Detection of antibody against ran protein from serum and tissue lysate

Abstract

To provide a method and a composition (e.g., kits) for detecting an anti-repeat-associated non-ATG (RAN) protein antibody in a subject (e.g., a subject being administered therapeutic anti-RAN protein antibodies or vaccines for a disease or disorder related to the expression, translation, and / or accumulation of RAN protein, such as amyotrophic lateral sclerosis (ALS) and / or frontotemporal dementia (FTD)), according to an aspect of the present disclosure.SOLUTION: In some aspects, a method described in the present disclosure includes detecting one or more anti-RAN protein antibodies in a biological sample obtained from a subject by an electrochemiluminescence-based immunoassay using one or more target diamino-repetition-peptides. In some aspects, the present disclosure relates to a kit containing one or more diamino-repetition-peptides and a plate and / or a reagent of an electrochemiluminescence-based immunoassay.SELECTED DRAWING: None

Description

[Technical field]

[0001] Federally Sponsored Research This invention was made with Government support under Grant No. R01 NS098819 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0002] Related Applications This application claims the benefit under 35 U.S.C. 119(e) of the filing date of U.S. Provisional Application Serial Number 62 / 903,689, entitled "DETECTION OF ANTIBODIES AGAINST RAN PROTEINS FROM SERUM AND TISSUE LYSATES," filed September 20, 2019, and Serial Number 62 / 904,612, entitled "VACCINE THERAPY FOR RAN PROTEIN DISEASES," filed September 23, 2019, the entire contents of each of which are incorporated herein by reference. [Background technology]

[0003] background Microsatellite repeat expansions are known to cause more than 40 neurodegenerative diseases and disorders. In a growing number of these diseases and disorders, the expansion mutations have been shown to undergo a novel type of protein translation that generates multiple reading frames and does not require the standard AUG start codon. This type of translation is called repeat-associated non-ATG (RAN) translation, and the proteins produced are called RAN proteins. There is growing evidence that RAN proteins are toxic and contribute to many more diseases and disorders, including, but not limited to, amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), myotonic dystrophy type 1 (DM1), myotonic dystrophy type 2 (DM2), Huntington's disease (HD), Alzheimer's disease (AD), and fragile X-associated tremor ataxia syndrome (FXTAS). Summary of the Invention

[0004] overview Aspects of the present disclosure relate to methods and kits for detecting anti-RAN protein antibodies in a sample. The antibodies detected by the methods described herein can be exogenously-administered antibodies (e.g., antibodies administered therapeutically to a subject) or endogenous antibodies (e.g., antibodies produced by cells of a subject). In some embodiments, the endogenous antibodies are autoantibodies. In some embodiments, the endogenous antibodies are produced by cells of a subject infected with an expression vector encoding the antibody. In some embodiments, the endogenous antibodies are produced by cells of a subject in response to administering a vaccine. In some embodiments, the endogenous antibodies are produced by cells of a subject in response to a subject having an expansion mutation that expresses one or more types of RAN protein (e.g., a subject with C9Orf72 ALS / FTD may produce antibodies such as autoantibodies against poly(GA), poly(GP), poly(GR), poly(PA), or poly(PR) proteins).

[0005] In some embodiments, the methods of detecting RAN protein antibodies described herein are useful for the diagnosis and treatment of certain neurological diseases and disorders associated with repeat-associated non-ATG (RAN) proteins (including, for example, polyserine [polySer], poly(proline-arginine) [poly(PR)], and poly(glycine-arginine) [poly(GR)], etc.). Certain repeat expansion mutations (e.g., CAGG, CCTG, GGGGCC, GGCCCC, GGGGCA, CAG, CTG, etc.) are associated with a number of different neurological diseases and disorders, such as amyotrophic lateral sclerosis (ALS); frontotemporal dementia (FTD); myotonic dystrophy type 1 (DM1); myotonic dystrophy type 2 (DM2); spinocerebellar ataxia (SCA) types 1, 2, 3, 6, 7, 8, 10, 12, 17, 31, and 36; spinal-bulbar muscular atrophy; dentatorubral-pallidoluysian atrophy (DRPLA); Huntington's disease (HD); and Alzheimer's disease (AD).

[0006] In some embodiments, it has been observed that the expansion mutations associated with certain diseases (e.g., ALS, FTD, FXTAS, HD, AD, SCA8, DM1, and DM2) undergo repeat-associated non-ATG (RAN) translation. This type of translation produces a protein called RAN protein. There is growing evidence that RAN protein is toxic and contributes to more diseases and disorders. Therefore, it is important to develop methods and kits for detecting, localizing, and identifying anti-RAN protein antibodies (e.g., potentially protective anti-RAN protein antibodies) in samples for both therapeutic and diagnostic use.

[0007] In some embodiments, an immunoassay (e.g., an electrochemiluminescence-based immunoassay) is used to detect or measure the level of one or more anti-RAN protein antibodies in a biological sample obtained from a subject (e.g., a blood, serum, or cerebrospinal fluid (CSF) sample).

[0008] The present disclosure is based in part on an immunoassay that can measure the level of one or more anti-RAN protein antibodies (also referred to as anti-RAN antibodies) from cells or tissues of a subject, which is more sensitive than the detection methods used so far. In some embodiments, the subject has or is suspected of having a RAN protein-related disease or disorder. In some embodiments, the subject has been administered a therapeutic agent for the treatment of a RAN protein-related disease or disorder. In some aspects, the method described by the present disclosure is useful for monitoring (e.g., measuring longitudinally) the level of one or more anti-RAN protein antibodies in a subject that has been administered or is being administered one or more therapeutic agents for the treatment of a disease or disorder (such as ALS or FTD) associated with RAN protein expression.

[0009] In some aspects, the disclosure relates to a method for detecting one or more anti-RAN protein antibodies using an electrochemiluminescence-based immunoassay, the method including: (1) contacting a target di-amino acid peptide repeat with a biological sample obtained from a subject to form an anti-RAN antibody-target peptide complex; (2) contacting the anti-RAN antibody-target peptide complex with an electrochemiluminescent detection agent to form a labeled complex; and (3) measuring the level of one or more anti-RAN protein antibodies present in the sample by detecting electrochemiluminescence of the labeled complex.

[0010] In some embodiments, the biological sample is a blood sample, a serum sample, or a tissue sample. In some embodiments, the tissue sample is a CNS tissue sample or a cerebrospinal fluid (CSF) sample. In some embodiments, the biological sample is diluted. In some embodiments, the biological sample is diluted prior to detecting one or more anti-RAN protein antibodies. In some embodiments, the biological sample is diluted with a diluent. In some embodiments, the diluent is phosphate buffered saline (PBS) or Tris buffered saline (TBST). In some embodiments, the biological sample is diluted to a biological sample:diluent ratio of 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:1250, 1:1500, 1:1750, or 1:2000, or any ratio contained herein.In some embodiments, the biological sample is diluted to a concentration of at least 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.011, 0.012, 0.013, 0.014, 0.015, 0.016, 0.107, 0.018, 0.019, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 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, or 99% or greater, or any value contained herein.

[0011] In some embodiments, the subject is a mammalian subject. In some embodiments, the subject is a human or a mouse. In some embodiments, the subject is a C9-BAC mouse.

[0012] In some embodiments, the anti-RAN antibody-targeting peptide conjugate is selected from the group consisting of anti-poly(CP), anti-poly(GA), anti-poly(GP), anti-poly(PR), anti-poly(GR), anti-poly(PA), anti-poly(A), anti-poly(G), anti-poly(S); anti-poly(C); anti-poly(Q); anti-poly(GD), anti-poly(GE), anti-poly(GQ), anti-poly(GT), anti-poly(L), anti-poly(LP), anti-poly(LPAC (SEQ ID NO: 1)), anti-poly(LS), anti-poly(P), anti-poly(QAGR (SEQ ID NO: 2)), anti-poly(RE), anti-poly(SP), anti-poly(VP), anti-poly(FP), or The antibodies include any one of anti-poly(GK) antibodies, and the target peptide includes any one of poly(CP), poly(GA), poly(GP), poly(PR), poly(GR), poly(PA), poly(A), poly(G), poly(S), poly(C), poly(Q), poly(GD), poly(GE), poly(GQ), poly(GT), poly(L), poly(LP), poly(LPAC (SEQ ID NO: 1)), poly(LS), poly(P), poly(QAGR (SEQ ID NO: 2)), poly(RE), poly(SP), poly(VP), poly(FP), or poly(GK) peptides. In some embodiments, the one or more anti-RAN protein antibodies present in the sample include one or more of anti-poly(CP), anti-poly(GA), anti-poly(GP), anti-poly(PR), anti-poly(GR), anti-poly(PA), anti-poly(A), anti-poly(G), anti-poly(S); anti-poly(C); anti-poly(Q); anti-poly(GD), anti-poly(GE), anti-poly(GQ), anti-poly(GT), anti-poly(L), anti-poly(LP), anti-poly(LPAC (SEQ ID NO: 1)), anti-poly(LS), anti-poly(P), anti-poly(QAGR (SEQ ID NO: 2)), anti-poly(RE), anti-poly(SP), anti-poly(VP), anti-poly(FP), or anti-poly(GK) antibodies.

[0013] In some embodiments, the subject has been administered a therapeutic anti-RAN antibody. In some embodiments, the therapeutic anti-RAN antibody is selected from the group consisting of anti-poly(cysteine-proline) [anti-poly(CP)]; anti-poly(glycine-proline) [anti-poly(GP)]; anti-poly(glycine-arginine) [anti-poly(GR)]; anti-poly(glycine) [anti-poly(G)]; anti-poly(alanine) [anti-poly(A)]; anti-poly(serine) [anti-poly(S)]; anti-poly(cysteine) [anti-poly(C)]; anti-poly(glutamine) [anti-poly(Q)]; anti-poly(glycine-alanine) [anti-poly(GA)]; anti-poly(glycine-aspartate) [anti-poly(GD)]; anti-poly(glycine-glutamate) [anti-poly(GE)]; anti-poly(glycine-glutamine) [anti-poly(GQ)]; anti-poly(glycine-threonine) [anti-poly(GT)]; anti-poly(leucine) [anti-polyLeu]; anti-poly(leucine-proline) [anti-poly(Poly) anti-poly(LP)]; anti-poly(leucine-proline-alanine-cysteine ​​(SEQ ID NO: 1)) [anti-poly(LPAC(SEQ ID NO: 1))]; anti-poly(leucine-serine) [anti-poly(LS)]; anti-poly(proline) [anti-poly(P)]; anti-poly(proline-alanine) [anti-poly(PA)]; anti-poly(proline-arginine) [anti-poly(PR)]; anti-poly(glutamine-alanine-glycine-arginine (SEQ ID NO: 2)) [anti-poly(QAGR(SEQ ID NO: 2))]; anti-poly(arginine-glutamate) [anti-poly(RE)]; anti-poly(serine-proline) [anti-poly(SP)]; anti-poly(valine-proline) [anti-poly(VP)]; anti-poly(phenylalanine-proline) [anti-poly(FP)]; or anti-poly(glycine-lysine) [anti-poly(GK)] antibodies, or any combination thereof.

[0014] In some embodiments, the subject has been administered a vaccine against a RAN protein-associated disease or disorder (e.g., the vaccine has been administered prior to obtaining the sample). In some embodiments, the vaccine elicits an immune response against one or more dipeptide repeat proteins. In some embodiments, the immune response includes endogenous production of anti-RAN protein antibodies. In some embodiments, the endogenous antibodies include one or more of anti-poly(CP), anti-poly(GA), anti-poly(GP), anti-poly(PR), anti-poly(GR), anti-poly(PA), anti-poly(A), anti-poly(G), anti-poly(S); anti-poly(C); anti-poly(Q); anti-poly(GD), anti-poly(GE), anti-poly(GQ), anti-poly(GT), anti-poly(L), anti-poly(LP), anti-poly(LPAC (SEQ ID NO: 1)), anti-poly(LS), anti-poly(P), anti-poly(QAGR (SEQ ID NO: 2)), anti-poly(RE), anti-poly(SP), anti-poly(VP), anti-poly(FP), or anti-poly(GK) antibodies, or any combination thereof.

[0015] In some embodiments, the vaccine comprises one or more peptide antigens. In some embodiments, the one or more peptide antigens comprise one or more immunogens. In some embodiments, the one or more peptide antigens comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 immunogens. In some embodiments, the one or more peptide antigens target one or more RAN proteins, optionally wherein the one or more RAN proteins are one or more of the following: poly(CP), poly(GA), poly(GP), poly(PR), poly(GR), poly(PA), poly(A), poly(G), poly(S), poly(C), poly(Q), poly(GD), poly(GE), poly(GQ), poly(GT), poly(L), poly(LP), poly(LPAC (SEQ ID NO: 1)), poly(LS), poly(P), poly(QAGR (SEQ ID NO: 2)), poly(RE), poly(SP), poly(VP), poly(FP), and / or poly(GK) RAN proteins.

[0016] In some embodiments, the one or more peptide antigens comprise one or more dipeptide repeat (DPR) peptide antigens, optionally wherein the one or more dipeptide repeat (DPR) peptide antigens are (CP) x (SEQ ID NO: 3), (A) x (SEQ ID NO: 9), (G) x (SEQ ID NO: 10), (S) x (SEQ ID NO: 11), (C) x (SEQ ID NO: 12), (Q) x (SEQ ID NO: 13), (GD) x (SEQ ID NO: 14), (GE) x (SEQ ID NO: 15), (GQ) x (SEQ ID NO: 16), (GT) x (SEQ ID NO: 17), (L) x (SEQ ID NO: 18), (LP) x (SEQ ID NO: 19), (LPAC) x (SEQ ID NO: 20), (LS) x (SEQ ID NO: 21), (P) x (SEQ ID NO: 22), (QAGR) x (SEQ ID NO: 23), (RE) x (SEQ ID NO: 24), (SP) x (SEQ ID NO: 25), (VP) x (SEQ ID NO: 26), (FP x (SEQ ID NO: 27), and / or (GK) x (SEQ ID NO:28), where x represents the number of repeating units in the antigen. In some embodiments, x is 5, 10, 15, 20, 25, 30, 35 or 40.

[0017] In some embodiments, the one or more peptide antigens are 10 (SEQ ID NO: 4), GA 15 (SEQ ID NO: 4), GR 25 (SEQ ID NO: 7), GP 10 (SEQ ID NO:5), PR 10 (SEQ ID NO:6), or one or more combinations thereof. In some embodiments, the one or more peptide antigens include one or more of [(GA) 15 (SEQ ID NO: 4) + (GR) 25 (SEQ ID NO: 7) + (PR) 10In some embodiments, the one or more peptide antigens include [(GA) 15 (SEQ ID NO: 4) + (GR) 25 (SEQ ID NO: 7) + (PR) 10 (SEQ ID NO:6)+(GP) 10 (SEQ ID NO:5)]. In some embodiments, the one or more peptide antigens comprise a B cell epitope.

[0018] In some embodiments, the one or more immunogens comprise one or more of keyhole limpet hemocyanin (KLH), Blue Carrier Immunogenic Protein (CCH), bovine serum albumin (BSA), ovalbumin (OVA), diphtheria toxin, measles virus fusion protein (MVF), hepatitis B virus surface antigen (HB-sAg), tetanus toxin (TT), pertussis toxin (PT), or a T cell helper epitope.

[0019] In some embodiments, the RAN protein-associated disease is amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Alzheimer's disease (AD), or Huntington's disease (HD).

[0020] In some embodiments, the target peptide comprises one or more of a dipeptide, tripeptide, tetrapeptide, pentapeptide, or homopolymeric repeat protein. In some embodiments, the one or more dipeptide repeat proteins are poly(CP), poly(GA), poly(GP), poly(PR), poly(GR), poly(PA), poly(A), poly(G), poly(S), poly(C), poly(Q), poly(GD), poly(GE), poly(GQ), poly(GT), poly(L), poly(LP), poly(LPAC (SEQ ID NO: 1), poly(LS), poly(P), poly(QAGR (SEQ ID NO: 2)), poly(RE), poly(SP), poly(VP), poly(FP), poly(GK), or any combination thereof. In some embodiments, the target amino acid repeat region of one or more RAN proteins comprises at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 1500, at least 2000, at least 3000, at least 4000, at least 5000, at least 10000, or more than 10000 amino acid residues. In some embodiments, the target amino acid repeat region of one or more RAN proteins comprises more than 200 amino acid residues (e.g., 500, 1000, 5000, 10000, or more than 10000, etc.). In some embodiments, the target amino acid repeat region of one or more RAN proteins is between 10 and 15,000 amino acids in length. In some embodiments, the target amino acid repeat region of one or more RAN proteins is between 50 and 12,000 amino acids in length. In some embodiments, the target amino acid repeat region of one or more RAN proteins is between 100 and 10,000 amino acids in length. In some embodiments, the target amino acid repeat region of one or more RAN proteins is between 150 and 8,000 amino acids in length. In some embodiments, the target amino acid repeat region of one or more RAN proteins is between 200 and 5,000 amino acids in length.In some embodiments, the target amino acid repeat region of one or more RAN proteins is between 300 and 2,000 amino acids in length. In some embodiments, the target amino acid repeat region of one or more RAN proteins is between 400 and 1,000 amino acids in length. In some embodiments, the target amino acid repeat region of one or more RAN proteins is between 500 and 750 amino acids in length. In some embodiments, the RAN protein has a polyamino acid repeat that is between 10 and 500 amino acid residues in length. In some embodiments, the RAN protein has a polyamino acid repeat that is between 20 and 300 amino acid residues in length. In some embodiments, the RAN protein has a polyamino acid repeat that is between 30 and 200 amino acid residues in length. In some embodiments, the RAN protein has a polyamino acid repeat that is between 40 and 100 amino acid residues in length. In some embodiments, the RAN protein has a polyamino acid repeat that is between 50 and 90 amino acid residues in length. In some embodiments, the RAN protein has a polyamino acid repeat that is between 60 and 80 amino acid residues in length.

[0021] In some embodiments, the electrochemiluminescent detecting agent comprises ruthenium(II) tris-bipyridine-(4-methylsulfone). In some embodiments, the electrochemiluminescent detecting agent is SULFO-TAG™. In some embodiments, the electrochemiluminescence of the labeled complex is measured by a Meso Scale Detection (MSD) assay.

[0022] In some embodiments, the methods described herein further comprise administering to the subject a second therapeutic agent (e.g., one or more antisense oligonucleotides, a vaccine, an exogenously administered anti-RAN antibody, another therapeutic agent, or a combination of two or more thereof) for the treatment of a RAN protein-associated neurological disease or disorder. In some embodiments, the second therapeutic agent is a small molecule, a nucleic acid, an interfering RNA, a protein, a peptide, an antibody, a vaccine, a gene therapy vector, or another immunogen. In some embodiments, a second biological sample is obtained from the subject after administration of the second therapeutic agent, and one or more anti-RAN protein antibodies are detected in the second biological sample using an electrochemiluminescence-based immunoassay.

[0023] In some embodiments, the one or more anti-RAN protein antibodies are anti-poly(CP), anti-poly(GA), anti-poly(GP), anti-poly(PR), anti-poly(GR), anti-poly(PA), anti-poly(A), anti-poly(G), anti-poly(S); anti-poly(C); anti-poly(Q); anti-poly(GD), anti-poly(GE), anti-poly(GQ), anti-poly(GT), anti-poly(L), anti-poly(LP), anti-poly(LPAC (SEQ ID NO: 1)), anti-poly(LS), anti-poly(P), anti-poly(QAGR (SEQ ID NO: 2)), anti-poly(RE), anti-poly(SP), anti-poly(VP), anti-poly(FP), or anti-poly(GK) antibodies, or any combination thereof.

[0024] In some aspects, the present disclosure relates to a method for measuring pharmacokinetic changes in anti-RAN protein antibody levels in a subject, the method comprising: (1) detecting one or more anti-RAN protein antibodies in a first biological sample obtained from the subject using an electrochemiluminescence-based immunoassay; (2) detecting one or more anti-RAN protein antibodies in a second biological sample obtained from the subject using an electrochemiluminescence-based immunoassay, where the second biological sample is obtained after administration of a therapeutic agent to the subject; and (3) determining that administration of a therapeutic agent to the subject results in a change in one or more anti-RAN protein antibody levels in the subject if the amount of anti-RAN protein antibodies detected in the first biological sample is different from the amount of anti-RAN protein antibodies detected in the second biological sample. In some embodiments, the second biological sample contains an elevated level of anti-RAN protein antibodies compared to the first biological sample. In some embodiments, the second biological sample contains a decreased level of anti-RAN protein antibodies compared to the first biological sample.

[0025] In some embodiments, the first biological sample is a control sample.In some embodiments, the control sample is a biological sample obtained from a healthy subject.In some embodiments, the control sample is a biological sample obtained from the same subject at an earlier time point.

[0026] In some embodiments, the first biological sample and / or the second biological sample is a blood sample, a serum sample, or a tissue sample. In some embodiments, the tissue sample is a sample of CNS tissue or cerebrospinal fluid (CSF).

[0027] In some embodiments, the one or more anti-RAN protein antibodies are anti-poly(CP), anti-poly(GA), anti-poly(GP), anti-poly(PR), anti-poly(GR), anti-poly(PA), anti-poly(A), anti-poly(G), anti-poly(S), anti-poly(C), anti-poly(Q), anti-poly(GD), anti-poly(GE), anti-poly(GQ), anti-poly(GT), anti-poly(L), anti-poly(LP), anti-poly(LPAC (SEQ ID NO: 1)), anti-poly(LS), anti-poly(P), anti-poly(QAGR (SEQ ID NO: 2)), anti-poly(RE), anti-poly(SP), anti-poly(VP), anti-poly(FP), or anti-poly(GK) antibodies, or any combination thereof.

[0028] In some embodiments, the present disclosure relates to a kit comprising one or more target diamino acid repeat peptides; and immunoassay plates and / or immunoassay reagents. In some embodiments, the one or more target diamino acid repeat peptides are selected from the following peptides: poly(CP), poly(GA), poly(GP), poly(PR), poly(GR), poly(PA), poly(A), poly(G), poly(S), poly(C), poly(Q), poly(GD), poly(GE), poly(GQ), poly(GT), poly(L), poly(LP), poly(LPAC (SEQ ID NO: 1), poly(LS), poly(P), poly(QAGR (SEQ ID NO: 2), poly(RE), poly(SP), poly(VP), poly(FP), and poly(GK). In some embodiments, the immunoassay plates and / or immunoassay reagents are MSD assay plates and / or MSD assay reagents. [Brief description of the drawings]

[0029] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] FIG. 1 shows a schematic diagram depicting one embodiment of an assay for the detection of RAN protein in a biological sample.

[0030] [Diagram 2]Figure 2 shows representative data for detection of anti-poly(GP) antibodies in serum obtained from C9 Bac mice treated with anti-RAN protein vaccine. Assay plates were coated with (GP)8 peptide and exposed to serum after mice were exposed to 2-3 doses of vaccine targeting the dipeptide repeats (DPRs) indicated as follows: GP10 (SEQ ID NO:5), GA15 (SEQ ID NO:4), GA10 (SEQ ID NO:4), GR25 (SEQ ID NO:7), PR10 (SEQ ID NO:6), (GA15 (SEQ ID NO:4) + GR25 (SEQ ID NO:7) + PR10 (SEQ ID NO:6)), (GA15 (SEQ ID NO:4) + GR25 (SEQ ID NO:7) + PR10 (SEQ ID NO:6) + GP10 (SEQ ID NO:4)). Data from adjuvant control mice are also shown.

[0031] [Diagram 3] Figure 3 shows representative data for detection of anti-poly(GA) antibodies in serum obtained from C9 Bac mice treated with anti-RAN protein vaccine. Assay plates were coated with (GA)8 (SEQ ID NO: 4) peptide and exposed to serum after mice were exposed to 2-3 doses of vaccine targeting the dipeptide repeats (DPRs) indicated as follows: GP10 (SEQ ID NO: 5), GA15 (SEQ ID NO: 4), GA10 (SEQ ID NO: 4), GR25 (SEQ ID NO: 7), PR10 (SEQ ID NO: 6), (GA15 (SEQ ID NO: 4) + GR25 (SEQ ID NO: 7) + PR10 (SEQ ID NO: 6)), (GA15 (SEQ ID NO: 4) + GR25 (SEQ ID NO: 7) + PR10 (SEQ ID NO: 6) + GP10 (SEQ ID NO: 4)). Data from adjuvant control mice are also shown.

[0032] [Figure 4]Figure 4 shows representative data for detection of anti-poly(GA) antibodies in lysates from brain tissue obtained from C9 Bac mice treated with anti-RAN protein vaccine. Assay plates were coated with (GA)8 (SEQ ID NO: 4) peptide and exposed to serum after mice were exposed to 2-3 doses of vaccines targeting the dipeptide repeats (DPRs) indicated as follows: GA15, GA10, (GA15 (SEQ ID NO: 4) + GR25 (SEQ ID NO: 7) + PR10 (SEQ ID NO: 6)). Data from adjuvant control mice are also shown.

[0033] [Diagram 5] 5 shows representative data for the detection of anti-poly(GA) antibodies in serum samples obtained from human C9+ALS patients. Assay plates were coated with (GA)8 (SEQ ID NO:4) peptide and exposed to serum. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] Detailed Description Aspects of the present disclosure relate to methods and compositions useful for detecting antibodies that bind to repeat-associated non-ATG proteins (RAN proteins) in biological samples obtained from subjects. The antibodies detected by the methods described herein can be exogenously administered antibodies (e.g., antibodies administered to a subject as a treatment) or endogenous antibodies (e.g., antibodies produced by cells of a subject). In some embodiments, endogenous antibodies are autoantibodies. In some embodiments, endogenous antibodies are produced by cells of a subject infected with an expression vector encoding an antibody. In some embodiments, endogenous antibodies are produced by cells of a subject in response to administering a vaccine. In some embodiments, endogenous antibodies are produced by cells of a subject in response to a subject having an expansion mutation that expresses one or more types of RAN proteins (e.g., a subject with C9Orf72 ALS / FTD may produce antibodies such as autoantibodies against poly(GA), poly(GP), poly(GR), poly(PA), or poly(PR) proteins).

[0035] In some embodiments, the methods of detecting RAN protein antibodies described herein are useful for the diagnosis and treatment of certain neurological diseases and disorders associated with repeat-associated non-ATG (RAN) proteins (e.g., including polyserine [polySer], poly(proline-arginine) [poly(PR)], and poly(glycine-arginine) [poly(GR)] RAN proteins, etc.). Mutations in certain repeat expansions (e.g., CAGG, CCTG, GGGGCC, GGCCCC, GGGGCA, CAG, and CTG) are associated with a number of different neurological diseases and disorders (e.g., amyotrophic lateral sclerosis (ALS), or frontotemporal dementia; myotonic dystrophy type 1 (DM1) and myotonic dystrophy type 2 (DM2); spinocerebellar ataxia types 1, 2, 3, 6, 7, 8, 10, 12, 17, 31, and 36; spinal-bulbar muscular atrophy; dentatorubral-pallidoluysian atrophy (DRPLA); Huntington's disease (HD); and Alzheimer's disease (AD)).

[0036] The present disclosure is based in part on longitudinal detection (e.g., over a period of time) of one or more anti-RAN protein antibody levels in a biological sample using an electrochemiluminescence-based immunoassay. In some embodiments, the methods described by the present disclosure are useful for indicating the effectiveness of a therapeutic agent that inhibits the expression, accumulation, and / or translation of RAN protein, for example, by indicating an increase in the concentration or production of anti-RAN protein antibody levels after administration of one or more therapeutic agents to a subject.

[0037] RAN protein "RAN proteins (repeat-associated non-ATG translated proteins)" are polypeptides translated from mRNA sequences that have a nucleotide stretch in the absence of an AUG start codon. In general, RAN proteins contain extended repeats of single, di, tri, or quad-amino acids (e.g., tetra-amino acids) referred to as polyamino acid repeats. RAN proteins can have polyamino acid repeats of at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 1500, at least 2000, at least 3000, at least 4000, at least 5000, at least 10000, or more than 10000 amino acid residues. In some embodiments, the RAN protein has a polyamino acid repeat of more than 200 amino acid residues in length (e.g., 500, 1000, 5000, 10,000, or more than 10,000, etc.). In some embodiments, the target amino acid repeat region of one or more RAN proteins is between 10 and 15,000 amino acids in length. In some embodiments, the target amino acid repeat region of one or more RAN proteins is between 50 and 12,000 amino acids in length. In some embodiments, the target amino acid repeat region of one or more RAN proteins is between 100 and 10,000 amino acids in length. In some embodiments, the target amino acid repeat region of one or more RAN proteins is between 150 and 8,000 amino acids in length. In some embodiments, the target amino acid repeat region of one or more RAN proteins is between 200 and 5,000 amino acids in length. In some embodiments, the target amino acid repeat region of one or more RAN proteins is between 300 and 2,000 amino acids in length. In some embodiments, the target amino acid repeat region of one or more RAN proteins is between 400 and 1,000 amino acids in length.In some embodiments, the target amino acid repeat region of one or more RAN proteins is between 500 and 750 amino acids in length. In some embodiments, the RAN protein has a polyamino acid repeat that is between 10 and 500 amino acid residues in length. In some embodiments, the RAN protein has a polyamino acid repeat that is between 20 and 300 amino acid residues in length. In some embodiments, the RAN protein has a polyamino acid repeat that is between 30 and 200 amino acid residues in length. In some embodiments, the RAN protein has a polyamino acid repeat that is between 40 and 100 amino acid residues in length. In some embodiments, the RAN protein has a polyamino acid repeat that is between 50 and 90 amino acid residues in length. In some embodiments, the RAN protein has a polyamino acid repeat that is between 60 and 80 amino acid residues in length.

[0038] The sequence encoding the RAN protein can be found in a number of loci in a subject's genome (e.g., the genome of a human subject), including, but not limited to, open reading frame 72 of chromosome 9 (C9orf72), open reading frame 80 of chromosome 2 (C2orf80), LRP8, CASP8, CRNDE, EXOC6B, SV2B, PPML1, ADARB2, GREB1, and MSMO1. The protein associated with C9orf72 is currently poorly characterized, but is known to be abundant in neurons, particularly in the cerebral cortex and motor neurons. C9orf72 protein is thought to be localized in presynaptic termini. C9orf72 protein likely influences RNA transcription, translation, and subcellular localization. The C9orf72 gene contains GGGGCC repeats (hexanucleotide repeats) that occur in variable repeat numbers.

[0039] In the context of ALS / FTD, which results from a repeat expansion of the hexanucleotide sequence GGGGCC in the C9Orf72 gene, RAN proteins have been identified that contain the following diamino acid repeats: poly(Gly-Ala), poly(Gly-Pro), poly(Gly-Arg), poly(Pro-Ala), or poly(Pro-Arg), each of which is also referred to as poly(GA), poly(GP), poly(GR), poly(PA), and poly(PR).ALS / FTD RAN proteins are generally described, for example, in International PCT Application No. PCT / US2014 / 022670, filed March 10, 2014 and published as WO2014 / 159247, and U.S. Application No. 14 / 775,278, filed September 11, 2015 and published as US2016 / 0025747, the entire contents of each of which are incorporated herein by reference.

[0040] In the context of SCA36, which results from a repeat expansion of the hexanucleotide sequence TGGGCC in the SCA36 gene, RAN proteins have been identified that contain the following diamino acid repeats: poly(GP) and poly(PR).

[0041] In the context of Huntington's disease (HD), translation of the RAN protein is triggered by a CAG·CTG expansion in the Htt gene, which results in translation of the RAN protein polyalanine, polyserine, polyleucine, and polycysteine ​​(polyAla, polySer, polyLeu, and polyCys) in addition to polyglutamine (polyGln or polyQ).

[0042] In the context of SCA8 and DM1, translation of the RAN protein is driven by a CTG·CAG repeat expansion. The SCA8 expansion mutation is bidirectionally transcribed to produce both CUG (ATXN8OS) and CAG (ATXN8) expanded RNAs, which are expressed in opposite directions across the expansion mutation. The CUG expansion transcripts form RNA foci, and the expanded CAG ATXN8 transcripts expressed in the opposite direction produce a nearly pure poly-Gln protein from an unusually short ORF that contains an AUG start codon directly upstream of the CAG repeat. This results in the translation of the RAN protein poly-alanine, poly-serine, poly-leucine, and poly-cysteine ​​(poly-Ala, poly-Ser, poly-Leu, and poly-Cys) in addition to poly-glutamine (poly-Gln or poly-Q).

[0043] In the context of fragile X syndrome (FXS) and fragile X-associated tremor / ataxia syndrome (FXTAS), translation of the RAN protein is triggered by a CGG·CCG expansion. Expansion of a CGG repeat tract in the 5' untranslated region (UTR) of the FMR1 gene causes two separate disorders depending on the length of the repeat. Longer expansions (>200 repeats) cause transcriptional silencing of the FMR1 gene resulting in FXS. In contrast, shorter alleles (55–200 repeats) within the premutation range cause the late-onset neurodegenerative disorder FXTAS. This results in translation of the RAN protein polyGly, polyPro, polyArg, and polyAla.

[0044] In the context of DM2, translation of the RAN protein is driven by an intronic CCTG·CAGG expansion mutation located in the cellular nucleic acid binding protein (CNBP) gene, which produces a tetrapeptide-extended protein in both the sense (poly(leucine-proline-alanine-cysteine ​​[LPAC])) and antisense (poly(glutamine-alanine-glycine-arginine [QAGR])) orientations.

[0045] In the context of SCA31, translation of the RAN protein is triggered by the accumulation of the Trp-Asn-Gly-Met-Glu (SEQ ID NO: 30) pentapeptide repeat protein (PPR) that encodes a UGGAA expansion. In the context of SCA31, translation of the RAN protein is triggered by the accumulation of the Trp-Asn-Gly-Met-Glu (SEQ ID NO: 30) pentapeptide repeat protein (PPR) that encodes a UGGAA expansion.

[0046] Other examples of RAN proteins may include poly(CP), poly(GD), poly(GE), poly(GQ), poly(GT), poly(LP), poly(LPAC (SEQ ID NO: 1)), poly(LS), poly(P), poly(RE), poly(SP), poly(VP), poly(FP), poly(GK), or any combination thereof. Additional examples of RAN proteins and methods of identifying RAN proteins are described, for example, in International PCT Application No. PCT / US2020 / 040725, filed July 2, 2020, the entire contents of which are incorporated herein by reference.

[0047] In some embodiments, the RAN protein is a member of the family of disorders that affect Huntington's disease (HD, HDL2), Alzheimer's disease (AD), Fragile X syndrome (FRAXA), Spinal-bulbar muscular atrophy (SBMA), Dentatorubral-pallidoluysian atrophy (DRPLA), Spinocerebellar ataxia type 1 (SCA1), Spinocerebellar ataxia type 2 (SCA2), Spinocerebellar ataxia type 3 (SCA3), Spinocerebellar ataxia type 6 (SCA6), Spinocerebellar ataxia type 7 (SCA7), Spinocerebellar ataxia type 8 (SCA8), Spinocerebellar ataxia type 9 (SCA9), Spinocerebellar ataxia type 10 (SCA10), Spinocerebellar ataxia type 11 (SCA11), Spinocerebellar ataxia type 12 (SCA22), Spinocerebellar ataxia type 13 (SCA13), Spinocerebellar ataxia type 14 (SCA14), Spinocerebellar ataxia type 15 (SCA15), Spinocerebellar ataxia type 16 (SCA16), Spinocerebellar ataxia type 17 (SCA17), Spinocerebellar ataxia type 18 (SCA18), Spinocerebellar ataxia type 19 (SCA19), Spinocerebellar ataxia type 20 (SCA20), Spinocerebellar ataxia type 21 (SCA21), Spinocerebellar ataxia type 22 (SCA22), Spinocerebellar ataxia type 23 (SCA23), Spinocerebellar ataxia type 24 (SCA24), Spinocerebellar ataxia type 25 (SCA25), Spino ), spinocerebellar ataxia type 12 (SCA12) or spinocerebellar ataxia type 17 (SCA17), amyotrophic lateral sclerosis (ALS), spinocerebellar ataxia type 36 (SCA36), spinocerebellar ataxia type 29 (SCA29), spinocerebellar ataxia type 10 (SCA10), myotonic dystrophy type 1 (DM1), myotonic dystrophy type 2 (DM2), or Fuchs' corneal dystrophy (e.g., CTG181).

[0048] Subjects and biological samples In some aspects, the present disclosure relates to a method for detecting one or more anti-RAN protein antibodies (e.g., detecting the level of one or more anti-RAN protein antibodies) in a biological sample obtained from a subject. The subject can be a mammal (e.g., a human, a mouse, a rat, a dog, a cat, or a pig). In some embodiments, the subject is a mammalian subject. In some embodiments, the subject is a human. In some embodiments, the subject is a mouse. In some embodiments, the subject is a C9-BAC mouse. The C9-BAC mouse model of ALS is described, for example, in International PCT Application No. PCT / US2014 / 022670, filed March 10, 2014 and published as WO2014 / 159247, and Liu et al., (2016) Neuron 90(3):521-34, the entire contents of each of which are incorporated herein by reference.

[0049] In some embodiments, the subject is characterized by a GGGGCC (e.g., G4C2) hexanucleotide sequence repeat expansion in the C9orf72 gene (e.g., a gene such as the human C9orf72 gene or a mouse gene corresponding to the human C9orf72 gene). In some embodiments, the human C9orf72 gene comprises or consists of a sequence represented by any one of NCBI reference sequence numbers NM_145005.6, NM_018325.4, and NM_001256054.2. In some embodiments, the subject is characterized by a TGGGCC hexanucleotide sequence repeat expansion in the SCA36 gene (e.g., a gene such as the human SCA36 gene or a mouse gene corresponding to the human SCA36 gene). In some embodiments, the human SCA36 gene comprises or consists of a sequence represented by any one of NCBI reference sequence numbers NM_006392.3, NR_027700.2, and NR_145428.1. In some embodiments, the subject has been determined to have a hexanucleotide sequence repeat expansion (e.g., a GGGGCC (e.g., G4C2) repeat expansion in C9Orf72 or a TGGGCC repeat expansion in SCA36) by a genetic assay (e.g., a DNA-based assay, e.g., a sequencing assay).

[0050] In some embodiments, the subject comprises at least 50, at least 100, at least 200, at least 500, at least 1000, or at least 5000 GGGGCC repeat expansions (e.g., the repeat expansion of C9Orf72). In some embodiments, the subject comprises at least 50, at least 100, at least 200, at least 500, at least 1000, or at least 5000 TGGGCC repeat expansions (e.g., the repeat expansion of SCA36).

[0051] In some embodiments, the subject is characterized by the presence of RNA aggregates that contain the translation of a RAN protein or a transcript that codes for a diamino acid repeat (DPR). In some embodiments, the RAN protein translated in the subject comprises one or more of the following: poly(GA), poly(GP), poly(GR), poly(PA), or poly(PR). In some embodiments, the RAN protein translated in the subject comprises one or more of the following: poly(CP), poly(GD), poly(GE), poly(GQ), poly(GT), poly(LP), poly(LPAC (SEQ ID NO: 1), poly(LS), poly(P), poly(QAGR (SEQ ID NO: 2)), poly(RE), poly(SP), poly(VP), poly(FP), poly(GK), or any combination thereof. In some embodiments, poly(GA), poly(GP), poly(GR), poly(PA), poly(PR) RAN proteins are expressed from the expanded repeats of C9Orf72, Htt, SCA36, LRP8, CASP8, and / or GREB1 of a subject. In some embodiments, poly(CP), poly(GD), poly(GE), poly(GQ), poly(GT), poly(LP), poly(LPAC (SEQ ID NO: 1), poly(LS), poly(P), poly(QAGR (SEQ ID NO: 2), poly(RE), poly(SP), poly(VP), poly(FP), poly(GK) proteins are expressed from the expanded repeats of C9Orf72, Htt, SCA36, LRP8, CASP8, and / or GREB1 of a subject.

[0052] Generally, the biological sample comprises blood, serum (e.g., plasma with clotting proteins removed), or cerebrospinal fluid (CSF). However, one of skill in the art will recognize other suitable biological samples, such as biological samples comprising CNS tissue (e.g., brain tissue, spinal tissue, etc.) and / or cells (e.g., brain cells, nerve cells, skin cells, etc.). In some embodiments, the biological sample comprises a blood sample or a tissue sample. In some embodiments, the blood sample comprises a sample of whole blood, plasma, or serum. In some embodiments, the tissue sample comprises a CNS tissue or a cerebrospinal fluid (CSF) sample. In some embodiments, the blood sample has been treated to remove white blood cells (e.g., leukocytes), such as the buffy coat of the sample.

[0053] In some embodiments, the biological sample is diluted. In some embodiments, the biological sample is diluted prior to detecting one or more anti-RAN protein antibodies. In some embodiments, the biological sample is diluted with a diluent. In some embodiments, the diluent is phosphate buffered saline (PBS) or Tris buffered saline (TBST). In some embodiments, the biological sample is diluted to a biological sample:diluent ratio of 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:1250, 1:1500, 1:1750, or 1:2000, or any ratio contained herein.In some embodiments, the biological sample is diluted to a concentration of at least 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.011, 0.012, 0.013, 0.014, 0.015, 0.016, 0.107, 0.018, 0.019, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 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, or 99% or greater, or any value contained herein.

[0054] In some embodiments, the biological sample obtained from the subject is stored prior to detection of one or more anti-RAN protein antibodies (e.g., prior to performing an assay). In some embodiments, the biological sample obtained from the subject is stored at a temperature between -80°C and about 23°C (e.g., room temperature). In some embodiments, the biological sample obtained from the subject is stored at a temperature between 0°C and about 23°C (e.g., about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23°C). In some embodiments, the biological sample obtained from the subject is stored at a temperature between 20°C and about 25°C (e.g., about 20, 21, 22, 23, 24, or 25°C).

[0055] In some embodiments, the biological sample is obtained from a subject having or suspected of having a disease or disorder associated with RAN protein expression, translation, and / or accumulation. In some embodiments, the subject exhibits one or more signs or symptoms of a disease or disorder associated with RAN protein expression, translation, and / or accumulation. "Subject having or suspected of having a disease (e.g., neurological disease) or disorder associated with RAN protein expression, translation, and / or accumulation" generally refers to a subject exhibiting one or more signs and symptoms of a neurodegenerative disease or disorder (including, but not limited to, memory deficits (e.g., short-term memory loss), confusion, deficits in executive function (e.g., attention, planning, adaptability, abstract thinking, etc.), aphasia, degeneration or loss of motor skills, etc.), or a subject having or identified as having one or more genetic mutations associated with RAN protein expression, translation, and / or accumulation.

[0056] In some embodiments, the disease or disorder associated with RAN protein expression, translation, and / or accumulation is any one of amyotrophic lateral sclerosis (ALS) or frontotemporal dementia; myotonic dystrophy type 1 (DM1) and myotonic dystrophy type 2 (DM2); spinocerebellar ataxia types 1, 2, 3, 6, 7, 8, 10, 12, 17, 31, and 36; spinal-bulbar muscular atrophy; dentatorubral-pallidoluysian atrophy (DRPLA); Huntington's disease (HD); Alzheimer's disease (AD); Fragile X-associated tremor ataxia syndrome (FXTAS); Fuchs endothelial corneal dystrophy (FECD); and Huntington's disease-like 2 syndrome (HDL2).

[0057] A "subject having or suspected of having ALS and / or FTD" may be a subject known or determined to have more than 30 GGGGCC repeats in the C9Orf72 gene or a subject exhibiting signs and symptoms of ALS / FTD, including but not limited to the following: motor dysfunction (e.g., spasticity), muscle atrophy, and / or neuropsychiatric manifestations (e.g., obsessive-compulsive behavior, lethargy, anxiety). In some embodiments, a subject with ALS is characterized by having one or more mutations in the C9Orf72 gene.

[0058] A "subject having or suspected of having Alzheimer's disease" may be a subject exhibiting one or more signs and symptoms of AD (including, but not limited to, the following: memory deficits (e.g., short-term memory loss), confusion, deficits in executive function (e.g., attention, planning, adaptability, abstract thinking, etc.), aphasia, decline or loss of motor skills, etc.) or a subject having or identified as having one or more genetic mutations associated with AD (e.g., mutations in certain genes including apolipoprotein (APP), presenilin genes (PSEN1 and PSEN2), or tau protein). In some embodiments, a subject having or suspected of having AD is characterized by the accumulation of β-amyloid (Aβ) peptides and hyperphosphorylated tau protein throughout the subject's brain tissue. In some embodiments, the subject has been diagnosed with AD by a health care professional according to the NINCDS-ADRDA Alzheimer's Criteria as described by McKhann et al., (1984) "Clinical diagnosis of Alzheimer's disease: report of the NINCDS-ADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer's Disease". Neurology, 34(7):939-44.

[0059] A "subject having or suspected of having myotonic dystrophy" (e.g., myotonic dystrophy type 1 (DM1) or myotonic dystrophy type 2 (DM2)) may be a subject who exhibits one or more signs or symptoms of DM1 and / or DM2 (including, but not limited to, the following: delayed muscle relaxation, muscle weakness, prolonged involuntary muscle contractions, or muscle loss; and / or abnormal heart rhythm, cataracts, or difficulty swallowing).

[0060] "Subjects having or suspected of having spinocerebellar ataxia" (e.g., spinocerebellar ataxia types 1, 2, 3, 6, 7, 8, 10, 12, 17, 31, or 36) are defined as having one or more signs or symptoms of spinocerebellar ataxia, including, but not limited to, speech and swallowing difficulties, muscle rigidity (e.g., spasticity), weakness of the muscles that control eye movement (e.g., ophthalmoplegia), rapid involuntary eye movements (e.g., nystagmus), uncoordinated movements and imbalance of balance (e.g., ataxia), muscle wasting, slow eye movements, dementia, uncontrolled muscle tension (e.g., dystonia), stiffness, tremors, bulging eyes, double vision, loss of coordination in the arms, progressive vision loss, blindness, changes in sensation or reflexes, trunk instability, hyperactive tendon reflexes, drawn-out slowness of The subject may exhibit scanning dysarthria characterized by impaired speech, cerebellar ataxia, unsteady gait, upper limb ataxia, dysphagia, gait dysfunction, extrapyramidal functions, pyramidal weakness, cognitive and behavioral disturbances, chorea, psychiatric disturbances, sensorineural hearing impairment, impaired vibration sense, rapid eye movements (e.g., saccades), trouble moving the eyes side-to-side (e.g., ocular lag), and / or droopy eyelids (e.g., ptosis).

[0061] A "subject having or suspected of having spinal-bulbar muscular atrophy" may be a subject who exhibits one or more signs or symptoms of spinal-bulbar muscular atrophy, including, but not limited to, the following: speech impairment, difficulty chewing and swallowing, impaired sleep, difficulty breathing, facial muscle weakness, difficulty communicating emotions, muscle weakness and atrophy of the arms and legs, muscle twitching and cramping, enlarged breasts (in male subjects), reduced fertility and atrophy (e.g., shrinkage) of the testes, abnormal processing of male hormones, muscle wasting, and / or difficulty walking.

[0062] A subject "having or suspected of having Dentatorubral-Pallidoluysian Atrophy (DRPLA)" may be a subject who exhibits one or more signs or symptoms of DRPLA, including but not limited to the following: ataxia, uncontrollable limb movements (e.g., choreoathetosis), psychiatric symptoms (e.g., delusions), and / or deterioration of intellectual function (e.g., dementia).

[0063] A subject "having or suspected of having Huntington's disease (HD)" may be a subject who exhibits one or more signs or symptoms of HD, including, but not limited to, the following: abnormal gait, increased muscle activity, involuntary movements, problems with coordination, muscle loss, muscle spasms, forgetfulness, delusions, lack of concentration, confusion, slowness of activity, difficulty thinking and understanding, obsessive-compulsive behavior, fidgetiness, irritability, immodesty, delirium, depression, hallucinations, paranoia, anxiety, lethargy, mood swings, difficulty speaking, memory loss, tremors, and / or weight loss.

[0064] A subject "has or is suspected of having a Fragile X disorder (e.g., Fragile X syndrome (FXS), Fragile X-associated tremor ataxia syndrome (FXTAS), or Fragile XE syndrome (FRAXE))" is a subject who has one or more signs or symptoms of FXS, FXTAS, and / or FRAXE, including, but not limited to, aggression, hyperactivity, impulsivity, nonsense word repetition, repetitive movements, self-injury, persistent repetitive words or movements, learning disability or speech delay (e.g., in children), flaccid muscles or problems with coordination, large ears, speech disorders, anxiety, double-jointed limbs, enlarged head, enlarged testicles, flat feet, weak eyesight, long thin face, prominent chin, scoliosis, single line on palm, or vertebral column. The subject may exhibit symptoms such as: weakness, loss of bladder or bowel function, speech delay, poor writing skills, hyperactivity, and / or moodiness.

[0065] A subject "having or suspected of having Huntington's disease-related syndrome type 2 (HDL2)" may be a subject who exhibits one or more signs or symptoms of HDL2, including but not limited to the following: progressive movement disorders (e.g., parkinsonism, chorea), cognitive and emotional decline (e.g., dementia, psychiatric disturbances), epileptic seizure(s), and / or any other sign or symptom associated with HD.

[0066] A subject "having or suspected of having Fuchs' endothelial corneal dystrophy (FECD)" may be a subject who exhibits one or more signs or symptoms of FECD, including, but not limited to, the following: blurred or cloudy vision (e.g., a general lack of clear vision), shimmering vision (e.g., symptoms worsening in the morning after waking and gradually improving throughout the day), permanently impaired vision, glare, seeing halos around lights, and / or pain or grittiness due to small blisters on the surface of the cornea.

[0067] Therapeutic Agents The methods of the present disclosure are useful in some embodiments for investigating the efficacy of therapeutic agents (e.g., candidate therapeutic agents) in animal models of diseases or disorders associated with RAN protein expression, translation, and / or accumulation. "Candidate therapeutic agent" generally refers to an agent (e.g., small molecules, nucleic acids, interfering RNA, proteins, peptides, antibodies, vaccines, gene therapy vectors, etc.) that is tested for its ability to reduce or inhibit RAN protein expression, translation, and / or accumulation, or to induce a protective immune response in a cell or subject. Therapeutic agents useful for treating diseases or disorders associated with RAN protein may target (e.g., reduce expression, activity, accumulation, aggregation, etc.) RAN protein or a nucleic acid encoding the RAN protein, and / or modulate the activity of another gene or gene product (e.g., a protein) that interacts with one or more RAN proteins.

[0068] In some embodiments, the therapeutic agent is an anti-RAN protein antibody. The anti-RAN protein antibody can be an exogenous antibody (e.g., an antibody administered to a subject as a treatment) or an endogenous antibody (e.g., an antibody produced by a subject's cells). In some embodiments, the endogenous antibody is an autoantibody. In some embodiments, the endogenous antibody is produced by a subject's cells that are infected with an expression vector that encodes the antibody. In some embodiments, the endogenous antibody is produced by a subject's cells in response to a vaccine being administered. In some embodiments, the endogenous antibody is produced by a subject's cells in response to a subject having an expansion mutation that expresses one or more types of RAN protein (e.g., a subject with C9Orf72 ALS / FTD may produce antibodies, such as autoantibodies, against poly(GA), poly(GP), poly(GR), poly(PA), or poly(PR) proteins). As used herein, "anti-RAN protein antibody" refers to both exogenously provided and endogenous anti-RAN protein antibodies. One of skill in the art will recognize that both exogenously provided and endogenous anti-RAN protein antibodies may be considered therapeutic agents.

[0069] In some embodiments, expression of the anti-RAN protein antibody reduces expression of the RAN protein compared to the expression level of the RAN protein present in the subject prior to expression of the anti-RAN protein antibody. In some embodiments, expression of the anti-RAN protein antibody reduces expression of the RAN protein by 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, up to 100-fold, or more than 100-fold, compared to the expression level of the RAN protein present in the subject prior to expression of the anti-RAN protein antibody. In some embodiments, expression of the anti-RAN protein antibody reduces expression of RAN protein by 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%, 102%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 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%, or 100%, or any percentage contained herein.

[0070] In some embodiments, expression of an anti-RAN protein antibody reduces RAN protein aggregation compared to the level of RAN protein aggregation present in the subject prior to expression of the anti-RAN protein antibody. In some embodiments, expression of an anti-RAN protein antibody reduces RAN protein aggregation by 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, up to 100-fold, or more than 100-fold, compared to the level of RAN protein aggregation present in the subject prior to expression of the anti-RAN protein antibody. In some embodiments, expression of an anti-RAN protein antibody reduces RAN protein aggregation by 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%, 102%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 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%, or 100%, or any percentage contained herein.

[0071] In some embodiments, the anti-RAN protein antibody is an anti-poly(CP), anti-poly(GA), anti-poly(GP), anti-poly(PR), anti-poly(GR), anti-poly(PA), anti-poly(A), anti-poly(G), anti-poly(S), anti-poly(C), anti-poly(Q), anti-poly(GD), anti-poly(GE), anti-poly(GQ), anti-poly(GT), anti-poly(L), anti-poly(LP), anti-poly(LPAC (SEQ ID NO: 1), anti-poly(LS), anti-poly(P), anti-poly(QAGR (SEQ ID NO: 2), anti-poly(RE), anti-poly(SP), anti-poly(VP), anti-poly(FP), or anti-poly(GK) antibody, or any combination thereof. The anti-RAN protein antibody may bind to extracellular RAN protein, intracellular RAN protein, or both extracellular and intracellular RAN protein.

[0072] In some embodiments, the anti-RAN protein antibody targets (e.g., specifically binds to) one or more amino acid repeat regions of the RAN protein selected from the following: poly(CP), poly(GA), poly(GP), poly(PR), poly(GR), poly(PA), poly(A), poly(G), poly(S), poly(C), poly(Q), poly(GD), poly(GE), poly(GQ), poly(GT), poly(L), poly(LP), poly(LPAC (SEQ ID NO: 1)), poly(LS), poly(P), poly(QAGR (SEQ ID NO: 2)), poly(RE), poly(SP), poly(VP), poly(FP), and / or poly(GK). In some embodiments, the target amino acid repeat region of one or more RAN proteins comprises at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 1500, at least 2000, at least 3000, at least 4000, at least 5000, at least 10000, or more than 10000 amino acid residues. In some embodiments, the target amino acid repeat region of one or more RAN proteins comprises more than 200 amino acid residues (e.g., 500, 1000, 5000, 10,000, or more than 10,000, etc.). In some embodiments, the target amino acid repeat region of one or more RAN proteins is between 10 and 15,000 amino acids in length. In some embodiments, the target amino acid repeat region of one or more RAN proteins is between 50 and 12,000 amino acids in length. In some embodiments, the target amino acid repeat region of one or more RAN proteins is between 100 and 10,000 amino acids in length. In some embodiments, the target amino acid repeat region of one or more RAN proteins is between 150 and 8,000 amino acids in length. In some embodiments, the target amino acid repeat region of one or more RAN proteins is between 200 and 5,000 amino acids in length.In some embodiments, the target amino acid repeat region of one or more RAN proteins is between 300 and 2,000 amino acids in length. In some embodiments, the target amino acid repeat region of one or more RAN proteins is between 400 and 1,000 amino acids in length. In some embodiments, the target amino acid repeat region of one or more RAN proteins is between 500 and 750 amino acids in length. In some embodiments, the RAN protein has a polyamino acid repeat that is between 10 and 500 amino acid residues in length. In some embodiments, the RAN protein has a polyamino acid repeat that is between 20 and 300 amino acid residues in length. In some embodiments, the RAN protein has a polyamino acid repeat that is between 30 and 200 amino acid residues in length. In some embodiments, the RAN protein has a polyamino acid repeat that is between 40 and 100 amino acid residues in length. In some embodiments, the RAN protein has a polyamino acid repeat that is between 50 and 90 amino acid residues in length. In some embodiments, the RAN protein has a polyamino acid repeat that is between 60 and 80 amino acid residues in length.

[0073] In some embodiments, the anti-RAN antibody targets (e.g., specifically binds to) any portion of the RAN protein that does not contain polyamino acid repeats, such as the C-terminus of the RAN protein (e.g., the C-terminus of poly(CP), poly(GA), poly(GP), poly(PR), poly(GR), poly(PA), poly(A), poly(G), poly(S), poly(C), poly(Q), poly(GD), poly(GE), poly(GQ), poly(GT), poly(L), poly(LP), poly(LPAC (SEQ ID NO: 1), poly(LS), poly(P), poly(QAGR (SEQ ID NO: 2)), poly(RE), poly(SP), poly(VP), poly(FP), and / or poly(GK)). Examples of anti-RAN antibodies that target the C-terminus of the RAN protein are disclosed, for example, in U.S. Publication No. 2013 / 0115603, the entire contents of which are incorporated herein by reference.

[0074] In some embodiments, a set (or combination) of anti-RAN antibodies (e.g., a combination of two or more anti-RAN antibodies selected from the following: poly(CP), poly(GA), poly(GP), poly(PR), poly(GR), poly(PA), poly(A), poly(G), poly(S), poly(C), poly(Q), poly(GD), poly(GE), poly(GQ), poly(GT), poly(L), poly(LP), poly(LPAC (SEQ ID NO: 1)), poly(LS), poly(P), poly(QAGR (SEQ ID NO: 2)), poly(RE), poly(SP), poly(VP), poly(FP), and poly(GK) antibodies, etc.) is administered to a subject for the purpose of treating a disease or disorder associated with RAN protein.

[0075] The "C-terminal portion" or "C-terminus" of a RAN protein includes the amino acid sequence encoded by the nucleotide sequence downstream of a polyamino acid repeat region in an intron of a gene (e.g., C9Orf72, HTT, DM1, SCA36, LRP8, CASP8, GREB1, etc.) for a sense transcript, or the nucleotide sequence downstream of a polyamino acid repeat region in an intron of a gene (e.g., C9Orf72, HTT, DM1, SCA36, LRP8, CASP8, GREB1, etc.) for an antisense transcript.

[0076] In some embodiments, the C-terminal portion of the RAN protein comprises one or more consecutive amino acids in the sequence encoded by the sense transcript of the gene (e.g., C9Orf72, HTT, DM1, SCA36, LRP8, CASP8, GREB1, etc.) beginning with the amino acid immediately following the polyamino acid repeat portion of the RAN protein. In some embodiments, the C-terminal portion of the RAN protein comprises one or more consecutive amino acids in the sequence encoded by the antisense transcript of the gene (e.g., C9Orf72, HTT, DM1, SCA36, LRP8, CASP8, GREB1, etc.) beginning with the amino acid immediately following the polyamino acid repeat portion of the RAN protein.

[0077] In some embodiments, the C-terminal portion of the RAN protein is 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, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 , 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, or more than 75 consecutive amino acids. In some embodiments, the C-terminal portion of the RAN protein comprises 1-5, 3-10, 5-15, 8-20, 10-25, 13-30, 15-35, 18-40, 20-45, 23-50, 25-55, 28-60, 30-65, 33-70, 35-75, or more than 75 consecutive amino acids in a sequence beginning with the amino acid immediately following the polyamino acid repeat portion of the RAN protein.

[0078] Anti-RAN antibodies can be polyclonal or monoclonal. Typically, polyclonal antibodies are produced by inoculation of a suitable mammal, such as a mouse, rabbit, or goat. Larger mammals are often preferred because the amount of serum that can be collected is greater. Antigen is injected into the mammal. This induces B lymphocytes that produce IgG immunoglobulins specific to the antigen. The polyclonal IgG is purified from the serum of the mammal. Monoclonal antibodies are generally produced by a single cell line (e.g., a hybridoma cell line). In some embodiments, anti-RAN antibodies are purified (e.g., isolated from serum).

[0079] In some embodiments, the therapeutic agent is a RAN protein vaccine. RAN protein vaccines generally include one or more peptide antigens that induce an immune response (e.g., the production of specific antibodies in a subject) against RAN protein. In some embodiments, the therapeutic candidate is a vaccine that is configured to induce an immune response against one or more RAN proteins expressed from the expanded repeat in a subject (e.g., poly(CP), poly(GA), poly(GP), poly(PR), poly(GR), poly(PA), poly(A), poly(G), poly(S), poly(C), poly(Q), poly(GD), poly(GE), poly(GQ), poly(GT), poly(L), poly(LP), poly(LPAC (SEQ ID NO: 1)), poly(LS), poly(P), poly(QAGR (SEQ ID NO: 2)), poly(RE), poly(SP), poly(VP), poly(FP), and / or poly(GK) RAN proteins, etc.). In some embodiments, the therapeutic agent comprises a peptide antigen that targets one or more RAN proteins (e.g., a RAN protein vaccine that targets one or more RAN proteins). In some embodiments, the peptide antigen targets one or more RAN proteins (e.g., including the amino acid sequences encoding them) selected from the following RAN proteins: poly(CP), poly(GA), poly(GP), poly(PR), poly(GR), poly(PA), poly(A), poly(G), poly(S), poly(C), poly(Q), poly(GD), poly(GE), poly(GQ), poly(GT), poly(L), poly(LP), poly(LPAC (SEQ ID NO: 1)), poly(LS), poly(P), poly(QAGR (SEQ ID NO: 2)), poly(RE), poly(SP), poly(VP), poly(FP), and / or poly(GK).

[0080] In some embodiments, administration of the RAN protein vaccine reduces the expression of the RAN protein compared to the expression level of the RAN protein present in the subject prior to administration of the RAN protein vaccine. In some embodiments, administration of the RAN protein vaccine reduces the expression of the RAN protein by 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or more than 100-fold compared to the expression level of the RAN protein present in the subject prior to administration of the RAN protein vaccine. In some embodiments, administration of the RAN protein vaccine reduces expression of RAN protein by 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%, or more compared to the expression level of RAN protein present in the subject prior to administration of the RAN protein vaccine. , 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%, or 100%, or any percentage contained herein.

[0081] In some embodiments, administration of the RAN protein vaccine reduces RAN protein aggregation compared to the level of RAN protein aggregation present in the subject prior to administration of the RAN protein vaccine. In some embodiments, administration of the RAN protein vaccine reduces RAN protein aggregation by 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, up to 100-fold, or more than 100-fold compared to the level of RAN protein aggregation present in the subject prior to administration of the RAN protein vaccine. In some embodiments, administration of the RAN protein vaccine reduces RAN protein aggregation by 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%, 102%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 1 , 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%, or 100%, or any percentage contained herein.

[0082] In some embodiments, one or more of the peptide antigens is a B cell epitope. In some embodiments, the peptide antigen is a dipeptide repeat (DPR) peptide antigen. In some embodiments, the DPR peptide antigen is a (GA) x (SEQ ID NO: 4), (GP x (SEQ ID NO: 5), (GR) x (SEQ ID NO: 7), (PA)x (SEQ ID NO: 8), where x represents the number of repeat units of the antigen. In some embodiments, each of the one or more DPR peptide antigens comprises a diamino acid repeat of (CP) x (SEQ ID NO: 3), (A) x (SEQ ID NO: 9), (G) x (SEQ ID NO: 10), (S) x (SEQ ID NO: 11), (C)x (SEQ ID NO: 12), (Q)x (SEQ ID NO: 13), (GD) x (SEQ ID NO: 14), (GE) x (SEQ ID NO: 15), (GQ) x (SEQ ID NO: 16), (GT) x (SEQ ID NO: 17), (L) x (SEQ ID NO: 18), (LP) x (SEQ ID NO: 19), (LPAC) x (SEQ ID NO: 20), (LS) x (SEQ ID NO: 21), (P) x (SEQ ID NO: 22), (QAGR) x (SEQ ID NO: 23), (RE) x (SEQ ID NO: 24), (SP) x (SEQ ID NO: 25), (VP) x (SEQ ID NO: 26), (FP x (SEQ ID NO: 27), and / or (GK) x(SEQ ID NO:28), where x represents the number of repeating units of the antigen. In some embodiments, "x" is 5, 10, 15, 20, 25, 30, 35, 40, or more than 40 (e.g., 5, 10, 15, 20, 25, 30, 35, 40, or more than 40 repeating units). In some embodiments, the peptide antigen (e.g., DPR peptide antigen) is between 2 and 150 (e.g., 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, 9, 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, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 1 In some embodiments, the peptide antigen (e.g., DPR peptide antigen) comprises more than 50 amino acid repeats (e.g., various homopolymeric amino acid repeats, diamino acid repeats, triamino acid repeats, tetraamino acid repeats, pentaamino acid repeats, or any combination of the above). In some embodiments, each DPR peptide antigen comprises between 10 and 25 diamino acid repeats. In some embodiments, the DPR peptide antigen comprises the following peptide antigens: (GA 10 (SEQ ID NO: 4), GA15 (SEQ ID NO: 4), GR 25 (SEQ ID NO: 7), GP 10 (SEQ ID NO:5), PR 10 (SEQ ID NO:6), or any combination thereof.

[0083] The number of peptide antigens in the RAN protein vaccine may vary (e.g., the RAN protein vaccine may be multivalent). In some embodiments, the RAN protein vaccine comprises peptide antigens for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more different amino acid repeats (e.g., different homopolymeric amino acid repeats, diamino acid repeats, triamino acid repeats, tetraamino acid repeats, pentaamino acid repeats, or any combination of the above). In some embodiments, the DPR peptide antigen is [(GA) 15 (SEQ ID NO: 4) + (GR) 25 (SEQ ID NO: 7) + (PR) 10 (SEQ ID NO: 6)], or [(GA) 15 (SEQ ID NO: 4) + (GR) 25 (SEQ ID NO: 7) + (PR) 10 (SEQ ID NO:6)+(GP) 10 (SEQ ID NO:5)].

[0084] Aspects of the present disclosure relate to compositions (e.g., RAN protein vaccines, etc.) that include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) additional immunogens. "Immunogen" refers to any antigen capable of inducing a humoral and / or cellular immune response rather than immunological tolerance. Examples of immunogens include, but are not limited to, keyhole limpet hemocyanin (KLH), blue carrier immunogenic protein (CCH), bovine serum albumin (BSA), ovalbumin (OVA), diphtheria toxin, measles virus fusion protein (MVF), hepatitis B virus surface antigen (HB-sAg), tetanus toxin (TT), pertussis toxin (PT), or portions of any of the above.

[0085] The immunogen may include a T helper cell epitope. "T helper cell epitope" or "Th epitope" refers to a T cell epitope that is presented on the surface of an antigen-presenting cell, where it is bound to an MHC class II molecule of 13-17 amino acids in length that is specifically recognized by T helper cells. In some embodiments, the inclusion of a Th epitope in a vaccine results in a biased Th2 type regulatory T cell response in the vaccinated subject, in preference to a Th1 proinflammatory T cell response. The inclusion of a Th epitope in a peptide vaccine and UBI Th epitopes are described, for example, in Wang et al. (2007) Vaccine 25(16):3041-52, and US 2019 / 0194280, the entire contents of which are incorporated herein by reference.

[0086] The peptide antigen and the additional immunogen (e.g., B cell epitope and T cell epitope) may be linked by any suitable modality. In some embodiments, the peptide antigen and the additional immunogen are covalently or non-covalently linked. In some embodiments, the peptide antigen and the additional immunogen are directly connected to each other. In some embodiments, the peptide antigen and the additional immunogen are linked via one or more (e.g., one, two, three, four, or more) linking molecules. In some embodiments, the conformational separation provided by one or more linking molecules allows more efficient interaction between the presented peptide antigen (e.g., DPR peptide antigen) and the appropriate Th cells and B cells, thereby enhancing the immunogenicity of the peptide antigen (e.g., DPR peptide antigen) or their cross-reactive functional immunological analogs.

[0087] The linking molecule may provide a chemical linkage (e.g., a linkage between one or more small molecules) or an amino acid linkage (e.g., a linkage between amino acids, reactive groups of amino acids, hybridization, etc.), or a combination thereof. In some embodiments, the linking molecule comprises an amino acid linker. In some embodiments, the linking molecule is selected from the group consisting of the amino acids Lys-, Gly-, Lys-Lys-Lys-, (α,ε-N)Lys, and ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 29).

[0088] In some embodiments, the RAN protein vaccine comprises one or more additional components, such as an N-terminal amide or signal peptide, or a C-terminal α-COOH or α-CONH2.

[0089] The RAN protein vaccine or RAN protein immunogen may be formulated in a pharmaceutical composition. In some embodiments, the pharmaceutical composition includes one or more pharma- ceutical acceptable excipients. Pharmaceutical excipients are known and described, for example, by Remington, JP (1965). Remington's Pharmaceutical Sciences, Easton, Pa: Mack Pub. Co., 19th ed., 1995. In some embodiments, the pharmaceutical composition, including the vaccine, includes one or more adjuvants. Adjuvants are pharmacological or immunological agents that modify the effect of other agents. Adjuvants are added to vaccines to boost immune responses, which may result in the production of more antibodies and longer-lasting immunity, thus minimizing the dose of antigen required. In some embodiments, the adjuvant is an inorganic salt of aluminum. In some embodiments, the adjuvant is alhydrogel (Al(OH)3) or adjuphos (AlPO4). Aluminum-based adjuvants are known, for example as described in Shardlow et al. (2018) Allergy, Asthma & Clinical Immunology, 14:80. In some embodiments, the pharmaceutical composition further comprises CpG. In some embodiments, the CpG is a CpG oligodeoxynucleotide (ODN), for example as described by Weiner, et al., (1997) PNAS 94(20):10833-7.

[0090] In some embodiments, one or more therapeutic molecules are administered to a subject to treat a disease or disorder associated with a RAN protein characterized by an expansion of a nucleic acid repeat (e.g., associated with a repeat-associated non-ATG translation). For example, in some embodiments, a subject is administered 2, 3, 4, 5, 6, 7, 8, 9, or 10 therapeutic agents (e.g., proteins, nucleic acids, small molecules, etc., or any combination thereof).

[0091] Anti-RAN protein antibody detection assay The present disclosure is based in part on the surprising discovery that certain immunoassays (e.g., electrochemiluminescence-based immunoassays) can be used to detect one or more anti-RAN protein antibodies in biological samples (e.g., blood, serum, CSF, etc.) obtained from a subject. In some embodiments, the time and conditions (e.g., incubation time and incubation temperature) under which the sample is processed affect the amount of background signal observed in a given blood sample. For example, if a serum sample is incubated (e.g., held or stored) at room temperature for more than 24 hours after being obtained from a subject, the RAN protein level in the sample is, in some embodiments, indistinguishable from a control sample due to high background signal.

[0092] In some embodiments, the immunoassay (e.g., an electrochemiluminescence-based immunoassay) is performed on the biological sample (e.g., a blood sample) within 2 days of being obtained from the subject. In some embodiments, the immunoassay (e.g., an electrochemiluminescence-based immunoassay) is performed on the biological sample (e.g., a blood sample) between about 1 minute and about 48 hours after being obtained from the subject. In some embodiments, the immunoassay (e.g., an electrochemiluminescence-based immunoassay) is performed on the biological sample (e.g., a blood sample) between about 60 minutes and about 24 hours after being obtained from the subject.

[0093] In some aspects, the disclosure provides kits comprising one or more target di-amino acid repeat peptides and an electrochemiluminescence-based immunoassay.

[0094] "Target diamino acid repeat peptide" refers to a peptide that comprises or consists of a diamino acid repeat sequence. In some embodiments, the target diamino acid repeat peptide comprises a repeat selected from the following: poly(GP), poly(GA), poly(GR), and poly(PR). In some embodiments, the target diamino acid repeat peptide comprises a repeat selected from the following: poly(CP), poly(GA), poly(GP), poly(PR), poly(GR), poly(PA), poly(A), poly(G), poly(S), poly(C), poly(Q), poly(GD), poly(GE), poly(GQ), poly(GT), poly(L), poly(LP), poly(LPAC (SEQ ID NO: 1), poly(LS), poly(P), poly(QAGR (SEQ ID NO: 2)), poly(RE), poly(SP), poly(VP), poly(FP), and poly(GK).

[0095] In some embodiments, the length of the target di-amino acid repeat peptide ranges from about 4 amino acid residues (e.g., 2 di-amino acid repeats such as "GPGP" (SEQ ID NO: 31)) to about 100 amino acid residues (e.g., 50 di-amino acid repeats such as "GPGPGPGPGPGPGPGPGPGPGPGPGPGPGPGPGPGPGPGPGPGPGPGPGPGPGPGPGPGPGPGPGPGPGPGPGPGPGPGPGP" (SEQ ID NO: 32)). In some embodiments, the target di-amino acid repeat peptide comprises between 3 and 25 amino acid repeats (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 di-amino acid repeats). In some embodiments, the target diamino acid repeat peptide comprises 8 diamino acid repeats (e.g., (GA)8 (SEQ ID NO:4), (GP)8 (SEQ ID NO:5), (GR)8 (SEQ ID NO:7), (PR)8 (SEQ ID NO:6), etc.). In some embodiments, the target diamino acid repeat peptide comprises at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 1500, at least 2000, at least 3000, at least 4000, at least 5000, at least 10000, or more than 10000 amino acid residues. In some embodiments, the target diamino acid repeat peptide comprises a polyamino acid repeat of more than 200 amino acid residues in length (e.g., 500, 1000, 5000, 10,000, or more than 10,000, etc.). In some embodiments, the target amino acid repeat region of one or more RAN proteins is between 10 and 15,000 amino acids in length. In some embodiments, the target amino acid repeat region of one or more RAN proteins is between 50 and 12,000 amino acids in length. In some embodiments, the target amino acid repeat region of one or more RAN proteins is between 100 and 10,000 amino acids in length.In some embodiments, the target amino acid repeat region of one or more RAN proteins is between 150 and 8,000 amino acids in length. In some embodiments, the target amino acid repeat region of one or more RAN proteins is between 200 and 5,000 amino acids in length. In some embodiments, the target amino acid repeat region of one or more RAN proteins is between 300 and 2,000 amino acids in length. In some embodiments, the target amino acid repeat region of one or more RAN proteins is between 400 and 1,000 amino acids in length. In some embodiments, the target amino acid repeat region of one or more RAN proteins is between 500 and 750 amino acids in length. In some embodiments, the RAN protein has a polyamino acid repeat that is between 10 and 500 amino acid residues in length. In some embodiments, the RAN protein has a polyamino acid repeat that is between 20 and 300 amino acid residues in length. In some embodiments, the RAN protein has a polyamino acid repeat that is between 30 and 200 amino acid residues in length. In some embodiments, the RAN protein has a polyamino acid repeat between 40 and 100 amino acid residues in length. In some embodiments, the RAN protein has a polyamino acid repeat between 50 and 90 amino acid residues in length. In some embodiments, the RAN protein has a polyamino acid repeat between 60 and 80 amino acid residues in length.

[0096] In general, "electrochemiluminescence-based immunoassay" refers to a biological assay in which the binding of one or more anti-RAN protein antibodies present in a biological sample to one or more target diamino acid repeat peptides bound to a substrate (such as a microplate) is detected using an electrochemiluminescent label (e.g., a detectable moiety) that emits light when stimulated by electricity in a suitable chemical environment (e.g., in the presence of tripropylamine (TPrA)). Electrochemiluminescent labels are described, for example, by Muzyka (2014) Biosens Bioelectron 15(54):393-407.

[0097] In some embodiments, the electrochemiluminescence-based immunoassay is Meso Scale Detection (MSD) assay.As used herein, the term "Meso Scale Detection (MSD) assay" refers to the immunoassay used for analyte detection by electrochemiluminescence (e.g., using one or more detectable reagents), such as SULFO-TAG™ label (e.g., label comprising one or more ruthenium complexes) that emits light when electrochemically stimulated, as described, for example, by Moxness et al. (2005) Clin.Chem.51(10):1983-5 and US Patent No. 7,008,796 (which are incorporated by reference for the description of MSD assay steps).

[0098] In general, an MSD assay involves contacting a solid substrate (e.g., a multi-well assay plate containing one or more target peptides (e.g., one or more target diamino acid repeat peptides) attached to a substrate) with a biological sample (e.g., a blood sample obtained from a subject containing one or more anti-RAN protein antibodies) under conditions in which anti-RAN protein antibodies present in the biological sample bind to one or more target peptides to form a complex, and then contacting the complex with one or more secondary antibodies (e.g., antibodies that bind to the anti-RAN protein antibody portion of the complex, such as anti-human antibodies, anti-mouse antibodies, anti-rabbit antibodies, or antibodies that bind to the target peptides, etc.) conjugated to a detectable reagent. In some embodiments, the detectable reagent comprises an electrochemiluminescent moiety, for example, as described in U.S. Pat. No. 5,310,687, which is incorporated herein by reference for its disclosure relating to such electrochemiluminescent moieties. In some embodiments, the detectable reagent comprises a ruthenium complex, for example, ruthenium(II) tris-bipyridine-(4-methylsulfone) (also [Ru(Bpy)3] +2 (also referred to as ) or a salt thereof.

[0099] A detectable reagent (e.g., a detectable moiety, e.g., a ruthenium complex such as SULFO-TAG™) is generally conjugated to a secondary antibody (e.g., a detection antibody, such as an antibody that binds to the anti-RAN protein antibody, such as an anti-human antibody, anti-mouse antibody, anti-rat antibody, anti-guinea pig antibody, or an antibody that binds to an antigen present in the species from which the sample was obtained, etc.).

[0100] In some aspects, the present disclosure provides methods for measuring one or more anti-RAN protein antibodies in a sample (e.g., using a kit as described herein), the methods including: (1) contacting a target di-amino acid repeat peptide with a biological sample obtained from a subject to form an anti-RAN antibody-target peptide complex; (2) contacting the anti-RAN antibody-target peptide complex with an electrochemiluminescent detection agent to form a labeled complex; and (3) measuring the level of one or more anti-RAN protein antibodies present in the sample by detecting electrochemiluminescence of the labeled complex.

[0101] In some embodiments, the subject has previously been administered a therapeutic agent (or candidate therapeutic agent) as described herein (e.g., a therapeutic anti-RAN protein antibody (e.g., a monoclonal antibody that specifically binds to RAN protein)) or the subject has been exposed to a vaccine configured to elicit an immune response in the subject against RAN protein (e.g., to induce the production of anti-RAN protein antibodies in the subject).

[0102] In some embodiments, the electrochemiluminescence-based immunoassay comprises contacting a sample with one or more target diamino acid repeat peptides to form a complex. In some embodiments, the one or more anti-RAN protein antibodies detected in the sample are selected from the following: anti-poly(GA), anti-poly(GP), anti-poly(GR), and anti-poly(PR). In some embodiments, the one or more anti-RAN protein antibodies detected in the sample are selected from anti-poly(CP), anti-poly(GA), anti-poly(GP), anti-poly(PR), anti-poly(GR), anti-poly(PA), anti-poly(A), anti-poly(G), anti-poly(S), anti-poly(C), anti-poly(Q), anti-poly(GD), anti-poly(GE), anti-poly(GQ), anti-poly(GT), anti-poly(L), anti-poly(LP), anti-poly(LPAC (SEQ ID NO: 1), anti-poly(LS), anti-poly(P), anti-poly(QAGR (SEQ ID NO: 2), anti-poly(RE), anti-poly(SP), anti-poly(VP), anti-poly(FP), and / or anti-poly(GK) antibodies. In some embodiments, two, three, or four anti-RAN protein antibodies are detected in the sample (e.g., poly(GP), poly(GR), poly(PR), and poly(GA), or any combination thereof).

[0103] The anti-RAN protein antibody can be a polyclonal or monoclonal antibody. In some embodiments, for example in the case of vaccination against a RAN protein-associated disease or disorder, the polyclonal antibody may circulate freely in the serum of the animal. In some embodiments, the therapeutic anti-RAN protein antibody is a monoclonal antibody. In some embodiments, the antigen is 12-20 amino acids. For antibodies against repeat motifs, the antigen is a repeat sequence. For antibodies against the C-terminal sequence of the RAN protein, the antigen is a C-terminal specific sequence. In some embodiments, the antigen is a portion of the C-terminal sequence, for example, a fragment of the C-terminal sequence that is 3-5 or 5-10 or more amino acids in length, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, or 50 amino acids in length.

[0104] Consequently, in some embodiments, the methods and kits described by the present disclosure are capable of measuring levels of anti-RAN protein antibodies in a subject over a specified period of time (e.g., over the course of longitudinal treatment), thereby providing an assessment of the therapeutic efficacy of certain treatments for diseases and disorders associated with RAN protein expression, translation, and / or accumulation (e.g., therapeutic agents for treating ALS / FTD). In some embodiments, the disease or disorder associated with RAN protein is selected from the group consisting of amyotrophic lateral sclerosis (ALS) or frontotemporal dementia; myotonic dystrophy type 1 (DM1) and myotonic dystrophy type 2 (DM2); spinocerebellar ataxia types 1, 2, 3, 6, 7, 8, 10, 12, 17, 31 and 36; spinal-bulbar muscular atrophy; dentatorubral-pallidoluysian atrophy (DRPLA); Huntington's disease (HD); Alzheimer's disease (AD); Fragile X-associated tremor ataxia syndrome (FXTAS); Fuchs endothelial corneal dystrophy (FECD); Huntington's disease-related syndrome type 2 (HDL2); Fragile X syndrome (FXS); folate-sensitive fragile site-associated disorder FRA7A at 7p11.2; folate-sensitive fragile site-associated disorder FRA2A at 2q11.2; and fragile XE syndrome (FRAXE).

[0105] Without wishing to be bound by any theory, measuring an increased level of one or more anti-RAN protein antibodies in a subject after administration of a therapeutic agent for treating a disease or disorder associated with RAN protein expression, translation, and / or accumulation (e.g., ALS / FTD) (e.g., compared to the level of anti-RAN protein antibodies measured in the subject prior to administration) suggests that the therapeutic agent for treating a disease or disorder associated with RAN protein expression, translation, and / or accumulation (e.g., ALS / FTD) effectively treats the subject. Measuring a decreased level of one or more anti-RAN protein antibodies in a subject after administration of a therapeutic agent for treating a disease or disorder associated with RAN protein expression, translation, and / or accumulation (e.g., ALS / FTD) (e.g., compared to the level of anti-RAN protein antibodies measured in the subject prior to administration) suggests that the therapeutic agent for treating a disease or disorder associated with RAN protein expression, translation, and / or accumulation (e.g., ALS / FTD) does not effectively treat the subject.

[0106] For example, in some embodiments, biological samples are obtained from a subject prior to the start of a therapeutic regimen and after the start of a therapeutic regimen (e.g., after 1 week, 2 weeks, 1 month, 6 months, or 1 year), and the amount of anti-RAN protein antibody detected in the samples is compared. In some embodiments, if the level (e.g., amount) of anti-RAN protein antibody in the post-treatment sample is increased compared to the pre-treatment level (e.g., amount) of anti-RAN protein antibody, the therapeutic regimen is successful. In some embodiments, if the level (e.g., amount) of anti-RAN protein antibody in the post-treatment sample is decreased compared to the pre-treatment level (e.g., amount) of anti-RAN protein antibody, the therapeutic regimen is not successful. In some embodiments, the level of anti-RAN protein antibody in the subject's biological samples (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 samples) is constantly monitored during the therapeutic regimen (e.g., measured at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 separate times).

[0107] In some aspects, the present disclosure provides a method for measuring pharmacokinetic changes in anti-RAN protein antibody levels in a subject, the method comprising: (1) detecting one or more anti-RAN protein antibodies in a first biological sample obtained from the subject using an electrochemiluminescence-based immunoassay; (2) detecting one or more anti-RAN protein antibodies in a second biological sample obtained from the subject using an electrochemiluminescence-based immunoassay, where the second biological sample is obtained after administration of a therapeutic agent to the subject; and (3) determining that administration of a therapeutic agent to the subject results in a change in one or more anti-RAN protein antibody levels in the subject if the amount of anti-RAN protein antibodies detected in the first biological sample is different from the amount of anti-RAN protein antibodies detected in the second biological sample. In some embodiments, the second biological sample contains an elevated level of anti-RAN protein antibodies compared to the first biological sample. In some embodiments, the second biological sample contains a decreased level of anti-RAN protein antibodies compared to the first biological sample.

[0108] As used herein, "elevated" means that the level of one or more anti-RAN antibodies present in a biological sample (e.g., a serum sample) is greater than a predetermined threshold or a control level, such as the level of one or more anti-RAN protein antibodies in a control sample. Controls and control levels include anti-RAN protein antibody levels obtained (e.g., detected) from subjects who do not have or are not suspected of having a disease or disorder associated with RAN protein expression, translation, and / or accumulation (e.g., ALS / FTD) (and / or subjects with 30 or fewer GGGGCC expansion repeats). In some embodiments, a control or control level includes anti-RAN protein antibody levels prior to administration of a therapeutic agent (e.g., a candidate therapeutic agent). Elevated levels include, for example, levels that are 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500%, or more above the control level. An elevated level also encompasses the phenomenon of increasing from a null state (e.g., no or undetectable expression or level of anti-RAN protein antibodies) to a non-null state (e.g., a level or detectable levels of expression or presence of anti-RAN protein antibodies). In some embodiments, an increase (e.g., an increase in the level of one or more anti-RAN protein antibody levels in a sample compared to a control or prior sample) can indicate the therapeutic efficacy of a therapeutic agent (e.g., the efficacy of a treatment in the subject from which the sample was obtained).

[0109] As used herein, "unchanged or reduced" means that the level of one or more anti-RAN protein antibodies is at or below a control level, such as a predefined threshold or the level of one or more anti-RAN protein antibodies in a control sample. Controls and control levels include anti-RAN protein antibody levels obtained (e.g., detected) from subjects who do not have or are not suspected of having a disease or disorder associated with RAN protein expression, translation, and / or accumulation (e.g., ALS / FTD) (and / or subjects with 30 or fewer GGGGCC expansion repeats). In some embodiments, a control or control level includes anti-RAN protein antibody levels prior to administration of a therapeutic agent (e.g., anti-RAN antibody or RAN protein vaccine). An unaltered level is the same level as a control level. Decreased levels include, for example, levels that are 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500%, or more than 500% below the control level. Decreased levels also include the phenomenon of decreasing from a non-zero state (e.g., a level or detectable level of expression or presence of anti-RAN protein antibodies) to a zero state (e.g., no or undetectable expression or level of anti-RAN protein antibodies). In some embodiments, the lack of change or decrease (e.g., the lack of a change or decrease in the level of one or more anti-RAN protein antibodies in a sample compared to a control or prior sample) may indicate a lack of therapeutic efficacy of a therapeutic agent (e.g., a lack of therapeutic efficacy in the subject from whom the sample was obtained).In some embodiments, measuring a lack of change or a decreased level of one or more anti-RAN protein antibodies in a subject after administration of a therapeutic agent for treating a disease or disorder associated with RAN protein expression, translation, and / or accumulation (e.g., ALS / FTD) (e.g., compared to the level of anti-RAN protein antibodies measured in the subject prior to administration) indicates that the therapeutic agent for a disease or disorder associated with RAN protein expression, translation, and / or accumulation (e.g., ALS / FTD) does not effectively treat the subject.

[0110] As used herein, an "alteration" in one or more anti-RAN protein antibody levels occurs in a subject when the amount of anti-RAN protein antibody detected in a first biological sample differs from the amount of anti-RAN protein antibody detected in a second biological sample. The amount of anti-RAN protein antibody detected in a first biological sample is considered to be "different" from the amount of anti-RAN protein antibody detected in a second biological sample when either an elevated or decreased level of one or more anti-RAN protein antibodies is observed in the second biological sample.

[0111] In some embodiments, the first biological sample is a control sample (e.g., control blood, serum or CSF sample).In some embodiments, the control sample is a prior sample (e.g., sample taken from the same subject 1 hour earlier, 1 day earlier, 2 days earlier, 3 days earlier, 4 days earlier, 5 days earlier, 6 days earlier, 1 week earlier, 2 weeks earlier, 3 weeks earlier, 1 month earlier, 2 months earlier, 3 months earlier, 6 months earlier, 1 year earlier, 2 years earlier, 3 years earlier, 4 years earlier, 5 years earlier, 10 years earlier, 20 years earlier, etc.) that is screened in the same subject that is suspected to have RAN protein-related disease or disorder. In some embodiments, the control sample is a later sample (e.g., a sample taken from the same subject 1 hour, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 10 years, 20 years, etc.) screened in the same subject with or suspected of having a RAN protein-related disease or disorder. In some embodiments, the control sample is taken from a subject (e.g., a healthy control subject) who has not been diagnosed with a RAN protein-related disease or disorder and has no obvious, visible, or otherwise phenotypic symptoms. In some embodiments, the control sample is a sample taken from a different subject with a RAN protein-related disease or disorder. In some embodiments, the control sample is a sample taken from a control subject matched (e.g., age-matched, sex-matched, etc.) to a subject having or suspected of having a RAN protein-associated disease or disorder.

[0112] The time interval during which the first and second biological samples are obtained may vary. In some embodiments, the first biological sample is obtained between 1 week and 1 minute prior to administration of the therapeutic agent (e.g., the first administration of the therapeutic agent). In some embodiments, the first biological sample is obtained between 1 day (e.g., 24 hours) and 1 minute prior to administration of the therapeutic agent (e.g., the first administration of the therapeutic agent). In some embodiments, the second biological sample is obtained between 1 minute and 6 months after administration of the therapeutic agent (e.g., the first administration of the therapeutic agent). In some embodiments, the second biological sample is obtained between 1 day and 1 week after administration of the therapeutic agent (e.g., the most recent or last administration of the therapeutic agent).

[0113] In some embodiments, the second biological sample may be collected about 1 hour, 5 hours, 10 hours, 24 hours (e.g., 1 day), 48 hours (e.g., 2 days), 120 hours (e.g., 5 days), 30 days, 45 days, or 6 months after administration of the therapeutic agent. In some embodiments, several biological samples (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more biological samples) are obtained from the subject, e.g., over a specified time frame (e.g., during a course of treatment), and one or more anti-RAN protein antibodies are detected.

[0114] In some embodiments, the therapeutic regimen is successful if the level (e.g., amount) of anti-RAN protein antibody detected in the post-treatment sample is increased compared to the pre-treatment level (e.g., amount) of anti-RAN protein antibody. In some embodiments, the therapeutic regimen is not successful if the level (e.g., amount) of anti-RAN protein antibody detected in the post-treatment sample is decreased compared to the pre-treatment level (e.g., amount) of anti-RAN protein antibody. In some embodiments, the level of anti-RAN protein antibody in biological samples (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 samples) obtained from the subject is constantly monitored during the therapeutic regimen (e.g., measured at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 separate times).

[0115] Methods for diagnosing and treating diseases and disorders associated with translation of RAN protein Aspects of the present disclosure relate to methods for identifying a subject (e.g., a human subject) as having or at risk of developing a neurodegenerative disease and disorder (e.g., ALS and / or FTD) associated with RAN protein expression, translation, and / or accumulation. In some embodiments, the present disclosure provides a method for identifying a subject as having a disease or disorder (e.g., ALS and / or FTD) associated with RAN protein expression, translation, and / or accumulation, comprising: detecting one or more anti-RAN protein antibodies in a biological sample obtained from the subject using an electrochemiluminescence-based immunoassay; and determining that the subject has a disease or disorder (e.g., ALS) associated with RAN protein expression, translation, and / or accumulation based on the presence of anti-RAN protein antibodies in the biological sample.

[0116] The present disclosure, in some aspects, relates to a method for monitoring the course of therapeutic treatment for a disease or disorder associated with RAN protein expression, translation, and / or accumulation (e.g., ALS / FTD, etc.). In some aspects, the present disclosure provides a method for treating a disease or disorder associated with RAN protein expression, translation, and / or accumulation, comprising administering an effective amount of a therapeutic agent to a subject determined to exhibit increased expression, translation, and / or accumulation of RAN protein (e.g., compared to a subject not having a disease or disorder associated with RAN protein translation), and determining (e.g., detecting) a level of one or more anti-RAN protein antibodies as measured by electrochemiluminescence immunoassay. In some embodiments, the subject has previously been administered a therapeutic agent (e.g., prior to determining). In some embodiments, the therapeutic agent administered to the subject is different from the previously administered therapeutic agent. In some embodiments, the subject is administered an increased or decreased dose of a therapeutic agent based on detection of an increased or decreased level of anti-RAN protein antibodies in a biological sample as measured by electrochemiluminescence immunoassay.

[0117] Methods of treating, or modifying current treatments of, diseases or disorders associated with RAN protein expression, translation, and / or accumulation are also contemplated by the present disclosure. In some embodiments, the disease or disorder associated with RAN protein is selected from the group consisting of amyotrophic lateral sclerosis (ALS) or frontotemporal dementia; myotonic dystrophy type 1 (DM1) and myotonic dystrophy type 2 (DM2); spinocerebellar ataxia types 1, 2, 3, 6, 7, 8, 10, 12, 17, 31 and 36; spinal-bulbar muscular atrophy; dentatorubral-pallidoluysian atrophy (DRPLA); Huntington's disease (HD); Alzheimer's disease (AD); Fragile X-associated tremor ataxia syndrome (FXTAS); Fuchs endothelial corneal dystrophy (FECD); Huntington's disease-related syndrome type 2 (HDL2); Fragile X syndrome (FXS); disorder associated with the folate-sensitive fragile site at 7p11.2 FRA7A; disorder associated with the folate-sensitive fragile site at 2q11.2 FRA2A; and fragile XE syndrome (FRAXE). In some embodiments, a subject (e.g., a human subject) identified by the methods described herein as having or at risk of developing a neurodegenerative disease or disorder associated with RAN protein expression, translation, and / or accumulation is administered a therapeutic agent useful for treating the disease or disorder associated with RAN protein.

[0118] In some embodiments, the methods described by the present disclosure include a step of administering (or continuing to administer) a therapeutic agent (e.g., an agent for treating a disease associated with RAN protein expression, translation, and / or accumulation (such as ALS / FTD) such as an anti-RAN antibody or a RAN protein vaccine) to the subject if the level of anti-RAN protein antibodies detected in the biological sample (e.g., the second biological sample) is elevated compared to the level of RAN protein detected in the control sample (e.g., the first biological sample). In some embodiments, the methods described by the present disclosure include a step of ceasing administration of a therapeutic agent (e.g., an agent for treating a disease or disorder associated with RAN protein expression, translation, and / or accumulation (such as ALS / FTD)) to the subject if the level of anti-RAN protein antibodies detected in the biological sample (e.g., the second biological sample) is decreased compared to the level of RAN protein detected in the control sample (e.g., the first biological sample).

[0119] As used herein, "treat" or "treatment" refers to (a) preventing or delaying the onset of a disease or disorder associated with RAN protein expression, translation, and / or accumulation; (b) reducing the severity of a disease or disorder associated with RAN protein expression, translation, and / or accumulation; (c) reducing or preventing the onset of symptoms characteristic of a disease or disorder associated with RAN protein expression, translation, and / or accumulation; (d) preventing the worsening of symptoms characteristic of a disease or disorder associated with RAN protein expression, translation, and / or accumulation; and / or (e) reducing or preventing the recurrence of symptoms in a subject who has previously exhibited symptoms of a disease or disorder associated with RAN protein expression, translation, and / or accumulation.

[0120] For example, in the context of ALS / FTD, "treat" or "treatment" refers to (a) preventing or delaying the onset of ALS and / or FTD; (b) reducing the severity of ALS and / or FTD; (c) reducing or preventing the onset of symptoms characteristic of ALS and / or FTD; (d) preventing the worsening of symptoms characteristic of ALS and / or FTD; and / or (e) reducing or preventing the recurrence of symptoms of ALS and / or FTD in a subject who has previously exhibited symptoms of ALS and / or FTD. Examples of therapeutic agents for the treatment of ALS / FTD include, but are not limited to, riluzole (Rilutek, Sanofi-Aventis), trazodone (Desyrel, Oleptro), selective serotonin reuptake inhibitors (SSRIs), baclofen, diazepam, phenytoin, trihexyphenidyl, amitriptyline, anti-RAN antibodies, and the like. Other therapeutic agents as described herein may also be used to treat ALS / FTD and any other disease or disorder associated with the expression, translation, and / or accumulation of RAN protein.

[0121] The subject may be administered a therapeutically effective amount of one or more therapeutic agents. As used herein, an "effective amount" is a dosage of a therapeutic agent sufficient to provide a medically desired result, such as treatment or amelioration of one or more signs or symptoms caused by a disease or disorder associated with the expression, translation, and / or accumulation of RAN protein (e.g., a neurodegenerative disease or disorder). In some embodiments, a therapeutically effective amount is an amount of a RAN protein vaccine sufficient to elicit the production of anti-RAN protein antibodies in a subject. In some embodiments, a therapeutically effective amount is an amount effective to increase the amount of anti-RAN antibodies present in a sample obtained from a subject receiving the treatment (e.g., compared to a subject not receiving the treatment). In some embodiments, a therapeutically effective amount is an amount effective to reduce repeat expansions in a subject. In some embodiments, a therapeutically effective amount is an amount effective to reduce the transcription of an RNA that produces a RAN protein in a subject. In some embodiments, a therapeutically effective amount is an amount effective to reduce the translation of a RAN protein in a subject. "Reducing" expression of a repeat sequence or translation of an RAN protein refers to a decrease in the amount or level of expression of a repeat sequence or translation of an RAN protein in a subject following administration of a therapeutic agent (and compared to the amount or level in the subject prior to administration).

[0122] In some embodiments, an effective amount is an amount effective to increase the amount of anti-RAN antibodies present in a sample obtained from a subject receiving treatment (e.g., the level of anti-RAN antibodies compared to the level of anti-RAN antibodies in a cell or subject not receiving a therapeutic agent) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98%. In some embodiments, an effective amount is an amount effective to reduce the level of RAN protein (e.g., the level of RAN protein compared to the level of RAN protein in a cell or subject not receiving a therapeutic agent) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98%. In some embodiments, an effective amount is an amount effective to reduce translation of RAN protein (e.g., the level of RAN protein compared to the level of RAN protein in a cell or subject that has not been administered the therapeutic agent) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98%.

[0123] The effective amount will vary depending on the age and physical condition of the subject being treated, the severity of the disease or disorder in the subject (e.g., the amount of accumulated RAN protein or the cytotoxicity caused by such accumulation), the duration of treatment, the nature of any concomitant treatment, the specific route of administration, and other factors within the knowledge and opinion of medical personnel. The effective amount may be included in a single dose (e.g., a single oral dose) or multiple doses (e.g., multiple oral doses).

[0124] In general, and as described elsewhere herein, the therapeutic agent may be a small molecule (e.g., metformin or a metformin derivative), an interfering RNA (e.g., dsRNA, siRNA, miRNA, amiRNA, ASO, aptamer, etc.), a protein or fragment thereof, a peptide, an antibody, such as an anti-RAN protein antibody, etc. In some embodiments, the therapeutic agent modulates the expression of RAN protein by modulating a pathway that controls the expression of RAN protein, such as, for example, the protein kinase R (PKR) pathway, the EIF2 pathway, or the EIF3 pathway. In some embodiments, the therapeutic agent is delivered by a viral vector, such as a lentiviral vector, a retroviral vector, an adenoviral vector, or an adeno-associated viral (AAV) vector. In some embodiments, an anti-RAN protein peptide vaccine is delivered by a viral vector. In some embodiments, one or more viral vectors encoding an anti-RAN protein antibody or fragment thereof are delivered by a viral vector. In some embodiments, the therapeutic agent is delivered to the subject in a recombinant adeno-associated viral (rAAV) particle.

[0125] Identification and selection of suitable additional therapeutic agents are within the capabilities of those skilled in the art and will depend on the disease or disorder suffered by the subject. In some embodiments, one or more additional therapeutic agents for the treatment of ALS / FTD (e.g., riluzole (Rilutek, Sanofi-Aventis), trazodone (Desyrel, Oleptro), selective serotonin reuptake inhibitors (SSRIs), baclofen, diazepam, phenytoin, trihexyphenidyl, amitriptyline, metformin, anti-RAN antibodies, etc.) are administered to the subject. In some embodiments, drugs are used for HD (e.g., tetrabenazine (Xenazine), baclofen, and deutetrabenazine (Austedo), etc.), for AD (e.g., cholinesterase inhibitors (Aricept®, Exelon®, Razadyne®), and memantine (Namenda®), etc.), for Fragile X Syndrome (e.g., selective serotonin reuptake inhibitors, carbamazepine, methylphenidate, trazodone, etc.), for spinocerebellar ataxia (e.g., baclofen ... The subject is administered one or more additional therapeutic agents, such as for myotonic dystrophy type 1 (e.g., tideglusib, mexiletine, etc.), for myotonic dystrophy type 2 (e.g., mexilitene, gabapentin, nonsteroidal anti-inflammatory drugs (NSAIDS), low dose thyroid replacement, low dose steroids, tricyclic antidepressants, etc.), for fragile X syndrome (e.g., sertraline, metformin, cannabidiol (CBD), acamprosate, lovastatin, minocycline, etc.), for myotonic dystrophy type 1 (e.g., tideglusib, mexiletine, etc.), or for myotonic dystrophy type 2 (e.g., mexilitene, gabapentin, nonsteroidal anti-inflammatory drugs (NSAIDS), low dose thyroid replacement, low dose steroids, tricyclic antidepressants, etc.).

[0126] Administration of the therapeutic agent may be accomplished by any method known in the art (see, for example, Harrison's Principle of Internal Medicine, McGraw Hill, Inc.). Administration may be local or systemic. The therapeutic agent may be administered by any route, including enteral (e.g., orally), parenteral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intratracheal, subcutaneous, intraventricular, transdermal, intradermal, ophthalmic, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and / or drops), mucosal, nasal, buccal, sublingual, intratracheal instillation, bronchial instillation, inhalation, as an oral spray, as a nasal spray, and / or as an aerosol. Systemic routes include oral and parenteral. Specifically contemplated routes are oral administration, intravenous administration (e.g., systemic intravenous injection), local administration via blood and / or lymphatic supply, and / or direct administration to the affected area. In some embodiments, the treatment as described by the present disclosure is administered to the subject by intramuscular injection. In general, the most appropriate administration route will depend on various factors, including the nature of the agent (e.g., its stability in the environment of the gastrointestinal tract) and / or the condition of the subject (e.g., whether the subject can tolerate oral administration). In some embodiments, the compounds or pharmaceutical compositions described herein are suitable for topical administration to the eye of a subject. Compositions for various administration routes are well known in the art (see, e.g., Remington's Pharmaceutical Sciences by EW Martin).

[0127] In some embodiments, the treatment for disease or disorder associated with the expression of RAN protein is administered to the central nervous system (CNS) of a subject in need thereof. As used herein, "central nervous system (CNS)" refers to all cells and tissues of the brain and spinal cord of a subject, including but not limited to neurons, glial cells, astrocytes, cerebrospinal fluid, etc. Modalities for administering therapeutic agents to the CNS of a subject include direct injection into the brain (e.g., intracerebral injection, intraventricular injection, intraparenchymal injection, etc.), direct injection into the spinal cord of a subject (e.g., intrathecal injection, lumbar injection, etc.), or any combination thereof.

[0128] In some embodiments, the treatment as described by the present disclosure is administered systemically to the subject, for example, by intravenous injection. The therapeutic molecule that is administered systemically can be modified in some embodiments to improve the delivery of the molecule to the CNS of the subject. Examples of modifications that improve the CNS delivery of therapeutic molecules include, but are not limited to, co-administration or conjugation with blood-brain barrier targeting agents (e.g., transferrin, melanotransferrin, low-density lipoprotein (LDL), angiopeps, RVG peptide, etc., as disclosed by Georgieva et al. Pharmaceuticals 6(4):557-583 (2014)), co-administration with BBB disrupting agents (e.g., bradykinin), and physical disruption of the BBB prior to administration (e.g., by MRI-guided focused ultrasound), etc.

[0129] The dosage will depend on the subject and the route of administration and can be determined by one skilled in the art. Without wishing to be bound by any particular theory, detection (e.g., quantification of anti-RAN protein antibodies) in a biological sample can be used to determine the effectiveness of a therapeutic agent or regimen in the subject from which the sample was obtained.

[0130] kit In some aspects, the present disclosure provides a kit comprising a first container containing one or more target diamino acid repeat peptides and a second container containing one or more detectable reagents. In some embodiments, the one or more anti-RAN antibodies bind to one or more target diamino acid repeat peptides selected from the following: poly(GP) peptides, poly(GR) peptides, poly(PR) peptides, and poly(PA) peptides. In some embodiments, the one or more anti-RAN antibodies bind to one or more target diamino acid repeat peptides selected from the following peptides: poly(CP), poly(GA), poly(GP), poly(PR), poly(GR), poly(PA), poly(A), poly(G), poly(S), poly(C), poly(Q), poly(GD), poly(GE), poly(GQ), poly(GT), poly(L), poly(LP), poly(LPAC (SEQ ID NO: 1), poly(LS), poly(P), poly(QAGR (SEQ ID NO: 2), poly(RE), poly(SP), poly(VP), poly(FP), and / or poly(GK). In some embodiments, the one or more detectable reagents are ruthenium complexes, such as ruthenium(II) tris-bipyridine-(4-methylsulfone) (also [Ru(Bpy)3] +2 In some embodiments, the kit comprises a third container containing a control sample. The control sample may be a negative control sample (e.g., a control sample that does not contain or is devoid of one or more anti-RAN protein antibodies) or a positive control sample (e.g., a control sample that includes one or more anti-RAN protein antibodies, optionally where the amount of one or more anti-RAN protein antibodies in the sample is known).

[0131] example This example describes a method for detecting anti-RAN protein antibodies in a biological sample obtained from a subject. The antibodies detected by the methods described herein can be exogenously administered antibodies (e.g., antibodies administered to a subject as a therapy) or endogenous antibodies (e.g., antibodies produced by cells of the subject).

[0132] Assay Method A schematic diagram depicting one embodiment of an assay for detecting RAN protein in a biological sample is shown in FIG. 1. In brief, a hydrophobic plate is coated with a target peptide (e.g., a target diamino acid repeat peptide such as a RAN peptide) and blocked with bovine serum albumin (BSA). A biological sample (e.g., a lysate or serum obtained from a subject) is added to the plate under conditions in which the antibody in the sample binds to the target peptide. The antibody-peptide complex is labeled with a secondary detection agent (e.g., a detectable secondary antibody). The labeled complex is then detected (e.g., using a plate reader such as a Meso Scale Discovery (MSD) plate reader).

[0133] One embodiment of the method for detecting RAN protein in biological samples includes the following steps: A multi-well assay plate (e.g., Meso Scale Discovery (MSD) assay plate) is coated with 40 μl of solution per well, containing target peptide (1 μg / ml) diluted in 1×PBS. In this example, the target peptides were diamino acid repeat peptides GP8 and GtapA8. After adding the solution, the plate is tapped to ensure that the solution is evenly distributed across the bottom of each well. The plate is then sealed with an adhesive seal and incubated overnight at 4° C. without shaking. The next day, the plate is shaken to remove the solution. The plate is washed (e.g., with 1×TBST) and tapped dry. Then, about 150 μl of blocking solution (e.g., 3% w / v BSA diluted in 1×TBST) is added to each well of the plate. The plate is sealed and incubated with shaking (e.g., at about 600 rpm) at room temperature for 1 hour. The plate is then washed with 1xTBST with shaking and tapped dry. Approximately 25 μl of biological sample (e.g., tissue lysate or serum) is added to each well of the plate, which is then sealed and incubated at room temperature for 2 hours with shaking (e.g., at about 600 rpm). The plate is then washed 3 times with 1xTBST with shaking and tapped dry. Approximately 25 μl of SULFO-TAG detection antibody diluted to 1 μg / ml in 1% BSA in TBST is added to each well of the plate and incubated at room temperature for 2 hours. The plate is then washed 3 times with 1xTBST with shaking and tapped dry. Approximately 150 μl per well of a Tris-based buffer (e.g., 1xMSD Read Buffer T) diluted in water is added to the wells. When adding the buffer, avoid creating bubbles by using reverse pipetting. The plate is then read immediately using a microplate reader.

[0134] Detection of therapeutic anti-RAN protein antibodies in biological samples A subject (e.g., a human, a non-human primate, a dog, a guinea pig, a rat, a mouse, etc.) is administered a therapeutic anti-RAN protein antibody, e.g., an antibody targeting poly(GP), poly(GA), poly(GR), or poly(PR) RAN protein, or a combination thereof. A biological sample, such as a serum sample, is collected after administration. A control subject is administered the delivery vehicle alone. The biological sample is assayed using the methods described in the examples above.

[0135] Detection of endogenous anti-RAN protein antibodies in biological samples A subject (e.g., a human, a non-human primate, a dog, a guinea pig, a rat, a mouse, etc.) is administered a viral vector encoding a therapeutic anti-RAN protein antibody (or a fragment thereof), e.g., an rAAV expressing an antibody targeting poly(GP), poly(GA), poly(GR), or poly(PR) RAN protein, or a combination thereof. A biological sample, such as a serum sample, is collected after administration. A control subject is administered the delivery vehicle alone. The biological sample is assayed using the methods described in the examples above.

[0136] Detection of anti-RAN protein antibodies in biological samples obtained from vaccinated subjects A subject (e.g., a human, a non-human primate, a dog, a guinea pig, a rat, a mouse, etc.) is administered one or more peptide vaccines comprising an antigen having one or more diamino acid repeat peptides (e.g., poly(GP), poly(GA), poly(GR), poly(PR), and combinations thereof). A biological sample, such as a serum sample, is collected after administration. A control subject is administered the delivery vehicle alone. The biological sample is assayed using the methods described in the examples above.

[0137] Detection of anti-RAN protein antibodies in biological samples obtained from mice C9Orf72 ALS mouse models (C9 Bac mice) were administered vaccines targeting one or more diamino acid repeat peptides (e.g., poly(GP), poly(GA), poly(GR), poly(PR), and combinations thereof). Animals were vaccinated 2-3 times prior to collection of biological samples. Control mice received adjuvant alone. Biological samples were assayed using the methods described in the examples above.

[0138] Figure 2 shows representative data for the detection of anti-poly(GP) antibodies in serum obtained from C9 Bac mice treated with an anti-RAN protein vaccine. Assay plates were coated with (GP)8 peptide and mice were treated with the following: GP 10 (SEQ ID NO:5), GA 15 (SEQ ID NO: 4), GA 10 (SEQ ID NO: 4), GR 25 (SEQ ID NO: 7), PR 10 (SEQ ID NO: 6), (GA 15 (SEQ ID NO: 4)+GR 25 (SEQ ID NO: 7)+PR 10 (SEQ ID NO: 6)), (GA 15 (SEQ ID NO: 4)+GR 25 (SEQ ID NO: 7)+PR 10 (SEQ ID NO:6)+GP 10 The data were exposed to serum following exposure to two or three doses of a vaccine targeting the dipeptide repeat (DPR) indicated (SEQ ID NO: 4). Data from adjuvant control mice are also shown.

[0139] Figure 3 shows representative data for the detection of anti-poly(GA) antibodies in serum obtained from C9 Bac mice treated with an anti-RAN protein vaccine. Assay plates were coated with (GA)8 (SEQ ID NO: 4) peptide, and the mice were treated with the following: GP 10 (SEQ ID NO:5), GA 15 (SEQ ID NO: 4), GA 10 (SEQ ID NO: 4), GR 25 (SEQ ID NO: 7), PR 10 (SEQ ID NO: 6), (GA 15 (SEQ ID NO: 4)+GR 25 (SEQ ID NO: 7)+PR10 (SEQ ID NO: 6)), (GA 15 (SEQ ID NO: 4)+GR 25 (SEQ ID NO: 7)+PR 10 (SEQ ID NO:6)+GP 10 The data were exposed to serum following exposure to two or three doses of a vaccine targeting the dipeptide repeat (DPR) indicated (SEQ ID NO: 4). Data from adjuvant control mice are also shown.

[0140] Figure 4 shows representative data for the detection of anti-poly(GA) antibodies in lysates from brain tissue obtained from C9 Bac mice treated with an anti-RAN protein vaccine. Assay plates were coated with (GA)8 (SEQ ID NO: 4) peptide, and the mice were treated with the following: GA 15 , G.A. 10 , (GA 15 (SEQ ID NO: 4)+GR 25 (SEQ ID NO: 7)+PR 10 The data were exposed to serum following exposure to two or three doses of a vaccine targeting the dipeptide repeat (DPR) indicated (SEQ ID NO: 6). Data from adjuvant control mice are also shown.

[0141] Detection of anti-RAN protein antibodies in biological samples obtained from ALS patients Serum samples were obtained from patients with ALS characterized by mutations in C9Orf72. Biological samples were assayed using the methods described in the above examples. The data indicate the presence of increased levels of anti-RAN protein antibodies in patient samples compared to control samples. Figure 5 shows representative data for the detection of anti-poly(GA) antibodies in serum samples obtained from human C9+ALS patients. Assay plates were coated with (GA)8 (SEQ ID NO: 4) peptide and exposed to serum.

Claims

1. 1. A method for detecting one or more anti-RAN protein antibodies using an electrochemiluminescence-based immunoassay, the method comprising: contacting the target diamino acid peptide repeat with a biological sample obtained from the subject, thereby forming an anti-RAN antibody-target peptide complex; contacting the anti-RAN antibody-target peptide complex with an electrochemiluminescent detection agent to form a labeled complex; and determining the level of one or more anti-RAN protein antibodies present in the sample by detecting electrochemiluminescence of the labeled complex; The method comprising:

2. The method of claim 1 , wherein the biological sample is a blood sample, a serum sample, or a tissue sample.

3. The method of claim 2, wherein the tissue sample is a CNS tissue or a cerebrospinal fluid (CSF) sample.

4. The method of any one of claims 1 to 3, wherein the subject is a mammalian subject.

5. The method of claim 4, wherein the subject is a human or a mouse.

6. The method of claim 4 or 5, wherein the subject has been administered a therapeutic anti-RAN protein antibody or a vaccine against a RAN protein-associated disease or disorder.

7. 7. The method of claim 6, wherein the RAN protein-related disease or disorder is amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), myotonic dystrophy type 1 (DM1), myotonic dystrophy type 2 (DM2), spinocerebellar ataxia types 1, 2, 3, 6, 7, 8, 10, 12, 17, 31 and 36, spinal-bulbar muscular atrophy, dentatorubral-pallidoluysian atrophy (DRPLA), Huntington's disease (HD), Alzheimer's disease (AD), fragile X-associated tremor ataxia syndrome (FXTAS), Fuchs endothelial corneal dystrophy (FECD), Huntington's disease-related syndrome type 2 (HDL2), fragile X syndrome (FXS), fragile XE syndrome (FRAXE), or a disorder associated with the folate-sensitive fragile site at 7p11.2 FRA7A or a disorder associated with the folate-sensitive fragile site at 2q11.2 FRA2A.

8. The method of claim 6 or 7, wherein the vaccine elicits an immune response in a subject against the one or more dipeptide repeat proteins.

9. 9. The method of claim 8, wherein the one or more dipeptide repeat proteins are poly(CP), poly(GA), poly(GP), poly(PR), poly(GR), poly(PA), poly(A), poly(G), poly(S), poly(C), poly(Q), poly(GD), poly(GE), poly(GQ), poly(GT), poly(L), poly(LP), poly(LPAC (SEQ ID NO: 1)), poly(LS), poly(P), poly(QAGR (SEQ ID NO: 2)), poly(RE), poly(SP), poly(VP), poly(FP), or poly(GK) repeat proteins, or any combination thereof.

10. The method of any one of claims 1 to 9, wherein the electrochemiluminescent detection agent comprises ruthenium(II) tris-bipyridine-(4-methylsulfone).

11. The method according to any one of claims 1 to 10, wherein electrochemiluminescence is measured by a Meso Scale Detection (MSD) assay.

12. A method according to any one of claims 1 to 11, wherein the subject is further administered a second therapeutic agent.

13. the second biological sample is obtained from the subject following administration of a second therapeutic agent; and The method of claim 12, wherein the one or more anti-RAN protein antibodies are detected in the second biological sample using an electrochemiluminescence-based immunoassay.

14. (i) one or more target diamino acid repeat peptides; and (ii) Immunoassay plates and / or reagents Including the kit.

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