Methods for treating and monitoring progranulin-associated disorders

Abstract

The present disclosure provides methods and materials for screening compounds for treating PGRN-related disorders, or for monitoring the response of a subject to a compound or administration regimen for treating PGRN-related disorders.Methods and materials for identifying and treating a subject with a PGRN-related disorder are also provided. TIFF2022513579000012.tif102153

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 746,293, filed October 16, 2018, and U.S. Provisional Application No. 62 / 884,492, filed August 8, 2019, the disclosures of which are incorporated by reference herein in their entireties for all purposes. [Background technology]

[0002] background Frontotemporal dementia (FTD) is a progressive neurodegenerative disorder that accounts for 5-10% of all patients with dementia and 10-20% of patients who develop dementia before the age of 65 (Rademakers et al., Nat Rev Neurol. 8(8):423-34, 2012). Although several genes have been linked to FTD, one of the most frequently mutated genes in FTD is GRN, which is located on human chromosome 17q21 and encodes the cysteine-rich protein progranulin (also known as proepithelin and acrogranin). Highly penetrant mutations in GRN were first reported in 2006 as a cause of an autosomal dominant form of familial FTD (Baker et al., Nature. 442(7105):916-9, 2006 (Non-Patent Document 2); Cruts et al., Nature. 2006 Aug 24; 442(7105):920-4 (Non-Patent Document 3); Gass et al., Hum. Mol. Genet. 15(20):2988-3001, 2006 (Non-Patent Document 4)). Recent estimates suggest that GRN mutations account for 5-20% of FTD patients with a positive family history and 1-5% of sporadic cases (Rademakers et al., supra). There is a need for new methods for evaluating compounds and related therapies for the treatment of PGRN-related disorders (including monitoring patient response to such treatments), as well as new methods for identifying subjects who may benefit from treatment of these disorders. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Rademakers et al., Nat Rev Neurol.8(8):423-34,2012 [Non-Patent Document 2] Baker et al.,Nature.442(7105):916-9,2006 [Non-Patent Document 3] Cruts et al.,Nature.2006 Aug 24;442(7105):920-4 [Non-Patent Document 4] Gass et al.,Hum.Mol.Genet.15(20):2988-3001,2006 Summary of the Invention

[0004] explanation In one aspect, the present disclosure provides a method for evaluating a compound for treating a progranulin (PGRN)-related disorder, or for monitoring the response of a subject to a compound, pharmaceutical composition, or their administration regimen for treating a PGRN-related disorder, the method comprising: (a) measuring the abundance of one or more bis(monoacylglycero)phosphate (BMP) species in a test sample from a subject having a PGRN-related disorder, wherein the test sample or subject is treated with a compound or its pharmaceutical composition; (b) comparing the abundance difference between the one or more BMP species measured in step (a) and one or more reference values; and (c) determining from the comparison whether the compound, pharmaceutical composition, or their administration regimen improves the one or more BMP species level for treating a PGRN-related disorder.

[0005] In some embodiments, the methods provided herein further include treating another test sample or subject with another compound and selecting a candidate compound that improves the level of one or more BMP species.

[0006] In some embodiments, the methods provided herein further include the steps of (d) maintaining or adjusting the amount or frequency of administration of the compound to the test sample or subject, and (e) administering the compound to the test sample or subject.

[0007] In one aspect, a method for identifying a subject having or at risk of having a PGRN-related disorder is provided, the method comprising: (a) measuring the abundance of one or more BMP species in a test sample from the subject; (b) comparing the abundance difference between the one or more BMP species measured in step (a) and one or more reference values; and (c) determining from the comparison whether the subject has a PGRN-related disorder.

[0008] In some embodiments, the method provided herein further comprises administering to the subject the compound for improving one or more BMP species levels for treating PGRN-related disorder.In some embodiments, at least one of one or more signs or symptoms of PGRN-related disorder improves after treatment.

[0009] In some embodiments, the treatment comprises administering PGRN, its derivatives, or pharmaceutical compositions thereof to a subject. In some embodiments, the PGRN derivatives contain chemical moieties or peptide fragments that allow PGRN to cross the blood-brain barrier. In some embodiments, the treatment comprises administering a library of compounds to a plurality of subjects or test samples.

[0010] In some embodiments, reference value is measured in a reference sample obtained from a reference subject or a reference subject population.In some embodiments, reference value is the abundance of one or more BMP species measured in a reference sample.In some embodiments, reference sample is the same type of cell, tissue or body fluid as test sample.In some embodiments, at least two reference values ​​are measured from different types of cell, tissue or body fluid.

[0011] In some embodiments, the reference sample is a healthy control.In some embodiments, the reference subject or the reference subject population does not have a PGRN-related disorder or does not have a reduced PGRN level.In certain embodiments, the reference subject or the reference subject population does not have any signs or symptoms of such disorders.

[0012] In some embodiments, BMP species levels are elevated in bone marrow-derived macrophages obtained in vitro from bone marrow cells of a subject having or at risk of having a PGRN-associated disorder compared to healthy controls or controls unrelated to a PGRN-associated disorder.

[0013] In some embodiments, BMP species levels are reduced in the liver, brain, cerebrospinal fluid, plasma, or urine of subjects having or at risk of having a PGRN-related disorder compared to healthy controls or controls unrelated to a PGRN-related disorder.

[0014] In some embodiments, the abundance of the BMP species in the test sample of a subject having or at risk of having a PGRN-related disorder has at least about a 1.2-fold, 1.5-fold, or 2-fold difference compared to a reference value of a control, such as a healthy control or a control unrelated to a PGRN-related disorder. In other embodiments, the abundance of the BMP species in the test sample of a subject having or at risk of having a PGRN-related disorder has about a 1.2-fold to about a 4-fold difference compared to a reference value of a control, such as a healthy control or a control unrelated to a PGRN-related disorder. In some embodiments, the difference compared to the reference value is about 2-fold to about 3-fold. In some embodiments, the subject has a disorder associated with a decrease in PGRN levels and / or one or more signs or symptoms of a disorder associated with a decrease in PGRN levels.

[0015] In some embodiments, the reference value is the value of the BMP species before treatment.In some embodiments, the subject is treated for the decrease of PGRN level or PGRN-related disorder, and the test sample comprises one or more pre-treatment test samples obtained from the subject before starting treatment, and one or more post-treatment test samples obtained from the subject after starting treatment.In some embodiments, the method further comprises determining that the subject is responsive to treatment if the abundance of at least one of the one or more post-treatment BMP species shows improvement with respect to the one or more pre-treatment BMP species compared with healthy control.

[0016] In some embodiments, the method includes the steps of: (a) measuring the abundance of one or more bis(monoacylglycero)phosphate (BMP) species in a test sample obtained from a subject; (b) treating the test sample or the subject with a compound, pharmaceutical composition, or an administration regimen thereof; (c) measuring the abundance of one or more BMP species in a test sample obtained from the treated subject; (d) comparing the abundance of one or more BMP species measured in steps (a) and (c); and (e) determining whether the compound or administration regimen improves BMP levels for the treatment of a PGRN-associated disorder.

[0017] In some embodiments, two or more post-treatment test samples are obtained at different time points after starting treatment, and the method further comprises determining that the subject is responsive to treatment if the abundance of at least one of the one or more BMP species measured in the post-treatment sample is a) lower in bone marrow derived macrophages (BMDM), or b) higher in liver, brain, cerebrospinal fluid, plasma, or urine than the abundance of the corresponding one or more BMP species measured in the pre-treatment sample.In some embodiments, the subject is determined to be responsive to treatment if the abundance of at least one of the one or more BMP species measured in the post-treatment sample is a) at least about 1.2-fold lower in BMDM, or b) at least about 1.2-fold higher in liver, brain, cerebrospinal fluid, plasma, or urine than the abundance of the corresponding one or more BMP species measured in the pre-treatment sample.

[0018] In some embodiments, the improved BMP species level is an improvement to the BMP species level before treatment compared to a reference value of a control, such as a healthy control or a control unrelated to PGRN-related disorder. In some embodiments, the improved BMP species level is closer to the control than the value of the BMP species level before treatment is to the control. In some embodiments, the improved BMP species level has a difference of less than 15%, 10%, or 5% compared to the control. In some embodiments, the improved BMP species level has a difference of less than 10% or 5% compared to the healthy control. In some embodiments, the improved BMP species level has a difference of less than 5% compared to the healthy control.

[0019] In some embodiments, the method further includes determining that the subject is responding to the treatment if the abundance of at least one of the one or more BMP species measured in at least one of the one or more post-treatment test samples is approximately the same as a corresponding reference value for a healthy control.

[0020] In some embodiments, the test sample, the reference sample, or one or more reference values ​​include or are associated with a cell, a tissue, whole blood, plasma, serum, cerebrospinal fluid, interstitial fluid, saliva, urine, stool, bronchoalveolar lavage fluid, lymph, semen, breast milk, amniotic fluid, or a combination thereof. In some embodiments, the cell is a peripheral blood mononuclear cell (PBMC), a bone marrow derived macrophage (BMDM), a retinal pigment epithelium (RPE) cell, a blood cell, an erythrocyte, a leukocyte, a neuron, a microglial cell, a brain cell, a cerebral cortex cell, a spinal cord cell, a bone marrow cell, a liver cell, a kidney cell, a spleen cell, a lung cell, an eye cell, a chorionic villus cell, a muscle cell, a skin cell, a fibroblast cell, a heart cell, a lymph node cell, or a combination thereof. In some embodiments, the cell is a cultured cell. In some embodiments, the cultured cell is a BMDM or a RPE cell.

[0021] In some embodiments, the tissue comprises brain tissue, cerebral cortex tissue, spinal cord tissue, liver tissue, kidney tissue, muscle tissue, heart tissue, eye tissue, retina tissue, lymph node, bone marrow, skin tissue, vascular tissue, lung tissue, spleen tissue, valve tissue, or a combination thereof. In some embodiments, the test sample and / or the reference sample are purified from cells and / or tissues and comprise endosomes, lysosomes, extracellular vesicles, exosomes, microvesicles, or a combination thereof.

[0022] In some embodiments, the one or more BMP species comprises two or more BMP species. In some embodiments, the one or more BMP species comprises BMP(16:0_18:1), BMP(16:0_18:2), BMP(18:0_18:0), BMP(18:0_18:1), BMP(18:1_18:1), BMP(16:0_20:3), BMP(18:1_20:2), BMP(18:0_20:4), BMP(16:0_22:5), BMP(20:4_20:4), BMP(22:6_22:6), BMP(20:4_20:5). ), BMP(18:2_18:2), BMP(16:0_20:4), BMP(18:0_18:2), BMP(18:0e_22:6), BMP(18:1e_20:4), BMP(18:3_22:5), BMP(20:4_22:6), BMP(18:0e_20:4), BMP(18:2_20:4), BMP(18:1_22:6), BMP(18:1_20:4), BMP(18:0_22:6), or any combination thereof.

[0023] In some embodiments, the one or more BMP species include BMP(18:1_18:1), BMP(18:0_20:4), BMP(20:4_20:4), BMP(22:6_22:6), BMP(20:4_22:6), BMP(18:1_22:6), BMP(18:1_20:4), BMP(18:0_22:6), BMP(18:3_22:5), or combinations thereof.

[0024] In some embodiments, the test sample comprises cultured cells and the one or more BMP species comprises BMP(18:1_18:1). In some embodiments, the test sample comprises plasma, tissue, urine, cerebrospinal fluid (CSF), and / or brain tissue or liver tissue and the one or more BMP species comprises BMP(22:6_22:6). In some embodiments, the test sample comprises liver tissue and the one or more BMP species comprises BMP(22:6_22:6), BMP(18:3_22:5), or a combination thereof. In some embodiments, the test sample comprises CSF or urine and the one or more BMP species comprises BMP(22:6_22:6). In some embodiments, the test sample comprises CSF and the one or more BMP species comprises BMP(18:1_18:1). In some embodiments, the test sample comprises microglia and the one or more BMP species comprises BMP(18:3_22:5).

[0025] In some embodiments, the abundance of one or more BMP species is measured using a method selected from the group consisting of liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-tandem mass spectrometry (LC-MS / MS), gas chromatography-mass spectrometry (GC-MS), gas chromatography-tandem mass spectrometry (GC-MS / MS), enzyme-linked immunosorbent assay (ELISA), and combinations thereof. In some embodiments, an internal BMP standard is used to measure the abundance of one or more BMP species in step (a) and / or determine a corresponding reference value. In some embodiments, the internal BMP standard comprises a BMP species that is not naturally occurring in the subject and / or reference subject or reference subject population. In some embodiments, the internal BMP standard comprises BMP(14:0_14:0).

[0026] In some embodiments, the PGRN-related disorder is a disorder associated with expression, processing, glycosylation, cellular uptake, transport, and / or function of PGRN. In some embodiments, the subject and / or the reference subject or the reference subject population has a reduced PGRN level and / or a disorder associated with reduced PGRN level, and the test sample is contacted with a candidate compound. In some embodiments, the subject and / or the reference subject or the reference subject population has one or more signs or symptoms of a disorder associated with reduced PGRN level. In some embodiments, the subject and / or the reference subject or the reference subject population has a mutation in the granulin (GRN) gene. In some embodiments, the mutation in the GRN gene reduces the expression and / or activity of PGRN. In some embodiments, the PGRN-related disorder is atherosclerosis, Gaucher disease, or age-related macular degeneration (AMD). In some embodiments, the PGRN-related disorder is Gaucher disease type 1. In some embodiments, the PGRN-related disorder is a disorder associated with TDP-43. In other embodiments, the TDP-43-related disorder is AD or ALS.

[0027] In some embodiments, the PGRN-related disorder is a neurodegenerative disease. In some embodiments, the neurodegenerative disease is selected from the group consisting of frontotemporal dementia (FTD), neuronal ceroid lipofuscinosis (NCL), Niemann-Pick disease type A (NPA), Niemann-Pick disease type B (NPB), Niemann-Pick disease type C (NPC), C9ORF72-associated amyotrophic lateral sclerosis (ALS) / FTD, sporadic ALS, Alzheimer's disease (AD), Gaucher disease types 2 and 3, and Parkinson's disease. In some embodiments, the subject and / or the reference subject is a human, a non-human primate, a rodent, a dog, or a pig.

[0028] In another aspect, the present disclosure provides a kit for monitoring PGRN levels in a subject. In some embodiments, the kit comprises a bis(monoacylglycero)phosphate (BMP) standard for measuring the abundance of one or more BMP species in a test sample obtained from a subject and / or a reference sample obtained from a reference subject or a reference subject population. In some embodiments, the BMP standard comprises a BMP species that does not naturally occur in the subject and / or the reference subject. In some embodiments, the BMP standard comprises BMP(14:0_14:0).

[0029] In some embodiments, the kit further comprises reagents for obtaining a sample from a subject and / or a reference subject, reagents for processing the sample, reagents for measuring the abundance of one or more BMP species, or a combination thereof, In some embodiments, the kit further comprises instructions for use. [The present invention 1001] 1. A method for evaluating a compound for the treatment of a progranulin (PGRN)-associated disorder or for monitoring a subject's response to a compound, pharmaceutical composition, or administration regimen thereof for the treatment of a PGRN-associated disorder, comprising: (a) measuring the abundance of one or more bis(monoacylglycero)phosphate (BMP) species in a test sample from a subject having a PGRN-associated disorder, wherein the test sample or the subject has been treated with the compound or a pharmaceutical composition thereof; (b) comparing the difference in abundance between the one or more BMP species measured in step (a) and one or more reference values; (c) determining from the comparison whether the compound, pharmaceutical composition, or administration regimen thereof improves the level of one or more BMP species for the treatment of a PGRN-associated disorder; The method comprising: [The present invention 1002] Treating another test sample or subject with another compound; selecting a candidate compound that improves the level of said one or more BMP species; The method of the present invention 1001 further comprises: [The present invention 1003] (d) maintaining or adjusting the amount or frequency of administration of said compound to said test sample or said subject; (e) administering said compound to said test sample or to said subject. The method of the present invention 1001 further comprises: [The present invention 1004] 1. A method for identifying a subject having or at risk of having a PGRN-associated disorder, comprising: (a) determining the abundance of one or more BMP species in a test sample from a subject; (b) comparing the difference in abundance between the one or more BMP species measured in step (a) and one or more reference values; (c) determining from the comparison whether the subject has a PGRN-associated disorder. The method comprising: [The present invention 1005] The method of any one of claims 10 to 14, further comprising administering to said subject a compound for improving said one or more BMP species levels for the treatment of a PGRN-associated disorder. [The present invention 1006] The method according to any one of claims 1001 to 1003 or 1005, wherein said compound is PGRN, a PGRN derivative, or a pharmaceutical composition thereof. [The present invention 1007] The method of any of the prior inventions, wherein said reference value is measured in a reference sample obtained from a reference subject or a reference subject population. [The present invention 1008] The method of claim 1007, wherein said reference subject or said reference subject population is a healthy control. [The present invention 1009] The method of claim 1007, wherein said reference subject or said reference subject population does not have a PGRN-related disorder or does not have reduced PGRN levels. [The present invention 1010] Any of the methods of the prior inventions, wherein a subject having or at risk of having a PGRN-related disorder has elevated levels of BMP species in bone marrow-derived macrophages compared to a healthy control or a control unrelated to a PGRN-related disorder. [The present invention 1011] Any of the methods of the prior inventions, wherein a subject having or at risk of having a PGRN-related disorder has reduced levels of BMP species in the liver, brain, cerebrospinal fluid, plasma, or urine compared to a healthy control or a control unrelated to a PGRN-related disorder. [The present invention 1012] Any of the methods of the prior inventions, wherein the abundance of a BMP species in a test sample from a subject having or at risk of having the PGRN-related disorder has a difference of at least about 1.2-fold, 1.5-fold, or 2-fold compared to a reference value of a control, such as a healthy control or a control unrelated to a PGRN-related disorder. [The present invention 1013] Any of the methods of the prior inventions, wherein the abundance of a BMP species in a test sample from a subject having or at risk of having a PGRN-related disorder has a difference of at least about 1.2-fold to about 4-fold compared to a reference value of a control, such as a healthy control or a control unrelated to a PGRN-related disorder. [The present invention 1014] The method according to any one of claims 1001 to 1007, wherein said reference value is the value of said BMP species before treatment. [The present invention 1015] The method of any of the prior inventions, wherein the improved BMP species level is an improvement relative to the BMP species level before treatment compared to a reference value of a control, such as a healthy control or a control unrelated to the PGRN-associated disorder. [The present invention 1016] The method of claim 1015, wherein said improved BMP species level has a difference of less than 15%, less than 10%, or less than 5% compared to said control. [The present invention 1017] The method of any of the prior inventions, wherein the test sample, the reference sample, or the one or more reference values ​​comprise or relate to a cell, a tissue, whole blood, plasma, serum, cerebrospinal fluid, interstitial fluid, saliva, urine, lymphatic fluid, or a combination thereof. [The present invention 1018] The method of the present invention, wherein the cell is a peripheral blood mononuclear cell (PBMC), a bone marrow derived macrophage (BMDM), a retinal pigment epithelial (RPE) cell, a blood cell, a red blood cell, a white blood cell, a neuronal cell, a microglial cell, a brain cell, a cerebral cortex cell, a spinal cord cell, a bone marrow cell, a liver cell, a kidney cell, a spleen cell, a lung cell, an eye cell, a chorionic villus cell, a muscle cell, a skin cell, a fibroblast cell, a cardiac cell, a lymph node cell, or a combination thereof. [The present invention 1019] The method of any one of claims 1017 to 1018, wherein the cell is a cultured cell. [The present invention 1020] The method of claim 1019, wherein the cultured cells are BMDM or RPE cells. [The present invention 1021] The method of claim 1017, wherein the tissue comprises brain tissue, cerebral cortex tissue, spinal cord tissue, liver tissue, kidney tissue, muscle tissue, heart tissue, eye tissue, retinal tissue, lymph node, bone marrow, skin tissue, vascular tissue, lung tissue, spleen tissue, valve tissue, or a combination thereof. [The present invention 1022] The method of any of the prior inventions, wherein the test sample comprises endosomes, lysosomes, extracellular vesicles, exosomes, microvesicles, or a combination thereof. [The present invention 1023] The method of any of the prior inventions, wherein the one or more BMP species comprises two or more BMP species. [The present invention 1024] The one or more BMP species are BMP(16:0_18:1), BMP(16:0_18:2), BMP(18:0_18:0), BMP(18:0_18:1), BMP(18:1_18:1), BMP(16:0_20:3), BMP(18:1_20:2), BMP(18:0_20:4), BMP(16:0_22:5), BMP(20:4_20:4), BMP(22:6_22:6), BMP(20:4_20:5), BM 20:4, BMP(18:0_22:6), BMP(18:1_20:4), BMP(20:4_22:6), BMP(18:0e_20:4), BMP(18:2_20:4), BMP(18:1_22:6), BMP(18:1_20:4), BMP(18:0_22:6), or a combination thereof. [The present invention 1025] The method of any of the prior inventions, wherein the one or more BMP species comprises BMP(18:1_18:1), BMP(18:0_20:4), BMP(20:4_20:4), BMP(22:6_22:6), BMP(20:4_22:6), BMP(18:1_22:6), BMP(18:1_20:4), BMP(18:0_22:6), BMP(18:3_22:5), or a combination thereof. [The present invention 1026] The method of any of the prior inventions, wherein the test sample comprises BMDM and the one or more BMP species comprises BMP(18:1_18:1). [The present invention 1027] The method of any of the prior inventions, wherein the test sample comprises plasma, urine, cerebrospinal fluid (CSF), and / or brain tissue or liver tissue, and the one or more BMP species comprises BMP(22:6_22:6). [The present invention 1028] The method of any of the prior inventions, wherein the test sample comprises liver tissue and the one or more BMP species comprises BMP(22:6_22:6), BMP(18:3_22:5), or a combination thereof. [The present invention 1029] The method of any of the prior inventions, wherein said test sample comprises CSF or urine and said one or more BMP species comprises BMP(22:6_22:6). [The present invention 1030] The method of any of the prior inventions, wherein said test sample comprises CSF and said one or more BMP species comprises BMP(18:1_18:1). [The present invention 1031] The method of any of the prior inventions, wherein the test sample comprises microglia and the one or more BMP species comprises BMP(18:3_22:5). [The present invention 1032] The method of any of the prior inventions, wherein the abundance of the one or more BMP species is measured using liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-tandem mass spectrometry (LC-MS / MS), gas chromatography-mass spectrometry (GC-MS), gas chromatography-tandem mass spectrometry (GC-MS / MS), enzyme-linked immunosorbent assay (ELISA), or a combination thereof. [The present invention 1033] The method of any of the prior inventions, wherein an internal BMP standard is used when determining the abundance of said one or more BMP species. [The present invention 1034] The method of any one of claims 10 to 33, wherein said internal BMP standard comprises a BMP species that does not naturally occur in said subject and / or said reference subject or said reference subject population. [The present invention 1035] The method of any one of claims 1033 to 1034, wherein the internal BMP standard comprises BMP(14:0_14:0). [The present invention 1036] The method of any of the prior inventions, wherein said PGRN-associated disorder is a disorder associated with expression, processing, glycosylation, cellular uptake, transport, and / or function of PGRN. [The present invention 1037] The method of any of the prior inventions, wherein the subject has one or more mutations in the granulin (GRN) gene. [The present invention 1038] The method of any of the prior inventions, wherein said PGRN-associated disorder is associated with reduced levels of PGRN. [The present invention 1039] The method of any of the prior inventions, wherein said PGRN-associated disorder is a neurodegenerative disease. [The present invention 1040] Any of the methods of the prior inventions, wherein the PGRN-related disease is a disease selected from the group consisting of frontotemporal dementia (FTD), neuronal ceroid lipofuscinosis (NCL), Niemann-Pick disease type A (NPA), Niemann-Pick disease type B (NPB), Niemann-Pick disease type C (NPC), C9ORF72-associated amyotrophic lateral sclerosis (ALS) / FTD, sporadic ALS, Alzheimer's disease (AD), atherosclerosis, Gaucher disease, Parkinson's disease, and age-related macular degeneration (AMD). [The present invention 1041] The method of any of the prior inventions, wherein said subject and / or said reference subject is a human, a non-human primate, a rodent, a dog, or a pig. [The present invention 1042] The method of any of the prior inventions, wherein the subject having a disorder associated with decreased PGRN levels is a PGRN knockout mouse or a PGRN knockout rat. [The present invention 1043] A kit for testing a compound or its administration regimen for the treatment of a PGRN-associated disorder, the kit comprising a BMP standard for measuring the abundance of one or more BMP species in a test sample from the subject. [The present invention 1044] The kit of the present invention, wherein the BMP standard comprises a BMP species that does not naturally occur in the subject. [The present invention 1045] The kit of the present invention 1043 or 1044, wherein the BMP standard comprises BMP(14:0_14:0). [The present invention 1046] Any of the kits of claims 1043 to 1045, further comprising a reagent for obtaining the sample from the subject, a reagent for processing the sample, a reagent for measuring the abundance of the one or more BMP species, or a combination thereof. [The present invention 1047] The kit of any one of claims 1043 to 1046, further comprising an instruction manual. [Brief description of the drawings]

[0030] [Figure 1] A and B show schematic diagrams of fusion proteins F1 and F2, respectively, each containing an Fc polypeptide dimer and PGRN. In A, the N-terminus of PGRN is fused to the C-terminus of the Fc polypeptide dimer via a linker of -GGGGSGGGGS- (SEQ ID NO: 1). In B, the C-terminus of PGRN is fused to the N-terminus of the Fc polypeptide dimer via a linker of (G4S)2 (SEQ ID NO: 1). Fusion proteins F1 and F2 are further described in Example 1. [Figure 2A] 1 shows that the levels of bis(monoacylglycero)phosphate (BMP) species in bone marrow-derived macrophages (BMDM) of Grn knockout mice are increased compared to wild type. 2 shows that the increase in BMP (18:1_18:1) levels was reduced by treating BMDM of Grn knockout mice with the PGRN fusion proteins F1 and F2 of Example 1. [Figure 2B] 1 shows that the levels of bis(monoacylglycero)phosphate (BMP) species in bone marrow-derived macrophages (BMDM) of Grn knockout mice are increased compared to wild type. 2 shows that the increase in BMP (20:4_20:4) levels was reduced by treating BMDM of Grn knockout mice with the PGRN fusion proteins F1 and F2 of Example 1. [Figure 2C]This shows that treatment of BMDM from Grn knockout mice with recombinant PGRN or PGRN expressed by lentivirus in Example 2 reduced the increase in BMP levels (total BMP as well as BMP (18:1_18:1)). Data for total BMP is shown. [Figure 2D] This shows that the BMDM of Grn knockout mice was treated with recombinant PGRN or PGRN expressed by lentivirus of Example 2, and the increase in BMP levels (total BMP and BMP (18:1_18:1)) was reduced. Data of BMP (18:1_18:1) is shown. [Figure 3A] 1 shows that BMP44:12 was reduced in peripheral liver, plasma, and urine of Grn knockout mice compared to wild type. [Figure 3B] 1 shows that BMP44:12 is reduced in the cerebrospinal fluid (CSF) and central nervous system (CNS) brain compared to wild type. [Figure 4] We show that Grn knockout mice ranging from 2 to 19 months of age exhibited an age-independent decrease in plasma BMP44:12 compared to wild-type mice. [Diagram 5] A and B show that treatment of Grn knockout mice with PGRN fusion proteins F1 and F2 increased BMP44:12 and 20:4_20:4 levels in the liver. [Figure 6] 1 shows that treatment of Grn knockout mice with PGRN fusion proteins F1 and F2 increased plasma BMP44:12 levels. [Figure 7] 1 shows that treatment of Grn knockout mice with PGRN fusion proteins F1 and F2 increased urinary BMP44:12 levels (normalized to creatine). [Figure 8] 1 shows that treatment of Grn knockout mice with PGRN fusion proteins F1 and F2 increased BMP44:12 levels in the CSF. [Figure 9]A and B show that treatment of Grn knockout mice with PGRN fusion proteins F1 and F2 increased brain BMP44:12 and 20:4_20:4 levels, respectively. [Figure 10A] FIG. 1 shows that BMP18:1_18:1 and 22:6_22:6 were decreased in sporadic and Grn FTD patient samples compared to clinically normal controls. [Figure 10B] FIG. 1 shows that BMP18:1_18:1 and 22:6_22:6 were decreased in sporadic and Grn FTD patient samples compared to clinically normal controls. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] definition As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "cell" may include two or more such cells, and the like.

[0032] As used herein, the terms "about" and "approximately," when used to modify a quantity specified in a numerical value or range, indicate that the numerical value as well as reasonable deviations from the value known to one of ordinary skill in the art, for example, ±20%, ±10%, or ±5%, are within the intended meaning of the recited value.

[0033] The term "abundance" refers to the amount or concentration of a molecule, compound, or agent (e.g., a bis(monoacylglycero)phosphate (BMP) molecule). The term includes absolute amount or concentration, as well as relative amount or concentration. In some embodiments, a reference standard (e.g., an internal BMP standard) is used for calibration to determine the absolute amount or concentration of a molecule, compound, or agent present (e.g., in a sample) and / or is normalized to a control to determine the relative amount or concentration of a molecule, compound, or agent present.

[0034] The term "PGRN level" refers to the amount, concentration, and / or activity level of progranulin present either in a subject or in a sample (e.g., a sample obtained from a subject). PGRN level can refer to the absolute amount, concentration, and / or activity level of PGRN present, or it can refer to the relative amount, concentration, and / or activity level. This term also refers to the amount or concentration of PGRN polypeptide and / or PGRN mRNA (e.g., expressed from the GRN gene) present.

[0035] The term "progranulin" or "PGRN" (also known as "proepithelin" and "acrogranin") is a cysteine-rich protein encoded by the gene GRN located on human chromosome 17q21. PGRN is a lysosomal protein and a secreted protein consisting of 7.5 tandem repeats of conserved granulin peptides, each of which is approximately 60 amino acids long and can be released by cleavage by various extracellular proteases (e.g., elastase) and lysosomal proteases (e.g., cathepsin L) (Kao et al., Nat Rev Neurosci. 18(6):325-333, 2017). In general, PGRN is thought to play both cell-autonomous and cell-non-autonomous roles in the control of innate immunity and in the function of lysosomes, where PGRN regulates the activity and levels of various cathepsins and other hydrolases (Kao et al., supra). PGRN also has a neurotrophic function and promotes neurite outgrowth and neuronal survival (Kao et al., supra). PGRN polypeptide may include human PGRN sequence. PGRN polypeptide may be immature PGRN, for example, where the first 17 amino acids constitute a signal peptide. PGRN polypeptide may also be mature PGRN, for example, where the signal peptide of 17 amino acids is truncated. As another non-limiting example, PGRN polypeptide may include sequences from non-human species, such as mouse (NCBI reference number NP_032201.2), rat (NCBI reference number NP_058809.2 or NP_001139314.1), or chimpanzee (NCBI reference number XP_016787144.1 or XP_016787145.1), either in immature or mature form. The term "Progranulin derivative" or "PGRN derivative" refers to modified PGRN. Modifications can be made to enhance the druggability of PGRN, such as modifications to target it to specific regions in the body, and / or to improve its pharmacokinetic or pharmacodynamic properties. Other modifications can be made to aid in its manufacture and / or shelf life.PGRN derivatives can include Fc fusion proteins that include PGRN attached to a dimer of an Fc polypeptide.

[0036] The term "PGRN-associated disorder" refers to any pathology associated with PGRN, including expression, processing, glycosylation, cellular uptake, transport, and / or function. The term "disorder associated with reduced PGRN levels" refers to any pathology that results directly or indirectly from insufficient (i.e., too low) levels of PGRN to enable normal physiological function in a cell, tissue, and / or subject, as well as precursors of such conditions. In some embodiments, the disorder is a neurodegenerative disease and / or a lysosomal storage disease.

[0037] The term "bone marrow-derived macrophage" or "BMDM" refers to macrophage cells generated in vitro or derived from mammalian bone marrow (e.g., bone marrow obtained from a subject). As a non-limiting example, BMDM can be generated by culturing undifferentiated bone marrow cells in the presence of cytokines such as macrophage colony-stimulating factor (M-CSF).

[0038] The terms "treatment", "treating" and the like are generally used herein to mean obtaining a desired pharmacological and / or physiological effect. "Treat" or "treatment" may refer to any indication of success in treating or ameliorating a disease, including neurodegenerative diseases (e.g., frontotemporal dementia (FTD), atherosclerosis, Gaucher disease, Alzheimer's disease (AD), neuronal ceroid lipofuscinosis (NCL), Niemann-Pick disease type A (NPA), Niemann-Pick disease type B (NPB), Niemann-Pick disease type C (NPC), C9ORF72-associated amyotrophic lateral sclerosis (ALS) / FTD, age-related macular degeneration (AMD), and Parkinson's disease), including any objective and subjective parameters, such as palliative, remission, improvement in patient survival, increased survival time or rate, reducing symptoms or making the disorder more tolerable to the patient, slowing the rate of degeneration or decline, or improving the patient's physical or mental well-being. The treatment or amelioration of symptoms can be based on objective or subjective parameters. The effect of treatment can be compared to an individual or pool of individuals not receiving treatment, or to the same patient before treatment or at different times during treatment.

[0039] As used interchangeably herein, the terms "subject," "individual," and "patient" refer to mammals, including, but not limited to, humans, non-human primates, rodents (e.g., rats, mice, and guinea pigs), rabbits, dogs, cows, pigs, horses, and other mammalian species. In one embodiment, the subject is a human.

[0040] As used herein, a "therapeutic amount" or a "therapeutically effective amount" of an agent is an amount of the agent that treats a symptom of the disease in a subject.

[0041] The term "administering" refers to a method of delivering an agent, compound, or composition to a desired site of biological action, including, but not limited to, topical, parenteral, intravenous, intradermal, intramuscular, subcutaneous, intrathecal, oral, colonic, rectal, or intraperitoneal delivery.

[0042] Bis(monoacylglycero)phosphate (BMP) Provided herein are methods for monitoring PGRN levels (e.g., in a sample, in a cell, in a tissue, and / or in a subject), where determining PGRN levels includes measuring the abundance of bis(monoacylglycero)phosphate (BMP) (e.g., in a sample, in a cell, in a tissue, and / or in a subject). BMP is a negatively charged glycerophospholipid (e.g., at pH typically present in late endosomes and lysosomes) having the structure shown in Formula I: TIFF0007624919000001.tif28128

[0043] The BMP molecule contains two fatty acid side chains. R and R' in formula I represent independently selected saturated or unsaturated aliphatic chains, each of which typically contains 14, 16, 18, 20, or 22 carbon atoms. When the fatty acid side chains are unsaturated, they can contain 1, 2, 3, 4, 5, or more than 6 carbon-carbon double bonds. Additionally, the BMP molecule can contain one or two alkyl ether substituents, where the carbonyl oxygen of one or both fatty acid side chains is replaced with two hydrogen atoms.

[0044] The nomenclature used herein to describe specific BMP species refers to species having two fatty acid side chains, where the structure of the fatty acid side chain is indicated in parentheses in the BMP format (e.g., BMP(18:1_18:1)). The numbers follow the standard fatty acid notation format of "number of fatty acid carbon atoms:number of double bonds". The prefix "e-" is used to indicate the presence of an alkyl ether substituent, where the carbonyl oxygen of the fatty acid side chain is replaced with two hydrogen atoms. For example, the "e" in "BMP(16:0e_18:0)" indicates that the side chain with 16 carbon atoms is an alkyl ether substituent.

[0045] BMPs are unusual in that they have a sn-1;sn-1' structural arrangement (i.e., based on phosphate-linked glycerol carbons) that is not observed in other glycerophospholipids. The synthesis of BMPs involves many acylation and diacylation steps, involving transacylase activity, which reorient the glycerol backbone to generate the unusual structural arrangement. The sn-1;sn-1' arrangement is thought to contribute to BMPs' resistance to cleavage by many phospholipases and their stability in late endosomes and lysosomes. Although BMPs are found in small amounts in many different cell types, BMP content is significantly higher in macrophages, as well as in lysosomes of the liver and other tissue types.

[0046] Consistent with their function as digestive organs, lysosomes contain large amounts of hydrolases at an acidic pH (i.e., a pH of about 4.6 to about 5). Various cellular components and foreign antigens are captured by receptors on the cell surface for uptake and delivery to lysosomes. Inside the cell, receptors such as the mannose-6-phosphate receptor bind and divert hydrolases from the biosynthetic pathway to the lysosome. The captured molecules pass through a set of intermediate heterogeneous organelles known as endosomes. Endosomes function as sorting stations that recycle receptors before sending hydrolases and other materials to lysosomes, where hydrolases are activated and unwanted materials are digested. In particular, the internal vesicles and / or membranes of mature or "late" endosomes and lysosomes contain large amounts of BMPs.

[0047] Negatively charged at lysosomal pH, BMPs can dock with luminal acid hydrolases that are positively charged at acidic pH and require a water-lipid interface for activation. By binding in this way, BMPs can stimulate many lysosomal lipid-degrading enzymes, including acid sphingomyelinase, acid ceramidase, acid phospholipase A2, and acid lipase, which has the ability to hydrolyze triacylglycerol and cholesterol esters.

[0048] Endosomal membranes are a continuation of lysosomal membranes, and they function to sort and recycle materials to the plasma membrane and the endoplasmic reticulum. Thus, low-density lipoproteins (LDL) taken up into cells reach late endosomes, where the constituent cholesterol esters and triglycerides are hydrolyzed by acid lipase. The distinctive network of BMP-rich membranes contained within late endosomes or lysosomes is a key component of cholesterol homeostasis in that it regulates cholesterol trafficking by functioning as a collection and redistribution point for free cholesterol. For example, incubation of lysosomal membranes with anti-BMP antibodies results in the accumulation of significant amounts of cholesterol.

[0049] In some embodiments of the method of the present disclosure, the abundance of a single BMP species is measured.In some embodiments, the abundance of two or more BMP species is measured.In some embodiments, the abundance of at least two, three, four, or five or more of the BMP species in Table 1 is measured.When the abundance of two or more BMP species is measured, any combination of different BMP species can be used.

[0050] In some embodiments, the abundance of two or more BMP species can be summed and the total abundance compared to a reference value. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 108, 109, 110, 111, 112, 113 8, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 9 The abundance of each of 4, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, or 147 or more BMP species (e.g., the BMP species listed in Table 1) can be summed and the total abundance then compared to a reference value.

[0051] In some cases, one or more BMP species may be differentially expressed (e.g., more abundantly or less abundantly) in one type of sample compared to another, such as, for example, a cell-based sample (e.g., cultured cells) versus a tissue-based sample or blood sample. Thus, in some embodiments, the selection of one or more BMP species (i.e., for measuring abundance) depends on the type of sample. In some embodiments, the one or more BMP species includes BMP (18:1_18:1), for example, when the sample (e.g., test sample and / or reference sample) is bone marrow-derived macrophage (BMDM). In other embodiments, the one or more BMP species includes BMP (22:6_22:6), for example, when the sample includes tissue (e.g., brain tissue, liver tissue), or plasma, urine, or CSF.

[0052] In some embodiments, an internal BMP standard (e.g., BMP (14:0_14:0)) is used to measure the abundance of one or more BMP species in a sample and / or to determine a reference value (e.g., to measure the abundance of one or more BMP species in a reference sample). For example, a known amount of an internal BMP standard can be added to a sample (e.g., a test sample and / or a reference sample) to act as a calibration point, thereby determining the amount of one or more BMP species present in the sample. In some embodiments, a reagent (e.g., methanol) used to extract or isolate BMP from a sample is "spiked" with the internal BMP standard. Typically, the internal BMP standard is one that does not naturally occur in a subject.

[0053] Identifying subjects with or at risk of having a PGRN-related disorder In some embodiments, a subject (e.g., a target subject) is determined to have a PGRN-related disease or reduced PGRN levels when the abundance of at least one (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, or 145 or more) BMP species (e.g., BMP species listed in Table 1) in a test sample is higher (if the test sample is BMDM) or lower (if the test sample is liver, brain, cerebrospinal fluid, plasma, or urine) than the reference value of the corresponding cell, tissue, or body fluid of a healthy control or a control unrelated to a PGRN-related disorder.

[0054] In some embodiments, the subject (e.g., the target subject) is administered BMP(16:0_18:1), BMP(16:0_18:2), BMP(18:0_18:0), BMP(18:0_18:1), BMP(18:1_18:1), BMP(16:0_20:3), BMP(18:1_20:2), BMP(18:0_20:4), BMP(16:0_22:5), BMP(16:0_22:6), BMP(16:0_22:7), BMP(16:0_22:8), BMP(16:0_22:9), BMP(16:0_22:1), BMP(16:0_22:2), BMP(16:0_22:3), BMP(16:0_22:4), BMP(16:0_22:5), BMP(16:0_22:1), BMP(16:0_22:2), BMP(16:0_22:3), BMP(16:0_22:2), BMP(16:0_22:3), BMP(16:0_22:4), BMP(16:0_22:5 ... ), BMP(20:4_20:4), BMP(22:6_22:6), BMP(20:4_20:5), BMP(18:2_18:2), BMP(16:0_20:4), BM P(18:0_18:2), BMP(18:0e_22:6), BMP(18:1e_20:4), BMP(20:4_22:6), BMP(18:0e_20:4), BMP( If the abundance of at least one of the BMP species selected from the group consisting of BMP(18:2_20:4), BMP(18:1_22:6), BMP(18:1_20:4), BMP(18:0_22:6), and BMP(18:3_22:5) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or all 23 species) is elevated in BMDM or decreased in liver, brain, cerebrospinal fluid, plasma, or urine compared to a reference value of the corresponding cell, tissue, or body fluid of a healthy control or a control unrelated to a PGRN-related disorder, the subject is determined to have a PGRN-related disease or to have a reduced PGRN level.

[0055] In some embodiments, a subject (e.g., a target subject) is administered at least one BMP species selected from the group consisting of BMP(18:1_18:1), BMP(18:0_20:4), BMP(20:4_20:4), BMP(22:6_22:6), BMP(20:4_22:6), BMP(18:1_22:6), BMP(18:1_20:4), BMP(18:0_22:6), and BMP(18:3_22:5). If the abundance of at least one (e.g., one, two, three, four, five, six, seven, or all eight) of the PGRN-associated proteins is elevated in BMDM or decreased in the liver, brain, cerebrospinal fluid, plasma, or urine compared to reference values ​​in corresponding cells, tissues, or body fluids of healthy controls or controls unrelated to a PGRN-associated disorder, the subject is determined to have a PGRN-associated disorder or to have reduced PGRN levels.

[0056] In some embodiments, a subject is determined to have a PGRN-related disorder or to have a reduced PGRN level if BMP (18:1_18:1) level is elevated in BMDM compared to a reference value of a healthy control or a control unrelated to PGRN-related disorder. In other embodiments, a subject is determined to have a PGRN-related disorder or to have a reduced PGRN level if BMP (22:6_22:6) is decreased in plasma, urine, cerebrospinal fluid (CSF), and / or brain or liver tissue compared to a reference value of a healthy control or a control unrelated to PGRN-related disorder. In other embodiments, a subject is determined to have a PGRN-related disorder or to have a reduced PGRN level if BMP (22:6_22:6) level and / or BMP (18:3_22:5) level are decreased in liver tissue. In other embodiments, a subject is determined to have a PGRN-associated disorder or to have reduced PGRN levels if BMP(18:3_22:5) levels are reduced in microglia.

[0057] In some embodiments, a subject (e.g., a target subject) is determined to have a PGRN-related disorder or reduced PGRN levels if the abundance of at least one BMP species (e.g., measured in a test sample) is at least about 1.1-fold (e.g., about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, or 10-fold or more) higher in BMDM or lower in liver, brain, cerebrospinal fluid, plasma, or urine compared to a reference value in a corresponding cell, tissue, or bodily fluid of a healthy control or a control unrelated to a PGRN-related disorder.

[0058] In some embodiments, a subject (e.g., a target subject) is determined to have a PGRN-associated disorder or reduced PGRN levels if the abundance of at least one BMP species (e.g., measured in a test sample) is at least about 1.2-fold to about 4-fold (e.g., at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3-fold, 3.1-fold, 3.2-fold, 3.3-fold, 3.4-fold, 3.5-fold, 3.6-fold, 3.7-fold, 3.8-fold, 3.9-fold, or 4-fold) greater than a reference value (e.g., a corresponding reference value). In some embodiments, a subject is determined to have a PGRN-associated disorder or reduced PGRN levels if the abundance of at least one BMP species is about 2-fold to about 3-fold (e.g., about 2-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, or 3-fold) higher in BMDM or lower in liver, brain, cerebrospinal fluid, plasma, or urine compared to a reference value in a corresponding cell, tissue, or bodily fluid of a healthy control or a control unrelated to a PGRN-associated disorder.

[0059] Monitoring response to treatment In one aspect, the present disclosure provides a method for monitoring the PGRN level in a subject (e.g., a target subject).In another aspect, a method is provided for monitoring the response of a subject to a compound, pharmaceutical composition, or their administration regimen, or to any therapy or therapeutic agent for treating PGRN-related disorder.

[0060] Typically, the abundance of each of one or more BMP species in test sample will be compared with one or more reference values ​​(e.g., corresponding reference values).In some embodiments, BMP value is measured before treatment and at one or more time points after treatment.The abundance value measured at later time points can be compared with the value before treatment and with a control value, such as the value of a healthy control or diseased control, to determine how the subject responds to therapy.The one or more reference values ​​can be from different cells, tissues, or body fluids that correspond to the cells, tissues, or body fluids of test sample.

[0061] In some embodiments, the reference value is the abundance of one or more BMP species measured in a reference sample. The reference value may be a measured abundance value (e.g., the abundance value measured in a reference sample) or may be derived or extrapolated from a measured abundance value. In some embodiments, the reference value is a range of values, for example, when the reference value is obtained from multiple samples or subject populations. Furthermore, the reference value can be expressed as a single value (e.g., the measured abundance value, the average value, or the median value) or as a range of values ​​with or without standard deviation or standard error.

[0062] When more than one test sample is obtained (e.g., from a subject), they are obtained at about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, , 53, 54, 55, 56, 57, 58, 59, or 60 minutes or more; about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours or more; about 1, 2, 3, 4, 5, 6, or 7 days or more; about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks or more; or even longer. When more than two test samples are obtained, the time intervals between when each test sample is obtained can all be the same, the intervals can all be different, or a combination thereof.

[0063] In some embodiments, both the first test sample and the second test sample are obtained from the subject (e.g., target subject) after the subject is treated. That is, the first test sample is obtained from the subject at an earlier time point during treatment than the second test sample. In some embodiments, the first test sample is obtained before the subject is treated for a disorder associated with a decrease in PGRN levels (i.e., pre-treatment test sample), and the second test sample is obtained after the subject is treated for a disorder associated with a decrease in PGRN levels (i.e., post-treatment test sample). In some embodiments, two or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) pre-treatment and / or post-treatment test samples are obtained from the subject. Moreover, the number of pre-treatment and post-treatment test samples obtained does not have to be the same.

[0064] In some embodiments, a subject may be determined to be not responding to treatment if the abundance of the measured BMP species is within about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the reference value.

[0065] If a subject (e.g., a target subject) is not responding to a treatment (e.g., for a disorder associated with decreased PGRN levels), in some embodiments, the dosage of one or more therapeutic agents (e.g., PGRN) is altered (e.g., increased) and / or the administration interval is altered (e.g., shortened the time between administrations). In some embodiments, a different therapeutic agent is selected if the subject is not responding to a treatment. In some embodiments, one or more therapeutic agents are discontinued if the subject is not responding to a treatment.

[0066] BMP detection techniques In some embodiments, the antibodies can be used to detect and / or measure the abundance of one or more BMP species. BMP species bound to the antibodies can be detected by microscopy or enzyme-linked immunosorbent assay (ELISA), for example.

[0067] In other embodiments, mass spectrometry (MS) is used to detect and / or measure the abundance of one or more BMP species according to the methods of the present disclosure. Mass spectrometry is a technique that ionizes compounds and selects the resulting ions by their mass-to-charge ratio (abbreviated as m / Q, m / q, m / Z, or m / z). A sample (e.g., a sample containing BMP molecules), which may be in gas, liquid, or solid form, is ionized, and the resulting ions are then accelerated through an electric and / or magnetic field, which separates the ions by their mass-to-charge ratio. The ions eventually impact an ion detector, which generates a mass spectrogram. The mass-to-charge ratio of the detected ions, together with their relative abundance, can be used to identify the parent compound(s), optionally by correlating known masses (e.g., known masses of whole or intact molecules) with the masses of the detected ions, and / or by recognizing patterns detected in the mass spectrogram.

[0068] A mass spectrometer typically includes at least four major components: (1) a sample introduction device (e.g., a vaporizer), (2) an ionization device, (3) an ion path, and (4) an ion detector. In addition, a mass spectrometer typically includes a device for converting the sample into a form suitable for the introduction device and / or separating compounds present in the sample, which in some embodiments may be a chromatographic device (e.g., liquid chromatography or gas chromatography) or a solid target for matrix-assisted laser desorption / ionization (MALDI) or another technique suitable for solid samples. A mass spectrometer also typically includes a device for signal processing of the detection signal (e.g., an analog-to-digital converter (ADC)), and / or software for processing, analyzing, and displaying the detection signal.

[0069] The sample introduction device facilitates the transfer of solid or liquid analytes to the gas phase, which is required for subsequent processing and analysis. Ionization devices can be used, for example, for hard ionization (e.g., electron ionization) or soft ionization (e.g., fast atom bombardment (FAB), chemical ionization (CI), electrospray ionization (ESI), MALDI, or atmospheric pressure chemical ionization (APCI)). ESI involves passing a solution through a length of capillary tubing, the end of which is applied with a high positive or negative potential. Upon reaching the end of the tube, the solution is vaporized into a jet or spray containing tiny droplets of the solution in solvent vapor. This spray of droplets flows through an evaporation chamber, which is slightly heated to prevent condensation and evaporate the solvent. As the droplets get smaller, the surface charge density increases until natural repulsion between like charges releases ions as well as neutral molecules.

[0070] In the ion path, the ions move from a near atmospheric pressure environment to the low pressure (e.g., high vacuum) environment of a mass spectrometer, which separates the ions according to their mass-to-charge ratio, and are transported towards an ion detector, typically an electron multiplier or microchannel plate that emits a cascade of electrons in response to being hit by an ion.

[0071] Different types of mass spectrometers can be used, examples of which include sector field mass spectrometers, time-of-flight (TOF) mass spectrometers, and quadrupole mass spectrometers. Sector field mass spectrometers use electrostatic and / or magnetic fields to modify the ion path and / or velocity, effectively bending the trajectory of the ion according to the mass-to-charge ratio of the ion. Ions of higher charge and / or lower mass are deflected more than ions of lower charge and / or higher mass. TOF mass spectrometers use electric fields to accelerate ions through a specific potential and measure the time it takes for the ions to strike the detector. If all ions have the same charge, their velocities will only differ as a function of their mass, and ions of lower mass will strike the detector first. Quadrupole mass spectrometers use one or more sets of four parallel bars (a set of four parallel bars is known as a quadrupole) to generate an oscillating electric field that stabilizes or destabilizes the path of the ions as they pass through a quadrupole field (e.g., a radio frequency (RF) quadrupole field) created between the four bars. Specifically, only ions within a particular range of mass-to-charge ratios can pass through the mass spectrometer at any given time. However, by varying the potential of the rods, a wide range of mass-to-charge ratios can be rapidly swept. The mass-to-charge ratio can be swept either continuously or by specifying discrete jumps.

[0072] In contrast to single mass spectrometry (MS), which uses a single mass analyzer (e.g., a quadrupole), tandem mass spectrometry (MS / MS) uses a series of mass analyzers (e.g., three mass analyzers) to perform multiple rounds of mass spectrometry, typically with molecular fragmentation steps between rounds. As a non-limiting example, MS / MS instruments generally use three quadrupole mass analyzers. The first quadrupole (Q1) can act as a first mass filter that separates the species or protein of interest from a larger heterogeneous population. The second quadrupole (Q2) can act as a collision chamber that stabilizes ions that have passed through Q1 and can be filled with low pressure gas against which the ions collide, causing ion fragmentation (collision induced fragmentation (CID)). The third quadrupole (Q3) can act as a second mass filter that separates the fragments generated in Q2 and sends them to a detector. Alternatively, instead of performing spatial tandem mass spectrometry, a single mass analyzer can be used to perform tandem mass spectrometry over time, such as when a quadrupole ion trap is used and the electric field is varied over time. Briefly, a quadrupole ion trap operates on the same physical principles as a quadrupole mass analyzer, but ions are trapped within the quadrupole (i.e., the electric field is changed faster than the time required for the ions to escape) and are selectively released over time by varying the electric field generated by the quadrupole.

[0073] Several methods can be used for fragmentation, including, but not limited to, CID, electron capture dissociation (ECD), electron transfer dissociation (ETD), infrared multiphoton dissociation (IRMPD), blackbody infrared radiation dissociation (BIRD), electron detachment dissociation (EDD), and surface induced dissociation (SID).

[0074] Tandem mass spectrometers can be used to perform different types of experiments, including full scan, product ion scan, precursor ion scan, neutral loss scan, and selected (or multiple) reaction monitoring (SRM or MRM) scan. In a full scan experiment, the entire mass range (or a portion thereof) of both mass analyzers (e.g., Q1 and Q3) is scanned, and the second mass analyzer (e.g., Q2) does not contain any collision gas. This allows all ions contained in the sample to be detected. In a product ion scan experiment, a specific mass-to-charge ratio is selected for the first mass analyzer (e.g., Q1), the second mass analyzer (e.g., Q2) is filled with collision gas to fragment ions with the selected mass-to-charge ratio, and then the entire mass range (or a portion thereof) of the third mass analyzer (e.g., Q3) is scanned. This allows all fragment ions of the selected precursor ion to be detected. In a precursor ion scan experiment, the entire mass range (or a portion thereof) of the first mass analyzer (e.g., Q1) is scanned, the second mass analyzer (e.g., Q2) is flooded with collision gas to fragment ions within the scan range, and a specific mass-to-charge ratio is selected for the third mass analyzer (e.g., Q3). By correlating the time between detection of product ions with the specific mass-to-charge ratio selected immediately prior to their detection, this type of experiment allows the user to determine which precursor ion(s) may have produced the product ion of interest. In a neutral loss scan experiment, the entire mass range (or a portion thereof) of the first mass analyzer (e.g., Q1) is scanned, the second mass analyzer (e.g., Q2) is flooded with collision gas to fragment all ions within the scan range, and the third mass analyzer (e.g., Q3) is scanned over a specific range corresponding to the fragmentation-induced loss of a single specific mass that occurred for all potential ions within the precursor scan range. This type of experiment allows the identification of all precursors that have in common the loss of a particular chemical group of interest (eg, a methyl group).In an MRM experiment, a particular mass-to-charge ratio is selected for a first mass spectrometer (e.g., Q1), a second mass spectrometer (e.g., Q2) is flooded with collision gas, and a third mass spectrometer (e.g., Q3) is set for another particular mass-to-charge ratio. This type of experiment allows for highly specific detection of molecules that are known to fragment into selected products in the third mass spectrometer. MS and MS / MS methods are further described in Grebe et al. Clin. Biochem. Rev. (2011) 32:5-31, which is incorporated herein by reference in its entirety for all purposes.

[0075] Additionally, MS and MS / MS techniques can be coupled with liquid chromatography (LC) or gas chromatography (GC) techniques, such that liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-tandem mass spectrometry (LC-MS / MS), gas chromatography-mass spectrometry (GC-MS), and gas chromatography-tandem mass spectrometry (GC-MS / MS) methods allow for enhanced mass resolution and mass measurement than is typically possible with MS or MS / MS alone.

[0076] Liquid chromatography refers to the process of selectively retarding one or more components of a fluid solution as the fluid permeates through a column of uniformly fine material or through a capillary passage. Retardation occurs as a result of the distribution of mixture components between one or more stationary phases and the bulk fluid (i.e., mobile phase) as the fluid moves relative to the stationary phase(s). High performance liquid chromatography (HPLC), sometimes known as "high pressure liquid chromatography," is a variant of LC in which separation is improved by forcing the mobile phase under pressure through a stationary phase, typically a densely packed column.

[0077] Furthermore, ultra-high performance liquid chromatography (UHPLC), also known as "ultra-high pressure liquid chromatography" or "ultra-performance liquid chromatography (UPLC)", is a variant of HPLC that is performed using much higher pressures than conventional HPLC techniques.

[0078] In some embodiments, the particle size in the column is less than about 2.0 μm (e.g., less than or equal to about 1.9 μm, 1.8 μm, 1.7 μm, 1.6 μm, or 1.5 μm). In some embodiments, the particle size is about 1.7 μm.

[0079] In some embodiments, the operating pressure of the column is from about 400 bar to about 1,000 bar (e.g., about 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1,000 bar).

[0080] In some embodiments, the temperature of the column during chromatography is maintained at a temperature between about 40° C. and about 60° C. (e.g., about 40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48° C., 49° C., 50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C., 59° C., or 60° C.). In some embodiments, the column is maintained at a temperature of about 55° C.

[0081] In some embodiments, the flow rate of the column is about 0.10 mL / min to about 0.50 mL / min (e.g., about 0.10 mL / min, 0.11 mL / min, 0.12 mL / min, 0.13 mL / min, 0.14 mL / min, 0.15 mL / min, 0.16 mL / min, 0.17 mL / min, 0.18 mL / min, 0.19 mL / min, 0.20 mL / min, 0.21 mL / min, 0.22 mL / min, 0.23 mL / min, 0.24 mL / min, 0.25 mL / min, 0.26 mL / min, 0.27 mL / min, 0.28 mL / min, 0.29 mL / min, 0.30 mL / min, 0.31 mL / min, 0.32 mL / min, 0.33 mL / min, 0.34 mL / min, 0.35 mL / min, 0.36 mL / min, 0.37 mL / min, 0.38 mL / min, 0.39 mL / min, 0.40 mL / min, 0.41 mL / min, 0.42 mL / min, 0.43 mL / min, 0.44 mL / min, 0.45 mL / min, 0.46 mL / min, 0.47 mL / min, 0.48 mL / min, 0.49 mL / min, 0.50 mL / min, 0.51 mL / min, 0.52 mL / min, 0.53 mL / min, 0.54 mL / min, 0.55 mL / min, 0.56 mL / min, 0.57 mL / min, 0.58 mL / min, 0.59 mL / min, 0.60 mL / min, 0.61 mL / min, 0.62 mL 8 mL / min, 0.29 mL / min, 0.30 mL / min, 0.31 mL / min, 0.32 mL / min, 0.33 mL / min, 0.34 mL / min, 0.35 mL / min, 0.36 mL / min, 0.37 mL / min, 0.38 mL / min, 0.39 mL / min, 0.40 mL / min, 0.41 mL / min, 0.42 mL / min, 0.43 mL / min, 0.44 mL / min, 0.45 mL / min, 0.46 mL / min, 0.47 mL / min, 0.48 mL / min, 0.49 mL / min, or 0.50 mL / min). In some embodiments, the flow rate is about 0.40 mL / min.

[0082] Gradient elution can be performed using two solvents (e.g., solvent A and solvent B). In some embodiments, solvent A is 10 mM ammonium formate in water + 0.1% formic acid, and solvent B is acetonitrile with 0.1% formic acid. In some embodiments, the gradient is generated in the following manner: 5% A and 95% B for 1 minute, change to 50% A and 50% B for 6 minutes, change to 5% A and 95% B for 0.1 minutes, then hold at 5% A and 95% B for 4.9 minutes. Once the compounds are separated by LC, HPLC, or UHPLC, they can be introduced into a mass spectrometer.

[0083] Gas chromatography refers to a method for separating and / or analyzing compounds that can be vaporized without decomposition. The mobile phase is a carrier gas, typically an inert gas (e.g., helium) or a non-reactive gas (e.g., nitrogen), and the stationary phase is typically a microscopic liquid or polymer layer disposed on an inert solid support inside a glass or metal tube that functions as a "column." As gaseous compounds of interest interact with the stationary phase in the column, they are differentially retarded and elute from the column at different times. The separated compounds can then be introduced into a mass spectrometer.

[0084] In some embodiments, antibody-based methods are used to detect and / or measure the abundance of one or more BMP species. Non-limiting examples of suitable methods include enzyme-linked immunosorbent assay (ELISA), immunofluorescence, and radioimmunoassay (RIA) techniques. Methods for performing ELISA, immunofluorescence, and RIA techniques are known in the art.

[0085] In the method of the present disclosure, any number of sample types can be used as test samples and / or reference samples, so long as the sample contains sufficient amount of BMP to be detected and thus its abundance can be measured.Non-limiting examples include cells, tissues, blood (e.g., whole blood, plasma, serum), bodily fluids (e.g., cerebrospinal fluid, urine, bronchoalveolar lavage fluid, lymph, semen, breast milk, amniotic fluid), stool, saliva, or any combination thereof.Non-limiting examples of suitable cell types include bone marrow derived macrophages (BMDMs), blood cells (e.g., peripheral blood mononuclear cells (PBMCs), red blood cells, white blood cells), neural cells (e.g., brain cells, cerebral cortex cells, spinal cord cells), bone marrow cells, liver cells, kidney cells, splenocytes, lung cells, eye cells (e.g., retinal cells such as retinal pigment epithelium (RPE) cells), chorionic villus cells, muscle cells, skin cells, fibroblasts, cardiac cells, lymph node cells, or any combination thereof.In some embodiments, the sample comprises a portion of a cell. In some embodiments, the sample is purified from a cell or tissue. Non-limiting examples of purified samples include endosomes, lysosomes, extracellular vesicles (e.g., exosomes, microvesicles), and combinations thereof.

[0086] In some embodiments, the sample (e.g., test sample and / or reference sample) comprises a cell that is a cultured cell. Non-limiting examples include BMDM and RPE cells. BMDM can be obtained, for example, by obtaining a sample that includes PBMC and culturing the monocytes contained therein.

[0087] Non-limiting examples of suitable tissue sample types include neural tissue (e.g., brain tissue, cerebral cortex tissue, spinal cord tissue), liver tissue, kidney tissue, muscle tissue, heart tissue, eye tissue (e.g., retinal tissue), lymph node, bone marrow, skin tissue, vascular tissue, lung tissue, spleen tissue, valve tissue, and combinations thereof. In some embodiments, the test sample and / or reference sample comprises brain tissue or liver tissue. In some embodiments, the test sample and / or reference sample comprises plasma.

[0088] Disorders associated with PGRN deficiency PGRN is involved in the maintenance of lysosomal homeostasis and the regulation of lipid metabolism, and PGRN deficiency is associated with lysosomal dysfunction (Evers et al. Cell Reports 20:2565-2574,2017). Similarly, many diseases can be attributed to or associated with lysosomal storage diseases, characterized by the accumulation of undigested or partially digested macromolecules, which ultimately leads to cellular and organismal dysfunction and clinical abnormalities. Lysosomal storage diseases are defined by the type of substrate stored and can be classified as cholesterol storage diseases, sphingolipidoses, oligosaccharidoses, mucolipidoses, mucopolysaccharidoses, lipoprotein storage diseases, neuronal ceroid lipofuscinosis, etc. In some cases, lysosomal storage diseases also include defects or abnormalities in proteins that result in the accumulation of macromolecules, such as proteins required for normal post-translational modification of lysosomal enzymes or proteins important for proper lysosomal trafficking. Non-limiting examples of diseases that may be caused by or associated with PGRN deficiency and result in lysosomal storage diseases include frontotemporal dementia (FTD) (e.g., GRN-FTD, MAPT-FTD, C9ORF72-ALS / FTD), atherosclerosis, Gaucher disease, Alzheimer's disease (AD), late-onset AD, neuronal ceroid lipofuscinosis (NCL), Niemann-Pick disease type A (NPA), Niemann-Pick disease type B (NPB), Niemann-Pick disease type C (NPC), C9ORF72-associated amyotrophic lateral sclerosis (ALS) / FTD, Parkinson's disease, and age-related macular degeneration (AMD).

[0089] Frontotemporal dementia (FTD) is a progressive neurodegenerative disorder. FTD includes a spectrum of clinically, pathologically, and genetically heterogeneous diseases that exhibit selective effects on the frontal and temporal lobes (Gazzina et al., Eur J Pharmacol. 817:76-85, 2017). Clinical symptoms of FTD include behavioral and personality changes, frontal executive dysfunction, and language dysfunction. Based on differences in clinical phenotypes, distinct conditions have been identified, such as behavioral FTD (bvFTD) and primary progressive aphasia (PPA), which can be either nonfluent / agrammatic PPA (avPPA) or semantic PPA (svPPA). These clinical symptoms may also overlap with atypical parkinsonisms such as corticobasal syndrome (CBS), progressive supranuclear palsy (PSP), and amyotrophic lateral sclerosis (ALS) (Gazzina et al., Eur J Pharmacol. 817:76-85, 2017). FTD is associated with a variety of neuropathological features, including tau pathology in neurons and astrocytes, or cytoplasmic ubiquitin inclusions in neurons. Transactivating DNA-binding protein with a molecular weight of 43 kDa (TDP-43) is the most prominent ubiquitinated protein lesion that accumulates in the majority of FTD cases as well as ALS (Petkau and Leavitt, Trends Neurosci. 37(7):388-98, 2014). FTD is a significant cause of early-onset dementia, with up to 80% of cases occurring between the ages of 45 and 64. The disease also exhibits a significant familial component, with a family history of the disease reported in approximately 30-50% of cases (Petkau and Leavitt, supra).

[0090] Although several genes have been linked to FTD, one of the most frequently mutated genes in FTD is GRN, which is located on human chromosome 17q21 and encodes the cysteine-rich protein PGRN (also known as proepithelin and acrogranin). Recent estimates suggest that GRN mutations account for 5-20% of FTD patients with a positive family history and 1-5% of sporadic cases (Rademakers et al., supra). Although the exact molecular and cellular mechanisms underlying the neurodegeneration and disease process in GRN-associated FTD are unknown, phenotypic characterization of Grn knockout mice combined with tissue analysis of patient brains suggests that both inflammation and lysosomal abnormalities are central to the disease (Kao et al., Nat Rev Neurosci. 18(6):325-333, 2017). Indeed, abundant gliosis is present in the cortical regions of patients (Lui et al., Cell. 165(4):921-35, 2016), and lipofuscin, a lysosomal pigment signifying lysosomal damage, has been reported in the eyes and cortex of GRN mutant carriers, including both presymptomatic individuals and patients (Ward et al., Sci. Transl. Med. 9(385), 2017).

[0091] Over 70 GRN disease mutations have been reported and mapped throughout the gene, resulting in confirmed or predicted loss-of-function (LOF) alleles (Ji et al. J Med Genet. 54:145-154, 2017). Most heterozygous mutations associated with FTD cause an approximately 50% reduction in mRNA levels, primarily as a result of nonsense mRNA decay, and a comparable reduction in PGRN protein levels (Petkau and Leavitt, supra; Kao et al., supra). Low levels of PGRN are also found in the blood (serum) and cerebrospinal fluid (CSF) of carriers, including presymptomatic individuals (Finch et al., Nat Rev Neurosci. 18(6):325-333, 2017; Goossens et al., Alzheimers Res. Ther. 10(1):31, 2018; Meeter et al., Dement. Geriatr. Cogn. Dis. Extra. 6(2):330-340, 2016). Thus, haploinsufficiency is thought to be the primary disease mechanism in GRN-related FTD, suggesting that therapeutic approaches to increase PGRN levels in carriers may delay the age of onset as well as progression of FTD (Petkau and Leavitt, supra; Kao et al., supra). This idea is supported by a human genetic study showing that variants in the gene TMEM106B increase PGRN levels by 25% and delay the age of onset of GRN-related FTD by 13 years (Nicholson and Rademakers, Acta Neuropathol. 132(5):639-651, 2016).

[0092] Homozygous GRN mutations have also been reported, but carriers exhibit a widely divergent clinical phenotype known as neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disorder (Batten disease, incidence 1-2.5 per 100,000 births; Cotman et al., Curr. Neurol. Neurosci. Rep. 13(8):366, 2013) (Smith et al., Am. J. Hum. Genet. 90(6):1102-7, 2012; Almeida et al., Neurobiol. Aging. 41:200.e1-200.e5, 2016). GRN is in fact one of 14 neuronal ceroid lipofuscinosis (CLN) genes reported to be associated with NCL; GRN is also known as CLN11 (Kollmann et al., Biochim. Biophys. Acta. 1832(11):1866-81, 2013).

[0093] Gaucher disease patients who carry homozygous mutations in the GBA gene have low levels of PGRN in their serum (Jian et al., EBioMedicine 11:127-137, 2016). Parkinson's disease patients who carry heterozygous mutations in GBA also have low levels of PGRN.

[0094] Variants in GRN have been associated with AD (Rademakers et al., supra; Brouwers et al., Neurology. 71(9):656-64, 2008; Lee et al., Neurodegener. Dis. 8(4):216-20, 2011; Viswanathan et al., Am. J. Med. Genet. B. Neuropsychiatr. Genet. 150B(5):747-50, 2009), and TDP-43 pathology is common in the brains of AD patients (Youmans and Wolozin, Exp. Neurol. 237(1):90-5, 2012). Delivery of the PGRN gene has also been shown to reduce amyloid burden in a mouse model of AD (van Kampen and Kay, PLoS One. 12(8):e0182896, 2017).

[0095] Niemann-Pick disease types A and B (NPA and NPB) are caused by mutations in the gene encoding acid sphingomyelinase (SMPD1), whereas Niemann-Pick disease type C (NPC) is caused by mutations in genes involved in cholesterol transport, namely NPC1 and NPC2 (Kolter and Sandhoff, Annu Rev Cell Dev. Biol. 21:81-103, 2005; Kobayashi et al., Nat Cell Biol. 1(2):113-8, 1999).

[0096] The majority of ALS cases exhibit TDP-43 pathology, which is also common in patients carrying GRN mutations (Petkau and Leavitt, Trends Neurosci. 37(7):388-98, 2014; Rademakers et al., Nat. Rev. Neurol. 8(8):423-34, 2012). Among all ALS cases, GGGGCC repeat expansion in the C9ORF72 gene is the most common cause of ALS and a significant cause of FTD. The average mutation frequency reported in North American and European populations is 37% in familial ALS patients, 6% in sporadic ALS patients, 21% in familial FTD patients, and 6% in sporadic FTD patients (Rademakers et al., supra). Furthermore, TMEM106B variants that are protective in GRN-related FTD are also protective in FTD patients carrying a repeat expansion within the C9ORF72 gene (van Blitterswijk et al., Acta Neuropathol. 127(3):397-406, 2014).

[0097] AMD is a degenerative disease and the leading cause of blindness in developed countries. AMD causes damage to the macula, a tiny spot near the center of the retina and part of the eye required for sharp central vision. Degenerative changes in the eye and loss of vision can be attributed to impaired lysosomal function and the accumulation of harmful proteins at the back of the retina (Viiri et al., PLoS One. 8(7):e69563, 2013). As the disease progresses, the retina's sensory cells in the central visual area are damaged, leading to loss of central vision.

[0098] kit In another aspect, the disclosure provides a kit for use in monitoring PGRN levels in a subject (e.g., a test subject and / or a reference subject or reference subject population). In some embodiments, the kit is for use in measuring or calibrating one or more bis(monoacylglycero)phosphate (BMP) species (e.g., in a test sample obtained from a subject and / or in a reference sample obtained from a reference subject or reference subject population). In some embodiments, the kit includes a BMP standard (e.g., an internal BMP standard) that can be used to measure or calibrate the abundance of one or more BMP species (e.g., in a sample such as a test sample and / or a reference sample). In some embodiments, the BMP standard includes a BMP species that does not naturally occur in a subject. In some embodiments, the BMP standard includes a BMP species that does not naturally occur in humans, non-human primates, rodents, dogs, and / or pigs. In some embodiments, the BMP standard includes a BMP species that does not naturally occur in humans. In some embodiments, the BMP standard includes BMP(14:0_14:0).

[0099] In some embodiments, the kit further comprises one or more reagents. For example, in some embodiments, the kit comprises reagents for obtaining a test sample (e.g., from a subject) and / or a reference sample (e.g., from a reference subject or reference subject population), reagents for processing the sample (e.g., isolating or purifying one or more BMP species from the test sample and / or reference sample), reagents for measuring the abundance of one or more BMP species in a sample (e.g., the test sample and / or the reference sample), and / or a sample for calibrating the abundance of one or more BMP species in a sample (e.g., the test sample and / or the reference sample).

[0100] In some embodiments, the kit further comprises instructional materials (e.g., instructions for using the kit to monitor PGRN levels in a subject (e.g., a test subject and / or a reference subject or reference population)) containing directions (e.g., protocols) for practicing the methods described herein. The instructional materials typically comprise written or printed material, but are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by the present disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, magnetic tapes, magnetic cartridges, magnetic chips), optical media (e.g., CD-ROMs), and the like. Such media may include addresses to internet sites that provide such instructional materials. EXAMPLES

[0101] The following examples are provided for illustrative purposes only and are not intended to limit the disclosure in any way.

[0102] Sample preparation Cells (e.g., bone marrow derived macrophages (BMDM)) were washed extensively with PBS and BMP species were extracted with methanol spiked with BMP (14:0_14:0) as an internal standard. After BMP species were extracted with methanol, the samples were vortex mixed and centrifuged at 14,000 rpm for 20 min at 4°C. The supernatant was then transferred to liquid chromatography-mass spectrometry vials for further analysis.

[0103] Tissue samples were weighed (e.g., 20 mg) and then homogenized in methanol (200 μL) spiked with BMP (14:0_14:0) using a TissueLyser homogenizer (Qiagen, Valencia, CA, USA). The homogenate was spun at 14,000 rpm for 20 min at 4° C. The supernatant was then transferred to liquid chromatography-mass spectrometry vials for further analysis.

[0104] Biofluids (10 μL) were protein precipitated with BMP(14:0_14:0) in methanol (100 μl) and spun at 14,000 rpm for 20 min at 4° C. The supernatant was then transferred to liquid chromatography-mass spectrometry vials for further analysis.

[0105] Lipidomics Procedure General Procedure: Animal groups and sample collections were randomized during lipid extraction.

[0106] Extraction of lipids and metabolites from urine: Urine was collected in Thermo Matrix tubes and stored at -80°C. Urine was thawed on ice and centrifuged at 1,000 x g for 10 min at 4°C to remove particulates. 10 μL of urine was transferred to a 2.0 mL Safe-Lock Eppendorf tube (Eppendorf catalogue no. 022600044) and 200 μL of MS grade ice-cold methanol containing internal standard mixture (2 μL per sample). Samples were vortexed at 2,500 rpm for 5 min. Samples were centrifuged at 21,000 x g for 20 min at 4°C. Methanol supernatant was transferred to LCMS glass vials in a 96-well plate. Samples were stored at -80°C until run on LCMS.

[0107] Extraction of lipids and metabolites from plasma: Plasma samples were thawed on ice. Plasma / serum (10 μL) or urine (20 μL) was transferred to 2 mL Safe-Lock Eppendorf tubes (Eppendorf catalog number 022600044). MS-grade ice-cold methanol (200 μL) containing internal standard mixture (2 μL per sample) was vortexed for 5 min and then centrifuged at 21,000×g for 20 min at 4° C. For lipidomics and metabolomics analysis, the methanolic supernatant was transferred to LCMS glass vials in a 96-well plate. Samples were stored at −80° C. until run on LCMS.

[0108] Lipid and metabolite extraction from brain: Frontal cortex of mouse brain (18-20 mg) was transferred to a 2 mL Safe-Lock Eppendorf tube (Eppendorf catalog no. 022600044) stored in dry ice containing 5 mm stainless steel beads (QIAGEN catalog no. 69989). MS-grade methanol (400 μL) containing an internal standard mixture (2 μL per sample) was added. The tissue was homogenized using a TissueLyser at 25 Hz for 30 s in a refrigerator and then centrifuged at 21,000 × g for 20 min at 4 °C (beads were left in the tube). The methanol supernatant was transferred to a new 1.5 mL Eppendorf vial and left at -20 °C for 1 h to further precipitate the proteins. The vials were centrifuged at 21,000×g for 20 min at 4° C. and the methanolic supernatant was transferred to LCMS glass vials for lipidomic and metabolomic analysis. Samples were stored at −80° C. until submitted to LCMS.

[0109] Extraction of lipids and metabolites from CSF: CSF was collected from mice by pipetting. CSF (5 μL) was added directly to MS-grade ice-cold MeOH (100 μL) containing an internal standard mixture (0.2 μL per sample) in a glass vial. The glass vial containing CSF and MeOH was vortexed for 5 min and directly submitted to LCMS.

[0110] Extraction of lipids and metabolites from liver: Liver (20 mg) was transferred to a 2 mL Safe-Lock Eppendorf tube (Eppendorf catalog number 022600044) stored in dry ice containing 5 mm stainless steel beads (QIAGEN catalog number 69989). MS-grade methanol (400 μL) containing an internal standard mixture (2 μL per sample) was then added. The tissue was homogenized using a TissueLyser (in a refrigerator) for 30 seconds at 25 Hz and centrifuged at 21,000×g for 20 minutes at 4° C. (beads were left in the tube). The methanol supernatant was transferred to a new 1.5 mL Eppendorf vial and left at −20° C. for 3 hours to further precipitate the proteins. The vials were centrifuged at 21,000×g for 20 minutes at 4° C., and the methanol supernatant was transferred to an LCMS glass vial for lipidomics and metabolomics analysis. Samples were stored at -80°C until submitted for LCMS.

[0111] Liquid chromatography-mass spectrometry BMP analysis was performed by liquid chromatography (Shimadzu Nexera X2 system, Shimadzu Scientific Instrument, Columbia, MD, USA) coupled with electrospray mass spectrometry (Sciex 6500+QTRAP, Sciex, Framingham, MA, USA). For each analysis, 5 μL of sample was injected onto a BEH amide 1.7 μm, 2.1×150 mm column (Waters Corporation, Milford, Massachusetts, USA) using a flow rate of 0.40 mL / min at 55° C. Mobile phase A consisted of water with 10 mM ammonium formate+0.1% formic acid. Mobile phase B consisted of acetonitrile with 0.1% formic acid. The gradient was programmed as follows: 0.0-1.0 min at 95% B, 1.0-7.0 min to 50% B, 7.0-7.1 min to 95% B, and 7.1-12.0 min to 95% B. Electrospray ionization was performed in negative ion mode using the following settings: curtain gas at 25, collision gas at medium, ion spray voltage at -4500, temperature at 600, ion source gas 1 at 50, ion source gas 2 at 60, collision energy at -50, CXP at -15, DP at -60, EP at -10, and dwell time at 20 ms. Data collection was performed using Analyst 1.6.3 (Sciex) in multiple reaction monitoring mode (MRM). BMP species were detected using the MRM transition parameters reported in Table 1. BMP species were quantified using BMP(14:0_14:0) as an internal standard. BMP species were identified based on their retention time and MRM characteristics. Quantification was performed using MultiQuant 3.02 (Sciex) after correction for isotopic overlap. BMP species were normalized to either total protein, tissue weight, or biofluid volume. Protein concentrations were measured using the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL, USA).

[0112] Precursor (Q1) [MH] -and product ion (Q3) m / z transitions were used to measure BMP species. BMP species were identified from their Q1 and Q3 values ​​according to Table 1. Abbreviations are used herein to refer to species with two side chains, with the structure of the fatty acid side chain shown in brackets in the BMP format (e.g., BMP(18:1_18:1)). Numbers follow the standard fatty acid notation format of number of fatty acid carbon atoms: number of double bonds. The prefix "e-" is used to indicate the presence of an alkyl ether substituent (e.g., BMP(16:0e_18:0)), where the carbonyl oxygen of the fatty acid side chain is replaced with two hydrogen atoms. Alternatively, BMP species can be referred to generically according to the total number of carbon atoms: total number of double bonds, and species with similar values ​​can be differentiated by their Q1 and Q3 values.

[0113] Table 1. BMP species and MRM transition parameters TIFF0007624919000002.tif72128TIFF0007624919000003.tif242108TIFF0007624919000004.tif242108 TIFF0007624919000005.tif242108TIFF0007624919000006.tif242108TIFF0007624919000007.tif94128

[0114] Example 1. Expression and isolation of recombinant fusion proteins To express the recombinant Fc dimer:PGRN fusion protein in Expi293 (Thermo-Fisher), cells were cultured at 2 × 10 cells using Expifectamine™ 293 / plasmid DNA complexes according to the manufacturer's instructions (Thermo-Fisher). 6Cells were transfected at a density of 1000 cells / mL. After transfection, cells were incubated at 37°C in a humidified atmosphere of 6-8% CO2 in an orbital shaker (Infors HT Multitron). The day after transfection, Expifectamine™ Transfection Enhancers 1 and 2 were added to the cultures. 96 hours after transfection, medium supernatants were collected by centrifugation. The clarified supernatants were supplemented with EDTA-free protease inhibitors (Roche) and stored at -80°C.

[0115] For isolation of the recombinant fusion protein, the clarified culture supernatant was loaded onto a HiTrap MabSelect SuRe Protein A affinity column (GE Healthcare Life Sciences) and washed with wash buffer I (PBS buffer pH 7.4) and wash buffer II (PBS buffer pH 7.4 and 150 mM NaCl). The fusion protein was eluted in 50 mM QB citrate buffer pH 3.0 containing 150 mM NaCl. Immediately after elution, arginine-succinate buffer (1 M arginine, 685 mM succinic acid, pH 5.0) was added to adjust the pH. Protein aggregates were separated from the monodisperse fusion protein by size exclusion chromatography (SEC) on a Superdex 200 increase 16 / 60 GL column (GE Healthcare Life Sciences). The mobile phase for SEC was maintained in arginine-succinate buffer pH 5.0. All chromatography steps were performed on an AKTA pure system or an AKTA Avant system (GE Healthcare Life Sciences).

[0116] PGRN fusion proteins F1 and F2 are shown in Figures 1A and 1B. In F1, PGRN was fused at its N-terminus to one of the Fc polypeptides via -Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Ser-((G4S)2) (SEQ ID NO: 1). In F2, PGRN was fused at its C-terminus to one of the Fc polypeptides via (G4S)2 (SEQ ID NO: 1). In both F1 and F2, the Fc polypeptide fused to Progranulin was covalently linked via a disulfide bond in the hinge region of another Fc polypeptide to form an Fc heterodimer.

[0117] Example 2. Treatment of BMDM from Grn knockout mice BMDMs were obtained in vitro from the bone marrow of wild-type and Grn knockout mice using a method similar to that described by Trouplin et al. (J. Vis. Exp. 2013 (81) 50966), except that recombinant M-CSF was added directly to the cell growth medium to induce differentiation. BMDMs were treated with 50 nM of PGRN fusion proteins F1 and F2 of Example 1, or RSV (respiratory syncytial virus) control for 72 h. Cellular lipids were extracted by addition of methanol containing an internal standard mixture, and BMP abundance was measured by liquid chromatography-mass spectrometry (LC-MS / MS) on a Q-trap 6500 (SCIEX). As shown in Figures 2A and 2B, the abundance of BMPs (18:1_18:1) and BMPs (20:4_20:4) was increased in BMDMs from untreated Grn knockout mice compared to wild-type controls. Furthermore, in separate experiments (three technical replicates) shown in Figures 2C and 2D, the abundance of total BMP and BMP(18:1_18:1) was decreased in Grn knockout BMDMs treated with either recombinant PGRN (Adipogen) or lentivirally expressed PGRN.

[0118] The BMP species that were found to be increased in abundance in BMDMs from granulin (GRN) knockout animals are shown in Table 2. Species that showed particularly significant increases in abundance are marked with an asterisk.

[0119] Table 2. Increased BMP species in BMDMs from GrnKO mice TIFF0007624919000008.tif170128

[0120] Example 3. Treatment of GrnKO mice to restore the phenotype Fusion 1 and Fusion 2 Progranulin fusion proteins from Example 1 were injected via the tail vein into Grn WT and Grn KO mice to determine whether the peripheral and CNS Grn KO phenotype could be rescued after a single 50 mg / kg injection.

[0121] Materials and Methods Animals: Mice used in this study were obtained from JAX Laboratories and consisted of 21 Grn KO mice (n = 12 males, n = 9 females) and 10 Grn WT mice (n = 5 males, n = 5 females) aged 3-5 months (Table 3). At least 7 days before the start of the study, animals were housed in a vivarium under standard conditions and had free access to food and water.

[0122] Table 3. Study design / experimental groups TIFF0007624919000009.tif46128

[0123] Allocation of animals to experimental groups: Mice were distributed equally to each experimental group, taking into account differences between litters, sex, and age.

[0124] Formulations: Fc dimer:PGRN fusion proteins, Fusion 1 and Fusion 2, were used at 5.05 mg / mL and 4.90 mg / mL in saline, respectively.

[0125] General Procedure: Experimental conditions were alternated when tissues were harvested. Animal groups and sample collections were randomized.

[0126] In-life procedures: Submandibular bleeding was performed using a 3 mm lancet (GoldenRod Animal Lancet). Plasma collection: Blood was collected into EDTA tubes (Sarstedt Microvette 500 K3E, ref. 201341102) and gently inverted 10 times. For small amounts of blood (<100 μL), blood was collected into EDTA tubes with capillary tubes (Sarstedt Microvette 100 K3E, ref. 201278100). EDTA tubes were immediately stored in the refrigerator until plasma preparation. The time between storage and preparation was 1 hour or less. Collection and processing times were consistent. Tubes were centrifuged at 12,700 rpm for 7 minutes at 4°C. Plasma (top layer) was transferred to 0.6 mL matrix tubes with rubber seals. Matrix tubes were flash frozen on dry ice before being transferred to -80°C. Urine collection: Urine was collected by restraining mice in plastic weighing boats and allowing most mice to urinate into the weighing boat. Urine was collected with a p200 pipette and transferred to 0.6 mL matrix tubes with rubber seals. Matrix tubes were flash frozen on dry ice and then transferred to -80°C.

[0127] Terminal fluid / tissue collection procedure: Animals were deeply anesthetized with an intraperitoneal (ip) injection of 2.5% Avertin and then tissues were collected in the order as described below.

[0128] Samples taken before intracardiac perfusion: Collection of plasma: Blood was collected by cardiac puncture using a 1 mL Terumo tuberculin syringe (reference number SS-01T2516) fitted with a 25 gauge needle (without pretreatment with EDTA). The needle was then removed and the blood transferred to an EDTA tube (Sarstedt Microvette 500 K3E, reference number 201341102) and gently inverted 10 times. After this procedure, the post-collection method was continued as previously described in the in-life procedure. Collection of cerebrospinal fluid: The three muscle layers of the posterior neck were peeled away with small scissors and forceps, the area around the exposed cisterna magna was cauterized to prevent contamination from blood, and the membrane was washed with a Q-tip and PBS. The membrane was dried and the cisterna magna was punctured with the tip of a 28 1 / 2 gauge insulin syringe needle. A p20 pipette was then quickly placed into the previously punctured hole and the CSF was drawn up into the pipette tip. The CSF was placed into a 0.5 mL Lo-bind Eppendorf tube and spun at 12,700 rpm for 7 min at 4°C. The CSF supernatant was transferred to a 0.5 mL Lo-bind Eppendorf tube and flash frozen on dry ice before being transferred to -80°C.

[0129] For samples taken after intracardial perfusion, animals were transcardially perfused with ice-cold PBS for 5 min at a flow rate of 5 mL / min using a peristaltic pump (Gilson Inc. Minipuls Evolution F110701). For tissues that could be dissected and pre-weighted during collection, samples were placed directly into a 1.5 mL Eppendorf tube. Tissues were collected in the following order: Liver (post-perfusion): Approximately 150 mg of liver was collected into a 1.5 mL Eppendorf tube. The Eppendorf tube was flash frozen on dry ice before being transferred to -80°C. Eyes: Both eyes were removed, the muscles and optic nerves were removed, and the eyes were placed into one 1.5 mL Eppendorf tube. The Eppendorf tube was flash frozen on dry ice before being transferred to -80°C. Brain: The right hemisphere, excluding the olfactory bulb and cerebellum, was collected, weighed, and placed into a 1.5 mL Eppendorf tube. Eppendorf tubes were flash frozen on dry ice and then transferred to -80°C.

[0130] As shown in Figures 3 to 9, administration of PGRN fusion proteins F1 and F2 increased and restored BMP levels in the liver, plasma, urine, CSF, and brain.

[0131] Example 4. BMP changes in CSF of FTD patient samples Samples from the National Cell Repository for Alzheimer's Disease (NCRAD), which is supported by government support under a Cooperative Agreement (U24 AG21886) awarded by the National Institute on Aging (NIA), were used in this study. CSF samples from sporadic FTD patients (n=25), GRN mutant FTD patients (n=16), and clinically normal control subjects (n=20) were blinded, randomized, metabolite / lipid extracted with methanol, and analyzed on a quantitative LC / MS / MS platform. Analyte identity was confirmed with authentic compounds, and the signal of each analyte was normalized to the corresponding internal standard. A decrease in BMP18:1_18:1 and 22:6_22:6 was observed in the CSF of sporadic and GRN FTD patient samples compared to clinically normal controls (Figures 10A and 10B).

[0132] It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light of them will be suggested to those skilled in the art and are to be included within the spirit and scope of this application and the scope of the appended claims. The sequences of the sequence accession numbers cited herein are incorporated herein by reference.

[0133] Informal sequence listing TIFF0007624919000010.tif26170

Claims

1. 1. A method for evaluating a compound for the treatment of frontotemporal dementia associated with progranulin (PGRN) deficiency or for monitoring a subject's response to a compound, a pharmaceutical composition thereof, or an administration regimen thereof for the treatment of frontotemporal dementia, comprising: (a) measuring the abundance of one or more bis(monoacylglycero)phosphate (BMP) species in a test sample from a subject having frontotemporal dementia associated with PGRN deficiency, wherein the test sample or the subject has been treated with the compound or a pharmaceutical composition thereof; (b) comparing the difference in abundance between the one or more BMP species measured in step (a) and one or more reference values; (c) determining from the comparison whether the compound, pharmaceutical composition thereof, or administration regimen thereof for treating frontotemporal dementia associated with PGRN deficiency improves the level of one or more BMP species; The method comprising:

2. The method of claim 1, further comprising the step of selecting a candidate compound that improves the level of said one or more BMP species after another test sample or subject is treated with another compound.

3. the improved BMP species level is an improvement over the BMP species level before treatment compared to a reference value of a healthy control or a control unrelated to frontotemporal dementia associated with PGRN deficiency; Optionally, the improved BMP species level has a difference of less than 15%, less than 10%, or less than 5% compared to the control. The method according to claim 1 or 2.

4. (d) maintaining or adjusting the amount or frequency of administration of said compound to said test sample; (e) administering said compound to said test sample; The method of claim 1, further comprising:

5. The method of any one of claims 1 to 4, wherein the compound is PGRN or a PGRN derivative.

6. The method according to any one of claims 1 to 5, wherein said reference value is the value of said BMP species before treatment.

7. 1. A method for identifying a subject having or at risk of having frontotemporal dementia associated with progranulin (PGRN) deficiency, comprising: (a) determining the abundance of one or more BMP species in a test sample from a subject; (b) comparing the difference in abundance between the one or more BMP species measured in step (a) and one or more reference values; wherein the difference in abundance indicates whether the subject has frontotemporal dementia associated with PGRN deficiency.

8. (a) the subject having or at risk of having frontotemporal dementia associated with PGRN deficiency has elevated levels of BMP species in bone marrow-derived macrophages compared to a healthy control or a control unrelated to frontotemporal dementia associated with PGRN deficiency; and / or (b) the subject having or at risk of having frontotemporal dementia associated with PGRN deficiency has decreased levels of BMP species in the liver, brain, cerebrospinal fluid, plasma, or urine compared to a healthy control or a control unrelated to frontotemporal dementia associated with PGRN deficiency; and / or (c) the abundance of the BMP species in the test sample of the subject having or at risk of having frontotemporal dementia associated with PGRN deficiency is at least about 1.2-fold, 1.5-fold, or 2-fold different from a reference value of a healthy control or a control unrelated to frontotemporal dementia associated with PGRN deficiency; and / or (d) the abundance of the BMP species in the test sample of the subject having or at risk of having frontotemporal dementia associated with PGRN deficiency is at least about 1.2-fold to about 4-fold different from a reference value of a healthy control or a control unrelated to frontotemporal dementia associated with PGRN deficiency; The method of claim 7.

9. (a) the test sample contains endosomes, lysosomes, extracellular vesicles, exosomes, microvesicles, or a combination thereof; and / or (b) the one or more BMP species are (i) two or more BMP species, and / or (ii) BMP (16:0_18:1), BMP (16:0_18:2), BMP (18:0_18:0), BMP (18:0_18:1), BMP (18:1_18:1), BMP (16:0_20:3) , BMP (18:1_20:2), BMP (18:0_20:4), BMP (16:0_22:5), BMP (20:4_20:4), BMP (22:6_22:6), BMP (20:4_20:5), BM P(18:2_18:2), BMP(16:0_20:4), BMP(18:0_18:2), BMP(18:0e_22:6), BMP(18:1e_20:4), BMP(20:4_22:6), BMP(18:0e_20:4), BMP(18:2_20:4), BMP(18:1_22:6), BMP(18:1_20:4), BMP(18:0_22:6), or combinations thereof, and / or (iii) BMP(18:1_18:1), BMP(18:0_20:4), BMP(20:4_20:4), BMP(22:6_22:6), BMP(20:4_22:6), BMP(18:1_22:6), BMP(18:1_20:4), BMP(18:0_22:6), BMP(18:3_22:5), or any combination thereof and / or (c) the test sample is (i) BMDM, wherein the one or more BMP species comprises BMP(18:1_18:1); and / or (ii) plasma, urine, cerebrospinal fluid (CSF), and / or brain or liver tissue, wherein the one or more BMP species comprises BMP(22:6_22:6); and / or (iii) comprising hepatic tissue, wherein the one or more BMP species comprises BMP(22:6_22:6), BMP(18:3_22:5), or a combination thereof; and / or (iv) CSF or urine, wherein the one or more BMP species comprises BMP(22:6_22:6); and / or (v) CSF, and the one or more BMP species comprises BMP (18:1_18:1); and / or (vi) microglia, and the one or more BMP species include BMP (18:3_22:5); The method according to any one of claims 1 to 8.

10. said reference value being measured in a reference sample obtained from a reference subject or a reference subject population; Optionally, the reference subject or the reference subject population is a healthy control, or the reference subject or the reference subject population does not have frontotemporal dementia associated with PGRN deficiency or does not have reduced PGRN levels. The method according to any one of claims 1 to 9.

11. the test sample, the reference sample, or the one or more reference values ​​comprise or relate to a cell, a tissue, whole blood, plasma, serum, cerebrospinal fluid, interstitial fluid, saliva, urine, lymphatic fluid, or a combination thereof; Optionally, the cell is a peripheral blood mononuclear cell (PBMC), a bone marrow derived macrophage (BMDM), a retinal pigment epithelial (RPE) cell, a blood cell, a red blood cell, a white blood cell, a neuronal cell, a microglial cell, a brain cell, a cerebral cortex cell, a spinal cord cell, a bone marrow cell, a liver cell, a kidney cell, a splenocyte, a lung cell, an eye cell, a chorionic villus cell, a muscle cell, a skin cell, a fibroblast cell, a cardiac cell, a lymph node cell, or a combination thereof; and / or The cells are cultured cells, and are optionally BMDM or RPE cells. The method of claim 10.

12. and / or the abundance of the one or more BMP species is measured using liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-tandem mass spectrometry (LC-MS / MS), gas chromatography-mass spectrometry (GC-MS), gas chromatography-tandem mass spectrometry (GC-MS / MS), enzyme-linked immunosorbent assay (ELISA), or a combination thereof; and / or When measuring the abundance of one or more BMP species, an internal BMP standard is used, and optionally the internal BMP standard comprises a BMP species that does not naturally occur in the subject and / or the reference subject or the reference subject population, and / or the internal BMP standard comprises BMP (14:0_14:0); The method of claim 10.

13. 12. The method of claim 11, wherein the tissue comprises brain tissue, cerebral cortex tissue, spinal cord tissue, liver tissue, kidney tissue, muscle tissue, heart tissue, eye tissue, retinal tissue, lymph node, bone marrow, skin tissue, vascular tissue, lung tissue, spleen tissue, valve tissue, or a combination thereof.

14. the subject has one or more mutations in the granulin (GRN) gene; and / or the PGRN deficiency-associated frontotemporal dementia is associated with reduced levels of PGRN; and / or the subject is a human, a non-human primate, a rodent, a dog, or a pig; and / or The subject has frontotemporal dementia associated with PGRN deficiency, which is associated with reduced PGRN levels, and is a PGRN knockout mouse or a PGRN knockout rat. The method according to any one of claims 1 to 13.

15. 1. A kit for testing a compound or a dosing regimen thereof for the treatment of frontotemporal dementia associated with progranulin (PGRN) deficiency, comprising a BMP standard for measuring the abundance of one or more BMP species in a test sample from a subject; Optionally, the BMP standard comprises a BMP species that does not naturally occur in the subject, and / or comprises BMP(14:0_14:0), and / or and / or further comprising reagents for obtaining said sample from said subject, reagents for processing said sample, reagents for measuring the abundance of said one or more BMP species, or a combination thereof; Further including instructions for use, The kit.

16. A pharmaceutical for use in a method for treating frontotemporal dementia associated with progranulin (PGRN) deficiency, the method comprising: (d) maintaining or adjusting the dosage or frequency of administration of the medicament to the subject after carrying out the method according to any one of claims 1 to 3 and 5 to 6; (e) administering the medicament to the subject; Including, Optionally, the medicament comprises PGRN or a PGRN derivative. Medicine.

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