiment is administered in the manner set forth in the FDA-approved labeling for one of its approved indications (e.g., in the manner approved for treating multiple myeloma, where the formulation is an injectable and is administered at a dose of 30 mg or 60 mg). In another preferred embodiment, the agent is bortezomib, which in one embodiment is administered in the manner set forth in the FDA-approved labeling for one of its approved indications (e.g., in the manner approved for treating multiple myeloma, where the formulation is an injectable and is administered at a dose of 3.5 mg or 1.3 mg / m 2In another preferred embodiment, the agent is bumetanide, which, in one embodiment, is administered in a manner set forth in the FDA-approved labeling for one of its approved indications (e.g., in a manner approved for treating edema, where the formulation is oral and administered at a dose of 0.5 mg, 1 mg, or 2 mg daily, every other day, or daily for 3-4 days, followed by a 1-2 day rest period). In another preferred embodiment, the agent is furosemide, which, in one embodiment, is administered in a manner set forth in the FDA-approved labeling for one of its approved indications (e.g., in a manner approved for treating edema or hypertension, where the formulation is oral and administered at a dose of 20 mg, 40 mg, 60 mg, or 80 mg per day (e.g., 40 mg twice daily)). In another preferred embodiment, the agent is torasemide, which in one embodiment is administered in the manner set forth in the FDA-approved labeling for one of its approved indications (e.g., in the manner approved for treating edema or hypertension, where the formulation is oral and administered at a dose of 5 mg, 10 mg, 15 mg, or 20 mg per day).

[0058] In another preferred embodiment, the agent is any of cisplatin, clozapine, cyclosporine A, dexamethasone, etanercept, filgrastim, haloperidol, heparin, infliximab, leflunomide, prednisolone, progesterone, tacrolimus, thalidomide, or calcitriol, which in one embodiment is administered in the manner set forth in the FDA-approved labeling for one of its approved indications.

[0059] For each of 10Z-hymenialdisine, JIB 04, CRT 0066101, celastrol, dihydroeponemycin, norepinephrine bitartrate, and other non-FDA-approved drugs, the preferred route of administration is oral, and the preferred dosage is 0.1 mg / kg to 100 mg / kg, 1 mg / kg to 5 mg / kg, 5 mg / kg to 10 mg / kg, 10 mg / kg to 15 mg / kg, or 15 mg / kg to 20 mg / kg.

[0060] The present invention will be better understood by reference to the following examples, although those skilled in the art will readily recognize that the detailed embodiments are merely illustrative of the invention, as more fully described in the claims that follow. [Example]

[0061] Example 1 - Study 1 [Table 1]

[0062] [Table 2]

[0063] [Table 3]

[0064] Example 2 - Study 2; Synchronized Cell Cycle Gene Expression Study for Alzheimer's Disease; Cross-Validation of Gene Differential Expression Patients with Alzheimer's disease (AD; n = 6) and patients with non-Alzheimer's dementia (non-ADD; The initial findings of differential expression of genes in synchronized skin fibroblasts between the first batch of samples (AD; n=2; non-AD n=3) were cross-correlated with the second batch of samples (AD; n=2; non-AD n=3). For the purposes of separating the two batches of samples, we designated the first set of samples as the "training set" and the second set of samples as the "validation set."

[0065] method The genes were ranked in order of decreasing statistical significance, i.e., with the highest statistical significance first (examples in Tables 4 and 5). The ranking was based on a t-test (two-tailed, unequal variance) for two groups of samples, AD and non-ADD. Comparison of the two lists of genes was performed as described below.

[0066] result The number of statistically significant genes was similar in the training and validation sets (Figure 8), with smaller differences at lower statistical significance (P<0.10) and larger differences at higher statistical significance (P<0.001). The larger differences at higher statistical significance (P<0.001) may be due not only to the different number of samples in the validation set (5) compared to the training set (8), but also to the different types of non-ADD samples in the two sets. This difference suggests a higher diversity of dysregulated pathways.

[0067] The majority of genes (n=53) shown in Tables 4 and 5 are under the highest statistical significance (P<0.001), and all of them are under high statistical significance (P<0.01). The presence of the top 40 genes from the training set (Table 4) was checked in the list of 2,077 genes from the validation set (P<0.10; Figure 8). Similarly, the presence of the top 40 genes from the validation set (Table 5) was checked in the list of 2,103 genes from the training set (P<0.10; Figure 8). Because the top 40 genes in Tables 4 and 5 are under the highest statistical significance, they are very likely to have the highest impact on Alzheimer's disease detection, treatment, and pathway dysregulation. The cross-correlation of the top 40 genes in each set was adjusted to accommodate for the diversity in non-ADD samples and to compensate for the different numbers of samples in the validation set (5) and training set (8). A gene pool was performed (P<0.10). Ultimately, however, only genes with similar statistical significance are considered to represent the core of AD dysregulation.

[0068] The results of these initial findings for the 40 most statistically significant genes suggest that approximately 81% of the genes dysregulated in the training set are also dysregulated in the validation set, but only approximately 7.5% of these genes show the same statistical significance in the training and validation sets (Table 6).

[0069] Those genes that show the same statistical significance in the training and validation sets are very likely to be at the core of the dysregulated pathway, at the core of genetic biomarkers for AD, and at the core of therapeutic targets for AD.

[0070] [Table 4-1] [Table 4-2]

[0071] [Table 5-1] [Table 5-2]

[0072] [Table 6-1] [Table 6-2] [Table 6-3]

[0073] [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6] [Table 7-7]

[0074] [Table 8-1] [Table 8-2]

[0075] Example 3 - Study 2; TPM Values Reference range Calculate the average and standard deviation of the transcripts per million (TPM) value of each gene for each of the two groups - Alzheimer's disease (AD) and non-Alzheimer's dementia (non-ADD).Then, calculate the reference interval according to Horn and Pesce (Reference Intervals: A User's Guide. Paul S. Horn and Amadeo J. Pesce. Washington, DC: AACC Press, 2005, ISBN 1-59425-035-9) as the average ± 2 standard deviation.The reference interval thus calculated ensures that 95% of all possible values in each group (AD or non-ADD) are taken into account.

[0076] The gap between AD and non-ADD If there is no overlap between the AD and non-ADD reference ranges, a gap exists between the two bell-shaped curves, indicating an unequivocal diagnosis.

[0077] If there is an overlap between the AD and non-ADD reference ranges (light gray in Table 9), the diagnosis may be false positive or false negative. Genes that show an overlap, i.e., no gap (light gray), in the reference ranges were excluded from the final vector diagnosis. Genes that show a mean of 0 in one of the groups (either the AD group or the non-ADD group) were also excluded.

[0078] Cutoff for each gene The cutoff for each of the remaining 26 genes (Table 10) was determined as the midpoint of the gap in the reference range.

[0079] Genetic vector AD diagnosis The AD diagnosis is based on the 26 components / genes of the vector. For each one of the components, greater than (>) or less than (<) the cutoff value is indicated for each gene in the last column.

[0080] [Table 9-1] [Table 9-2] [Table 9-3]

[0081] [Table 10-1] [Table 10-2]

[0082] References 1. Chen M et al. “Serum Starvation Induced Cell Cycle Synchronization Facilitates Human Somatic Cells Reprogramming”, PLoS ONE 7(4) (2012). 2. Baghdadchi N. “The Effects of Serum Starvation on Cell Cycle Synchronization”, OSR Journal of Student Research (2013). 3. Hayes O et al. “Cell confluency is as efficient as serum starvation for inducing arrest in the G0 / G1 phase of the cell cycle in granulosa and fibroblast cells of cattle”, Anim. Reprod. Sci.87(3-4):181-92 (2005). 4. Spellman PT et al, ”Comprehensive identification of cell cycle-regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization” Mol. Biol. Cell. 9(12):3273-97(1998).

Claims

[Claim 1] The invention as set forth in the drawings.

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