Methods for treating ran protein-associated neurological diseases

Abstract

To provide compositions and methods for the diagnosis and / or treatment of certain neurodegenerative diseases, e.g., diseases associated with repeat-associated non-ATG (RAN) translation proteins, such as Alzheimer's disease (AD).SOLUTION: In some embodiments, the disclosure relates to identifying a subject having a RAN protein-associated disease by detecting expression or activity of repeat-associated non-ATG (RAN) translation proteins (e.g., RAN proteins). In some embodiments, the disclosure relates to methods of treating a RAN protein-associated disease by administering, to a subject in need thereof, an agent that reduces expression or activity of RAN proteins.SELECTED DRAWING: Figure 7

Description

[Technical field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application Serial No. 62 / 871,031, entitled "METHODS FOR TREATING ALZHEIMER'S DISEASE," filed on July 5, 2019, and U.S. Provisional Application Serial No. 63 / 025,096, entitled "METHODS FOR TREATING RAN PROTEIN-ASSOCIATED NEUROLOGICAL DISEASES," filed on May 14, 2020, the entire contents of each of which are incorporated herein by reference. [Background technology]

[0002] background Microsatellite repeat expansions are known to cause over 40 neurodegenerative disorders. Molecular features common to many of these disorders include the accumulation of RNA foci containing sense and antisense expanded transcripts, and protein accumulation from translation of repeat-associated non-AUG (RAN). Translation of RAN can occur over a wide range of repeat lengths, from premutation length (approximately 30-40 repeats) to full expansion (up to 10,000 repeats). Repetitive elements make up a large proportion of the human genome, while detection of repeat and repeat expansion mutations has been challenging. Summary of the Invention

[0003] Abstract Described herein are compositions and methods for the diagnosis and treatment of certain neurological diseases associated with repeat-associated non-ATG (RAN) proteins, including, for example, polyserine [polySer], poly(proline-arginine) [poly(PR)], and poly(glycine-arginine) [poly(GR)]. Mutations in certain repeat expansions (e.g., CAGG, CCTG, GGGGCC, GGCCCC, GGGGCA, CAG and CTG) are associated with a number of different neurological diseases (e.g., amyotrophic lateral sclerosis (ALS), or frontotemporal dementia; myotonic dystrophy type 1 (DM1) and myotonic dystrophy type 2 (DM2); spinocerebellar degeneration 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); fragile X tremor ataxia syndrome (FXTAS); Fuchs endothelial corneal dystrophy (FECD); Huntington's disease type 2 (HDL2); fragile X syndrome (FXS); disorders related to folate-sensitive fragile site 7pl l.2 FRA7A; folate-sensitive fragile site 2ql 1). Disorders related to FRA2A; and associated with Fragile XE syndrome (FRAXE).

[0004] In an increasing number of these diseases, including but not limited to ALS or FTD, FXTAS, HD, SCA8, DM1 and DM2, expansion mutations have been shown to undergo a novel type of protein translation that occurs in multiple reading frames and does not require a canonical AUG start codon.This type of translation is called repeat-associated non-ATG (RAN) translation, and the protein that is produced is called RAN protein.There is increasing evidence that RAN protein is toxic and causes an increasing number of diseases.Therefore, it is important to develop a therapeutic strategy to reduce the level of RAN protein in order to treat neurological diseases caused by repeat expansion mutations.

[0005] In some embodiments, compositions and methods are disclosed for the diagnosis and treatment of certain neurological diseases associated with RAN protein.In some embodiments, the neurological diseases associated with RAN protein are 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 degeneration type 1, 2, 3, 6, 7, 8, 10, 12, 17, 31 and 36; spinobulbar muscular atrophy; dentatorubral-pallidoluysian atrophy (DRPLA); Huntington's disease (HD); fragile X tremor ataxia syndrome (FXTAS); Fuchs endothelial corneal dystrophy (FECD); Huntington's disease type 2 syndrome (HDL2); fragile X syndrome (FXS); 7pl l.2 folate-sensitive fragile site FRA7A-related disorder; folate-sensitive fragile site 2ql 1 FRA2A-related disorder; and fragile XE syndrome (FRAXE). In a particular embodiment, the neurological disease associated with RAN protein is Alzheimer's disease (AD).

[0006] Aspects of the present disclosure relate to methods and compositions for the diagnosis and treatment of certain RAN protein-related diseases, such as Alzheimer's disease and other neurological diseases or disorders.The present disclosure is based, in part, on the discovery that certain RAN proteins, including, for example, polyserine [polySer], poly(proline-arginine) [poly(PR)], and poly(glycine-arginine) [poly(GR)], accumulate in the brains of certain subjects with such diseases, and that these RAN proteins can be detected in biological samples (e.g., blood, serum, or cerebrospinal fluid (CSF)) of subjects at risk of developing AD.Further non-limiting examples of RAN proteins that accumulate in the brains of certain subjects with AD and that can be detected in biological samples from subjects at risk for developing AD include poly(cysteine-proline) [poly(CP)]; poly(glycine-proline) [poly(GP)]; poly(glycine) [poly(G)]; poly(alanine) [polyAla]; poly(glycine-alanine) [poly(GA)]; poly(glycine-aspartic acid) [poly(GD)]; poly(glycine-glutamic acid) [poly(GE)]; poly(glycine-glutamine) [poly(GQ)]; poly(glycine-threonine) [poly(GT)]; poly(leucine) [polyLeu]; poly(leucine-proline) [poly(LP)]; poly(leucine-proline-alanine-cysteine) [poly(LPAC)] (SEQ ID NO: 260); poly(leucine-serine) [poly(LPAC)] (SEQ ID NO: 260); poly(LS)]; poly(proline) [poly(P)]; poly(proline-alanine) [poly(PA)]; poly(glutamine-alanine-glycine-arginine) [poly(QAGR)] (SEQ ID NO: 261); poly(arginine-glutamic acid) [poly(RE)]; poly(serine-proline) [poly(SP)], poly(valine-proline) [poly(VP)], poly(phenylalanine-proline) [poly(FP)], poly(glycine-lysine) poly(GK)], poly(FTPLSLPV) (SEQ ID NO:262), poly(LLPSPSRC) (SEQ ID NO:263), poly(YSPLPPGV) (SEQ ID NO:264), poly(HREGEGSK) (SEQ ID NO:255), poly(TGRERGVN) (SEQ ID NO:265), poly(PGGRGE) (SEQ ID NO:258), poly(GRQRGVNT) (SEQ ID NO:266), and / or poly(GSKHREAE) (SEQ ID NO:267).

[0007] In some embodiments, enzyme-linked immunosorbent assay (ELISA), electrochemiluminescence immunoassay (Meso Scale Discovery, MSD), digital ELISA technology (Single molecule array, SIMOA), and / or dot blot assay are used to detect RAN protein. In some embodiments, rolling circle amplification-based ELISA (RCA-based ELISA) is used to detect RAN protein. In some embodiments, real-time PCR-based ELISA (rtPCR-based ELISA) is used to detect RAN protein. In some embodiments, the assay comprises an antibody against the repeat motif of RAN protein described herein. In some embodiments, the assay comprises an antibody against the C-terminal specific sequence of RAN protein. In some embodiments, an assay is used to detect the expansion mutation. In some embodiments, the assay for detecting the expansion mutation is repeat prime PCR, long range PCR, and / or Southern blot. These assays use primers that bind to DNA sequences in, upstream and / or downstream of the repeat, or adjacent to the repeat.

[0008] In some embodiments, the RAN protein present in a subject having or at risk of developing a neurological disease associated with RAN protein is not transcribed from the C9orf72 locus in the subject. In some embodiments, the accumulation of repeats comprising sense or antisense RNAs comprising repeat expansions may be detected using fluorescent in situ hybridization probes to detect accumulating RNA. In some embodiments, the RNA accumulation present in a subject having or at risk of developing a neurological disease associated with RAN protein is not transcribed from the C9orf72 locus in the subject. In some embodiments, the gene that produces the novel RAN protein includes chromosome 2 open reading frame 80 (C2orf80), LRP8, CASP8, CRNDE, EXOC6B, SV2B, PPML1, ADARB2, GREB1, and / or MSMO1. In some embodiments, the assay is used to determine whether the RAN protein is expressed from one or more of C2orf80, LRP8, CASP8, CRNDE, EXOC6B, SV2B, PPML1, ADARB2, GREB1 and MSMO1. In some embodiments, the assay for identifying RNA aggregates is fluorescent in situ hybridization; probe = DNA sequence complementary to the repeat sequence in the extended locus and labeled with a fluorophore (e.g., Cy3, Cy5, A555, A549, A488). In some embodiments, the assay for identifying RNA aggregates is inactivated Cas9-based hybridization; probe = DNA sequence complementary to the repeat sequence in the extended locus and fluorophore-labeled inactivated Cas9.

[0009] In some aspects, the present disclosure provides a method for treating a RAN protein-associated neurological disease by administering a therapeutic agent (e.g., one or more antisense oligonucleotides, anti-RAN antibodies, other therapeutic agents, or a combination of two or more of these) to a subject diagnosed as having or at risk for a neurological disease associated with a RAN protein for the treatment of the neurological disease associated with a RAN protein, wherein the subject is characterized as having a neurological disease associated with a RAN protein by detection of at least one RAN protein and / or at least one RNA encoding a RAN protein in a biological sample obtained from the subject. The subject may have more than one of the RAN proteins expressed from an expansion mutation. In some embodiments, the antibodies targeting the RAN protein disclosed herein may target one or more RAN proteins. In some embodiments, an individual antibody may target one or more RAN proteins. In some embodiments, a combination of two or more different antibodies may be used, where each antibody targets a different RAN protein. In some embodiments, the RAN protein is expressed in all three reading frames from both the sense and antisense transcripts.

[0010] In some embodiments, the RAN protein is poly(GR), poly(PR) and / or polySer. In some embodiments, the RAN protein is poly(CP), poly(GP), poly(G), poly(A), poly(GA), poly(GD), poly(GE), poly(GQ), poly(GT), poly(L), poly(LP), poly(LPAC) (SEQ ID NO: 260), poly(LS), poly(P), poly(PA), poly(QAGR) (SEQ ID NO: 261), poly(RE), poly(SP), poly(VP), poly(FP), poly(GK), poly(GT ... ), poly(FTPLSLPV) (SEQ ID NO: 262), poly(LLPSPSRC) (SEQ ID NO: 263), poly(YSPLPPGV) (SEQ ID NO: 264), poly(HREGEGSK) (SEQ ID NO: 255), poly(TGRERGVN) (SEQ ID NO: 265), poly(PGGRGE) (SEQ ID NO: 258), poly(GRQRGVNT) (SEQ ID NO: 266), and / or poly(GSKHREAE) (SEQ ID NO: 267). In some embodiments, the RAN protein is not transcribed from the C9orf72 locus of the subject.

[0011] In some embodiments, at least one RAN protein is encoded by a gene that contains between 2 and 10,000 repeats of a sequence selected from Table 1, Table 2 or Table 3. In some embodiments, the therapeutic agent is a small molecule, an interfering nucleic acid, a modified interfering nucleic acid, a DNA aptamer, an RNA aptamer, a peptide, a protein, an antibody, an antibody drug conjugate, another large molecule, gene therapy (including gene therapy designed to deliver one or more of the other listed types of therapeutic agents), a natural product, or a herbal medicine.

[0012] In some embodiments, the small molecule is a eukaryotic initiation factor 2 (eIF2), eukaryotic initiation factor 3 (eIF3), protein kinase R (PKR), p62 (sequestosome-1 or ubiquitin-binding protein), LC3 (microtubule-associated protein 1 light chain 3) I subunit, LC3 II subunit, or a modulator of Toll-like receptor 3 (TLR3).

[0013] In some embodiments, the small molecule is metformin, or its pharma- ceutically acceptable salt, cocrystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative or prodrug.In some embodiments, the small molecule is buformin, phenformin, metformin, or its derivative or functional analog.In some embodiments, the small molecule is an inhibitor of PKR, such as TARBP2.

[0014] In some embodiments, the interfering nucleic acid is dsRNA, siRNA, shRNA, miRNA, artificial miRNA (amiRNA) or antisense oligonucleotide (ASO).In some embodiments, the interfering nucleic acid modifies the expression of eukaryotic initiation factor 2 (eIF2), eukaryotic initiation factor 3 (eIF3), protein kinase R (PKR), p62, LC3 I subunit, LC3 II subunit or Toll-like receptor 3 (TLR3).

[0015] In some embodiments, the interfering nucleic acid modulates the expression of eIF2A or eIF2α. In some embodiments, the interfering nucleic acid inhibits the expression of one or more eIF3 subunits selected from the group consisting of eIF3a, eIF3b, eIF3c, eIF3d, eIF3e, eIF3f, eIF3g, eIF3h, eIF3i, eIF3j, eIF3k, eIF3l and eIF3m. In some embodiments, the interfering nucleic acid inhibits the expression of protein kinase R (PKR). In some embodiments, the interfering nucleic acid inhibits the expression of a gene comprising a nucleic acid repeat as described in any one of Tables 1, 2 and 3. In some embodiments, the interfering nucleic acid directly binds to a repeat sequence as described in any one of Tables 1, 2 and 3 (e.g., a microsatellite extension comprising any one of the sequences as described in Tables 1, 2 and 3).

[0016] In some embodiments, the protein (e.g., a therapeutic protein) modifies eukaryotic initiation factor 2 (eIF2), eukaryotic initiation factor 3 (eIF3), protein kinase R (PKR), p62, LC3 I subunit, LC3 II subunit, or Toll-like receptor 3 (TLR3).

[0017] In some embodiments, the protein (e.g., a therapeutic protein) is a dominant negative variant of protein kinase R (PKR) or a dominant negative variant of TLR3 protein. In some embodiments, the dominant negative variant comprises a mutation at amino acid position 296. In some embodiments, the mutation is K296R.

[0018] In some embodiments, the therapeutic agent (e.g., nucleic acid encoding a therapeutic protein, interfering nucleic acid, etc.) is delivered to the subject by a vector. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a recombinant adeno-associated virus (rAAV). In some embodiments, the rAAV comprises AAV8 capsid protein or a variant thereof.

[0019] In some embodiments, the therapeutic protein is an anti-RAN protein vaccine. In some embodiments, the anti-RAN protein vaccine is a poly(proline-arginine) [poly(PR)]; poly(glycine-arginine) [poly(GR)]; poly(serine) [polySer]; poly(cysteine-proline) [poly(CP)]; poly(glycine-proline) [poly(GP)]; poly(glycine) [poly(G)]; poly(alanine) [polyAla]; poly(glycine-alanine) [poly(GA)]; poly(glycine-Ala) poly(glycine-glutamic acid) [poly(GE)]; poly(glycine-glutamine) [poly(GQ)]; poly(glycine-threonine) [poly(GT)]; poly(leucine) [poly(Leu)]; poly(leucine-proline) [poly(LP)]; poly(leucine-proline-alanine-cysteine) [poly(LPAC)] (SEQ ID NO: 260); poly(leucine-serine) [poly(LS)]; poly(proline) [ poly(P)]; poly(proline-alanine) [poly(PA)]; poly(glutamine-alanine-glycine-arginine) [poly(QAGR)] (SEQ ID NO: 261); poly(arginine-glutamic acid) [poly(RE)]; poly(serine-proline) [poly(SP)], poly(valine-proline) [poly(VP)], poly(phenylalanine-proline) [poly(FP)], poly(glycine-lysine) [poly(GK)], poly(FTPLSL The peptide antigen comprises an amino acid repeat sequence selected from poly(PV) (SEQ ID NO:262), poly(LLPSPSRC) (SEQ ID NO:263), poly(YSPLPPGV) (SEQ ID NO:264), poly(HREGEGSK) (SEQ ID NO:255), poly(TGRERGVN) (SEQ ID NO:265), poly(PGGRGE) (SEQ ID NO:258), poly(GRQRGVNT) (SEQ ID NO:266), and poly(GSKHREAE) (SEQ ID NO:267).

[0020] In some embodiments, the therapeutic protein is an antibody. In some embodiments, the antibody targets eukaryotic initiation factor 2 (eIF2), eukaryotic initiation factor 3 (eIF3), protein kinase R (PKR), p62, LC3 I subunit, LC3 II subunit, or Toll-like receptor 3 (TLR3). Such antibodies are known in the art (see, for example, Duffy et al. Cell Immunol. 2007 Aug;248(2):103-14. PubMed PMID: 18048020). Those skilled in the art will understand how to bind the antibody to the listed protein targets and how to screen for the desired modification of the function of the target protein.

[0021] In some embodiments, the antibody is an anti-RAN protein antibody. In some embodiments, the anti-RAN protein antibody targets any one or more of the following: poly(proline-arginine) [poly(PR)]; poly(glycine-arginine) [poly(GR)]; poly(serine) [polySer]; poly(cysteine-proline) [poly(CP)]; poly(glycine-proline) [poly(GP)]; poly(glycine) [poly(G)]; poly(alanine) [polyAla]; poly(glycine-alanine) [poly(GA)]; poly(glycine-aspartic acid) [poly(GD)]; poly(glycine-G poly(glutamic acid) [poly(GE)]; poly(glycine-glutamine) [poly(GQ)]; poly(glycine-threonine) [poly(GT)]; poly(leucine) [poly(Leu)]; poly(leucine-proline) [poly(LP)]; poly(leucine-proline-alanine-cysteine) [poly(LPAC)] (SEQ ID NO: 260); poly(leucine-serine) [poly(LS)]; poly(proline) [poly(P)]; poly(proline-alanine) [poly(PA)]; poly(glutamine-alanine-glycine-arginine) [poly(QAGR)] (SEQ ID NO: 261); Column number 261; poly(arginine-glutamic acid) [poly(RE)]; poly(serine-proline) [poly(SP)], poly(valine-proline) [poly(VP)], poly(phenylalanine-proline) [poly(FP)], poly(glycine-lysine) [poly(GK)], poly(FTPLSLPV) (SEQ ID NO: 262), poly(LLPSPSRC) (SEQ ID NO: 263), poly(YSPLPPGV) (SEQ ID NO: 264), poly(HREGEGSK) (SEQ ID NO: 255), poly(TGRERGVN) (SEQ ID NO: 265), poly(PGG Poly(RGE) (SEQ ID NO: 258), poly(GRQRGVNT) (SEQ ID NO: 266), and poly(GSKHREAE) (SEQ ID NO: 267). In some embodiments, the anti-RAN protein antibody specifically binds to a poly-amino acid repeat of the RAN protein. In some embodiments, the anti-RAN protein antibody specifically binds to the C-terminus of the RAN protein. In some embodiments, the anti-RAN protein antibody is a monoclonal antibody. In some embodiments, the anti-RAN protein antibody is a polyclonal antibody.In some embodiments, anti-RAN antibodies are generated that have binding activity to newly identified RAN proteins that occur in neurological diseases associated with RAN proteins (i.e., AD) as predicted by the sequence of novel enriched repeat expansion mutations. In some embodiments, the locus contains known risk factors for neurological diseases associated with RAN proteins that have now been identified as containing novel repeat expansion mutations that can generate one or more types of RAN proteins.

[0022] In some embodiments, the locus comprises the LRP8 gene and / or the CASP8 gene. In some embodiments, the LRP8 gene repeat expansion motif comprises a (sense-antisense) GGGGCA-TGCCCC (SEQ ID NO: 1) repeat motif, which encodes a protein containing proline-arginine (PR), glycine-arginine (GR), glycine-aspartic acid (GD), glycine-threonine (GT), valine-proline (VP) and serine-proline (SP) dipeptide repeat motifs from the sense and antisense transcripts. In some embodiments, the repeat expansion mutation in the CASP8 locus contains a GAGAGG·CCTCTC (SEQ ID NO:2) repeat motif, which can generate novel RAN proteins containing glycine-arginine (GR), glycine-glutamic acid (GE), arginine-glutamic acid (RE), serine-proline (SP), leucine-proline (LP) and leucine-serine (LS) dipeptide repeat motifs from sense and antisense transcripts. In some embodiments, the repeat expansion mutation in the C2orf80 locus contains a GAGAGG repeat motif, which can generate novel RAN proteins containing glycine-arginine (GR), glycine-glutamic acid (GE), arginine-glutamic acid (RE), serine-proline (SP), leucine-proline (LP) and leucine-serine (LS) dipeptide repeat motifs.

[0023] In some embodiments, the locus comprises the GREB1 gene. In some embodiments, the repeat expansion mutation in the GREB1 locus comprises a GGGGCA repeat motif, which can generate novel RAN proteins containing glycine-arginine (GR), glycine-alanine (GA), glycine-glutamine (GQ), proline-alanine (PA), leucine-proline (LP) and cysteine-proline (CP) dipeptide repeat motifs from sense and antisense transcripts.

[0024] In some embodiments, the methods described by the present disclosure further include administering a second therapeutic agent to the subject (e.g., a therapeutic agent approved by the FDA for the treatment of Alzheimer's disease). In some embodiments, the second therapeutic agent is selected from donepezil, galantamine, memantine, rivastigmine, or a combination thereof. In some embodiments, the biological sample is blood, serum, or cerebrospinal fluid (CSF).

[0025] In some embodiments, the detection of one or more RAN proteins comprises performing a binding assay (e.g., an antibody-based binding assay), a hybridization assay, an immunoblot analysis, a Western blot analysis, immunohistochemistry, and / or an ELISA (e.g., an RCA-based ELISA, an rtPCR-based ELISA, etc.). In some embodiments, the hybridization assay comprises contacting the sample with one or more detectable nucleic acid probes (e.g., a detectable nucleic acid probe that specifically binds to a sequence that encodes a RAN protein). In some embodiments, the hybridization assay comprises Fluorescence In Situ Hybridization (FISH) and / or dCas9-based enrichment. In some embodiments, the detection of RAN proteins further comprises nucleic acid sequencing, e.g., next-generation sequencing (NGS), which may or may not include performing an enrichment step (e.g., dCas9-based enrichment) on the sample.

[0026] In some embodiments, the detection of one or more RAN proteins comprises next-generation sequencing (NGS), which may or may not comprise performing an enrichment step (e.g., dCas9-based enrichment) on the sample using guide RNA. In some embodiments, the guide RNA used in enrichment targets the repeat containing NGG protospacer adjacent motif (PAM). In some embodiments, the guide RNA used in enrichment targets non-NGG PAM-containing repeat. In some embodiments, the non-NGG PAM-containing repeat comprises CAG and CTG expansion repeat (e.g., GGGGCC in ALS / FTD and CCTG in DM2). In some embodiments, the guide RNA used in enrichment enriches for non-NGG PAM-containing repeat expansions that are longer than the corresponding normal allele (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100 repeats longer). In some embodiments, the guide RNA used in enrichment simultaneously identifies multiple repeat expansions (including sequences with non-NGG PAMs in some embodiments).

[0027] In some embodiments, dCas9-based enrichment is performed using a Streptococcus pyogenes-derived dCas9 (spdCas9) molecule. In some embodiments, dCas9-based enrichment is performed on Staphylococcus aureus, Streptococcus pyogenes, Campylobacter jejuni, Corynebacterium diphtheria, Eubacterium ventriosum, Streptococcus pasteurianus, Lactobacillus farciminis, Sphaerochaeta globus, Azospirillum (e.g., strain B510), Gluconacetobacter diazotrophicus, Neisseria cinerea-, Roseburia intestinalis, Parvibaculum lavamentivorans, Nitratifractor salsuginis (e.g., strain DSM 16511), Campylobacter lari (e.g., strain CF89-12), or Streptococcus In yet other embodiments, the dCas9 molecule is a mutant of a wild-type Cas9 molecule, e.g., one whose Cas9 nuclease activity is inactivated. In some embodiments, the mutant Cas9 molecule comprises a mutation that inactivates Cas9 nuclease activity, e.g., a mutation in the DNA cleavage domain of the Cas9 molecule. In some embodiments, the mutant Cas9 molecule comprises a mutation that inactivates Cas9 nuclease activity, e.g., a mutation in the RuvC domain and / or a mutation in the HNH domain.

[0028] In some aspects, the present disclosure provides a method for diagnosing a RAN protein-associated disease by detecting at least one RAN protein in a sample obtained from a subject; determining that at least one RAN protein is not transcribed from the C9orf72 locus of the subject; and diagnosing the subject as having a RAN protein-associated disease based on the presence of at least one RAN protein that is not transcribed from the C9orf72 locus. In some embodiments, the method includes determining the presence of a repeat expansion in one or more specific loci. In some embodiments, determining the presence of a repeat expansion in a specific locus is performed using repeat prime PCR, long range PCR, and / or Southern blot, and primers specific for each locus. In some aspects, the present disclosure provides a method for aiding in the diagnosis of a RAN protein-associated disease by performing an assay on a biological sample obtained from a subject to determine whether a RAN protein is present in the biological sample; and identifying the subject as at risk for a RAN protein-associated disease if a RAN protein is present in the biological sample.

[0029] In some embodiments, the sample is central nervous system (CNS) tissue, blood, or cerebrospinal fluid (CSF). In some embodiments, detecting comprises contacting (e.g., incubating) the sample with an anti-RAN antibody. In some embodiments, the anti-RAN protein antibody targets any one or more of the following: poly(proline-arginine) [poly(PR)]; poly(glycine-arginine) [poly(GR)]; poly(serine) [polySer]; poly(cysteine-proline) [poly(CP)]; poly(glycine-proline) [poly(GP)]; poly(glycine) [poly(G)]; poly(alanine) [polyAla]; poly(glycine-alanine) [poly(GA)]; poly(glycine-aspartate) [poly(G)]; poly(alanine) [poly(Ala)]; poly(glycine-alanine) [poly(GA)]; poly(glycine-aspartate) [poly(G)]; poly(alanine) [poly(G ... poly(glycine-glutamic acid) [poly(GE)]; poly(glycine-glutamine) [poly(GQ)]; poly(glycine-threonine) [poly(GT)]; poly(leucine) [poly(Leu)]; poly(leucine-proline) [poly(LP)]; poly(leucine-proline-alanine-cysteine) [poly(LPAC)] (SEQ ID NO: 260); poly(leucine-serine) [poly(LS)]; poly(proline) [poly(P)]; poly(proline-alanine) poly(glutamine-alanine-glycine-arginine) [poly(QAGR)] (SEQ ID NO: 261); poly(arginine-glutamic acid) [poly(RE)]; poly(serine-proline) [poly(SP)], poly(valine-proline) [poly(VP)], poly(phenylalanine-proline) [poly(FP)], poly(glycine-lysine) [poly(GK)], poly(FTPLSLPV) (SEQ ID NO: 262), poly(LLPSPSRC) (SEQ ID NO: 263), poly(PGK) In some embodiments, the detecting comprises performing dCas9-based enrichment on the sample. In some embodiments, the detecting further comprises nucleic acid sequencing.In some embodiments, the sequencing is next generation sequencing (NGS).

[0030] In some aspects, the disclosure provides a method for increasing proteasome activity in a cell, the method comprising administering an anti-RAN protein antibody in an amount sufficient to reduce RAN protein aggregation in the cell. In some embodiments, the anti-RAN protein antibody targets one or more of the following: RAN protein poly(proline-arginine) [poly(PR)]; poly(glycine-arginine) [poly(GR)]; poly(serine) [polySer]; poly(cysteine-proline) [poly(CP)]; poly(glycine-proline) [(poly(GP)]; poly(glycine) [poly(G)]; poly(alanine) [polyAla]; poly(glycine-alanine) [poly(GA)]; poly(glycine-aspartic acid) [poly(GD)]; poly(glycine-glutamic acid) [poly(GE)]; poly(glycine-glutamine) [poly(GQ)]; poly(glycine-threonine) [poly(GT)]; poly(leucine) [polyLeu]; poly(leucine-proline) [poly(LP)]; poly(leucine-proline-alanine-cysteine) [poly(LPAC)] (SEQ ID NO: 260); poly(leucine) poly(serine-serine) [poly(LS)]; poly(proline) [poly(P)]; poly(proline-alanine) [poly(PA)]; poly(glutamine-alanine-glycine-arginine) [poly(QAGR)] (SEQ ID NO: 261); poly(arginine-glutamic acid) [poly(RE)]; poly(serine-proline) [poly(SP)], poly(valine-proline) [poly(VP)], poly(phenylalanine-proline) [poly(FP)], poly( glycine-lysine) [poly(GK)], poly(FTPLSLPV) (SEQ ID NO:262), poly(LLPSPSRC) (SEQ ID NO:263), poly(YSPLPPGV) (SEQ ID NO:264), poly(HREGEGSK) (SEQ ID NO:255), poly(TGRERGVN) (SEQ ID NO:265), poly(PGGRGE) (SEQ ID NO:258), poly(GRQRGVNT) (SEQ ID NO:266), and poly(GSKHREAE) (SEQ ID NO:267).

[0031] In some aspects, the disclosure provides a method for vaccinating a subject against a RAN protein disease, the method comprising administering to the subject a peptide antigen targeting one or more RAN proteins. In some embodiments, the peptide antigen is a RAN protein: poly(proline-arginine) [poly(PR)]; poly(glycine-arginine) [poly(GR)]; poly(serine) [polySer]; poly(cysteine-proline) [poly(CP)]; poly(glycine-proline) [poly(GP)]; poly(glycine) [poly(G)]; poly(alanine) [polyAla]; poly(glycine-alanine) [poly(GA)]; poly(glycine-Ala ... poly(glycine-glutamic acid) [poly(GE)]; poly(glycine-glutamine) [poly(GQ)]; poly(glycine-threonine) [poly(GT)]; poly(leucine) [poly(Leu)]; poly(leucine-proline) [poly(LP)]; poly(leucine-proline-alanine-cysteine) [poly(LPAC)] (SEQ ID NO: 260); poly(leucine-serine) [poly(LS)]; poly(proline) [poly(Poly(P))] poly(arginine-glutamic acid) [poly(RE)]; poly(serine-proline) [poly(SP)], poly(valine-proline) [poly(VP)], poly(phenylalanine-proline) [poly(FP)], poly(glycine-lysine) [poly(GK)], poly(FTPLSLPV). (SEQ ID NO:262), poly(LLPSPSRC) (SEQ ID NO:263), poly(YSPLPPGV) (SEQ ID NO:264), poly(HREGEGSK) (SEQ ID NO:255), poly(TGRERGVN) (SEQ ID NO:265), poly(PGGRGE) (SEQ ID NO:258), poly(GRQRGVNT) (SEQ ID NO:266), and poly(GSKHREAE) (SEQ ID NO:267).

[0032] In some embodiments, the cell is a mammalian cell (e.g., a human cell, a mouse cell, a rat cell, a cat cell, a dog cell, a guinea pig cell, a pig cell, a monkey cell, etc.). In some embodiments, the cell is a neuronal cell, an astrocyte cell, or a glial cell. In some embodiments, the cell comprises a gene having a nucleic acid sequence comprising at least 35 repeats of a sequence set forth in any one of Tables 1, 2, and 3. In some embodiments, the anti-RAN protein antibody is a monoclonal antibody.

[0033] In some embodiments, administration of anti-RAN protein antibody results in increased proteasome activity in cells compared to the proteasome activity in cells before administration.In some embodiments, increased proteasome activity is indicated by a decrease in the level or content or activity of P62 subunit in cells.In some embodiments, increased proteasome activity can be detected by a diffuse signal compared to the typically punctate signal of proteasome subunits sequestered by RAN protein (e.g., by poly(GA)).In some embodiments, increased proteasome activity can be measured in protein lysate or in cells using a fluorescence-based method.In some embodiments, after administration of anti-RAN antibody, improvement of disease-mediated dysregulation of extracellular proteasome system can be measured.

[0034] Also disclosed herein are antibodies and / or antigen-binding fragments that specifically bind to any one or more of the following: polySer, poly(PR), poly(GR), poly(CP), poly(GP); poly(G), poly(A), poly(GA), poly(GD), poly(GE), poly(GQ), poly(GT), poly(L), poly(LP), poly(LPAC) (SEQ ID NO: 260), poly(LS), poly(P), poly(PA), poly(QAGR) (SEQ ID NO: 261), poly(A), poly(A), poly(B), poly(C), poly(D), poly(D), poly(E), poly(E), poly(F ... (RE), poly(SP), poly(VP), poly(FP), poly(GK), poly(FTPLSLPV) (SEQ ID NO: 262), poly(LLPSPSRC) (SEQ ID NO: 263), poly(YSPLPPGV) (SEQ ID NO: 264), poly(HREGEGSK) (SEQ ID NO: 255), poly(TGRERGVN) (SEQ ID NO: 265), poly(PGGRGE) (SEQ ID NO: 258), poly(GRQRGVNT) (SEQ ID NO: 266), and / or poly(GSKHREAE) (SEQ ID NO: 267). For example, an antibody or antibody fragment may bind to only one type of RAN protein (e.g., polySer or polyGA), or an antibody or antibody fragment may bind to multiple RAN proteins (e.g., with different affinities). In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to a RAN protein, and the antibody or antigen-binding fragment thereof, comprises a heavy chain variable region (VH) comprising: (i) a CDR1 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 117, 119, 121, and 123; (ii) a CDR2 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 125, 127, 129, and 131; and / or (iii) a CDR3 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 133, 135, 137, and 139.

[0035] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to a RAN protein, and the antibody or antigen-binding fragment thereof, comprises a light chain variable region (VL) comprising: (i) a CDR1 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 118, 120, 122, and 124; (ii) a CDR2 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 126, 128, 130, and 132; and / or (iii) a CDR3 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 134, 136, 138, and 140.

[0036] In some embodiments, the antibody or antigen-binding fragment comprises a variable heavy chain amino acid sequence set forth in SEQ ID NO: 109, 111, 113 or 115. In some embodiments, the antibody or antigen-binding fragment comprises a variable light chain amino acid sequence set forth in SEQ ID NO: 110, 112, 114 or 116. In some embodiments, the antibody binds to polyGA. In some embodiments, the antibody binds to polySer. In some embodiments, the antibody binds to polyPR.

[0037] Also disclosed herein is a composition comprising an antibody or antigen-binding fragment disclosed herein and a pharma- ceutically acceptable carrier and / or a pharma-ceutically acceptable buffer. In some embodiments, the composition is for use in treating a repeat expansion disease. In some embodiments, the composition is for use in treating a repeat expansion disease 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 degeneration 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); fragile X tremor ataxia syndrome (FXTAS); Fuchs endothelial corneal dystrophy (FECD); Huntington's disease type 2 syndrome (HDL2); fragile X syndrome (FXS); disorders related to 7pl l.2 folate-sensitive fragile site FRA7A; disorders related to folate-sensitive fragile site 2ql 1 FRA2A; and fragile XE syndrome (FRAXE). In some embodiments, the composition is for use in treating Alzheimer's disease.

[0038] Further disclosed herein is an isolated nucleic acid molecule encoding the antibody or antigen-binding fragment disclosed herein. Also provided is a cell transformed with the disclosed nucleic acid. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell.

[0039] Also provided herein is a method for treating RAN protein-associated disease in a subject.In some embodiments, the method comprises administering any one of the antibodies disclosed herein or a combination of two or more antibodies disclosed herein (e.g., 1, 2, 3, 4, 5, 6, 5-10, 10-15 or more antibodies), wherein the subject is characterized as having RAN protein-associated disease by detecting at least one RAN protein in a biological sample obtained from the subject. [Brief description of the drawings]

[0040] [Figure 1A-B] Figures 1A-1C show screening for repeat expansions and RNAi aggregates associated with Alzheimer's disease (AD). Figure 1A shows a schematic diagram depicting analysis of samples for RAN protein expression using an anti-RAN protein antibody, RNA aggregate screening, and dCas9 pull-down enrichment. Figure 1B shows a schematic diagram depicting dCas9 repeat expansion enrichment. [Figure 1C] Figures 1A-1C show a screen for repeat expansions and RNAi aggregates associated with Alzheimer's disease (AD). Figure 1C shows data validating enrichment of C9orf72 G4C2 repeats using sgRNA-dCas9 complexes.

[0041] [Figure 2A] 2A-2C show the screening data of poly(GR) and poly(PR) proteins. FIG. 2A shows the data of dot blot screening for RAN-protein positive samples. Anti-poly(GR) antibody was used for screening. The data of blot screening is shown. Anti-poly(Ser) antibody was used for screening. [Figure 2B] Figures 2A-2C show the screening data for poly(GR) and poly(PR) proteins. Figure 2B shows the dot blot screening data for RAN-protein positive samples. Anti-poly(Ser) antibody was used for screening. [Figure 2C] Figures 2A-2C show the screening data for poly(GR) and poly(PR) proteins. Figure 2C shows histological staining of samples for RAN proteins (poly(GR) and poly(PR) proteins) and phosphorylated TDP-43 for RAN-positive (top), RAN-negative (bottom), and healthy control brain tissue (middle).

[0042] [Figure 3A] Figures 3A-3C show immunofluorescence data indicating that RAN protein localization can be distinguished from typical AD proteins such as 3R tau. Figure 3A shows staining for poly(PR) and poly(GR) that can be distinguished from 3R tau. [Figure 3B] Figures 3A-3C show immunofluorescence data indicating that RAN protein localization can be distinguished from typical AD proteins such as 3R tau, and Figure 3B shows that anti-poly(PR) and anti-poly(GR) antibodies do not cross-react with 3R tau antibodies. [Figure 3C] Figures 3A-3C show immunofluorescence data indicating that RAN protein localization can be distinguished from typical AD proteins such as 3R tau. Figure 3C shows positive control cells expressing the GR60 construct (left) or the PR60 construct (right).

[0043] [Figure 4] Figure 4 shows Fluorescence In Situ Hybridization (FISH) screening of cells with a GC-rich DNA probe. Staining of RNA aggregates was present in AD cases characterized by RAN protein translation (top) but was absent in RAN-negative cases (bottom).

[0044] [Figure 5A] Figures 5A-5B show the effect of RAN protein translation on cellular proteasomes and autophagy. Figure 5A shows sequestration of LC3B and 26S subunits by poly(GA)RAN protein in cells transformed with GFP-GA60. [Figure 5B] Figures 5A-5B show the effect of RAN protein translation on cellular proteasomes and autophagy. Figure 5B shows that reduced proteasome activity in poly(GA)RAN protein-expressing cells is rescued by treatment with anti-poly(GA) antibodies.

[0045] [Figure 6] FIG. 6 shows a schematic diagram of the molecular pathways regulating autophagy that are affected by the expression of RAN protein.

[0046] [Figure 7] Figure 7 shows dCas9-based enrichment and detection of repeat expansions (dCas9READ). Preferential binding of the sgRNA-dCas9 complex to repeat expansions allows enrichment and identification of repeats and unique adjacent sequences.

[0047] [Figure 8A] Figures 8A-8D show enrichment of C9ALS / FTD and DM2 extensions using dCas9READ. Figure 8A shows qPCR showing enrichment of G4C2 CCTG using dCas9READ. Mean + / - SEM, unpaired two-tailed t-test, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 8B] Figures 8A-8D show enrichment of C9ALS / FTD and DM2 expansions using dCas9READ. Figure 8B shows qPCR showing enrichment of G4C2 expansion mutations using dCas9READ. Mean + / - SEM, unpaired two-tailed t-test, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 8C] Figures 8A-8D show enrichment of C9ALS / FTD and DM2 extensions using dCas9READ. Figure 8C shows identification of flanking sequences mapping to the C9orf72 and CNBP (DM2) loci. [Figure 8D]Figures 8A-8D show enrichment of C9ALS / FTD and DM2 extensions using dCas9READ. Figure 8D shows total reads showing enrichment of C9orf72 and CNBP from patient versus control DNA. Means + / - SEM, unpaired two-tailed t-test, **p<0.01, ***p<0.001, ****p<0.0001.

[0048] [Figure 9A-B] Figures 9A-9G show positive RAN protein signals in AD. Figure 9A shows the antibodies used to screen for RAN protein in AD. Figure 9B shows an example of a-GR screening dot blots and quantification of a-GR signals (unpaired two-tailed t-test). Mean values ​​+ / - SEM, **p<0.01, ***p<0.001. [Figure 9C] Figures 9A-9G show positive RAN protein signals in AD. Figure 9C shows representative positive IHC staining of GR and PR compared to p-tau, Aβ and pTDP43 in the CA1 region of an AD autopsy brain. [Fig. 9D-E] Figures 9A-9G show positive RAN protein signals in AD. Figure 9D shows quantification of PR signals in late-onset AD cases and controls (one-way ANOVA with Tukey analysis for multiple comparisons). Figure 9E shows staining of α-GR and α-PR in T98 cells expressing GR60 and PR60 proteins. Mean values ​​+ / - SEM, **p<0.01, ***p<0.001. [Figure 9F] Figures 9A-9G show positive RAN protein signals in AD, and Figure 9F shows α-PR staining in cells expressing 3-repeat tau protein with or without PR60 or GR60. [Figure 9G] Figures 9A-9G show positive RAN protein signals in AD. Figure 9G shows α-GR staining in cells expressing 3-repeat tau protein with or without PR60 or GR60.

[0049] [Figure 10A]Figures 10A-C show RNA aggregates and accumulation detected in AD cases. Figure 10A shows RNA aggregates detected by DNA probes for C4G2 and C4GT in AD, and quantification of aggregates in AD samples with positive dot blot signals for α-GR, α-GA, or α-GP. Data represent mean + / - SEM, *<0.05, **p<0.01. [Figure 10B] Figure 10A-C shows RNA aggregates and accumulation detected in AD cases. Figure 10B shows dsRNA signals in the dentate gyrus (DG) region in postmortem tissues of AD and quantification comparing cognitive healthy controls, SCA controls and AD cases (one-way ANOVA with Turkey analysis for multiple comparisons). Data represent mean + / - SEM, *<0.05, **p<0.01. [Figure 10C] Figures 10A-C show RNA aggregates and accumulation detected in AD cases. Figure 10C shows dsRNA staining in a control experiment in which tissue was treated with RNAse A (unpaired two-tailed t-test). Data represent mean + / - SEM, *<0.05, **p<0.01.

[0050] [Figure 11] Figure 11 shows the Pathology-to-Genetics strategy. Patient tissues will be screened with α-RAN antibodies. RAN repeat motifs will be used to determine possible repeat motifs for RNA aggregate screening and sgRNAs for repeat identification using dCas9READ. Novel antibodies against RAN repeats and the corresponding unique C-terminal region will be used to confirm putative expansion mutations and test pathology.

[0051] [Figure 12]Figures 12A-12B show expanded loci of CASP8 and ADARB2. Figure 12A shows the CASP8 repeat sequence confirmed by Sanger sequencing of long-range PCR products. Figure 12B shows expanded or normal alleles at the ADARB2 locus in LOAD cases and non-AD controls. Each lane represents an individual AD patient or control sample. Yellow asterisks indicate the size of the allele as reported in the reference genome. Red asterisks indicate expanded alleles.

[0052] [Figure 13A] Figures 13A-13E show immunofluorescence data validating the generated anti-RAN antibodies in transfected cells expressing recombinant proteins. Figure 13A shows the validation of anti-polyER antibodies. HEK293T cells were transfected with either 3xFlag-(ER)30 or control plasmid. [Figure 13B] Figures 13A-13E show immunofluorescence data validating the generated anti-RAN antibodies in transfected cells expressing recombinant proteins. Figure 13B shows validation of anti-polyEG antibodies. HEK293T cells were transfected with either 3xFlag-(EG)30 or control plasmid. [Figure 13C] Figures 13A-13E show immunofluorescence data validating the generated anti-RAN antibodies in transfected cells expressing recombinant proteins. Figure 13C shows validation of anti-polyLS antibodies. HEK293T cells were transfected with either 3xFlag-(LS)30 or control plasmid. [Fig. 13D-E] Figures 13A-13E show immunofluorescence data validating the generated anti-RAN antibodies in transformed cells expressing recombinant proteins. Figure 13D shows validation of anti-GAGAGG-ASF1 antibodies. HEK293T cells were transformed with either CMV-3xFlag-ASF2 or control plasmid. Figure 13E shows validation of anti-GAGAGG-ASF2 antibodies. HEK293T cells were transformed with either CMV-3xFlag-ASF2 or control plasmid. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] Detailed Description In some aspects, the present disclosure relates to methods and compositions that are useful for the diagnosis and / or treatment of subjects having or at risk of developing a disease associated with RAN protein expression, translation and / or accumulation (e.g., a neurological disease). In some embodiments, the disease associated with RAN protein expression, translation and / or accumulation 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 degeneration 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); fragile X tremor ataxia syndrome (FXTAS); Fuchs endothelial corneal dystrophy (FECD); Huntington's disease type 2 (HDL2); fragile X syndrome (FXS); disorders related to 7pl l.2 folate-sensitive fragile site FRA7A; disorders related to folate-sensitive fragile site 2ql 1 FRA2A; and fragile XE syndrome (FRAXE). In some embodiments, the neurological disease associated with RAN protein is Alzheimer's disease (AD).

[0054] In some aspects, the disclosure relates to methods for the diagnosis and / or treatment of a subject having or at risk of developing a disease associated with expression, translation and / or accumulation of a RAN protein (e.g., a neurological disease). The disclosure relates, in part, to methods for the diagnosis and / or treatment of a subject having or at risk of developing a disease associated with expression, translation and / or accumulation of a RAN protein (e.g., a neurological disease). The disclosure relates to methods for the diagnosis and / or treatment of a subject having or at risk of developing a disease associated with expression, translation and / or accumulation of a RAN protein (e.g., a neurological disease), the ... poly(glycine-glutamic acid) [poly(GE)]; poly(glycine-glutamine) [poly(GQ)]; poly(glycine-threonine) [poly(GT)]; poly(leucine) [poly(Leu)]; poly(leucine-proline) [poly(LP)]; poly(leucine-proline-alanine-cysteine) [poly(LPAC)] (SEQ ID NO: 260); poly(leucine-serine) [poly(LS)]; poly(proline) [poly( poly(proline-alanine) [poly(PA)]; poly(glutamine-alanine-glycine-arginine) [poly(QAGR)] (SEQ ID NO: 261); poly(arginine-glutamic acid) [poly(RE)]; poly(serine-proline) [poly(SP)], poly(valine-proline) [poly(VP)], poly(phenylalanine-proline) [poly(FP)], poly(glycine-lysine) [poly(GK)], poly(FTPLSLPV) ( In some embodiments, the patient is characterized by the expression and accumulation of poly(GSKHREAE) (SEQ ID NO:267), poly(GSKHREAE) (SEQ ID NO:268), poly(GSKHREAE) (SEQ ID NO:269), poly(GSKHREAE) (SEQ ID NO:270), poly(GSKHREAE) (SEQ ID NO:271), poly(GSKHREAE) (SEQ ID NO:272), poly(GSKHREAE) (SEQ ID NO:273), poly(GSKHREAE) (SEQ ID NO:274), poly(GSKHREAE) (SEQ ID NO:275), poly(GSKHREAE) (SEQ ID NO:276), poly(GSKHREAE) (SEQ ID NO:277), poly(GSKHREAE) (SEQ ID NO:278), poly(GSKHREAE) (SEQ ID NO:279), poly(GSKHREAE

[0055] Aspects of the disclosure include, but are not limited to, the synthesis and / or synthesis of certain repeat-associated non-ATG (RAN) proteins (e.g., polyserine [polySer], poly(proline-arginine) [poly(PR)], and poly(glycine-arginine) [poly(GR)]; poly(cysteine-proline) [poly(CP)]; poly(glycine-proline) [poly(GP)]; poly(glycine) [poly(G)]; poly(alanine) [polyAla]; poly(glycine-alanine) [poly(GA)]; poly(glycine-aspartic acid) [poly(GD)]; poly(G Poly(lysine-glutamic acid) [poly(GE)]; Poly(glycine-glutamine) [poly(GQ)]; Poly(glycine-threonine) [poly(GT)]; Poly(leucine) [poly(Leu)]; Poly(leucine-proline) [poly(LP)]; Poly(leucine-proline-alanine-cysteine) [poly(LPAC)] (SEQ ID NO: 260); Poly(leucine-serine) [poly(LS)]; Poly(proline) [poly(P)]; Poly(proline-alanine) [poly(PA)]; Poly(glutamine-alanine-glycine-alanine) poly(arginine-glutamic acid) [poly(RE)]; poly(serine-proline) [poly(SP)], poly(valine-proline) [poly(VP)], poly(phenylalanine-proline) [poly(FP)], poly(glycine-lysine) [poly(GK)], poly(FTPLSLPV) (SEQ ID NO: 262), poly(LLPSPSRC) (SEQ ID NO: 263), poly(YSPLPPGV) (SEQ ID NO: 264), poly(HREGEGSK) (SEQ ID NO: 255), poly (TGRERGVN) (SEQ ID NO: 265), poly(PGGRGE) (SEQ ID NO: 258), poly(GRQRGVNT) (SEQ ID NO: 266), and / or poly(GSKHREAE) (SEQ ID NO: 267)), which are expressed from a subject's locus that is not C9orf72 and are detectable in a biological sample from a subject having or suspected of having Alzheimer's disease (AD) or another disease associated with RAN protein expression, translation and / or accumulation (e.g., a neurological disease).The biological sample may be any specimen derived from or obtained from a subject having or suspected of having a disease (e.g., a neurological disease) associated with the expression, translation and / or accumulation of RAN protein, such as AD. In some embodiments, the biological sample is blood, serum (e.g., plasma with clotting proteins removed) or cerebrospinal fluid (CSF). In some embodiments, the biological sample is a tissue sample, such as central nervous system (CNS) tissue, such as brain tissue or spinal cord tissue. Those skilled in the art will recognize other biological samples, such as cells (e.g., brain cells, nerve cells, skin cells, etc.), suitable for the methods described by this disclosure.

[0056] A "subject having or suspected of having a disease (e.g., a neurological disease) associated with RAN protein expression, translation and / or accumulation" generally refers to a subject who exhibits one or more signs and symptoms of a neurodegenerative disease (including, but not limited to, memory deficits (e.g., short-term memory loss), confusion, deficits in executive function (e.g., attention, planning, flexibility, abstract thinking, etc.), aphasia, degeneration or loss of motor skills, etc.) or who has or has been identified as having one or more genetic mutations associated with RAN protein expression, translation and / or accumulation.

[0057] 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, memory deficits (e.g., short-term memory loss), confusion, deficits in executive function (e.g., attention, planning, flexibility, 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 AD, such as mutations in specific 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 as having AD by a medical 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. The subject may 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 non-human animal, such as a mouse, a rat, a guinea pig, a cat, a dog, a horse, a camel, etc. In some embodiments, the subject is a human.

[0058] RAN protein "RAN protein (repeat-associated non-ATG translated protein)" is a polypeptide translated from a sense or antisense RNA sequence transcribed bidirectionally in the absence of an AUG start codon from a repeat expansion mutation. Sequences encoding RAN proteins can be found in the genome at multiple loci, including but not limited to open reading frame 72 on chromosome 9 (C9orf72), open reading frame 80 on chromosome 2 (C2orf80), LRP8, CASP8, CRNDE, EXOC6B, SV2B, PPML1, ADARB2, GREB1, and MSMO1. Proteins associated with C9orf72 are currently poorly characterized, but are known to be abundant in neurons, particularly in the cerebral cortex and motor neurons. C9orf72 protein is believed to localize in presynaptic terminals. C9orf72 protein may affect RNA transcription, translation, and subcellular localization. The C9orf72 gene contains GGGGCC repeats. This hexanucleotide repeat occurs in variable repeat numbers, with low numbers of repeats not being associated with any pathology.

[0059] The protein related to C2orf80 is an uncharacterized protein whose expression is known to be localized in brain tissue and, to a lesser extent, in testis tissue. The C2orf80 locus contains a GAGAGG repeat motif, which, depending on the reading frame in which translation is initiated, can generate novel RAN proteins containing poly(GR), poly(GE), poly(RE), poly(SP), poly(LP) and poly(LS) dipeptide repeat motifs. When translation is initiated in the reading frame that produces C2orf80 poly(leucine-proline)RAN, the C-terminal sequence PCSSPVHLIPDLFVVEFREWSEMDRVGKKGEREEGSLFFQLWALSCNVQSEEKI (SEQ ID NO: 203) is also translated to produce the amino acid sequence (LP) nIf translation is initiated in the reading frame that gives rise to C2orf80 poly(serine-proline)RAN, the C-terminal sequence LLLPCPSDS (SEQ ID NO:205) will also be translated, resulting in the full-length RAN protein having the amino acid sequence (SP n If translation is initiated in the reading frame that gives rise to the C2orf80 poly(serine-leucine)RAN, the C-terminal sequence PAPPLSI (SEQ ID NO:207) will also be translated, resulting in the full-length RAN protein having the amino acid sequence (SL) n If translation is initiated in the reading frame that gives rise to the C2orf80 poly(glycine-glutamic acid)RAN, the C-terminal sequence GDFKQEKRKLLLLREGSRIETFGIQKLIQTFSSTCLFASTE (SEQ ID NO:209) will also be translated, resulting in the amino acid sequence (GE) n GDFKQEKRKLLLLREGSRIETFGIQKLIQTFSSTCLFASTE (SEQ ID NO: 210), where n is the number of incorporated GE repeats. If translation were to begin in a reading frame that would result in C2orf80 poly(glycine-arginine)RAN, it is unlikely that additional amino acids would be translated, and therefore the full-length RAN protein would simply have the sequence (GR) n (SEQ ID NO:27), where n is the number of incorporated GR repeats. If translation is initiated in the reading frame that gives rise to C2orf80 poly(arginine-glutamic acid)RAN, the C-terminal sequence TLSRKKENYYC (SEQ ID NO:212) will also be translated to give the amino acid sequence (RE) n This will likely result in a full-length RAN protein having the sequence TLSRKKENYYC (sequence number 213), where n is the number of RE repeats incorporated.

[0060] LRP8 (low density lipoprotein receptor-related protein 8) serves as a key component of the Reelin pathway that governs the stratification of neurons in the forebrain during brain development, among other known functions. The LRP8 gene contains a repeat expansion motif that includes the (sense-antisense) GGGGCA-TGCCCC (SEQ ID NO: 1) repeat motif, which, depending on the reading frame initiated, can encode a RAN protein containing poly(PR), poly(GR), poly(GD), poly(GT), poly(VP), and poly(SP) dipeptide repeat motifs from the sense and antisense transcripts. When translation is initiated in the reading frame that gives rise to LRP8 poly(glycine-alanine)RAN, the C-terminal sequence DSTSRKALGPLLSLAPSCQAPSIPKPCHNPMLQEVFLQPTPAHLPPS (SEQ ID NO: 214) is also translated, resulting in the amino acid sequence (GA) n If translation is initiated in the reading frame that gives rise to the LRP8 poly(glycine-glutamine)RAN, the C-terminal sequence IPPPEKPLGPSSALPPPAKPPVSPSPATIPCSRRSSSSPPQPTSHPPDSDVLSPAKPCDLEEVIFLLCKWGMRTTCLTG (SEQ ID NO:216) will also be translated, resulting in the full-length RAN protein having the amino acid sequence (GQ) n It is likely that this will result in a full-length RAN protein having the amino acid sequence (GR) IPPPEKPLGPSSALPPPAKPPVSPSPATIPCSRRSSSSPPQPTSHPPDSDVLSPAKPCDLEEVIFLLCKWGMRTTCLTG (SEQ ID NO: 217), where n is the number of incorporated GQ repeats. If translation is initiated in the reading frame that results in LRP8 poly(glycine-arginine)RAN, the C-terminal sequence FHLQKSPWAPPQPCPLLPSPQYPQALPQSHAPGGLPPAHPSPPPTLLTQMFCLLLSRVTWRKSFFSSVNGG (SEQ ID NO: 218) will also be translated, resulting in the amino acid sequence (GR) nIf translation is initiated in the reading frame that produces the LRP8 poly(cysteine-proline)RAN, the C-terminal sequence PSPSLPSPHAVSKWIPDTKTPTGVVPEVRPVNLGPRALPVPLKARVWVCSGAELKAAKLTGKVSPVIRDVQGYGWGRGDSHLW (SEQ ID NO: 220) will also be translated, resulting in the full-length RAN protein having the amino acid sequence (CP) n If translation is initiated in the reading frame that gives rise to the LRP8 poly(proline-alanine)RAN, the C-terminal sequence RLFPLPMLSPNGSLTPRLQLG (SEQ ID NO: 222) will also be translated, resulting in the full-length RAN protein having the amino acid sequence (PA) PSPSLPSPHAVSKWIPDTKTPTGVVPEVRPVNLGPRALPVPLKARVWVCSGAELKAAKLTGKVSPVIRDVQGYGWGRGDSHLW (SEQ ID NO: 221), where n is the number of incorporated CP repeats. n It is likely that this will result in a full-length RAN protein having the amino acid sequence (LP): RLFPLPMLSPNGSLTPRLQLG (SEQ ID NO: 223), where n is the number of PA repeats incorporated. If translation is initiated in the reading frame that gives rise to the LRP8 poly(leucine-proline)RAN, the C-terminal sequence QPVSSLSPCCLQMDP (SEQ ID NO: 224) will also be translated, resulting in the amino acid sequence (LP): n This will likely result in a full length RAN protein having the sequence QPVSSLSPCCLQMDP (sequence number 225), where n is the number of incorporated LP repeats.

[0061] CASP8 (caspase-8) is expressed in a variety of tissues and serves as the most upstream protease in the activation of the caspase cascade during TNFRSF6 / FAS-mediated and / or TNFRSF1A-induced cell death. The CASP8 gene contains a GAGAGG·CCTCTC (SEQ ID NO:2) repeat motif, which can generate novel RAN proteins containing poly(GR), poly(GE), poly(RE), poly(SP), poly(LP), and poly(LS) dipeptide repeat motifs from sense and antisense transcripts, depending on the reading frame in which translation is initiated. When translation is initiated in the reading frame that gives rise to CASP8 poly(leucine-serine)RAN, the C-terminal sequence PSPSPSPSPRLPLPLMPSQSWTVLLPSRLTATSLPDSPASACRVPAIAGARRHA (SEQ ID NO:226) is also translated to produce the amino acid sequence (LS) n If translation is initiated in the reading frame that gives rise to the CASP8 poly(leucine-proline)RAN, the C-terminal sequence PVSLSLSLSPSPSPSHAEPKLDGTAAISAHCNLPA (SEQ ID NO:228) will also be translated, resulting in the full-length RAN protein having the amino acid sequence (LP): PSPSPSPSPRLPLPLMPSQSWTVLLPSRLTATSLPDSPASACRVPAIAGARRHA (SEQ ID NO:227), where n is the number of incorporated LS repeats. n If translation is initiated in the reading frame that gives rise to the CASP8 poly(proline-serine)RAN, the C-terminal sequence PRLPLPLPLPVSLSLSCRAKAGRYCCHLGSLQPPCLILLPQPAECLRLQARAATPDWFSFFFWWRWGFAVLAGLVSSS (SEQ ID NO: 230) will also be translated, resulting in the amino acid sequence (PS nIt is likely that this will result in a full-length RAN protein having the amino acid sequence (GR) PRLPLPLPVSLSLSCRAKAGRYCCHLGSLQPPCLILLPQPAECLRLQARAATPDWFSFFFWWRWGFAVLAGLVSSS (SEQ ID NO: 231), where n is the number of PS repeats incorporated. If translation is initiated in the reading frame that results in CASP8 poly(glycine-arginine) RAN, the C-terminal sequence GVKFLSINVMPTVLSSCGL (SEQ ID NO: 232) will also be translated, resulting in a full-length RAN protein having the amino acid sequence (GR) n If translation is initiated in the reading frame that gives rise to the CASP8 poly(arginine-glutamic acid)RAN, the C-terminal sequence RGQILIYQCYAHCALQLWSVNYCGIT (SEQ ID NO:234) will also be translated, resulting in the full-length RAN protein having the amino acid sequence (RE) GVKFLSINVMPTVLSSCGL (SEQ ID NO:233), where n is the number of incorporated GR repeats. n If translation is initiated in the reading frame that gives rise to the CASP8 poly(glycine-glutamic acid)RAN, the C-terminal sequence GSNSYLSMLCPLCSPAVVCELLWYNVTVQISLFRGFDHDL (SEQ ID NO:236) will also be translated, resulting in the full-length RAN protein having the amino acid sequence (GE) n This is likely to result in a full-length RAN protein having the sequence GSNSYLSMLCPLCSPAVVCELLWYNVTVQISLFRGFDHDL (sequence number 259), where n is the number of incorporated GE repeats.

[0062] The Colorectal Neoplasia Differentially Expressed (CRNDE) locus is thought to be transcribed into multiple transcript variants, some of which may function as non-coding RNAs. One transcript variant encodes a putative short protein that is localized to the nucleus. Expression of CRNDE is increased in proliferating tissues, such as colorectal adenomas and adenocarcinomas. The CRNDE gene contains a GGGGGC (G5C) repeat motif, which can generate novel RAN proteins, including poly(glycine) [polyGly] and poly(proline) [polyPro] proteins, as well as the dipeptide repeats poly(GA), poly(GR), poly(PA) and poly(PR), from sense and antisense transcripts, depending on the reading frame in which translation is initiated. When translation is initiated in a reading frame that gives rise to CRNDE polyGlyRAN, typically based on the nucleic acid sequence located 3' of the repeat motif, the C-terminal sequence RKRGTAGVAG (SEQ ID NO:3) is also translated to produce the amino acid sequence (G) n If translation is to begin in a reading frame that results in CRNDE polyProRAN, the C-terminal sequence RGLFVGCFFNFFNPFSCTVFFLVSGAGETPARY (SEQ ID NO:5) will also be translated to give the amino acid sequence (P) n If translation is initiated in the reading frame that gives rise to CRNDE poly(GA)RAN, the C-terminal sequence GVGGESAGLPEWQDDVMRMSV (SEQ ID NO:7) will also be translated, resulting in the full-length RAN protein having the amino acid sequence (GA) nIt is likely that this will result in a full-length RAN protein having the amino acid sequence GVGGESAGLPEWQDDVMRMSV (SEQ ID NO:8), where n is the number of incorporated glycine-alanine repeats. If translation is initiated in the reading frame that results in CRNDEpoly(GR)RAN, the C-terminal sequence GWGEKARDCRSGRMM (SEQ ID NO:9) will also be translated, resulting in the amino acid sequence (GR) n It is likely that this will result in a full-length RAN protein having the amino acid sequence (PR) GWGEKARDCRSGRMM (SEQ ID NO: 10), where n is the number of incorporated glycine-arginine repeats. If translation is initiated in the reading frame that results in CRNDE poly(PR)RAN, the C-terminal sequence PVACLLVVFLIFLTPFLVLSSFWCQGLERLLQDIEAFRMYGSV (SEQ ID NO: 11) will also be translated, resulting in the amino acid sequence (PR) n It is likely that this will result in a full-length RAN protein having the amino acid sequence PVACLLVVFLIFLTPFLVLSSFWCQGLERLLQDIEAFRMYGSV (SEQ ID NO: 12), where n is the number of incorporated proline-arginine repeats. If translation is initiated in the reading frame that results in CRNDE poly(PA)RAN, the C-terminal sequence PWLVCWLFF (SEQ ID NO: 13) will also be translated, resulting in the amino acid sequence (PA) n This will likely result in a full-length RAN protein having the sequence PWLVCWLFF (SEQ ID NO: 14), where n is the number of incorporated proline-alanine repeats.

[0063] EXOC6B (exocyst complex component 6B) is a part of the exocyst complex involved in docking exocytic vesicles to fusion sites on the plasma membrane. The EXOC6B gene contains a GGGGCA (G4CA) repeat motif, which can generate novel RAN proteins, including the dipeptide repeats poly(glycine-alanine) [poly(GA)], poly(glycine-glutamine) [poly(GQ)], poly(glycine-arginine) [poly(GR)], poly(cysteine-proline) [poly(CP)], poly(proline-alanine) [poly(PA)], and poly(leucine-proline) [poly(LP)], from sense and antisense transcripts, depending on the reading frame in which translation is initiated. If translation is initiated in the reading frame that gives rise to EXOC6B poly(glycine-alanine)RAN, the C-terminal sequence GGRRREEVVLIPHFWLPEKALWQRDAGASKLKVQSACTEGKN (SEQ ID NO: 15) is also translated, resulting in the amino acid sequence (GA n If translation is initiated in the reading frame that gives rise to EXOC6B poly(glycine-glutamine)RAN, the C-terminal sequence GAGGEKRWY (SEQ ID NO:17) will also be translated, resulting in the full-length RAN protein having the amino acid sequence (GQ n If translation is initiated in the reading frame that gives rise to EXOC6B poly(glycine-arginine)RAN, the C-terminal sequence GQEERRGGINSPFLASRESPLAKRCRC (SEQ ID NO:19) will also be translated, resulting in the amino acid sequence (GR nIf translation is initiated in the reading frame that gives rise to EXOC6B poly(proline-cysteine)RAN, the C-terminal sequence PSSPQLITHGSWCINTSASLPTRKEDGVEYL (SEQ ID NO:22) will also be translated, resulting in the full-length RAN protein having the amino acid sequence (PC) GQEERRGGINSPFLASRESPLAKRCRC (SEQ ID NO:20), where n is the number of incorporated glycine-arginine repeats. n If translation is initiated in the reading frame that gives rise to EXOC6B poly(proline-alanine)RAN, the C-terminal sequence PVVLSS (SEQ ID NO:24) will also be translated, resulting in the full-length RAN protein having the amino acid sequence (PA) PSSPQLITHGSWCINTSASLPTRKEDGVEYL (SEQ ID NO:23), where n is the number of incorporated proline-cysteine ​​repeats. n It is likely that this will result in a full-length RAN protein having PVVLSS (SEQ ID NO:25), where n is the number of incorporated proline-alanine repeats. If translation were to begin in a reading frame that would result in EXOC6B poly(proline-lysine)RAN, it is unlikely that additional amino acids would be translated, and therefore the full-length RAN protein would simply be (PL) n (SEQ ID NO:26), where n is the number of incorporated proline-lysine repeats.

[0064] SV2B (synaptic vesicle glycoprotein 2B) is thought to play a role in the control of secretion from neuronal and endocrine cells. In the former, it is a component of the pathology of botulism and acts as a receptor for the C. botulinum neurotoxin. The SV2B gene also contains a G4CA repeat motif and can therefore generate the RAN protein described for the same motif in the EXOC6B gene above. If translation is initiated in the reading frame that gives rise to the SV2B poly(glycine-arginine)RAN, it is unlikely that additional amino acids will be translated and therefore the full length RAN protein will simply be expressed as (GR) n(SEQ ID NO:27), where n is the number of incorporated glycine-arginine repeats. If translation is initiated in the reading frame that gives rise to the SV2B poly(glycine-alanine)RAN, the C-terminal sequence GDSNTTSAKSQDTASLQM (SEQ ID NO:28) will also be translated to give the amino acid sequence (GA n If translation is initiated in the reading frame that gives rise to the SV2B poly(glycine-glutamine)RAN, the C-terminal sequence GTVTQHLPRVKTQPLCKCRQAMLRCV (SEQ ID NO:30) will also be translated, resulting in the full-length RAN protein having the amino acid sequence (GQ n It is likely that this will result in a full length RAN protein having the sequence GTVTQHLPRVKTQPLCKCRQAMLRCV (SEQ ID NO:31), where n is the number of incorporated glycine-glutamine repeats. If translation is initiated in a reading frame that results in SV2B poly(proline-alanine)RAN, it is unlikely that additional amino acids will be translated, and therefore the full length RAN protein will simply have the sequence (PA) n (SEQ ID NO:32), where n is the number of incorporated proline-alanine repeats. If translation is initiated in the reading frame that gives rise to the SV2B poly(proline-lysine)RAN, the C-terminal sequence PSHNSLTLVSSLTLPLDTIGTDPQQSA (SEQ ID NO:33) will also be translated to give the amino acid sequence (PL n It is likely that this will result in a full-length RAN protein having the amino acid sequence (PC) PSHNSLTLVSSLTLPLDTIGTDPQQSA (SEQ ID NO: 34), where n is the number of incorporated proline-lysine repeats. If translation is initiated in the reading frame that gives rise to the SV2B poly(proline-cysteine)RAN, the C-terminal sequence PHIIL will also be translated, resulting in a full-length RAN protein having the amino acid sequence (PC) PSHNSLTLVSSLTLPLDTIGTDPQQSA (SEQ ID NO: 34). n This will likely result in a full-length RAN protein having PHIIL (SEQ ID NO: 35), where n is the number of incorporated proline-cysteine ​​repeats.

[0065] Methylsterol monooxygenase 1, the protein encoded by the MSMO1 locus, is an enzyme localized to the endoplasmic reticulum where it is part of the catalytic pathway that removes the methyl group from 4,4-dimethylzymosterol, thus contributing to the biosynthesis of zymosterol, which is part of steroid biosynthesis. The MSMO1 gene also contains a G5C repeat motif and is therefore capable of producing the RAN protein described for the same motif as in the CRNDE locus above. When translation is initiated in the reading frame that gives rise to the MSMO1 poly(proline-alanine)RAN, the C-terminal sequence PGHSSSSTTIATTPGRSLPM (SEQ ID NO: 36) is also translated to produce the amino acid sequence (PA) n If translation were to begin in the reading frame that would yield the MSMO1 polyProRAN, the C-terminal sequence GTHRPAQQLQQHLGVLCPCSEVKVLGAELSLDVQSF (SEQ ID NO:38) would likely be translated to yield the full-length RAN protein with the amino acid sequence (P)PGHSSSSTTIATTPGRSLPM (SEQ ID NO:37), where n is the number of incorporated proline-alanine repeats. n If translation is initiated in the reading frame that produces the MSMO1 poly(proline-arginine)RAN, the C-terminal sequence ALIVQHNNCNNTWAFSAHVARSRSWEPNSPLMFNLFKSFPAFISHLQNDDNRI (SEQ ID NO:40) will also be translated, resulting in the amino acid sequence (PR): nIf translation is initiated in the reading frame that gives rise to the MSMO1 poly(glycine-arginine)RAN, the C-terminal sequence GLDAGLCSSKAQFTPSLNIKILCTGV (SEQ ID NO:42) will also be translated, resulting in the full-length RAN protein having the amino acid sequence (GR). n It is likely that this will result in a full-length RAN protein having the amino acid sequence (G) of GLDAGLCSSKAQFTPSLNIKILCTGV (SEQ ID NO: 43), where n is the number of incorporated glycine-arginine repeats. If translation is initiated in the reading frame that results in the MSMO1 polyGlyRAN, the C-terminal sequence LTQVFAALKLSLHHLSTLKYCVLGFNNKTVQM (SEQ ID NO: 44) will also be translated, resulting in the amino acid sequence (G) n LTQVFAALKLSLHHLSTLKYCVLGFNNKTVQM (SEQ ID NO: 45), where n is the number of incorporated glycines. Finally, if translation is initiated in a reading frame that results in MSMO1 poly(glycine-alanine)RAN, it is unlikely that additional amino acids will be translated, and therefore the full-length RAN protein will simply have the sequence (GA) n (SEQ ID NO: 46), where n is the number of glycine-alanine repeats incorporated.

[0066] Protein phosphatase 1L, a protein encoded by the PPM1L locus, is a magnesium or manganese-requiring phosphatase and is involved in signal transduction pathways. The protein downregulates apoptosis signal-regulating kinase 1, a protein involved in apoptosis following cytotoxic stress. The protein is an endoplasmic reticulum transmembrane protein that also helps regulate ceramide transport from the ER to the Golgi apparatus. The PPM1L gene contains a GGGGAA repeat motif. When translation is initiated in the reading frame that gives rise to PPM1L poly(phenylalanine-proline)RAN, the C-terminal sequence PLPLPLPFPLPRSLPLPLPSPPLFDRVSLVTQSGVHWHNLGSLQPPPPRFR (SEQ ID NO: 237) is also translated to give the amino acid sequence (FP) n If translation were to begin in a reading frame that would yield PPM1L poly(serine-proline)RAN, the C-terminal sequence FLSPSPFLSPSPFPFPFPFPFPFPVPFPFPSPPLPFLTESHWSPSLECTGTILAHCNLRLPGSGDC (SEQ ID NO:239) would likely be translated to yield the full-length RAN protein having the amino acid sequence (SP n If translation is initiated in the reading frame that gives rise to PPM1L poly(leucine-proline)RAN, the C-terminal sequence CPFPLPLSFPLPLSFPLPLSPSPSPSLSPSPSPSPPSPPLPSPF (SEQ ID NO: 241) will also be translated, resulting in the full-length RAN protein having the amino acid sequence (LP) nIf translation is initiated in the reading frame that gives rise to the PPML1 poly(glycine-glutamic acid)RAN, the C-terminal sequence GDREGKKVSSAEYISRLRSHHSKHYCSSDMLKQNSQTLLSLVTSKSK (SEQ ID NO:243) will also be translated, resulting in the full-length RAN protein having the amino acid sequence (GE) n If translation is initiated in the reading frame that gives rise to PPML1 poly(glycine-lysine)RAN, the C-terminal sequence TGRERKFQALNIFQD (SEQ ID NO:245) will also be translated, resulting in the full-length RAN protein having the amino acid sequence (GK). n It is likely that this will result in a full-length RAN protein with the amino acid sequence (GR) TGRERKFQALNIFQD (SEQ ID NO: 246), where n is the number of incorporated glycine-lysine repeats. Finally, if translation is initiated in the reading frame that gives rise to PPML1 poly(glycine-arginine)RAN, the C-terminal sequence GQGGKESFKR (SEQ ID NO: 247) will also be translated, resulting in the amino acid sequence (GR) n This is likely to result in a full-length RAN protein having the sequence GQGGKESFKR (sequence number 248), where n is the number of incorporated glycine-arginine repeats.

[0067] ADARB2 (Adenosine Deaminase RNA Specific B2) encodes a member of the double-stranded RNA adenosine deaminase family of RNA editing enzymes, and may play a regulatory role in RNA editing, but is thought to lack editing activity itself, preventing the binding of other ADAR enzymes and reducing the efficiency of these enzymes. The ADARB2 gene contains a TTTACTCCCCTCTCCCTCCCGGTG (SEQ ID NO: 21) repeat motif. In some embodiments, the ADARB2 gene encodes an RAN protein that contains poly(GRQRGVNT) (SEQ ID NO: 266) repeats and / or poly(GSKHREAE) (SEQ ID NO: 267) repeats. If translation is initiated in the reading frame that gives rise to the ADARB2 poly(FTPLSLPV) (SEQ ID NO:262)RAN, the C-terminal sequence PLPPGAYSPLPPGVYSPLLPGVYSLCPGVYSPASWPSTFCRSCCFHTFCPMGDGLCSVGP (SEQ ID NO:249) is also translated to give the amino acid sequence (FTPLSLPV): n If translation is initiated in the reading frame that produces the ADARB2 poly(LLPSPSRC) (SEQ ID NO:263)RAN, the C-terminal sequence AGLAVFTRSAPWVMDCVLWGPEITQATEQTFSPQEVLAASSSLPASVPALCPQPPSPTAPAASPRTLGKCIPSLGPGTGPVSHVAALDPPSPVLVPHAGQASGAPVCGPPQLVAQHQACNQLLVNIGPVAFSDTNKSEGSW (SEQ ID NO:251) will also be translated to produce the amino acid sequence (LLPSPSRC) nAGLAVFTRSAPWVMDCVLWGPEITQATEQTFSPQEVLAASSSLPASVPALCPQPPSPTAPAASPRTLGKCIPSLGPGTGPVSHVAALDPPSPVLVPHAGQASGAPVCGPPQLVAQHQACNQLLVNIGPVAFSDTNKSEGSW (SEQ ID NO: 252), where n is the number of incorporated LLPSPSRC (SEQ ID NO: 269) repeats. If translation is initiated in the reading frame that yields ADARB2 poly(YSPLPPGV) (SEQ ID NO: 264)RAN, the C-terminal sequence CLLPSPSRCLLPSASRCLLPSPSRCLLPSSSRCLLPSASRCLLPSASRCLLPVSWCLLPCFLAIYLLPVLLFSHVLPHG (SEQ ID NO: 253) will also be translated to yield the amino acid sequence (YSPLPPGV) n If translation is initiated in a reading frame that results in ADARB2 poly(HREGEGSK) (SEQ ID NO:255)RAN, it is unlikely that additional amino acids will be translated, and therefore the full length RAN protein will simply have the sequence (HREGEGSK) n (SEQ ID NO:255), where n is the number of incorporated HREGEGSK (SEQ ID NO:271) repeats. If translation is initiated in the reading frame that gives rise to the ADARB2 poly(TGRERGVN) (SEQ ID NO:265)RAN, the C-terminal sequence GKHRRRKCRHVRSAPTPMGQRWGCSAPASQLVGVLQQANHSPSERLGTLPPHLGHGWMQKESRFATVLHSHLCW (SEQ ID NO:256) will also be translated to give the amino acid sequence (TGRERGVN) nGKHRRRKCRHVRSAPTPMGQRWGCSAPASQLVGVLQQANHSPSERLGTLPPHLGHGWMQKESRFATVLHSHLCW (SEQ ID NO: 257), where n is the number of incorporated TGRERGVN (SEQ ID NO: 272) repeats. If translation were to begin in a reading frame that would yield ARARB2 poly(PGGRGE) (SEQ ID NO: 258)RAN, it is unlikely that additional amino acids would be translated, and therefore the full-length RAN protein would simply have the sequence (PGGRGE) n (SEQ ID NO: 258), where n is the number of PGGRGE (SEQ ID NO: 273) repeats incorporated.

[0068] GREB1 (growth regulating estrogen receptor binding 1) is an estrogen-responsive gene and an early response gene in the pathway regulated by the estrogen receptor. It is thought to play an important role in hormone-responsive tissues and cancer, and it encodes the GREB1 protein. The GREB1 gene contains a GGGGCA repeat motif. When translation is initiated in the reading frame that gives rise to GREB1 poly(glycine-arginine)RAN, the C-terminal sequence DRMPSVGEGAEG (SEQ ID NO: 128) is also translated to give the amino acid sequence (GR) n It is likely that this will result in a full-length RAN protein having the amino acid sequence (GA) DRMPSVGEGAEG (SEQ ID NO: 136), where n is the number of incorporated glycine-arginine repeats. If translation is initiated in the reading frame that results in GREB1 poly(glycine-alanine)RAN, the C-terminal sequence GTGCLQWVKVQKGRSEEVGMEEGEEGGGEELRK (SEQ ID NO: 182) will also be translated, resulting in the amino acid sequence (GA) nIf translation is initiated in the reading frame that gives rise to the GREB1 poly(glycine-glutamine)RAN, the C-terminal sequence DAFSG (SEQ ID NO: 186) will also be translated, resulting in the full-length RAN protein having the amino acid sequence (GQ) n If translation is initiated in the reading frame that gives rise to GREB1 poly(proline-alanine)RAN, the C-terminal sequence WARGSLSSSRSPLTSLPWGLPQTQVSPRHTLHLCGASPDGP (SEQ ID NO:211) will also be translated, resulting in the amino acid sequence (PA) n If translation is initiated in the reading frame that gives rise to the GREB1 poly(leucine-proline)RAN, the C-terminal sequence GHVAHSLPPGLL (SEQ ID NO:275) will also be translated, resulting in the full-length RAN protein with the amino acid sequence WARGSLSSSRSPLTSLPWGLPQTQVSPRHTLHLCGASPDGP (SEQ ID NO:274), where n is the number of incorporated proline-alanine repeats. n If translation is initiated in the reading frame that produces the GREB1 poly(cysteine-proline)RAN, the C-terminal sequence CLGTWLTLFLQVSFNITSLGTPTDSGLPATHTSPLWCFSRWSLTSHVSNICPSCWAGYS (SEQ ID NO:277) will also be translated, resulting in the amino acid sequence (CP) n This is likely to result in a full-length RAN protein having the sequence CLGTWLTLFLQVSFNITSLGTPTDSGLPATHTSPLWCFSRWSLTSHVSNICPSCWAGYS (sequence number 278), where n is the number of cysteine-proline repeats incorporated.

[0069] Generally, RAN proteins contain extended repeats of single, di-, tri-, or 4-amino acids (e.g., tetra-amino acids) referred to as polyamino acid repeats. For example, "AAAAAAAAAAAAAAAAAAAAA" (poly-alanine) (SEQ ID NO: 47), "LLLLLLLLLLLLLLLLLL" (poly-leucine) (SEQ ID NO: 48), "SSSSSSSSSSSSSSSSSSSS" (poly-serine) (SEQ ID NO: 49), or "CCCCCCCCCCCCCCCCCCCC" (poly-cysteine) (SEQ ID NO: 50) are polyamino acid repeats that are each 20 amino acid residues in length. Examples of di-amino acid RAN proteins include GPGPGPGPGPGPGPGPGPGP (poly-GP) (SEQ ID NO:51), GAGAGAGAGAGAGAGAGA (poly-GA) (SEQ ID NO:52), GRGRGRGRGRGRGRGRGRGR (poly-GR) (SEQ ID NO:53), PAPAPAPAPAPAPAPAPAPA (poly-PA) (SEQ ID NO:54) and PRPRPRPRPRPRPRPRPRPR (poly-PR) (SEQ ID NO:55). Examples of tetra-amino acid repeats include LPACLPACLPAC (e.g., poly-LPAC) (SEQ ID NO:56) and QAGRQAGRQAGR (e.g., poly-QAGR) (SEQ ID NO:57). RAN proteins may have polyamino acid repeats 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, or at least 200 amino acid residues in length. In some embodiments, the RAN protein has polyamino acid repeats greater than 200 amino acid residues in length (e.g., 500, 1000, 5000, 10,000, etc.).

[0070] Generally, RAN proteins are translated from abnormal repeat expansions of DNA (e.g., TCT repeats, hexanucleotide repeats, etc.). The present disclosure relates, in part, to one or more (e.g., 2, 3, 4, 5, or more) RAN proteins, such as poly(proline-arginine) [poly(PR)]; poly(glycine-arginine) [poly(GR)]; poly(serine) [polySer]; poly(cysteine-proline) [poly(CP)]; poly(glycine-proline) [poly(GP)]; poly(glycine) [poly(G)]; poly(alanine) [polyAla]; poly(glycine-alanine) [poly(Ala)]; poly(glycine-alanine) [poly(Ala)]; poly(glycine-alanine) [poly(Ala)]; poly(glycine-alanine) [poly(Ala)]; poly(glycine-alanine) [poly(Ala)]; poly(glycine-alanine) [poly(Ala)]; poly(glycine-alanine) [poly(Ala)]; poly(glycine-alanine) [poly(Ala)]; poly(glycine-alanine) [poly(Ala)]; poly(glycine-argin ... ) [poly(GA)]; poly(glycine-aspartic acid) [poly(GD)]; poly(glycine-glutamic acid) [poly(GE)]; poly(glycine-glutamine) [poly(GQ)]; poly(glycine-threonine) [poly(GT)]; poly(leucine) [poly(Leu)]; poly(leucine-proline) [poly(LP)]; poly(leucine-proline-alanine-cysteine) [poly(LPAC)] (SEQ ID NO: 260); poly(leucine-serine) [ poly(LS)]; poly(proline) [poly(P)]; poly(proline-alanine) [poly(PA)]; poly(glutamine-alanine-glycine-arginine) [poly(QAGR)] (SEQ ID NO: 261); poly(arginine-glutamic acid) [poly(RE)]; poly(serine-proline) [poly(SP)], poly(valine-proline) [poly(VP)], poly(phenylalanine-proline) [poly(FP)], poly(glycine-lysine) [poly(GK)], poly(FTPLSLPV) (SEQ ID NO: 262), poly(LLPSPSRC) (SEQ ID NO: 263), poly(YSPLPPGV) (SEQ ID NO: 264), poly(HREGEGSK) (SEQ ID NO: 255), poly(TGRERGVN) (SEQ ID NO: 265), and / or poly(PGGRGE) (SEQ ID NO: 258).In some embodiments, the disease state of a subject having or suspected of having a RAN protein-associated disease is classified according to the number and / or type of microsatellite repeats present (e.g., detected) in the subject (e.g., in the subject's genome or in the subject's genes). In some embodiments, subjects having fewer than 10 repeat sequences do not exhibit signs or symptoms of a RAN protein-associated disease characterized by RAN protein translation. In some embodiments, subjects having 10-40 repeats may or may not exhibit one or more signs or symptoms of a RAN protein-associated disease characterized by RAN protein translation. In some embodiments, subjects having more than 40 trinucleotide repeats exhibit one or more signs or symptoms of a RAN protein-associated disease characterized by RAN protein translation. In certain cases, a subject is identified as having a RAN protein-associated disease characterized by a large (>100) number of repeats. Microsatellite repeat sequences encoding RAN proteins are commonly known. In some embodiments, the RAN protein-associated disease is Alzheimer's disease.

[0071] In some embodiments, a subject having or suspected of having a RAN protein-associated disease has one or more microsatellite repeat sequences encoding poly(PR)RAN protein. Examples of microsatellite repeat sequences encoding poly(PR) proteins include CCTCGT (SEQ ID NO:58), CCCCGT (SEQ ID NO:59), CCACGT (SEQ ID NO:60), CCGCGT (SEQ ID NO:61), CCTCGC (SEQ ID NO:62), CCCCGC (SEQ ID NO:63), CCACGC (SEQ ID NO:64), CCGCGC (SEQ ID NO:65), CCTCGA (SEQ ID NO:66), CCCCGA (SEQ ID NO:67), CCACGA (SEQ ID NO:68), CCGCGA (SEQ ID NO:69), CCTCGG (SEQ ID NO:70), CCCCGG (SEQ ID NO:71), CCACGG (SEQ ID NO:72), CCGCGG (SEQ ID NO:73), CCTAGA (SEQ ID NO:74), CCCAGA (SEQ ID NO:75), CCAAGA (SEQ ID NO:76), CCGAGA (SEQ ID NO:77), CCTAGG (SEQ ID NO:78), CCCAGG (SEQ ID NO:79), CCAAGG (SEQ ID NO:80), and CCGAGG (SEQ ID NO:81).

[0072] In some embodiments, a subject having or suspected of having a RAN protein-associated disease has one or more microsatellite repeat sequences encoding poly(GR)RAN protein. Examples of microsatellite repeat sequences encoding poly(GR) proteins include GGTCGT (SEQ ID NO:82), GGCCGT (SEQ ID NO:83), GGACGT (SEQ ID NO:84), GGGCGT (SEQ ID NO:85), GGTCGC (SEQ ID NO:86), GGCCGC (SEQ ID NO:87), GGACGC (SEQ ID NO:88), GGGCGC (SEQ ID NO:89), GGTCGA (SEQ ID NO:90), GGCCGA (SEQ ID NO:91), GGACGA (SEQ ID NO:92), GGGCGA (SEQ ID NO:93), GGTCGG (SEQ ID NO:94), GGCCGG (SEQ ID NO:95), GGACGG (SEQ ID NO:96), GGGCGG (SEQ ID NO:97), GGTAGA (SEQ ID NO:98), GGCAGA (SEQ ID NO:99), GGAAGA (SEQ ID NO:100), GGGAGA (SEQ ID NO:101), GGTAGG (SEQ ID NO:102), GGCAGG (SEQ ID NO:103), GGAAGG (SEQ ID NO:104), and GGGAGG (SEQ ID NO:105).

[0073] Following the above list of possible repeat motifs that can give rise to the RAN proteins poly(PR) and poly(GR), the skilled artisan can easily elucidate other RAN proteins, e.g. poly(serine) [polySer]; poly(cysteine-proline) [poly(CP)]; poly(glycine-proline) [poly(GP)]; poly(glycine) [poly(G)]; poly(alanine) [polyAla]; poly(glycine-alanine) [poly(GA)]; poly(glycine-asparagine) [poly(G)]; poly(glycine-glutamic acid) [poly(GE)]; poly(glycine-glutamine) [poly(GQ)]; poly(glycine-threonine) [poly(GT)]; poly(leucine) [poly(Leu)]; poly(leucine-proline) [poly(LP)]; poly(leucine-proline-alanine-cysteine) [poly(LPAC)] (SEQ ID NO: 260); poly(leucine-serine) [poly(LS)]; poly(proline) [poly(P) ]; poly(proline-alanine) [poly(PA)]; poly(glutamine-alanine-glycine-arginine) [poly(QAGR)] (SEQ ID NO: 261); poly(arginine-glutamic acid) [poly(RE)]; poly(serine-proline) [poly(SP)], poly(valine-proline) [poly(VP)], poly(phenylalanine-proline) [poly(FP)], poly(glycine-lysine) [poly(GK)], poly(FTPLSLPV) (SEQ ID NO: 262). One will understand how to derive possible hexanucleotide repeats that can result in poly(HREGEGSK) (SEQ ID NO:255), poly(TGRERGVN) (SEQ ID NO:265), poly(PGGRGE) (SEQ ID NO:258), poly(GRQRGVNT) (SEQ ID NO:266), and / or poly(GSKHREAE) (SEQ ID NO:267).

[0074] In some embodiments, the subject with or suspected of having RAN protein-related disease has one or more microsatellite repeat sequences that code polySerRAN protein.Examples of the microsatellite repeat sequences that code polySer protein include TCT, TCC, TCA, TCG, AGT and AGC.

[0075] In some aspects, the present disclosure provides methods for producing RAN proteins (e.g., poly(proline-arginine) [poly(PR)]; poly(glycine-arginine) [poly(GR)]; poly(serine) [polySer]; poly(cysteine-proline) [poly(CP)]; poly(glycine-proline) [poly(GP)]; poly(glycine) [poly(G)]; poly(alanine) [polyAla]; poly(glycine-alanine) [poly(GA)]; poly(glycine-aspartic acid) [poly(GD)]; poly(glycine-glutamic acid) [poly(GE)]; poly( Poly(glycine-glutamine) [poly(GQ)]; Poly(glycine-threonine) [poly(GT)]; Poly(leucine) [poly(Leu)]; Poly(leucine-proline) [poly(LP)]; Poly(leucine-proline-alanine-cysteine) [poly(LPAC)] (SEQ ID NO: 260); Poly(leucine-serine) [poly(LS)]; Poly(proline) [poly(P)]; Poly(proline-alanine) [poly(PA)]; Poly(glutamine-alanine-glycine-arginine) [poly(QAGR)] (SEQ ID NO: 261); Poly(arginine-glutamic acid ) [poly(RE)]; poly(serine-proline) [poly(SP)], poly(valine-proline) [poly(VP)], poly(phenylalanine-proline) [poly(FP)], poly(glycine-lysine) [poly(GK)], poly(FTPLSLPV) (SEQ ID NO: 262), poly(LLPSPSRC) (SEQ ID NO: 263), poly(YSPLPPGV) (SEQ ID NO: 264), poly(HREGEGSK) (SEQ ID NO: 255), poly(TGRERGVN) (SEQ ID NO: 265), poly(PGGRGE) (SEQ ID NO: 258), poly(GRQRGVNT) (SEQ ID NO: 266), poly(PGGRGE) (SEQ ID NO: 259), poly(PGGRGE) (SEQ ID NO: 267), poly(PGGRGE) (SEQ ID NO: 269), poly(PGGRGE) (SEQ ID NO: 270), poly(PGGRGE) (SEQ ID NO: 271), poly(PGGRGE) (SEQ ID NO: 272), poly(PGGRGE) (SEQ ID NO: 273), poly(PGGRGE) (SEQ ID NO: 274), poly(PGGRGE) (SEQ ID NO: 275), poly(PGGRGE) (SEQ ID NO: 276), poly(PGGRGE) (SEQ ID NO: 277), poly(PGGRGE) (SEQ ID NO: 278), poly(PGGRGE) (SEQ ID NO: 279), poly(PGGRGE) (SEQ ID NO: 280), poly(PGGRGE) (SEQ ID NO: 281), poly(PGGRGE) (SEQ ID NO: 282), poly(PGGRGE) (SEQ ID NO: 283), poly(PGGRGE) (SEQ ID NO: 284), poly(PGGRGE) (SEQ The present invention relates to the finding that the aggregation pattern of poly(GSKHREAE) (SEQ ID NO: 266) and / or poly(GSKHREAE) (SEQ ID NO: 267) is length-dependent. For example, RAN proteins with polyamino acid repeats of >20, >48, or >80 residues in length aggregate differently in the brain of a subject. In general, the differential aggregation properties of RAN proteins with different lengths can be used to detect RAN proteins in biological samples. Longer RAN proteins are found at higher levels in biological samples such as blood, serum, or CSF.In some embodiments, RAN proteins having polyamino acid repeats >40, >50, >60, >70 or >80 amino acid residues in length are detectable in a biological sample.

[0076] Methods for detecting RAN protein The present disclosure is based in part on the discovery that certain biological sample processing methods (e.g., antibody-based capture, hybridization-based assay, dCas9-based enrichment, or a combination thereof) allow for reproducible detection of one or more RAN proteins in a biological sample. In some embodiments of the methods described by this disclosure, a sample (e.g., a biological sample) is treated with an antibody-based capture process to isolate one or more RAN proteins in the sample. Typically, the antibody-based capture method includes contacting the sample with one or more (e.g., 2, 3, 4, 5 or more) anti-RAN protein antibodies. In some embodiments, the one or more anti-RAN antibodies are conjugated to a solid support (e.g., a scaffold, a resin bead, etc.). In some embodiments, the antibody-based capture method includes physically separating and / or isolating the RAN protein bound by the anti-RAN antibody, for example, by eluting the RAN protein by a chromatographic method such as affinity chromatography or ion exchange chromatography.

[0077] The biological sample may be subjected to an antigen retrieval procedure before being contacted with anti-RAN antibodies. As used herein, "antigen retrieval" (also referred to as epitope retrieval or antigen unmasking) refers to a process of treating a biological sample (e.g., blood, serum, CSF, etc.) under conditions that expose antigens (e.g., epitopes) that were previously inaccessible to detection agents (e.g., antibodies, aptamers, and other binding molecules). In general, antigen retrieval methods include steps including, but not limited to, heating, pressure treatment, enzyme digestion, treatment with reducing agents, treatment with oxidizing agents, treatment with crosslinking agents, treatment with denaturing agents (e.g., detergents, ethanol, acids), or pH changes, or any combination of the above. Several antigen retrieval methods are known in the art, including, but not limited to, protease-induced epitope detection (PIER) and heat-induced epitope detection (HIER). In some embodiments, antigen retrieval procedures reduce background and increase the sensitivity of detection techniques (eg, immunohistochemistry (IHC), immunoblot (such as Western blot), ELISA, etc.).

[0078] The detection of RAN protein in biological samples may be performed by Western blot. Western blot generally uses the use of a detection agent or probe to identify the presence of protein or peptide. In some embodiments, the detection of one or more RAN proteins is performed by immunoblot (e.g., dot blot, 2-D gel electrophoresis, Western blot, etc.), immunohistochemistry (IHC), ELISA (e.g., RCA-based ELISA or rtPCR-based ELISA), label-free immunoassay such as surface plasmon resonance biolayer interferometry, immunoquantitative PCR, mass spectrometry such as GC-MS, LC-MS, MALDI-TOF-MS, bead-based immunoassay, immunoprecipitation, immunostaining, or immunoelectrophoresis. In some embodiments, the detection agent is an antibody. In some embodiments, the antibody is an anti-RAN protein antibody, such as anti-polySer, anti-poly(GR), anti-poly(PR), anti-poly(CP), anti-poly(GP), anti-poly(G), anti-poly(A), anti-poly(GA), anti-poly(GD), anti-poly(GE), anti-poly(GQ), anti-poly(GT), anti-poly(L), anti-poly(LP), anti-poly(LPAC) (SEQ ID NO: 260), anti-poly(LS), anti-poly(P), anti-poly(PA), anti-poly(QAGR) (SEQ ID NO: 261), anti-poly(RE), anti-poly(SP), anti-poly(VP), anti-poly(FP), anti-poly( GK), anti-poly(FTPLSLPV) (SEQ ID NO:262), anti-poly(LLPSPSRC) (SEQ ID NO:263), anti-poly(YSPLPPGV) (SEQ ID NO:264), anti-poly(HREGEGSK) (SEQ ID NO:255), anti-poly(TGRERGVN) (SEQ ID NO:265), anti-poly(PGGRGE) (SEQ ID NO:258), anti-poly(GRQRGVNT) (SEQ ID NO:266), and / or anti-poly(GSKHREAE) (SEQ ID NO:267) (also referred to as α-polySer, α-poly(PR), α-poly(GR), etc.). In some embodiments, the anti-RAN protein antibody targets (e.g., specifically binds to) an amino acid repeat region of RAN protein (e.g., PRPRPRPRPR (SEQ ID NO:106), GRGRGRGRGR (SEQ ID NO:107), SSSSSSSSS (SEQ ID NO:108), etc.).In some embodiments, the anti-RAN protein antibody targets (e.g., specifically binds) an epitope that includes amino acids at the 3' translated C-terminus specific to the unique reading frame of the repeated amino acid. In some embodiments, the anti-RAN protein antibody targets (e.g., specifically binds) an epitope that includes amino acids that bridge the C-terminus of the amino acid repeat region and the 3' N-terminus of the translated C-terminus specific to the unique reading frame of the repeated amino acid.

[0079] In some embodiments, the anti-RAN antibody is directed 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., poly(GR), poly(PR), polySer, poly(CP), poly(GP), poly(G), poly(A), poly(GA), poly(GD), poly(GE), poly(GQ), poly(GT), poly(L), poly(LP), poly(LPAC) (SEQ ID NO: 260), poly(LS), poly(P), poly(PA), poly(QAGR) (SEQ ID NO: 261), poly(RE), poly(SP), poly(SEQ ID NO: 262), poly(SEQ ID NO: 263), poly(SEQ ID NO: 264), poly(SEQ ID NO: 265), poly(SEQ ID NO: 266), poly(SEQ ID NO: 267), poly(SEQ ID NO: 268), poly(SEQ ID NO: 269), poly(SEQ ID NO: 270), poly(SEQ ID NO: 271), poly(SEQ ID NO: 272), poly(SEQ ID NO: 273), poly(SEQ ID NO: 274), poly(SEQ ID NO: 275), poly(SEQ ID NO: 276), poly(SEQ ID NO: 277), poly(SEQ ID NO: 278), poly(SEQ ID NO: 279), poly(SEQ ID NO: 280), poly(SEQ ID NO: 281), poly(SEQ ID NO: 282), poly(SEQ ID NO: 283), poly(SEQ ID NO: 284), poly(SEQ ID NO: 285), poly(SEQ ID NO: 286), poly(SEQ ID NO: 287), poly(SEQ ID NO: 288), poly(SEQ ID NO: 289), poly(SEQ ID NO: 290), poly(SEQ The anti-RAN antibody targets (e.g., specifically binds) the C-terminus of poly(VP), poly(FP), poly(GK), poly(FTPLSLPV) (SEQ ID NO: 262), poly(LLPSPSRC) (SEQ ID NO: 263), poly(YSPLPPGV) (SEQ ID NO: 264), poly(HREGEGSK) (SEQ ID NO: 255), poly(TGRERGVN) (SEQ ID NO: 265), poly(PGGRGE) (SEQ ID NO: 258) protein), poly(GRQRGVNT) (SEQ ID NO: 266), and / or poly(GSKHREAE) (SEQ ID NO: 267). Examples of anti-RAN antibodies targeting RAN protein polyamino acid repeats are disclosed, for example, in International Application Publication No. WO2014 / 159247, the entire contents of which are incorporated herein by reference. 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 by reference herein.In some embodiments, a set (or combination) of anti-RAN antibodies (e.g., anti-polySer, anti-poly(GR), anti-poly(PR), anti-poly(CP), anti-poly(GP), anti-poly(G), anti-poly(A), anti-poly(GA), anti-poly(GD), anti-poly(GE), anti-poly(GQ), anti-poly(GT), anti-poly(L), anti-poly(LP), anti-poly(LPAC) (SEQ ID NO: 260), anti-poly(LS), anti-poly(P), anti-poly(PA), anti-poly(QAGR) (SEQ ID NO: 261), anti-poly(RE), anti-poly(SP), anti-poly(VP), anti-poly(FP), anti-poly(GK), anti-poly(GT ... A combination of two or more anti-RAN antibodies selected from anti-poly(FTPLSLPV) (SEQ ID NO: 262), anti-poly(LLPSPSRC) (SEQ ID NO: 263), anti-poly(YSPLPPGV) (SEQ ID NO: 264), anti-poly(HREGEGSK) (SEQ ID NO: 255), anti-poly(TGRERGVN) (SEQ ID NO: 265), anti-poly(PGGRGE) (SEQ ID NO: 258), anti-poly(GRQRGVNT) (SEQ ID NO: 266), and / or anti-poly(GSKHREAE) (SEQ ID NO: 267) is used to detect one or more RAN proteins in a biological sample.

[0080] Anti-RAN antibodies may 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 of the greater amount of serum that can be recovered. An antigen is injected into the mammal. This induces B lymphocytes to produce IgG immunoglobulins specific for the antigen. This polyclonal IgG is purified from the mammal's serum. Monoclonal antibodies are generally produced by a single cell line (e.g., a hybridoma cell line). In some embodiments, the anti-RAN antibody is purified (e.g., isolated from serum). In some embodiments, the antigen is 12-20 amino acids. For antibodies against a repeat motif, the antigen is a repeat sequence. For antibodies against a 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, e.g., a fragment of the C-terminal sequence that is 3-5 or 5-10, or more, amino acids in length, e.g., 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 (e.g., from one of the C-terminal sequences described herein).

[0081] In some embodiments, the disclosure provides a method of generating an antibody, the method comprising administering to a subject an antibody to a RAN protein repeat sequence, e.g., anti-polySer, anti-poly(GR), anti-poly(PR), anti-poly(CP), anti-poly(GP), anti-poly(G), anti-poly(A), anti-poly(GA), anti-poly(GD), anti-poly(GE), anti-poly(GQ), anti-poly(GT), anti-poly(L), anti-poly(LP), anti-poly(LPAC) (SEQ ID NO: 260), anti-poly(LS), anti-poly(P), anti-poly(PA), anti-poly(QAGR) (SEQ ID NO: 261), anti-poly(RE), anti-poly(GQ), anti-poly(GT), anti-poly(L), anti-poly(LP), anti-poly(LPAC) (SEQ ID NO: 262), anti-poly(LS), anti-poly(P), anti-poly(PA), anti-poly(QAGR) (SEQ ID NO: 263), anti-poly(RE), anti-poly(GQ ... The method includes administering a peptide antigen comprising: anti-poly(SP), anti-poly(VP), anti-poly(FP), anti-poly(GK), anti-poly(FTPLSLPV) (SEQ ID NO: 262), anti-poly(LLPSPSRC) (SEQ ID NO: 263), anti-poly(YSPLPPGV) (SEQ ID NO: 264), anti-poly(HREGEGSK) (SEQ ID NO: 255), anti-poly(TGRERGVN) (SEQ ID NO: 265), anti-poly(PGGRGE) (SEQ ID NO: 258), anti-poly(GRQRGVNT) (SEQ ID NO: 266), and / or anti-poly(GSKHREAE) (SEQ ID NO: 267). In some embodiments, the subject is a mammal, e.g., a non-human primate, a rodent (e.g., a rat, a hamster, a guinea pig, etc.). In some embodiments, the subject is a human (e.g., the subject is injected with a peptide antigen for the purpose of eliciting a host antibody response to the peptide antigen, e.g., a RAN protein). In some embodiments, antibodies are produced by expressing one or more RAN proteins or RAN protein repeat sequences in a cell (eg, a B cell, a hybridoma cell, etc.).

[0082] Numerous methods can be used to obtain anti-RAN antibodies. For example, antibodies can be produced using recombinant DNA methods. Monoclonal antibodies can also be produced by hybridoma production according to known methods (see, for example, Kohler and Milstein (1975) Nature, 256: 495-499). Hybridomas formed in this manner are then screened using standard methods, such as enzyme-linked immunosorbent assay (ELISA; for example, RCA-based ELISA or rtPCR-based ELISA) and surface plasmon resonance (for example, OCTET or BIACORE) analysis, to identify one or more hybridomas that produce antibodies that specifically bind to the identified antigen. Any form of the identified antigen (e.g., RAN protein), such as recombinant antigen, naturally occurring form, any variant or fragment thereof, can be used as an immunogen. One exemplary method of producing antibodies includes screening a protein expression library, such as a phage or ribosome display library, that expresses antibodies or fragments thereof (e.g., scFv). Phage display is described, for example, in Ladner et al., U.S. Pat. No. 5,223,409; Smith (1985) Science 228:1315-1317; Clackson et al. (1991) Nature, 352: 624-628; Marks et al. (1991) J. Mol. Biol., 222: 581-597; WO92 / 18619; WO91 / 17271; WO92 / 20791; WO92 / 15679; WO93 / 01288; WO92 / 01047; WO92 / 09690; and WO90 / 02809.

[0083] In addition to using a display library, the identified antigen (e.g., one or more RAN proteins) may be used to immunize a non-human animal, such as a rodent, such as a mouse, hamster, or rat. In one embodiment, the non-human animal is a mouse.

[0084] In another embodiment, monoclonal antibodies are obtained from non-human animals and then modified, for example, chimerized, using recombinant DNA techniques known in the art.Various approaches to make chimeric antibodies have been described.See, for example, Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851, 1985;Takeda et al., Nature 314:452, 1985;Cabilly et al., U.S. Patent No. 4,816,567;Boss et al., U.S. Patent No. 4,816,397;Tanaguchi et al., European Patent Publication EP171496;European Patent Publication 0173494, British Patent GB2177096B.

[0085] Antibodies can also be humanized by methods known in the art. For example, monoclonal antibodies with desired binding specificity can be commercially humanized (Scotgene, Scotland; and Oxford Molecular, Palo Alto, Calif.). Fully humanized antibodies, such as those expressed in transgenic animals, are within the scope of the present invention (see, for example, Green et al. (1994) Nature Genetics 7, 13; and U.S. Patent Nos. 5,545,806 and 5,569,825).

[0086] For additional antibody production techniques, see Antibodies: A Laboratory Manual, 2nd Edition, edited by Edward A. Greenfield, Dana-Farber Cancer Institute (C) (2014). The present disclosure is not necessarily limited to any particular source, method of production, or other particular characteristics of the antibodies.

[0087] In some embodiments, methods for detecting one or more RAN proteins in a biological sample are useful for monitoring the progression of diseases associated with the expression, translation and / or accumulation of RAN proteins. In some embodiments, the disease 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 degeneration type 1, 2, 3, 6, 7, 8, 10, 12, 17, 31 and 36; spinobulbar muscular atrophy; dentatorubral-pallidoluysian atrophy (DRPLA); Huntington's disease (HD); fragile X tremor ataxia syndrome (FXTAS); Fuchs endothelial corneal dystrophy (FECD); Huntington's disease type 2 syndrome (HDL2); fragile X syndrome (FXS); 7pl l.2 folate-sensitive fragile site FRA7A-related disorder; folate-sensitive fragile site 2ql 1 FRA2A-related disorder; and fragile XE syndrome (FRAXE). In certain embodiments, the neurological disease associated with RAN protein is Alzheimer's disease (AD). For example, in some embodiments, biological samples are obtained from subjects before and after the start of a therapeutic regimen (e.g., 1 week, 2 weeks, 1 month, 6 months, or 1 year later), and the amount of RAN protein detected in the samples is compared.In some embodiments, if the level (e.g., amount) of RAN protein in the post-treatment sample is reduced compared to the level (e.g., amount) of RAN protein before treatment, the therapeutic regimen is successful.In some embodiments, the level of RAN protein in the subject's biological sample (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more samples) is continuously monitored during the therapeutic regimen (e.g., measured at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more separate occasions).

[0088] In some embodiments, the detection agent is an aptamer (e.g., an RNA aptamer, a DNA aptamer, a peptide aptamer). In some embodiments, the aptamer is a RAN protein (e.g., polySer, poly(PR), poly(GR), poly(CP), poly(GP), poly(G), poly(A), poly(GA), poly(GD), poly(GE), poly(GQ), poly(GT), poly(L), poly(LP), poly(LPAC) (SEQ ID NO: 260), poly(LS), poly(P), poly(PA), poly(QAGR) (SEQ ID NO: 261), poly(RE), poly(SP), poly(VP), poly(V ... (FP), poly(GK), poly(FTPLSLPV) (SEQ ID NO:262), poly(LLPSPSRC) (SEQ ID NO:263), poly(YSPLPPGV) (SEQ ID NO:264), poly(HREGEGSK) (SEQ ID NO:255), poly(TGRERGVN) (SEQ ID NO:265), poly(PGGRGE) (SEQ ID NO:258), poly(GRQRGVNT) (SEQ ID NO:266), and / or poly(GSKHREAE) (SEQ ID NO:267)).

[0089] Aspects of the present disclosure relate to nucleic acid hybridization-based methods for identifying the presence of a RAN protein or a microsatellite repeat sequence encoding a RAN protein in a biological sample (e.g., a biological sample obtained from a subject). The present disclosure is based, in part, on methods for detecting a nucleic acid sequence encoding a RAN protein by a detectable nucleic acid probe (e.g., a fluorophore-conjugated DNA probe). In general, a "detectable nucleic acid probe" refers to a nucleic acid sequence that specifically binds (e.g., hybridizes to) a target sequence and includes a detectable moiety, such as a fluorescent moiety, a radioactive moiety, a chemiluminescent moiety, an electroluminescent moiety, biotin, a peptide tag (e.g., poly-His tag, FLAG-tag, etc.), and the like. In some embodiments, the detectable nucleic acid probe includes a region of complementarity to (e.g., a nucleic acid sequence that is complementary to and can hybridize to) a nucleic acid sequence encoding one or more RAN proteins. The region of complementarity can range from about 2 nucleotides in length to about 100 nucleotides in length (e.g., any number of nucleotides from 2 to 100, inclusive). In some embodiments, the nucleic acid probe comprises a region of complementarity with a sequence described in any one of Tables 1, 2 and 3, or a repeat sequence comprising multiple repeats of a sequence described in any one of Tables 1, 2 and 3. In some embodiments, the detectable nucleic acid probe is a DNA probe. In some embodiments, the DNA probe is conjugated with a fluorophore.

[0090] The biological sample may also be contacted with a plurality of detectable nucleic acid probes. The number of the plurality of nucleic acid probes may vary. In some embodiments, the plurality of nucleic acid probes may range from 2 to 100 (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, 108, 110, 111 In some embodiments, the plurality comprises more than 100 probes. The nucleic acid probes may be the same or different sequences. In some embodiments, the plurality of detectable nucleic acid probes comprises a probe that hybridizes to a nucleic acid sequence encoding a poly(GR)RAN protein (e.g., the repeat sequence described in Table 1). In some embodiments, the plurality of detectable nucleic acid probes comprises a probe that hybridizes to a nucleic acid sequence encoding a poly(PR)RAN protein (e.g., a repeat sequence described in Table 2). In some embodiments, the plurality of detectable nucleic acid probes comprises a probe that hybridizes to a nucleic acid sequence encoding a polySerRAN protein (e.g., a repeat sequence described in Table 3).In some embodiments, the plurality of detectable nucleic acid probes are poly(proline-arginine) [poly(PR)]; poly(glycine-arginine) [poly(GR)]; poly(serine) [polySer]; poly(cysteine-proline) [poly(CP)]; poly(glycine-proline) [poly(GP)]; poly(glycine) [poly(G)]; poly(alanine) [polyAla]; poly(glycine-alanine) [poly(GA)]; poly(glycine-Ala) poly(glycine-glutamic acid) [poly(GE)]; poly(glycine-glutamine) [poly(GQ)]; poly(glycine-threonine) [poly(GT)]; poly(leucine) [poly(Leu)]; poly(leucine-proline) [poly(LP)]; poly(leucine-proline-alanine-cysteine) [poly(LPAC)] (SEQ ID NO: 260); poly(leucine-serine) [poly(LS)]; poly(proline) [ poly(P)]; poly(proline-alanine) [poly(PA)]; poly(glutamine-alanine-glycine-arginine) [poly(QAGR)] (SEQ ID NO: 261); poly(arginine-glutamic acid) [poly(RE)]; poly(serine-proline) [poly(SP)], poly(valine-proline) [poly(VP)], poly(phenylalanine-proline) [poly(FP)], poly(glycine-lysine) [poly(GK)], poly(FTPLSLP Poly(V) (SEQ ID NO:262), poly(LLPSPSRC) (SEQ ID NO:263), poly(YSPLPPGV) (SEQ ID NO:264), poly(HREGEGSK) (SEQ ID NO:255), poly(TGRERGVN) (SEQ ID NO:265), poly(PGGRGE) (SEQ ID NO:258), poly(GRQRGVNT) (SEQ ID NO:266), and / or poly(GSKHREAE) (SEQ ID NO:267).

[0091] In some embodiments, the detectable nucleic acid probes are useful for localizing RAN protein translation by Fluorescence In Situ Hybridization (FISH). The method for detecting one or more RAN proteins may include an enrichment step. "Enrichment" refers to the process of increasing the amount and / or concentration of target nucleic acid in a sample relative to other nucleic acids in the sample. In general, enrichment can occur by increasing the number of target nucleic acid sequences in a sample (e.g., by amplifying target sequences, such as by polymerase chain reaction (PCR)) or by decreasing the amount or concentration of non-target nucleic acid sequences in a sample (e.g., by separating or isolating target nucleic acid sequences from non-target sequences).

[0092] In some embodiments, the methods described herein include enriching the biological sample for nucleic acid sequences (e.g., microsatellite repeat sequences) encoding RAN proteins. In some embodiments, enrichment includes contacting the biological sample with 1) a labeled (e.g., biotinylated) dCas9 protein, and 2) one or more single-stranded guide RNAs (sgRNAs) that specifically bind to the nucleic acid repeat sequences encoding RAN proteins. In some embodiments, the labeled dCas9 protein and one or more sgRNAs are provided together as a single molecule (e.g., a dCas9-sgRNA complex). In some embodiments, after contacting the biological sample with the labeled dCas9 protein and one or more sgRNAs, the nucleic acid sequences encoding one or more RAN proteins are isolated from the labeled dCas9 protein and sgRNAs by affinity chromatography, e.g., as described by Liu et al. (2017) Cell 170: 1028-1043.

[0093] In some embodiments, the detection of one or more RAN proteins comprises next-generation sequencing (NGS). In some embodiments, the enrichment step (e.g., dCas9-based enrichment) is performed on the sample using guide RNA. In some embodiments, the guide RNA used in enrichment targets repeats that contain NGG protospacer adjacent motifs (PAMs). In other embodiments, the guide RNA used in enrichment targets non-NGG PAM-containing repeats. In some embodiments, the non-NGG PAM-containing repeats comprise CAG and CTG expansion repeats (e.g., GGGGCC in ALS / FTD and CCTG in DM2). In some embodiments, the guide RNA used in enrichment enriches for non-NGG PAM-containing repeat expansions that are longer than the corresponding normal allele (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100 repeats longer). In some embodiments, the guide RNA used in enrichment simultaneously identifies multiple repeat expansions, which in some embodiments include sequences with non-NGG PAMs.

[0094] Treatment method The present disclosure also contemplates the method of treating the disease associated with the expression, translation and / or accumulation of RAN protein.In some embodiments, the disease 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 degeneration type 1, 2, 3, 6, 7, 8, 10, 12, 17, 31 and 36; spinobulbar muscular atrophy; dentatorubral-pallidoluysian atrophy (DRPLA); Huntington's disease (HD); fragile X tremor ataxia syndrome (FXTAS); Fuchs endothelial corneal dystrophy (FECD); Huntington's disease type 2 syndrome (HDL2); fragile X syndrome (FXS); 7pl l.2 folate-sensitive fragile site FRA7A-related disorder; folate-sensitive fragile site 2ql 1 FRA2A-related disorder; and fragile XE syndrome (FRAXE). In certain embodiments, the neurological disease associated with RAN protein is Alzheimer's disease (AD).In some embodiments, the subject diagnosed with a disease associated with RAN protein by the method described in the present disclosure is administered a therapeutic agent useful for treating the disease associated with RAN protein.

[0095] "Treating" a disease (e.g., AD), as the term is used herein, means reducing the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject. The compositions described above or elsewhere herein are typically administered to a subject in an effective amount, which is an amount capable of producing a desired result. The desired result will depend on the active agent being administered. For example, an effective amount of rAAV particles may be the amount of particles capable of delivering an expression construct to a host cell, tissue, or organ. A therapeutically acceptable amount of an anti-RAN protein antibody may be an amount capable of treating a disease, e.g., Alzheimer's disease, by reducing the expression and / or aggregation of RAN protein, and / or the appearance or number of RNA aggregates that contain microsatellite repeat sequences encoding RAN protein. As is well known in the medical and veterinary fields, the dosage for any one individual subject depends on many factors, including the subject's size, body surface area, age, the particular composition to be administered, the active ingredients in the composition, the time and route of administration, general health, and other drugs being administered at the same time.

[0096] Therapeutic agents useful for treating diseases associated with RAN proteins may be small molecules, proteins, peptides, nucleic acids (e.g., interfering nucleic acids), or gene therapy vectors (e.g., viral vectors encoding therapeutic proteins and / or interfering nucleic acids). Therapeutic agents useful for treating diseases associated with RAN proteins may target (e.g., reduce the expression, activity, accumulation, aggregation, etc.) RAN proteins or nucleic acids encoding RAN proteins, and / or modulate the activity of another gene or gene product (e.g., protein) that interacts with one or more RAN proteins. Examples of genes and gene products that interact with one or more RAN proteins include eukaryotic initiation factor 2 (eIF2), eukaryotic initiation factor 3 (eIF3), protein kinase R (PKR), p62, LC3 I subunit, LC3 II subunit, and Toll-like receptor 3 (TLR3). In some embodiments, the Therapeutic Agent inhibits the expression or activity of one or more of eukaryotic initiation factor 2 (eIF2), eukaryotic initiation factor 3 (eIF3), protein kinase R (PKR), p62, LC3 I subunit, LC3 II subunit, and Toll-like receptor 3 (TLR3).

[0097] In some embodiments, the therapeutic agent is a small molecule. In some embodiments, the small molecule inhibits the expression or activity of one or more RAN proteins. In some embodiments, the small molecule is an inhibitor of eIF3 (or eIF3 subunits). Examples of small molecule inhibitors of eIF3 include, but are not limited to, mTOR inhibitors (e.g., rapamycin, PP242), S6 kinase (S6K) inhibitors, etc.

[0098] In some embodiments, the small molecule inhibits the expression or activity of eukaryotic initiation factor 2A (eIF2A) or eIF2α. Examples of small molecule inhibitors of eIF2A include, but are not limited to, salubrinal, Sal003, ISRIB, etc. In some embodiments, the small molecule is an inhibitor of TARBP2. Examples of TARBP2 inhibitors include anti-TARBP2 antibodies, interfering RNAs (e.g., dsRNA, siRNA, shRNA, miRNA, etc.) that target TARBP2, peptide inhibitors of TARBP2, and small molecule inhibitors of TARBP2. In some embodiments, the small molecule is metformin, also known as N,N-dimethylbiguanide (IUPAC N,N-Dimethylimidodicarbonimidic diamide and CAS 657-24-9), or chloroguanide [1-[amino-(4-chloroanilino)methylidene]-2-propan-2-yl-guanidine, CAS 500-92-5], chlorproguanil [1-[amino-(3,4-dichloroanilino)methylidene]-2-propan-2-ylguanidine, CAS 537-21-3], buformin [N-butylimidodicarbonimidic diamide, CAS 692-13-7], or phenformin [2-(N-phenethylcarbamimidoyl)guanidine, CAS 692-13-7]. 114-86-3], or an alternative biologically active biguanide, including a pharma- ceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug of any of the biguanides.

[0099] The term "pharmaceutically acceptable salt" refers to a salt that is suitable, within the scope of sound medical judgment, for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, etc., commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, which is incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by using other methods known in the art, such as ion exchange. Other pharma- ceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogensulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, and the like. Salts derived from suitable bases include sulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Salts derived from suitable bases include alkali metal, alkaline earth metal, ammonium and N + (C 1-4 Alkyl) 4-Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. Further pharma- ceutically acceptable salts include non-toxic ammonium, quaternary ammonium and amine cations, formed where appropriate with counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkylsulfonates, and arylsulfonates.

[0100] The term "solvate" refers to a form of a compound that is associated with a solvent, usually through a solvolysis reaction. This physical association may include hydrogen bonding. Conventional solvents include water, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, and the like. Metformin can be prepared, for example, in crystalline form and can be solvated. Suitable solvates include pharmaceutically acceptable solvates, and further include both stoichiometric and non-stoichiometric solvates. In some cases, for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid, the solvate will be capable of being isolated. "Solvate" also encompasses both solution-phase and insoluble solvates. Representative solvates include hydrates, ethanolates, and methanolates.

[0101] The term "hydrate" refers to a compound associated with water. Typically, the number of water molecules contained in a hydrate of a compound is in a fixed ratio to the number of compound molecules in the hydrate. Thus, a hydrate of a compound can be represented, for example, by the general formula R·xH2O, where R is a compound and where x is a number greater than 0. A given compound can form more than one type of hydrate, including, for example, monohydrates (x is 1), lower hydrates (x is a number greater than 0 and less than 1; e.g., hemihydrates (R·0.5H2O)), and polyhydrates (x is a number greater than 1; e.g., dihydrates (R·2H2O) and hexahydrates (R·6H2O)).

[0102] The term "tautomer" or "tautomeric" refers to two or more interconvertible compounds resulting from the formal shift of at least one hydrogen atom and at least one change in valency (e.g., from a single bond to a double bond, a triple bond to a single bond, or vice versa). The exact ratio of tautomers depends on several factors, including temperature, solvent, and pH. Tautomerization (i.e., the reaction that provides a tautomeric pair) can be catalyzed by acid or base. Exemplary tautomerizations include keto to enol, amide to imide, lactam to lactim, enamine to imine, and enamine to (different enamine) tautomerization.

[0103] It should also be understood that compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or the arrangement of their atoms in space are termed "isomers." Isomers that differ in the arrangement of their atoms in space are termed "stereoisomers."

[0104] Stereoisomers that are not mirror images of one another are called "diastereomers" and are superimposable. Those that are mirror images of each other are called "enantiomers". If a compound is bonded to four different groups, for example, a pair of enantiomers is possible. Enantiomers can be characterized by the absolute configuration of their chiral centers. The molecules are described by the R and S ordering rules of Cahn and Prelog, or by the As dextrorotatory or levorotatory depending on the way it rotates through the plane (i.e., (+) or (-) respectively). Chiral compounds are represented as individual enantiomers, or They can exist as mixtures thereof. Mixtures containing equal proportions of enantiomers Such a substance is called a "racemic mixture."

[0105] The term "prodrug" refers to a compound that has a cleavable group and becomes a compound described herein that is pharmacologic in vivo by solvolysis or under physiological conditions. Examples of such include, but are not limited to, choline ester derivatives, N-alkylmorpholine esters, and the like. Other derivatives of the compounds described herein are active in their acid and acid derivative forms, but often offer the advantage of solubility, tissue compatibility, or delayed release in the mammalian organism in the acid-sensitive form (see Bundgard, H., Design of Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam 1985). Prodrugs include acid derivatives well known to those skilled in the art, such as esters prepared by reacting the parent acid with a suitable alcohol, or amides, anhydrides, or mixed anhydrides prepared by reacting the parent acid compound with a substituted or unsubstituted amine. Simple aliphatic or aromatic esters, amides and anhydrides derived from the acidic groups pendant on the compounds described herein are particular prodrugs. In some cases, it is desirable to prepare double ester type prodrugs, such as (acyloxy)alkyl esters or ((alkoxycarbonyl)oxy)alkyl esters. The C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, aryl, C7-C8 alkyl, C8-C9 alkyl, C9-C10 alkyl, C10-C12 alkyl, C10-C14 alkyl, C10-C16 alkyl, C10-C18 ... 12 Substituted aryl, and C7-C 12 Arylalkyl esters may be preferred. In some embodiments, the small molecule is buformin or phenformin.

[0106] The therapeutic agent may be an anti-RAN protein antibody. In some embodiments, the anti-RAN protein antibody is selected from the group consisting of anti-poly-serine, anti-poly(GR), anti-poly(PR), anti-poly(CP), anti-poly(GP), anti-poly(G), anti-poly(A), anti-poly(GA), anti-poly(GD), anti-poly(GE), anti-poly(GQ), anti-poly(GT), anti-poly(L), anti-poly(LP), anti-poly(LPAC) (SEQ ID NO: 260), anti-poly(LS), anti-poly(P), anti-poly(PA), anti-poly(QAGR) (SEQ ID NO: 261), anti-poly(RE), anti-poly(SP), anti-poly(VP), anti-poly(FP), anti-poly(GK). , anti-poly(FTPLSLPV) (SEQ ID NO: 262), anti-poly(LLPSPSRC) (SEQ ID NO: 263), anti-poly(YSPLPPGV) (SEQ ID NO: 264), anti-poly(HREGEGSK) (SEQ ID NO: 255), anti-poly(TGRERGVN) (SEQ ID NO: 265), anti-poly(PGGRGE) (SEQ ID NO: 258), anti-poly(GRQRGVNT) (SEQ ID NO: 266), and / or anti-poly(GSKHREAE) (SEQ ID NO: 267) antibodies (also referred to as α-polySer, α-poly(PR), α-poly(GR), etc.). The anti-RAN protein antibody may bind to extracellular RAN protein, intracellular RAN protein, or both extracellular and intracellular RAN protein.

[0107] In some embodiments, the anti-RAN protein antibody targets (e.g., specifically binds to) an amino acid repeat region of the RAN protein (e.g., PRPRPRPRPR (SEQ ID NO: 106), GRGRGRGRGR (SEQ ID NO: 107), SSSSSSSS (SEQ ID NO: 108), etc.). Examples of anti-RAN antibodies that target RAN protein polyamino acid repeats are disclosed, for example, in International Application Publication No. WO2014 / 159247, the entire contents of which are incorporated herein by reference.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 list: poly(proline-arginine) [poly(PR)]; poly(glycine-arginine) [poly(GR)]; poly(serine) [polySer]; poly(cysteine-proline) [poly(CP)]; poly(glycine-proline) [poly(GP)]; poly(glycine) [poly (G)]; poly(alanine) [polyAla]; poly(glycine-alanine) [poly(GA)]; poly(glycine-aspartic acid) [poly(GD)]; poly(glycine-glutamic acid) [poly(GE)]; poly(glycine-glutamine) [poly(GQ)]; poly(glycine-threonine) [poly(GT)]; poly(leucine) [polyLeu]; poly(leucine-proline) [poly(LP)]; poly(leucine-proline-alanine-cysteine) [poly(L poly(PAC)] (SEQ ID NO:260); poly(leucine-serine) [poly(LS)]; poly(proline) [poly(P)]; poly(proline-alanine) [poly(PA)]; poly(glutamine-alanine-glycine-arginine) [poly(QAGR)] (SEQ ID NO:261); poly(arginine-glutamic acid) [poly(RE)]; poly(serine-proline) [poly(SP)], poly(valine-proline) [poly(VP)], poly(phenylalanine-proline) [poly(P)] poly(FP)], poly(glycine-lysine) [poly(GK)], poly(FTPLSLPV) (SEQ ID NO:262), poly(LLPSPSRC) (SEQ ID NO:263), poly(YSPLPPGV) (SEQ ID NO:264), poly(HREGEGSK) (SEQ ID NO:255), poly(TGRERGVN) (SEQ ID NO:265), poly(PGGRGE) (SEQ ID NO:258), poly(GRQRGVNT) (SEQ ID NO:266), and poly(GSKHREAE) (SEQ ID NO:267).

[0108] In some embodiments, the anti-RAN antibody is directed to any portion of the RAN protein that does not contain the polyamino acid repeats, such as the C-terminus of the RAN protein (e.g., poly(CP), poly(GP), poly(G), poly(A), poly(GA), poly(GD), poly(GE), poly(GQ), poly(GR), poly(GT), poly(L), poly(LP), poly(LPAC) (SEQ ID NO: 260), poly(LS), poly(P), poly(PA), poly(PR), poly(QAGR) (SEQ ID NO: 261), poly(RE), polySer, poly(SP), poly(Ser ... The anti-RAN antibody targets (e.g., specifically binds to) the C-terminus of poly(VP), poly(FP), poly(GK), poly(FTPLSLPV) (SEQ ID NO: 262), poly(LLPSPSRC) (SEQ ID NO: 263), poly(YSPLPPGV) (SEQ ID NO: 264), poly(HREGEGSK) (SEQ ID NO: 255), poly(TGRERGVN) (SEQ ID NO: 265), poly(PGGRGE) (SEQ ID NO: 258) protein, poly(GRQRGVNT) (SEQ ID NO: 266), and / or poly(GSKHREAE) (SEQ ID NO: 267). Examples of anti-RAN antibodies that target the C-terminus of RAN protein are disclosed, for example, in U.S. Publication No. 2013 / 0115603, the entire contents of which are incorporated herein by reference.In some embodiments, a set (or combination) of anti-RAN antibodies (e.g., poly(CP), poly(GP), poly(G), poly(A), poly(GA), poly(GD), poly(GE), poly(GQ), poly(GR), poly(GT), poly(L), poly(LP), poly(LPAC) (SEQ ID NO: 260), poly(LS), poly(P), poly(PA), poly(PR), poly(QAGR) (SEQ ID NO: 261), poly(RE), polySer, poly(SP), poly(VP), poly(FP), poly(GK), poly(FTP), poly(LPS), poly(LPAC ... A combination of two or more anti-RAN antibodies selected from poly(PV) (SEQ ID NO: 262), poly(LLPSPSRC) (SEQ ID NO: 263), poly(YSPLPPGV) (SEQ ID NO: 264), poly(HREGEGSK) (SEQ ID NO: 255), poly(TGRERGVN) (SEQ ID NO: 265), poly(PGGRGE) (SEQ ID NO: 258), poly(GRQRGVNT) (SEQ ID NO: 266), and poly(GSKHREAE) (SEQ ID NO: 267), etc., is administered to a subject for the purpose of treating a disease associated with the RAN protein.

[0109] Anti-RAN antibodies may 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 of the greater amount of serum that can be collected. Antigen is injected into the mammal. This induces B lymphocytes to produce IgG immunoglobulin specific for the antigen. The polyclonal IgG is purified from the mammal's serum. 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).

[0110] The therapeutic molecule may be an antisense oligonucleotide (ASO). Generally, antisense oligonucleotides block translation of a target protein by hybridizing to an mRNA sequence encoding the target protein, thereby inhibiting protein synthesis by the ribosomal machinery. In some embodiments, the antisense oligonucleotide (ASO) targets a gene that contains a microsatellite repeat sequence. In some embodiments, the antisense oligonucleotide inhibits translation of one or more RAN proteins. Those skilled in the art will understand how to construct antisense oligonucleotides that contain short (about 15-30) nucleotides with a base sequence complementary to the RAN mRNA. Those skilled in the art will understand that the complementarity of RNA to mRNA can be established using canonical nucleotides, including ribose, phosphate, and one of the bases adenine, guanine, cytosine and uracil bridged by phosphodiester bridges, which typically represent naturally occurring nucleic acids, or that some of the nucleotides can be modified by replacing ribose with alternative sugar moieties, such as 2'-deoxyribose or 2'-O-(2-methoxyethyl)ribose, and / or that some or all of the nucleotides can be modified by methylation, and / or that some or all of the phosphodiester bonds between nucleotides can be replaced by phosphorothioate bridges. Those skilled in the art will understand that modification of some nucleotides at both the 3' and 5' ends of antisense oligonucleotides to inhibit degradation by ubiquitous terminally active RNA nucleases will improve the stability of antisense oligonucleotides, thereby improving their half-life. However, one of skill in the art will understand that it may be desirable for at least a portion of the antisense oligos to promote the activity of RNase H after complexing with the RAN mRNA and promote the enzymatic degradation of the RAN mRNA after it is complexed with the antisense oligos.

[0111] In some embodiments, the therapeutic agent is an inhibitory nucleic acid. In some embodiments, the inhibitory nucleic acid is an interfering RNA selected from the group consisting of dsRNA, siRNA, shRNA, miRNA and ami-RNA. In some embodiments, the inhibitory nucleic acid is a nucleic acid aptamer (e.g., RNA aptamer or DNA aptamer). In general, the inhibitory RNA molecule can be unmodified or modified. In some embodiments, the inhibitory RNA molecule comprises one or more modified oligonucleotides, such as phosphorothioate-, 2'-O-methyl-, etc. modified oligonucleotides, because such modifications are recognized in the art as improving the stability of oligonucleotides in vivo.

[0112] In some embodiments, the therapeutic agent is an effective amount of an agent that inhibits eukaryotic initiation factor 2 (eIF2) or an agent that inhibits protein kinase R (PKR) (e.g., an inhibitor of eIF2 and / or PKR). In some embodiments, the inhibitor of eIF2 is an inhibitor of serine / threonine kinase. Examples of serine / threonine kinase include, but are not limited to, protein kinase A (PKA), protein kinase C (PKC), Mos / Raf kinase, mitogen-activated protein kinase (MAPK), protein kinase B (AKT kinase), and the like. In some embodiments, the eIF2 inhibitor is a protein kinase R (PKR) inhibitor. Inhibitors of eIF2 and PKR are described, for example, in International Application Publication No. WO2018 / 195110, the entire contents of which are incorporated herein by reference.

[0113] In some embodiments, the therapeutic agent is a protein kinase R (PKR) variant that functions in a dominant negative manner to inhibit the phosphorylation of eIF2α.As used herein, "protein kinase R (PKR) variant" refers to a protein that comprises an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to wild-type protein kinase R (PKR) (e.g., GenBank accession number NP_002750.1), where the variant protein comprises at least one amino acid variation (also sometimes referred to as "mutation") compared to the amino acid sequence of wild-type PKR.

[0114] In some embodiments, the amino acid sequence of the PKR variant is at least 75%, at least 85%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical to the amino acid sequence of wild-type PKR. In some embodiments, the amino acid sequence is about 95-99.9% identical to the amino acid sequence of wild-type PKR. In some embodiments, the protein comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 different amino acid sequence variations compared to the sequence of amino acids set forth in the amino acid sequence of wild-type PKR. In some embodiments, the PKR variant comprises a mutation at position 296 (e.g., position 296 of human wild-type PKR). In some embodiments, the mutation at position 296 is K296R.

[0115] An eIF2 inhibitor may be a direct inhibitor or an indirect inhibitor. In general, a direct regulator functions by interacting with (e.g., interacting with or binding to) a gene encoding eIF2 (or eIF2α) or an eIF2 protein complex. In general, an indirect regulator functions by interacting with a gene or protein that regulates the expression or activity of eIF2 or eIF2α (e.g., does not directly interact with a gene or protein encoding eIF2 or eiF2α).

[0116] In some embodiments, the inhibitor of eIF2 or PKR is a selective inhibitor. "Selective inhibitor" refers to an inhibitor of eIF2 or PKR that preferentially inhibits the activity or expression of one type of eIF2 subunit compared to other types of eIF2 subunits, or that preferentially inhibits the activity or expression of PKR compared to other kinases. In some embodiments, the inhibitor of eIF2 is a selective inhibitor of eIF2α. In some embodiments, the inhibitor of eIF2 is a selective inhibitor of eIF2A. In some embodiments, the inhibitor of eIF2 is a selective inhibitor of protein kinase R (PKR), such as a selective PKR inhibitor.

[0117] Examples of proteins that inhibit eiF2 (e.g., eIF2 subunits) include, but are not limited to, polyclonal anti-eIF2 antibodies, monoclonal anti-eIF2 antibodies, etc. Examples of nucleic acid molecules that inhibit eiF2 (e.g., eIF2 subunits) include, but are not limited to, dsRNA, siRNA, miRNA, etc. that target genes that code for eIF2 subunits (e.g., genes that code for the mRNAs described in GenBank Accession No. NM_004094.4). Examples of small molecule inhibitors of eIF2 include, but are not limited to, LY 364947, eIF-2α inhibitor II Sal003, etc.

[0118] Examples of proteins that inhibit PKR include, but are not limited to, certain dominant-negative PKR variants (e.g., K296R PKR mutant), TARBP2, etc. Examples of nucleic acid molecules that inhibit PKR include, but are not limited to, dsRNA, siRNA, miRNA, etc. that target the gene encoding PKR. Examples of small molecule inhibitors of PKR include, but are not limited to, 6-amino-3-methyl-2-oxo-N-phenyl-2,3-dihydro-1H-benzo[d]imidazole-1-carboxamide, N-[2-(1H-indol-3-yl)ethyl]-4-(2-methyl-1H-indol-3-yl)pyrimidin-2-amine, metformin, buformin, phenformin, etc.

[0119] Examples of nucleic acid molecules that inhibit eIF2A include, but are not limited to, dsRNA, siRNA, miRNA, etc. that target the gene that codes for eIF2A (e.g., the gene that codes for the mRNA described in GenBank Accession No. NM_032025.4). Examples of small molecule inhibitors of eIF2A include, but are not limited to, salubrinal, Sal003, ISRIB, etc.

[0120] In some embodiments, the eIF2 inhibitor or PKR inhibitor is an interfering (e.g., inhibitory) nucleic acid. In some embodiments, the inhibitory nucleic acid is an interfering RNA selected from the group consisting of dsRNA, siRNA, shRNA, mi-RNA and ami-RNA. In some embodiments, the inhibitory nucleic acid is an antisense nucleic acid (e.g., an antisense oligonucleotide (ASO) or a nucleic acid aptamer (e.g., an RNA aptamer). In general, the inhibitory RNA molecule may be unmodified or modified. In some embodiments, the inhibitory RNA molecule comprises one or more modified oligonucleotides, such as phosphorothioate-, 2'-O-methyl-, etc.-modified oligonucleotides, since such modifications are recognized in the art as improving the stability of oligonucleotides in vivo.

[0121] In some embodiments, the interfering RNA comprises a sequence that is complementary to 5 to 50 contiguous nucleotides (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, about 30, about 35, about 40 or about 50 contiguous nucleotides) of a nucleic acid sequence (e.g., an RNA sequence) encoding an eIF2 subunit or a nucleic acid sequence (e.g., an RNA sequence) encoding PKR.

[0122] In some embodiments, the therapeutic agent is an inhibitor of eukaryotic initiation factor 3 (eIF3), which is a multiprotein complex involved in the initiation phase of eukaryotic protein translation. Generally, in humans, eIF3 contains 13 non-identical subunits (e.g., eIF3a-m). Mammalian eIF3, the largest and most complex initiation factor, contains up to 13 non-identical subunits. Typically, eIF3f is involved in many steps of translation initiation, including stabilizing the tertiary complex, mediating the binding of mRNA to the 40S subunit, and promoting the dissociation of the 40S and 60S ribosomal subunits. In some embodiments, a therapeutic agent that inhibits the expression or activity of a subunit of eIF3 (e.g., eIF3f, eIF3m, eIF3h, or other eIF3 subunits) can be used to reduce or inhibit RAN translation in a cell or in a subject (e.g., a subject with Alzheimer's disease, which is characterized by RAN protein translation). Inhibitors of the eIF3 subunit are further described, for example, in International Application Publication No. WO2017 / 176813, the entire contents of which are incorporated herein by reference.

[0123] An eIF3 inhibitor may be a direct inhibitor or an indirect inhibitor. In general, a direct regulator functions by interacting with (e.g., interacting with or binding to) a gene encoding eIF3 (or an eIF3 subunit), or an eIF3 protein complex, or an eIF3 subunit. In general, an indirect regulator functions by interacting with a gene or protein that regulates the expression or activity of eIF3 or an eIF3 subunit (e.g., does not directly interact with a gene or protein encoding eIF3 or an eIF3 subunit). In some embodiments, an inhibitor of eIF3 is a selective inhibitor. A "selective inhibitor" refers to a regulator of eIF3 that preferentially inhibits the activity or expression of one type of eIF3 subunit compared to other types of eIF3 subunits. In some embodiments, an inhibitor of eIF3 is a selective inhibitor of eIF3f.

[0124] The eIF3 inhibitor may be a protein (e.g., an antibody), a nucleic acid, or a small molecule. Examples of proteins that inhibit eiF3 (e.g., eIF3 subunits) include, but are not limited to, polyclonal anti-eIF3 antibodies, monoclonal anti-eIF3 antibodies, measles virus N protein, viral stress-inducible protein p56, and the like. Examples of nucleic acid molecules that inhibit eiF3 (e.g., eIF3 subunits) include, but are not limited to, dsRNA, siRNA, miRNA, amiRNA, and the like that target genes encoding eIF3 subunits. Examples of small molecule inhibitors of eIF3 include, but are not limited to, mTOR inhibitors (e.g., rapamycin, PP242), S6 kinase (S6K) inhibitors, and the like.

[0125] In some embodiments, the interfering RNA comprises a sequence that is complementary to between 5 and 50 contiguous nucleotides (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, about 30, about 35, about 40 or about 50 contiguous nucleotides) of a nucleic acid sequence (e.g., an RNA sequence) encoding an eIF3 subunit. Examples of nucleic acid sequences encoding eIF3 subunits include GenBank Accession No. NM_003750.2 (eIF3a), GenBank Accession No. NM_003751.3 (eIF3b), GenBank Accession No. NM_003752.4 (eIF3c), GenBank Accession No. NM_003753.3 (eIF3d), GenBank Accession No. NM_001568.2 (eIF3e), GenBank Accession No. NM_003754.2 (eIF3f), GenBank Accession No. NM_003755.2 (eIF3g), GenBank Accession No. NM_003756.2 (eIF3h), GenBank Accession No. NM_003757.2 (eIF3i), GenBank Accession No. NM_003758.2 (eIF3j), GenBank Accession No. NM_003759.2 (eIF3k), GenBank Accession No. NM_003751.3 (eIF3l), GenBank Accession No. NM_003752.4 (eIF3j), GenBank Accession No. NM_003753.3 (eIF3k), GenBank Accession No. NM_003754.2 (eIF3k), GenBank Accession No. NM_003755.2 (eIF3i), GenBank Accession No. NM_003756.2 (eIF3k), GenBank Accession No. NM_003757.2 (eIF3k), GenBank Accession No. NM_003758.2 (eIF3k), GenBank Accession No. NM_003759.2 (eIF3k), GenBank Accession No. NM_003751.3 (eIF3l), GenBank Accession No. NM_003751 Accession No. NM_003755.4 (eIF3g), GenBank Accession No. NM_003756.2 (eIF3h), GenBank Accession No. NM_003757.3 (eIF3i), GenBank Accession No. NM_003758.3 (eIF3j), GenBank Accession No. NM_013234.3 (eIF3k), GenBank Accession No. NM_016091.3 (eIF3l), GenBank Accession No. NM_006360.5 (eIF3m), and the like. In some embodiments, the interfering RNA is siRNA. In some embodiments, siRNA of eIF3f is administered (e.g., Dharmacon Cat # J-019535-08). In some embodiments, siRNA of eIF3m is administered (e.g., Dharmacon Cat # J-016219-12). In some embodiments, an siRNA of eIF3h is administered (eg, Dharmacon Cat # J-003883-07).

[0126] In some embodiments, eIF3f is a negative regulator of RAN translation, and the reduction in the level of human eIF3f is associated with the reduction in the accumulation of RAN protein in cells.In some embodiments, RAN translation (e.g., in cells expressing RAN protein) is sensitive to eIF3f knockdown, unlike the translation from nearby cognates or the translation of AUG.In some embodiments, the translation machinery used for RAN translation can be distinguished from the translation machinery of AUG and near AUG in cells.

[0127] In some embodiments, the therapeutic agent is an inhibitor of TLR3. The inhibitor of TLR3 can be a protein (e.g., an antibody), a nucleic acid, or a small molecule. Examples of proteins that inhibit TLR3 include, but are not limited to, polyclonal anti-TLR3 antibodies, monoclonal anti-TLR3 antibodies, and the like. Examples of nucleic acid molecules that inhibit TLR3 include, but are not limited to, dsRNA, siRNA, miRNA, amiRNA, and the like that target the gene that codes for TLR3. Examples of small molecule inhibitors of TLR3 are described, for example, in Cheng et al. (2011) J Am Chem Soc 133(11):3764-7.

[0128] In some embodiments, the therapeutic agent is an inhibitor of p62 protease. The inhibitor of p62 can be a protein (e.g., an antibody), a nucleic acid, or a small molecule. Examples of proteins that inhibit p62 include, but are not limited to, polyclonal anti-p62 antibodies, monoclonal anti-p62 antibodies, and the like. Examples of nucleic acid molecules that inhibit p62 include, but are not limited to, dsRNA, siRNA, miRNA, amiRNA, and the like that target the gene encoding p62. In some embodiments, the therapeutic agent is an agent that increases proteasome activity, for example, as described in Leestemaker et al. (2017) Cell Chemical Biology 24, 725-736.

[0129] In some embodiments, the therapeutic agent comprises a peptide antigen that targets one or more RAN proteins (e.g., is a RAN protein vaccine that targets one or more RAN proteins). In some embodiments, the peptide antigen targets (e.g., comprises an amino acid sequence encoding) one or more of the following RAN proteins: poly(proline-arginine) [poly(PR)]; poly(glycine-arginine) [poly(GR)]; poly(serine) [polySer]; poly(cysteine-proline) [poly(CP)]; poly(glycine-proline) [poly(GP)]; poly(glycine) [poly(G)]; poly(alanine) [PolyAla]; Poly(glycine-alanine) [Poly(GA)]; Poly(glycine-aspartic acid) [Poly(GD)]; Poly(glycine-glutamic acid) [Poly(GE)]; Poly(glycine-glutamine) [Poly(GQ)]; Poly(glycine-threonine) [Poly(GT)]; Poly(leucine) [PolyLeu]; Poly(leucine-proline) [Poly(LP)]; Poly(leucine-proline-alanine-cysteine) [Poly(LPAC)] (Sequence No. No. 260; poly(leucine-serine) [poly(LS)]; poly(proline) [poly(P)]; poly(proline-alanine) [poly(PA)]; poly(glutamine-alanine-glycine-arginine) [poly(QAGR)] (SEQ ID NO: 261); poly(arginine-glutamic acid) [poly(RE)]; poly(serine-proline) [poly(SP)], poly(valine-proline) [poly(VP)], poly(phenylalanine-proline) [poly(FP )], poly(glycine-lysine) [poly(GK)], poly(FTPLSLPV) (SEQ ID NO:262), poly(LLPSPSRC) (SEQ ID NO:263), poly(YSPLPPGV) (SEQ ID NO:264), poly(HREGEGSK) (SEQ ID NO:255), poly(TGRERGVN) (SEQ ID NO:265), poly(PGGRGE) (SEQ ID NO:258), poly(GRQRGVNT) (SEQ ID NO:266), and poly(GSKHREAE) (SEQ ID NO:267).

[0130] In some embodiments, one or more therapeutic molecules are administered to a subject to treat a disease associated with a RAN protein characterized by an expansion of nucleic acid repeats (e.g., associated with 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).

[0131] Monoclonal antibodies Various aspects of the present disclosure relate to antibodies and antigen-binding fragments that specifically bind to RAN protein, as well as methods of making and using the same. In some embodiments, the antibody or antigen-binding fragment specifically binds to one or more of the following: poly(glycine-alanine) [poly(GA)], poly(proline-arginine) [poly(PR)], poly(glycine-arginine) [poly(GR)], poly-serine (polySer), poly(glycine-proline) [poly(GP)], poly-leucine (polyLeu), poly-alanine (polyAla), poly(leucine-proline-alanine-cysteine) [poly(LPAC)] (SEQ ID NO: 260), and poly(glutamine-alanine-glycine-arginine) [poly(QAGR)] (SEQ ID NO: 261). In some embodiments, the antibody or antigen-binding fragment specifically binds to poly(GA). In some embodiments, the antibody or antigen-binding fragment specifically binds to polySer. In some embodiments, the antibody or antigen-binding fragment specifically binds to poly(PR). In some embodiments, the antibody or antigen-binding fragment specifically binds to poly(GR). In some embodiments, the antibody or antigen-binding fragment specifically binds to poly-Leu. In some embodiments, the antibody or antigen-binding fragment specifically binds to poly-Ala. In some embodiments, the antibody or antigen-binding fragment specifically binds to poly(LPAC) (SEQ ID NO: 260). In some embodiments, the antibody or antigen-binding fragment specifically binds to poly(QAGR) (SEQ ID NO: 261). In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody or antigen-binding fragment specifically binds to poly(CP). In some embodiments, the antibody or antigen-binding fragment specifically binds to poly(GP). In some embodiments, the antibody or antigen-binding fragment specifically binds to poly(G). In some embodiments, the antibody or antigen-binding fragment specifically binds to poly(GD). In some embodiments, the antibody or antigen-binding fragment specifically binds to poly(GE).In some embodiments, the antibody or antigen-binding fragment specifically binds to poly(GQ). In some embodiments, the antibody or antigen-binding fragment specifically binds to poly(GT). In some embodiments, the antibody or antigen-binding fragment specifically binds to poly(LP). In some embodiments, the antibody or antigen-binding fragment specifically binds to poly(LS). In some embodiments, the antibody or antigen-binding fragment specifically binds to poly(P). In some embodiments, the antibody or antigen-binding fragment specifically binds to poly(PA). In some embodiments, the antibody or antigen-binding fragment specifically binds to poly(RE). In some embodiments, the antibody or antigen-binding fragment specifically binds to poly(SP). In some embodiments, the antibody or antigen-binding fragment specifically binds to poly(VP). In some embodiments, the antibody or antigen-binding fragment specifically binds to poly(FP). In some embodiments, the antibody or antigen-binding fragment specifically binds to poly(GK). In some embodiments, the antibody or antigen-binding fragment specifically binds to poly(FTPLSLPV) (SEQ ID NO: 262). In some embodiments, the antibody or antigen-binding fragment specifically binds to poly(LLPSPSRC) (SEQ ID NO: 263). In some embodiments, the antibody or antigen-binding fragment specifically binds to poly(YSPLPPGV) (SEQ ID NO: 264). In some embodiments, the antibody or antigen-binding fragment specifically binds to poly(HREGEGSK) (SEQ ID NO: 255). In some embodiments, the antibody or antigen-binding fragment specifically binds to poly(TGRERGVN) (SEQ ID NO: 265). In some embodiments, the antibody or antigen-binding fragment specifically binds to poly(PGGRGE) (SEQ ID NO: 258). In some embodiments, the antibody or antigen-binding fragment specifically binds to poly(GRQRGVNT) (SEQ ID NO: 266). In some embodiments, the antibody or antigen-binding fragment specifically binds to poly(GSKHREAE) (SEQ ID NO: 267).

[0132] Antibody, as used herein, broadly refers to an immunoglobulin molecule or any functional mutant, variant or derivative thereof. The functional mutant, variant and derivative thereof, as well as the antigen-binding fragment, desirably retain the essential epitope-binding properties of an Ig molecule. An antibody can specifically bind to a target through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. Generally, a complete or full-length antibody comprises two heavy chains and two light chains. Each heavy chain comprises a heavy chain variable region (VH) and a first, second and third constant region (CH1, CH2 and CH3). Each light chain comprises a light chain variable region (VL) and a constant region (CL). The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). The CDR components on the heavy chain are referred to as CDRH1, CDRH2 and CDRH3, while the CDR components on the light chain are referred to as CDRL1, CDRL2 and CDRL3.

[0133] CDR typically refers to Kabat CDR as described in the sequence of Proteins of Immunological Interest (US Department of Health and Human Services (1991), eds. Kabat et al.). Another standard for characterizing antigen-binding sites should refer to the hypervariable loops as described by Chothia. See, for example, Chothia, D. et al. (1992) J. Mol. Biol. 227:799-817; and Tomlinson et al. (1995) EMBO J. 14:4628-4638. Yet another standard is the AbM definition used by Oxford Molecular's AbM antibody modeling software. See generally, for example, Protein Sequence and Structure Analysis of Antibody Variable Domains (in Antibody Engineering Lab Manual (Duebel, S and Kontermann, R. eds., Springer-Verlag, Heidelberg)). The embodiments described with respect to the Kabat CDRs can alternatively be implemented with respect to the Chothia hypervariable loops, or similarly as described with respect to the AbM defined loops, or using any combination of these methods.

[0134] Each VH and VL consists of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A full-length antibody can be of any class, such as IgD, IgE, IgG, IgA or IgM (or subclasses thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant domain of its heavy chain, an immunoglobulin can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma and mu, respectively. The subunit structures and three-dimensional conformations of the different classes of immunoglobulins are well known.

[0135] The term "antigen-binding fragment" refers to any derivative of an antibody that is less than the full length and can specifically bind to a target. Preferably, the antigen-binding fragment provided herein retains the ability to specifically bind to an RAN protein. The antigen-binding fragment may include a heavy chain variable region (VH), a light chain variable region (VL), or both. Each of the VH and VL typically includes three complementarity determining regions CDR1, CDR2, and CDR3.

[0136] Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, scFv, Fv, dsFv, diabody, affibody (affibody®) and Fd fragments. Antigen-binding fragments can be produced by any suitable means. For example, antigen-binding fragments may be produced by enzymatic or chemical fragmentation of intact antibodies, or they may be produced recombinantly from genes encoding partial antibody sequences. Alternatively, antigen-binding fragments may be wholly or partially synthetically produced. Antigen-binding fragments may be single-chain antibody fragments. Alternatively, the fragments may comprise multiple chains linked together, for example, by disulfide bridges. Antigen-binding fragments may also optionally be multimolecular complexes. Functional antigen-binding fragments will typically contain at least about 50 amino acids, more typically at least about 200 amino acids.

[0137] Single-chain Fv (scFv) is a recombinant antigen-binding fragment consisting of only a variable light chain (VL) and a variable heavy chain (VH) covalently linked to each other by a polypeptide linker. Either VL or VH can be an NH2-terminal domain. The polypeptide linker can be of various lengths and compositions, so long as the two variable domains are bridged without significant steric interference. Typically, the linker is composed mainly of a stretch of glycine and serine residues, with some glutamic acid or lysine residues interspersed for solubility. ScFv is encompassed in the term "antigen-binding fragment".

[0138] Diabodies are dimeric scFvs. Diabody components typically have shorter peptide linkers than most scFvs, and they show a preference for associating as dimers (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al. (1994) Structure 2: 1121-1123). Diabodies are also encompassed within the term "antigen-binding fragment".

[0139] Fv fragment is an antigen-binding fragment consisting of one VH and one VL domain held together by non-covalent interactions. The two domains of Fv fragment, VL and VH, may be encoded by separate genes, but they may be linked by a synthetic linker that allows them to be made into a single protein chain by recombinant methods, where the pair of VL and VH domains forms a monovalent molecule (known as single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed in the term "antigen-binding fragment" of an antibody. The term dsFv is used herein to refer to an Fv with an intermolecular disulfide bond engineered to stabilize the VH-VL pair. dsFv are also encompassed within the term "antigen-binding fragment."

[0140] F(ab')2 fragment is an antigen-binding fragment essentially equivalent to that obtained by digestion of immunoglobulin (typically IgG) with the enzyme pepsin at pH 4.0-4.5. The fragment may be recombinantly produced. F(ab')2 is also encompassed within the term "antigen-binding fragment."

[0141] A Fab fragment is an antigen-binding fragment essentially equivalent to that obtained by reduction of the disulfide bridges linking the two heavy chain pieces in a F(ab')2 fragment. Fab' fragments may be recombinantly produced. Fab' is also encompassed within the term "antigen-binding fragment."

[0142] A Fab fragment is an antigen-binding fragment essentially equivalent to that obtained by digestion of an immunoglobulin (typically an IgG) with the enzyme pepsin. A Fab fragment may be produced recombinantly. The heavy chain segment of a Fab fragment is the Fd piece. A Fab fragment is also encompassed in the term "antigen-binding fragment".

[0143] Affibodies are small proteins that contain a bundle of three helices that function as antigen-binding molecules (e.g., antibody mimics). In general, affibodies are about 58 amino acids long and have a molar mass of about 6 kDa. Affibody molecules with unique binding properties are obtained by randomization of 13 amino acids located in two alpha helices that are involved in the binding activity of the parent protein domain. Specific affibody molecules that bind to the desired target protein can be isolated from a pool (library) containing billions of different variants using methods such as phage display. Affibodies are also encompassed in the term "antigen-binding fragments."

[0144] The term "human antibody" refers to an antibody having variable and constant regions that substantially correspond to or are derived from an antibody obtained from a human subject, for example, encoded by a human germline immunoglobulin sequence or a variant thereof. A human antibody may contain one or more amino acid residues not encoded by a human germline immunoglobulin sequence (e.g., mutations induced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). Such mutations may be present in one or more of the CDRs, particularly CDR3, or in one or more of the framework regions. In some embodiments, a human antibody may have at least one, two, three, four, five, or more positions replaced with an amino acid residue not encoded by a human germline immunoglobulin sequence. However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0145] The term "recombinant human antibody," as used herein, refers to any human antibody that is prepared, expressed, generated or isolated by recombinant means, e.g., antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library (Hoogenboom HR, (1997) TIB Tech. 15:62-70; Azzazy H., and Highsmith WE, (2002) Clin. Biochem. 35:425-445; Gavilondo JV, and Larrick JW (2002) BioTechniques 29: 128-145; Hoogenboom H., and Chames P. (2000) Immunology Today 21:371-378), antibodies isolated from animals (e.g., mice) transgenic for human immunoglobulin genes (e.g., Taylor, LD, et al. (1992) Immunology Today 21:371-378), and antibodies isolated from animals (e.g., mice) transgenic for human immunoglobulin genes (e.g., Taylor, LD, et al. (1992) Immunology Today 21:371-378). Nucl. Acids Res. 20:6287-6295; Kellermann SA., and Green LL (2002) Current Opinion in Biotechnology 13:593-597; Little M. et al (2000) Immunology Today 21:364-370), or any other means including splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions as defined above. In certain embodiments, however, such recombinant human antibodies may be subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis) such that the amino acid sequences of the VH and VL regions of the recombinant antibodies, while derived from and related to human germline VH and VL sequences, may not naturally occur in the human antibody germline repertoire in vivo.

[0146] In some embodiments, the antibody or antigen-binding fragment specifically binds to an amino acid sequence as set forth in any one or more of SEQ ID NOs: 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, and 56.

[0147] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain constant region comprising the amino acid sequence represented by SEQ ID NO: 195. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain constant region comprising the amino acid sequence represented by SEQ ID NO: 197. In some embodiments, the anti-RAN antibodies and antigen-binding fragments of the present disclosure comprise a light chain constant region comprising the amino acid sequence represented by SEQ ID NO: 196. In some embodiments, the anti-RAN antibodies and antigen-binding fragments of the present disclosure comprise a light chain constant region comprising the nucleic acid sequence represented by SEQ ID NO: 198.

[0148] In some embodiments, the anti-RAN antibody or antigen-binding fragment may or may not include an antibody framework region, such as a framework region amino acid sequence set forth in SEQ ID NOs: 155-186. In some embodiments, the anti-RAN antibody is a murine antibody. In some embodiments, the anti-RAN antibody is a chimeric or humanized antibody.

[0149] In some embodiments, the antibody or antigen-binding fragment comprises a VH sequence as set forth in SEQ ID NO: 109, 111, 113, or 115. In some embodiments, the antibody or antigen-binding fragment comprises a VH sequence as set forth in SEQ ID NO: 110, 112, 114, or 116. In some embodiments, the antibody or antigen-binding fragment comprises a VH sequence as set forth in SEQ ID NO:109, and a VL sequence as set forth in SEQ ID NO:110.

[0150] In some embodiments, the antibody or antigen-binding fragment comprises a VH sequence as set forth in SEQ ID NO:111, and a VL sequence as set forth in SEQ ID NO:112. In some embodiments, the antibody or antigen-binding fragment comprises a VH sequence as set forth in SEQ ID NO:113, and a VL sequence as set forth in SEQ ID NO:114.

[0151] In some embodiments, the antibody or antigen-binding fragment comprises a VH sequence as set forth in SEQ ID NO:115, and a VL sequence as set forth in SEQ ID NO:116. In some embodiments, the antibody or antigen-binding fragment comprises six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3, where CDRH1 comprises the sequence as set forth in SEQ ID NO: 117, CDRH2 comprises the sequence as set forth in SEQ ID NO: 125, CDRH3 comprises the sequence as set forth in SEQ ID NO: 133, CDRL1 comprises the sequence as set forth in SEQ ID NO: 118, CDRL2 comprises the sequence as set forth in SEQ ID NO: 126, and CDRL3 comprises the sequence as set forth in SEQ ID NO: 134.

[0152] In some embodiments, the antibody or antigen-binding fragment comprises six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3, where CDRH1 comprises the sequence as set forth in SEQ ID NO: 119, CDRH2 comprises the sequence as set forth in SEQ ID NO: 127, CDRH3 comprises the sequence as set forth in SEQ ID NO: 135, CDRL1 comprises the sequence as set forth in SEQ ID NO: 120, CDRL2 comprises the sequence as set forth in SEQ ID NO: 128, and CDRL3 comprises the sequence as set forth in SEQ ID NO: 136.

[0153] In some embodiments, the antibody or antigen-binding fragment comprises six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3, where CDRH1 comprises the sequence as set forth in SEQ ID NO: 121, CDRH2 comprises the sequence as set forth in SEQ ID NO: 129, CDRH3 comprises the sequence as set forth in SEQ ID NO: 137, CDRL1 comprises the sequence as set forth in SEQ ID NO: 122, CDRL2 comprises the sequence as set forth in SEQ ID NO: 130, and CDRL3 comprises the sequence as set forth in SEQ ID NO: 138.

[0154] In some embodiments, the antibody or antigen-binding fragment comprises six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3, where CDRH1 comprises the sequence as set forth in SEQ ID NO: 123, CDRH2 comprises the sequence as set forth in SEQ ID NO: 131, CDRH3 comprises the sequence as set forth in SEQ ID NO: 139, CDRL1 comprises the sequence as set forth in SEQ ID NO: 124, CDRL2 comprises the sequence as set forth in SEQ ID NO: 132, and CDRL3 comprises the sequence as set forth in SEQ ID NO: 140.

[0155] It should be understood that in some embodiments, the present disclosure contemplates variants (e.g., homologs) of amino acid and nucleic acid sequences for the heavy and light chain variable regions of the antibody. "Homology" refers to the percentage of identity between two polynucleotides or two polypeptide moieties. The term "substantial homology," when referring to a nucleic acid or a fragment thereof, indicates that when optimally aligned with another nucleic acid (or its complementary strand) by appropriate nucleotide insertion or deletion, there is nucleotide sequence identity in about 90-100% of the aligned sequence. For example, in some embodiments, nucleic acid sequences that share substantial homology are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity. The term "substantial homology" when referring to a polypeptide or fragment thereof indicates that when aligned with another optimally with appropriate gaps, insertions or deletions, there is nucleotide sequence identity in about 90-100% of the aligned sequences. The term "highly conserved" means at least 80% identity, preferably at least 90% identity, and more preferably greater than 97% identity. For example, in some embodiments, highly conserved proteins share at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity. In some cases, highly conserved may refer to 100% identity. Identity is readily determined by one of skill in the art, for example, using algorithms and computer programs known to those of skill in the art.

[0156] In some embodiments, the RAN antibodies of the present disclosure have high affinity, e.g., 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 Lower than M, 10 -11The anti-RAN protein antibody can bind to the RAN protein with a Kd of 1000 nM or less. For example, the anti-RAN antibody or antigen-binding fragment can bind to the RAN protein with an affinity of 5 pM to 500 nM, e.g., 50 pM to 100 nM, e.g., 500 pM to 50 nM. The present disclosure also includes antibodies or antigen-binding fragments that compete for binding to the RAN protein with any of the antibodies described herein and have an affinity of 50 nM or less (e.g., 20 nM or less, 10 nM or less, 500 pM or less, 50 pM or less, or 5 pM or less). The affinity and binding kinetics of the anti-RAN protein antibody can be tested using any method known in the art, including, but not limited to, biosensor technology (e.g., OCTET or BIACORE).

[0157] In some embodiments, the anti-RAN antibodies of the disclosure comprise a VH, a VL and a CDR, and the amino acid sequences are shown in Table 4 below. Table 4 - Amino acid sequences of anti-RAN antibodies [Table A-1] [Table A-2]

[0158] Table 5 - Nucleic acid sequences of anti-RAN antibodies [Table B-1] [Table B-2] [Table B-3] Table 6 - Nucleic acid and amino acid framework sequences of anti-RAN antibodies [Table C-1] [Table C-2] [Table C-3] Table 7 - Constant region sequences [Table D-1] [Table D-2] [Table D-3]

[0159] In some embodiments, antibody clone 27B11.A7 binds to polyGA. In some embodiments, clone 27B11.A7 is an IgG1 antibody. In some embodiments, antibody clone 23H2.D1.B5 binds to polyGA. In some embodiments, antibody clone 23H2.D1.B5 is an IgG3 antibody. In some embodiments, antibody clone 16A3.C8 binds to polySer. In some embodiments, antibody clone 16A3.C8 is an IgG1 antibody. In some embodiments, antibody clone HL2362-2G4 binds to polyPR. In some embodiments, antibody clone HL2362-2G4 is an IgG2A kappa antibody.

[0160] Anti-RAN antibody can be used to treat or assist in the treatment of one or more symptoms of disease associated with RAN protein.In some embodiments, the disease 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 degeneration type 1, 2, 3, 6, 7, 8, 10, 12, 17, 31 and 36; spinobulbar muscular atrophy; dentatorubral-pallidoluysian atrophy (DRPLA); Huntington's disease (HD); fragile X tremor ataxia syndrome (FXTAS); Fuchs endothelial corneal dystrophy (FECD); Huntington's disease type 2 syndrome (HDL2); fragile X syndrome (FXS); 7pl l.2 folate-sensitive fragile site FRA7A-related disorder; folate-sensitive fragile site 2ql 1 FRA2A-related disorder; and fragile XE syndrome (FRAXE). In a particular embodiment, the neurological disease associated with RAN protein is Alzheimer's disease (AD).

[0161] In some embodiments, anti-RAN antibodies can be used to treat or aid in the treatment of one or more symptoms of a disease associated with RAN protein, for example, by administering a therapeutically effective amount of one or more anti-RAN antibodies to a subject diagnosed as having one or more symptoms of a disease associated with RAN protein (e.g., early stage Alzheimer's disease) or at risk of developing a disease associated with RAN protein (e.g., based on one or more assays described herein). In some embodiments, one or more of the anti-RAN antibodies or antigen-binding fragments disclosed herein are administered to a subject, where the subject has been characterized as having a disease associated with RAN protein by detection of at least one RAN protein in a biological sample obtained from the subject.

[0162] Generation of anti-RAN antibodies Typically, polyclonal antibodies are produced by inoculation of a suitable mammal, such as a mouse, rabbit, or goat. An antigen is injected into the mammal. This induces B lymphocytes to produce IgG immunoglobulin 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).

[0163] Exemplary anti-RAN antibodies disclosed herein were generated using the antigens set forth in Table 8. In some embodiments, the antigen comprises a RAN protein repeat sequence selected from: poly(proline-arginine) [poly(PR)]; poly(glycine-arginine) [poly(GR)]; poly(serine) [polySer]; poly(cysteine-proline) [poly(CP)]; poly(glycine-proline) [(poly(GP)]; poly(glycine) [poly(G)]; poly(alanine) [polyAla]; poly(glycine-alanine) [poly(GA)]; poly(glycine-aspartic acid) [poly(GD)]; poly(glycine-glutamic acid) [poly(GE)]; poly(glycine-glutamine) [poly(GQ)]; poly(glycine-threonine) [poly(GT)]; poly(leucine) [polyLeu]; poly(leucine-proline) [poly(LP)]; poly(leucine-proline-alanine-cysteine) [poly(LPAC)] (SEQ ID NO: 260); poly(leucine) -serine) [poly(LS)]; poly(proline) [poly(P)]; poly(proline-alanine) [poly(PA)]; poly(glutamine-alanine-glycine-arginine) [poly(QAGR)] (SEQ ID NO: 261); poly(arginine-glutamic acid) [poly(RE)]; poly(serine-proline) [poly(SP)], poly(valine-proline) [poly(VP)], poly(phenylalanine-proline) [poly(FP)], poly(glutamic acid) [poly(P ... lysine-lysine) [poly(GK)], poly(FTPLSLPV) (SEQ ID NO:262), poly(LLPSPSRC) (SEQ ID NO:263), poly(YSPLPPGV) (SEQ ID NO:264), poly(HREGEGSK) (SEQ ID NO:255), poly(TGRERGVN) (SEQ ID NO:265), poly(PGGRGE) (SEQ ID NO:258), poly(GRQRGVNT) (SEQ ID NO:266), and poly(GSKHREAE) (SEQ ID NO:267).

[0164] Table 8 - Antigens for generating RAN antibodies [Table E]

[0165] Numerous methods can be used to obtain anti-RAN antibodies. For example, antibodies can be produced using recombinant DNA methods. Monoclonal antibodies can also be produced by hybridoma production according to known methods (see, for example, Kohler and Milstein (1975) Nature, 256: 495-499). Hybridomas formed in this manner are then screened using standard methods, such as enzyme-linked immunosorbent assay (ELISA; for example, RCA-based ELISA or rtPCR-based ELISA) and surface plasmon resonance (for example, OCTET or BIACORE) analysis, to identify one or more hybridomas that produce antibodies that specifically bind to a particular antigen. Any form of the identified antigen (e.g., RAN protein), such as recombinant antigen, naturally occurring form, any variant or fragment thereof, can be used as an immunogen. One exemplary method of producing antibodies includes screening a protein expression library, such as a phage or ribosome display library, that expresses antibodies or fragments thereof (e.g., scFv). Phage display is described, for example, in Ladner et al., U.S. Pat. No. 5,223,409; Smith (1985) Science 228: 1315-1317; Clackson et al. (1991) Nature, 352: 624-628; Marks et al. (1991) J. Mol. Biol., 222: 581-597; WO92 / 18619; WO91 / 17271; WO92 / 20791; WO92 / 15679; WO93 / 01288; WO92 / 01047; WO92 / 09690; and WO90 / 02809.

[0166] In another embodiment, monoclonal antibodies are obtained from non-human animals and then modified, for example, chimerized, using recombinant DNA techniques known in the art.Various approaches to produce chimeric antibodies have been described.See, for example, Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851, 1985;Takeda et al., Nature 314:452, 1985;Cabilly et al., U.S. Patent No. 4,816,567;Boss et al., U.S. Patent No. 4,816,397;Tanaguchi et al., European Patent Publication EP171496;European Patent Publication 0173494, British Patent GB2177096B.

[0167] Antibodies can also be humanized by methods known in the art. For example, monoclonal antibodies with desired binding specificity can be commercially humanized (Scotgene, Scotland; and Oxford Molecular, Palo Alto, Calif.). Fully humanized antibodies, such as those expressed in transgenic animals, are within the scope of the present invention (see, for example, Green et al. (1994) Nature Genetics 7, 13; and U.S. Pat. Nos. 5,545,806 and 5,569,825). For further antibody production techniques, see Antibodies: A Laboratory Manual, Second Edition. Edited by Edward A. Greenfield, Dana-Farber Cancer Institute(C) (2014). This disclosure is not necessarily limited to any particular source, method of production, or particular characteristics of the antibody.

[0168] Some aspects of the disclosure relate to isolated cells (e.g., host cells) transformed with a polynucleotide or vector. The host cell may be a prokaryotic or eukaryotic cell. The polynucleotide or vector present in the host cell may be integrated into the genome of the host cell or maintained extrachromosomally. The host cell may be any prokaryotic or eukaryotic cell, such as a bacterial, insect, fungal, plant, animal or human cell. In some embodiments, the fungal cell is, for example, of the genus Saccharomyces, particularly of the species S. cerevisiae. The term "prokaryotic" includes all bacteria that can be transformed or transfected with DNA or RNA molecules for the expression of an antibody or the corresponding immunoglobulin chain. Prokaryotic hosts may include gram-negative as well as gram-positive bacteria, such as, for example, E. coli, S. typhimurium, Serratia marcescens and Bacillus subtilis. The term "eukaryotic" includes yeast, higher plants, insects and vertebrate cells, such as mammalian cells, such as NSO and CHO cells. Depending on the host used in a recombinant production procedure, the antibody or immunoglobulin chain encoded by the polynucleotide may be glycosylated or non-glycosylated. The antibody or the corresponding immunoglobulin chain may also include an initial methionine amino acid residue.

[0169] In some embodiments, after the vector is incorporated into a suitable host, the host may be maintained under suitable conditions for high level expression of the nucleotide sequence, followed by recovery and purification of immunoglobulin light chains, heavy chains, light / heavy chain dimers, or complete antibodies, antigen-binding fragments, or other immunoglobulin forms, if desired; see Beychok, Cells of Immunoglobulin Synthesis, Academic Press, NY, (1979). Thus, the polynucleotide or vector is introduced into a cell, which then produces the antibody or antigen-binding fragment. Furthermore, transgenic animals, preferably mammals, containing the aforementioned host cells may be used for the production of antibodies or antibody fragments on a large scale.

[0170] The transformed host cells can be grown in fermenters and cultured to achieve optimal cell growth according to techniques known in the art. Once expressed, complete antibodies, their dimers, individual light and heavy chains, other immunoglobulin forms, or antigen-binding fragments can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis, and the like; see Scopes, "Protein Purification", Springer Verlag, NY (1982). The antibodies or antigen-binding fragments can then be isolated from the growth medium, cell lysates, or cell membrane fractions. Isolation and purification of antibodies or antigen-binding fragments, e.g., expressed by microorganisms, can be by any conventional means, such as, for example, preparative chromatographic separation and immunological separation (e.g., including the use of monoclonal or polyclonal antibodies against the constant regions of the antibodies).

[0171] Aspects of the present disclosure relate to hybridomas, which provide a source of monoclonal antibodies that are indefinitely extended. As used herein, "hybridoma cells" refer to immortalized cells derived from the fusion of B lymphoblast cells with myeloma fusion partners. To prepare monoclonal antibody-producing cells (e.g., hybridoma cells), individual animals (e.g., mice) whose antibody titers have been confirmed are selected, and their spleens or lymph nodes are harvested 2 to 5 days after the final immunization, and the antibody-producing cells contained therein are fused with myeloma cells to prepare hybridomas that are producers of the desired monoclonal antibodies. Measurement of antibody titers in antisera can be performed, for example, by reacting the antisera with a labeled protein as described later in this specification, and then measuring the activity of the labeling agent bound to the antibody. Cell fusion can be performed according to known methods, for example, the method described by Kochler and Milstein (Nature 256:495 (1975)). Fusion promoters, such as polyethylene glycol (PEG) or Sendai virus (HVJ), are used.

[0172] Examples of myeloma cells include NS-1, P3U1, SP2 / 0, and AP-1. The ratio of the number of antibody-producing cells (spleen cells) to be used to the number of myeloma cells is preferably about 1:1 to about 20:1. PEG (preferably PEG1000 to PEG6000) is added at a concentration of preferably about 10% to about 80%. Cell fusion can be efficiently carried out by incubating a mixture of both cells at about 20°C to about 40°C, preferably about 30°C to about 37°C, for about 1 to 10 minutes.

[0173] A variety of methods can be used to screen for hybridomas that produce antibodies (e.g., against tumor antigens or autoantibodies of the present invention).For example, here, the supernatant of hybridoma is added to the solid phase (e.g., microplate) on which the antibody is adsorbed directly or together with a carrier, and then the anti-immunoglobulin antibody (when mouse cells are used in cell fusion, anti-mouse immunoglobulin antibody is used) or protein A that is labeled with radioactive substance or enzyme is added to detect the monoclonal antibody against the protein that is bound to the solid phase. Alternatively, the supernatant of hybridoma is added to the solid phase on which the anti-immunoglobulin antibody or protein A is adsorbed, and then the protein that is labeled with radioactive substance or enzyme is added to detect the monoclonal antibody against the protein that is bound to the solid phase.

[0174] Selection of monoclonal antibodies can be performed according to any known method or a modified method thereof. Usually, a medium for animal cells supplemented with HAT (hypoxanthine, aminopterin, thymidine) is used. Any selection and growth medium can be used as long as the hybridoma can grow. For example, RPMI 1640 medium containing 1% to 20%, preferably 10% to 20% fetal bovine serum, GIT medium containing 1% to 10% fetal bovine serum, serum-free medium for culturing hybridomas (SFM-101, Nissui Seiyaku), etc. can be used. Usually, the culture is performed at 20°C to 40°C, preferably 37°C, for about 5 days to 3 weeks, preferably 1 week to 2 weeks, under about 5% CO2 gas. The antibody titer of the supernatant of the hybridoma culture can be measured according to the same manner as described above for the antibody titer of the antiprotein in the antiserum.

[0175] As an alternative to obtaining immunoglobulins directly from hybridoma cultures, immortalized hybridoma cells may be used as a source for subsequent expression and / or genetic manipulation of rearranged heavy and light chain loci. Rearranged antibody genes may be reverse transcribed from appropriate mRNA to generate cDNA. If desired, the heavy chain constant region may be replaced with that of a different isotype or removed entirely. Variable regions may be linked to encode a single chain Fv region. Multiple Fv regions may be linked or heavy and light chain combinations may be used to confer binding capacity to more than one target. Any suitable method may be used for cloning antibody variable regions and generating recombinant antibodies.

[0176] In some embodiments, the appropriate nucleic acid encoding the variable region of the heavy and / or light chain is obtained and inserted into an expression vector that can be transfected into a standard recombinant host cell. A variety of such host cells can be used. In some embodiments, mammalian host cells may be advantageous for efficient processing and production. Exemplary mammalian cell lines useful for this purpose include CHO cells, 293 cells, or NSO cells. Antibodies or antigen-binding fragments can be produced by culturing the modified recombinant host under appropriate conditions for host cell growth and expression of the coding sequence. Antibodies or antigen-binding fragments can be recovered by isolating them from the culture. The expression system may be designed to include a signal peptide so that the resulting antibody is secreted into the medium; however, intracellular production is also possible.

[0177] The present disclosure also includes a polynucleotide encoding at least the variable region of an immunoglobulin chain of an antibody described herein. In some embodiments, the variable region encoded by the polynucleotide comprises at least one complementarity determining region (CDR) of the VH and / or VL of the variable region of an antibody produced by any one of the hybridomas described above.

[0178] The polynucleotide encoding the antibody or antigen-binding fragment may be, for example, DNA, cDNA, RNA, or synthetically produced DNA or RNA, or recombinantly produced chimeric nucleic acid molecule, comprising any of these polynucleotides alone or in combination. In some embodiments, the polynucleotide is part of a vector. Such vectors may contain additional genes, such as marker genes, that allow the selection of the vector in a suitable host cell and under suitable conditions.

[0179] In some embodiments, the polynucleotide is operably linked to an expression control sequence that allows expression in prokaryotic or eukaryotic cells. Expression of the polynucleotide includes transcription of the polynucleotide into a translatable mRNA. Regulatory elements that ensure expression in eukaryotic cells, preferably mammalian cells, are well known to those skilled in the art. They may include regulatory sequences that facilitate initiation of transcription, and optionally polyA signals that facilitate termination of transcription and stabilization of the transcript. Further regulatory elements may include transcriptional and translational enhancers, and / or naturally associated or heterologous promoter regions. Possible regulatory elements that allow expression in prokaryotic host cells include, for example, PL, Lac, Trp or Tac promoters in E. coli, and examples of regulatory elements that allow expression in eukaryotic host cells are AOX1 or GAL1 promoters in yeast, or CMV-promoter, SV40-promoter, RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40-enhancer or globin intron in mammalian and other animal cells.

[0180] Besides the elements responsible for the initiation of transcription, such regulatory elements may also contain transcription termination signals downstream of the polynucleotide, such as the SV40-polyA site or the tk-polyA site. Furthermore, depending on the expression system used, leader sequences capable of directing the polypeptide to a cellular compartment or secreting it into the medium may be added to the coding sequence of the polynucleotide, which are well known in the art. The leader sequence is assembled with the translation, initiation and termination sequences at the appropriate time, and preferably the leader sequence is capable of directing the secretion of the translated protein or a portion thereof, for example, into the extracellular medium. Optionally, heterologous polynucleotide sequences may be used that code for fusion proteins containing C- or N-terminal identification peptides that confer desired characteristics, such as stabilization or convenient purification of the expressed recombinant product.

[0181] In some embodiments, the polynucleotides encoding at least the variable domains of the light and / or heavy chains may encode the variable domains of both immunoglobulin chains or only one of them. Similarly, the polynucleotides may be under the control of the same promoter or may be separately controlled for expression. Furthermore, some aspects relate to vectors, particularly plasmids, cosmids, viruses and bacteriophages routinely used in genetic engineering, that contain a polynucleotide encoding a variable domain of an immunoglobulin chain of an antibody or antigen-binding fragment (optionally in combination with a polynucleotide encoding a variable domain of the other immunoglobulin chain of the antibody).

[0182] In some embodiments, the expression control sequences are provided as eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells, although control sequences for prokaryotic hosts can also be used. Expression vectors derived from viruses such as retroviruses, vaccinia viruses, adeno-associated viruses, herpes viruses, or bovine papilloma viruses can be used for delivery of polynucleotides or vectors into target cell populations (e.g., to engineer cells to express antibodies or antigen-binding fragments). A variety of suitable methods can be used to construct recombinant viral vectors. In some embodiments, polynucleotides and vectors can be reconstituted into liposomes for delivery to target cells. Vectors containing polynucleotides (e.g., heavy and / or light chain variable domains and expression control sequences of sequences encoding immunoglobulin chains) can be transferred into host cells by suitable methods that vary depending on the type of cellular host.

[0183] qualification Some aspects of the present disclosure relate to antibody-drug conjugates targeted to one or more RAN proteins. As used herein, "antibody-drug conjugate" refers to a molecule comprising an antibody or an antigen-binding fragment thereof linked to a targeted molecule (e.g., a biologically active molecule, such as a therapeutic molecule, and / or a detectable label). Thus, in some embodiments, the antibody or antigen-binding fragment of the present disclosure may be modified with a detectable label, including, but not limited to, an enzyme, a prosthetic group, a fluorescent material, a luminescent material, a bioluminescent material, a radioactive material, a positron-emitting metal, a non-radioactive paramagnetic metal ion, and an affinity label, for the detection and isolation of one or more RAN proteins. The detectable substance can be coupled or conjugated to the polypeptide of the present disclosure directly or indirectly through an intermediate (e.g., a linker known in the art, etc.) using techniques known in the art. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, or acetylcholinesterase; non-limiting examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; non-limiting examples of suitable fluorescent materials include biotin, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; an example of a luminescent material includes luminol; non-limiting examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive materials include radioactive metal ions, such as alpha emitters or other radioisotopes, such as iodine ( 131 I, 125 I, 123 I, 121 I), Carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), Indium ( 115 mIn, 113mIn, 112 In, 111 In), and technetium ( 99 Tc, 99 mTc), thallium ( 201 Ti), Gallium ( 68 Ga, 67 Ga), palladium ( 103 Pd), Molybdenum ( 99 Mo), Xenon ( 133 Xe), fluorine ( 18 F), 153 Sm, Lu, 159 Gd, 149 Pm, 140 La, 175 Yb, 166 Ho, 90 Y, 47 Sc, 86 R, 188 Re, 142 Pr, 105 Rh, 97 Ru, 68 Ge, 57 Co, 65 Zn, 85 Sr, 32 P, 153 Gd, 169 Yb, 51 Cr, 54 Mn, 75 Se, and tin ( 113 Sn, 117 Sn). The detectable substance can be coupled or conjugated to the anti-RAN antibody or antigen-binding fragment of the present disclosure directly or indirectly through an intermediate (e.g., a linker known in the art, etc.) using techniques known in the art. The anti-RAN antibody conjugated to the detectable substance can be used for diagnostic assays as described herein.

[0184] In some embodiments, the antibody or antigen-binding fragment of the present disclosure may be modified with a therapeutic moiety (e.g., a therapeutic agent). In some embodiments, the antibody is coupled to the targeted agent via a linker. As used herein, the term "linker" refers to a molecule or sequence, such as an amino acid sequence, that attaches one molecule or sequence to another, such as in a crosslinker. "Linked," "conjugated," or "coupled" means attached or bound by a covalent bond, or a non-covalent bond, or other bond, such as van der Waals forces. The antibody described by the present disclosure can be linked to the targeted agent (e.g., a therapeutic or detectable moiety) directly, for example, as a fusion protein with a protein or peptide detectable moiety (with or without any linking sequence, such as a flexible linker sequence), or via a chemical coupling moiety. Many such coupling moieties are known in the art, for example, peptide linkers or chemical linkers, such as those described in International Patent Application Publication No. WO2009 / 036092. In some embodiments, the linker is a flexible amino acid sequence. Examples of flexible amino acid sequences include glycine and serine-rich linkers, which include stretches of two or more glycine residues. In some embodiments, the linker is a photolinker. Examples of photolinkers include ketyl-reactive benzophenone (BP), anthraquinone (AQ), nitrene-reactive nitrophenyl azide (NPA), and carbene-reactive phenyl-(trifluoromethyl)diazirine (PTD).

[0185] Pharmaceutical Compositions In some aspects, the disclosure relates to pharmaceutical compositions comprising anti-RAN antibodies or antigen-binding fragments. In some embodiments, the compositions comprise anti-RAN antibodies and a pharma- ceutically acceptable carrier. As used herein, the term "pharma- ceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharma- ceutical active substances is well known in the art. Except insofar as a conventional media or agent is incompatible with the active compound, its use in the composition is contemplated. Supplementary active compounds can also be incorporated into the composition. The pharmaceutical compositions can be prepared as described below. The active ingredient can be mixed or combined with any conventional pharma- ceutical acceptable carrier or excipient. The compositions can be sterile.

[0186] Typically, pharmaceutical compositions are formulated to deliver an effective amount of an agent (e.g., an anti-RAN antibody). In general, an "effective amount" of an active agent refers to an amount sufficient to induce a desired biological response (e.g., ameliorating one or more symptoms of Alzheimer's disease). The effective amount of an agent can vary depending on factors such as the desired biological endpoint, the pharmacokinetics of the compound, the disease being treated (e.g., Alzheimer's disease, repeat expansion disease), the mode of administration, and the patient.

[0187] A composition is said to be a "pharmaceutically acceptable carrier" if its administration can be tolerated by a recipient patient. Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier. Other suitable carriers are well known in the art. See, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (1990).

[0188] It will be understood by those skilled in the art that any route of administration, vehicle or carrier that is commonly used and inert with respect to the active agent may be utilized to prepare and administer the pharmaceutical composition of the present disclosure. Illustrative of such methods, vehicles and carriers are described, for example, in Remington's Pharmaceutical Sciences, 4th Edition (1970), the disclosure of which is incorporated herein by reference. A person skilled in the art who is informed by the principles of the present disclosure will have no difficulty in determining suitable and appropriate vehicles, excipients and carriers, or in combining the active ingredient therewith to form the pharmaceutical composition of the present disclosure.

[0189] An effective amount of a compound (e.g., an anti-RAN antibody), also referred to as a therapeutically effective amount, is an amount effective to ameliorate at least one adverse effect associated with a disease associated with a RAN protein, such as memory loss, cognitive impairment, loss of coordination, speech disorders, etc. In some embodiments, the neurological disease 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 degeneration type 1, 2, 3, 6, 7, 8, 10, 12, 17, 31 and 36; spinobulbar muscular atrophy; dentatorubral-pallidoluysian atrophy (DRPLA); Huntington's disease (HD); fragile X tremor ataxia syndrome (FXTAS); Fuchs endothelial corneal dystrophy (FECD); Huntington's disease type 2 syndrome (HDL2); fragile X syndrome (FXS); 7pl l.2 folate-sensitive fragile site FRA7A-related disorder; folate-sensitive fragile site 2ql 1 FRA2A-related disorder; and fragile XE syndrome (FRAXE). In certain embodiments, the neurological disease associated with RAN protein is Alzheimer's disease (AD). The therapeutically effective amount to be included in the pharmaceutical composition depends on several factors, such as the type, size and condition of the patient to be treated, the intended mode of administration, the ability of the patient to incorporate the intended dosage form, etc. In general, the amount of active agent is included in each dosage form to provide about 0.1 to about 250 mg / kg, and preferably about 0.1 to about 100 mg / kg. Those skilled in the art will be able to empirically determine the appropriate therapeutically effective amount.

[0190] In combination with the teachings provided herein, by selecting from among a variety of active compounds and weighing factors such as potency, relative bioavailability, patient weight, severity of adverse side effects, and the mode of administration selected, an effective prophylactic or therapeutic treatment regimen can be designed that does not cause substantial toxicity but is overall effective for treating a particular subject.The effective amount for any particular application may vary depending on factors such as the disease or condition being treated, the particular therapeutic agent being administered, the size of the subject, or the severity of the disease or condition.Those skilled in the art can empirically determine the effective amount of a particular nucleic acid and / or other therapeutic agent without the need for undue experimentation.

[0191] In some cases, the compounds of the present disclosure are prepared in a colloidal dispersion system. Colloidal dispersion systems include liquid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. In some embodiments, the colloidal system of the present disclosure is a liposome. Liposomes are artificial membrane vessels useful as delivery vectors in vivo or in vitro. Large unilamellar vesicles (LUVs), ranging in size from 0.2 to 4.0 μm, have been shown to be capable of encapsulating large macromolecules.

[0192] Liposomes can be targeted to specific tissues by coupling them to specific ligands, such as monoclonal antibodies, sugars, glycolipids, or proteins.Ligands that may be useful for targeting liposomes, for example, to smooth muscle cells, include, but are not limited to, complete molecules or fragments of molecules, such as antibodies, that interact with smooth muscle cell-specific receptors and molecules, and complete molecules or fragments of molecules, such as antibodies, that interact with cell surface markers of cancer cells.Such ligands can be easily identified by binding assays well known to those skilled in the art.In yet other embodiments, liposomes can be targeted to tissues by coupling them to antibodies known in the art.

[0193] The compounds described by the present disclosure can be administered alone (e.g., in saline or buffer) or with any delivery vehicle known in the art.For example, the following delivery vehicles have been described: cochleates; emulsomes; ISCOMs; liposomes; live bacterial vectors (e.g., Salmonella, Escherichia coli, Bacillus Calmette-Guerin, Shigella, Lactobacillus); live viral vectors (e.g., vaccinia, adenovirus, herpes simplex); microspheres; nucleic acid vaccines; polymers (e.g., carboxymethylcellulose, chitosan); polymer rings; proteasomes; sodium fluoride; transgenic plants; virosomes; and virus-like particles.

[0194] The formulations of the present disclosure are administered in a pharma- ceutically acceptable solution, which may routinely contain pharma- ceutically acceptable concentrations of salts, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients.

[0195] The term pharmaceutically acceptable carrier means one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to humans or other vertebrates.The term carrier refers to a natural or synthetic organic or inorganic component with which active component is combined to facilitate application.The components of pharmaceutical composition can also be mixed with the compounds of the present disclosure and with each other in such a manner that there is no interaction that would substantially impair the desired pharmaceutical efficiency.

[0196] The core of the sugar-coated tablet is provided with a suitable coating agent.For this purpose, concentrated sugar solution can be used, which can optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, and suitable organic solvent or solvent mixture.For identification or to characterize various combinations of doses of active compound, dyes or pigments can be added to the tablet or sugar-coated tablet coating.

[0197] In addition to the formulations described herein, the compounds may also be formulated as depot preparations. Such long-acting formulations can be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, e.g., as a sparingly soluble salt.

[0198] The pharmaceutical compositions may also comprise suitable solid- or gel-phase carriers or excipients, examples of which include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

[0199] Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous or saline solutions for inhalation, microencapsulated, encochleated, coated on microscopic gold particles, contained in liposomes, nebulized, aerosolized, pelleted for implantation in the skin, or dried on a sharp object for scratching into the skin. Pharmaceutical compositions are also available in the form of granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops, or preparations with delayed release of active compounds (in which excipients and additives and / or adjuvants such as disintegrants, binders, coating agents, swelling agents, lubricants, flavoring agents, sweetening agents or solubilizers are customarily used as described above). Pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of methods for drug delivery, see Langer R (1990) Science 249:1527-1533, which is incorporated herein by reference.

[0200] The compound may be administered per se (neat) or in the form of a pharma- ceutically acceptable salt. When used in medicine, the salt should be pharma- ceutically acceptable, but non-pharma-ceutically acceptable salts may be used conventionally to prepare their pharma-ceutically acceptable salts. Such salts include, but are not limited to, those prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, p-toluenesulfonic acid, tartaric acid, citric acid, methanesulfonic acid, formic acid, malonic acid, succinic acid, naphthalene-2-sulfonic acid, and benzenesulfonic acid. Such salts may also be prepared as alkali metal or alkaline earth metal salts, such as sodium, potassium, or calcium salts of the carboxylic acid group.

[0201] Suitable buffering agents include acetic acid and salts (1-2% w / v), citric acid and salts (1-3% w / v), boric acid and salts (0.5-2.5% w / v), and phosphoric acid and salts (0.8-2% w / v).Suitable preservatives include benzalkonium chloride (0.003-0.03% w / v), chlorobutanol (0.3-0.9% w / v), parabens (0.01-0.25% w / v), and thimerosal (0.004-0.02% w / v).

[0202] The composition may be conveniently presented in unit dosage form and may be prepared by any method well known in the field of pharmacy.All methods include the step of associating the compound with a carrier that constitutes one or more accessory ingredients.Generally, the composition is prepared by uniformly and intimately associating the compound with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.Liquid dosage units are vials or ampoules.Solid dosage units are tablets, capsules and suppositories.

[0203] Administration The therapeutic agent can be delivered by any suitable modality known in the art.In some embodiments, the therapeutic agent (e.g., protein, antibody, interfering nucleic acid, etc.) is delivered to the subject by vector, such as viral vector (e.g., adenoviral vector, recombinant adeno-associated viral vector (rAAV vector), lentiviral vector, etc.) or plasmid-based vector.In some embodiments, the therapeutic agent is delivered to the subject (e.g., the subject with Alzheimer's disease characterized by the expression of one or more RAN proteins) in recombinant adeno-associated viral (rAAV) particles.

[0204] In some embodiments, the recombinant rAAV particle comprises a nucleic acid vector, such as a single-stranded (ss) or self-complementary (sc) AAV nucleic acid vector. In some embodiments, the nucleic acid vector comprises a transgene encoding a therapeutic agent (e.g., a protein, an antibody, an interfering nucleic acid, etc.) as described herein, and one or more regions comprising an inverted terminal repeat (ITR) sequence (e.g., a wild-type ITR sequence or an engineered ITR sequence) flanking the expression construct. In some embodiments, the nucleic acid is encapsidated by a viral capsid. In some embodiments, the transgene is operably linked to a promoter, such as a constitutive promoter or an inducible promoter. In some embodiments, the promoter is a tissue-specific (e.g., CNS-specific) promoter. In some embodiments, the rAAV particle comprises a viral capsid having tropism for CNS tissue, such as an AAV9 capsid protein or an AAV.PHPB capsid protein.

[0205] Aspects of the present disclosure relate to the delivery of a therapeutically effective amount of a therapeutic agent to a subject. In some embodiments, a therapeutically effective amount is an amount effective in reducing repeat expansion in a subject. In some embodiments, a therapeutically effective amount is an amount effective in reducing the transcription of the RNA that produces RAN protein in a subject. In some embodiments, a therapeutically effective amount is an amount effective in reducing the translation of RAN protein in a subject. In some embodiments, a therapeutically effective amount is an amount effective for treating Alzheimer's disease associated with repeat expansion. "Reducing" the expression of a repeat sequence or the translation of a RAN protein refers to reducing the amount or level of the expression of a repeat sequence or the translation of a RAN protein in a subject after administration of a therapeutic agent (and compared to the amount or level in the subject before administration).

[0206] In some embodiments, an effective amount is an amount effective in decreasing the level of RAN protein 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% (e.g., the level of RAN protein compared to the level of RAN protein in a cell or subject not administered a therapeutic agent). In some embodiments, an effective amount is an amount effective in decreasing the translation of RAN protein 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% (e.g., the level of RAN protein compared to the level of RAN protein in a cell or subject not administered a therapeutic agent).

[0207] The pharmaceutical composition described herein can be prepared by any method known in the pharmaceutical art.In general, such preparation method includes combining the compound described herein (i.e., "active ingredient") with carrier or excipient, and / or one or more other accessory ingredients, and then, if necessary and / or desired, shaping and / or packaging the product into desired single or multiple dose units.

[0208] The pharmaceutical composition can be prepared, packaged, and / or sold in bulk as a single unit dose and / or as a plurality of single unit doses. A "unit dose" is a discrete amount of the pharmaceutical composition comprising a predetermined amount of an active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject, and / or a convenient fraction of such a dosage, such as one-half or one-third of such a dosage.

[0209] The relative amounts of active ingredient, pharma- ceutically acceptable excipient and / or any additional materials in the pharmaceutical compositions described herein will vary depending on the identity, size and / or condition of the subject being treated, and also depending on the route by which the composition is administered. The compositions may contain from 0.1% to 100% (w / w) active ingredient.

[0210] Pharmaceutically acceptable excipients used in the manufacture of the provided pharmaceutical compositions include inert diluents, dispersing and / or granulating agents, surfactants and / or emulsifying agents, disintegrating agents, binders, preservatives, buffers, lubricants and / or oils. Excipients such as cocoa butter and suppository waxes, colorants, coating agents, sweeteners, flavoring agents, and aromatics may also be present in the compositions.

[0211] Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and mixtures thereof.

[0212] Exemplary granulating and / or dispersing agents include potato starch, corn starch, tapioca starch, sodium starch glycolate, clay, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose, and wood products, natural sponge, cation exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethylcellulose, cross-linked sodium carboxymethylcellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethylcellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and mixtures thereof.

[0213] Exemplary surfactants and / or emulsifiers include natural emulsifiers (e.g., gum arabic, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g., bentonite (aluminum silicate) and Veegum (magnesium aluminum silicate)), long chain amino acid derivatives, polymeric alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxypolymethylene, polyacrylic acid, acrylic acid polymers, and carboxyvinyl polymers), carrageenan, cellulose derivatives (e.g., sodium carboxymethylcellulose, powdered cellulose, hydroxymethylcellulose, hydroxypropyl ... cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylene sorbitan monolaurate (Tween® 20), polyoxyethylene sorbitan (Tween® 60), polyoxyethylene sorbitan monooleate (Tween® 80), sorbitan monopalmitate (Span® 40), sorbitan monostearate (Span® 60), sorbitan tristearate (Span® 65), glyceryl monooleate, sorbitan monooleate (Span® 80), polyoxyethylene esters (e.g. polyoxyethylene monostearate (Myrj® 45), polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate and Solutol®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. Cremophor®), polyoxyethylene ethers,(e.g., polyoxyethylene lauryl ether (Brij® 30)), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic® F-68, poloxamer P-188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, sodium docusate, and / or mixtures thereof.

[0214] Exemplary binders include starches (e.g., corn starch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, and the like), natural and synthetic gums (e.g., gum arabic, sodium alginate, Irish moss extract, panwar gum, ghatti gum, isapol bark mucilage, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum®), and larch arabinogalactan), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohol, and / or mixtures thereof.

[0215] Exemplary preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, antiprotozoal preservatives, alcohol preservatives, acidic preservatives, and other preservatives. In some embodiments, the preservative is an antioxidant. In other embodiments, the preservative is a chelating agent.

[0216] Exemplary antioxidants include alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium disulfite, and sodium sulfite.

[0217] Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and its salts and hydrates (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, etc.), citric acid and its salts and hydrates (e.g., citric acid monohydrate), fumaric acid and its salts and hydrates, malic acid and its salts and hydrates, phosphoric acid and its salts and hydrates, and tartaric acid and its salts and hydrates. Exemplary antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.

[0218] Exemplary antifungal preservatives include butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.

[0219] Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoic acid, and phenylethyl alcohol.

[0220] Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.

[0221] Other preservatives include tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium disulfite, potassium sulfite, potassium metabisulfite, Glydant® Plus, Phenonip®, methylparaben, Germall® 115, Germaben® II, Neolone®, Kathon®, and Euxyl®.

[0222] Exemplary buffering agents include citrate buffer, acetate buffer, phosphate buffer, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium phosphate hydroxide, potassium acetate, potassium chloride, potassium gluconate, potassium mixture, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixture, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixture, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and mixtures thereof.

[0223] Exemplary lubricants include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behenate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and mixtures thereof.

[0224] Exemplary natural fats and oils include almond oil, apricot kernel oil, avocado oil, bassau oil, bergamot oil, black currant seed oil, borage oil, cade oil, chamomile oil, canola oil, caraway oil, carnauba wax oil, castor oil, cinnamon bark oil, cocoa butter, coconut oil, cod liver oil, coffee oil, corn oil, cottonseed oil, emu oil, eucalyptus oil, evening primrose oil, fish oil, linseed oil, geraniol oil, gourd oil, grape seed oil, hazel nut oil, hyssop oil, isopropyl myristate, jojoba oil, kukui nut oil, lavandin oil, lavender oil, lemon oil, blue Examples of oils that may be used include oleander oil, macadamia nut oil, mallow oil, mango seed oil, meadowfoam seed oil, mink oil, nutmeg oil, olive oil, orange oil, orange roughy oil, coconut oil, palm kernel oil, peach kernel oil, peanut oil, poppy seed oil, pumpkin seed oil, rapeseed oil, rice bran oil, rosemary oil, safflower oil, sandalwood oil, sasquana oil, savoury oil, sea buckthorn oil, sesame oil, shea butter oil, silicone oil, soybean oil, sunflower oil, tea tree oil, thistle oil, camellia oil, vetiver oil, walnut oil and wheat germ oil. Exemplary synthetic oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and mixtures thereof.

[0225] Liquid dosage forms for oral and parenteral administration include pharma- ceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.In addition to active ingredients, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (such as cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan, and mixtures thereof.In addition to inert diluents, oral compositions may contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavorings and aromatics. In certain embodiments for parenteral administration, the conjugates described herein are mixed with solubilizing agents such as Cremophor®, alcohols, fats, modified oils, glycols, polysorbates, cyclodextrins, polymers, and mixtures thereof. Exemplary liquid dosage forms in certain embodiments are formulated for ease of swallowing or for administration via a feeding tube.

[0226] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert pharma- ceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate, and / or (a) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol and silicic acid, (b) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and gum arabic, (c) humectants, such as glycerol, (d) disintegrants, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate, (e) solution retarders, such as paraffin. (f) absorption enhancers such as quaternary ammonium compounds, (g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, (h) absorbents such as kaolin and bentonite clay, and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

[0227] Similar type solid compositions can be used as fillers in soft and hard filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols. Tablet, dragee, capsule, pill and granule solid dosage forms can be prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical art. They can optionally contain opacifying agents and can be of a composition that releases the active ingredient only, or preferentially, in a certain part of the intestinal tract, optionally in a delayed manner. Examples of encapsulating compositions that can be used include polymeric substances and waxes. Similar type solid compositions can be used as fillers in soft and hard filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols.

[0228] The active ingredient can be microencapsulated with one or more excipients as mentioned above. The solid dosage forms of tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings, release-controlling coatings and other coatings well known in the field of pharmaceutical formulation. In such solid dosage forms, the active ingredient can be mixed with at least one inert diluent, such as sucrose, lactose or starch. Such dosage forms may contain additional substances other than the inert diluent, such as tableting lubricants and other tableting aids, as is customary, such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also contain buffering agents. They may optionally contain opacifying agents and may be of a composition that releases the active ingredient only or preferentially in a certain part of the intestinal tract, optionally in a delayed manner. Examples of encapsulating agents that can be used include polymeric substances and waxes.

[0229] Although the description of pharmaceutical compositions provided herein is directed primarily to pharmaceutical compositions suitable for administration to humans, one skilled in the art will understand that such compositions are generally suitable for administration to animals of all kinds. Modifications of pharmaceutical compositions suitable for administration to humans to make the compositions suitable for administration to a variety of animals are well understood, and a veterinary pharmacologist of ordinary skill can design and / or implement such modifications with routine experimentation.

[0230] The therapeutic agent described herein is typically formulated in dosage unit form for ease of administration and uniformity of dosage.However, it will be understood that the total daily usage of the composition described herein will be determined by a physician within the scope of sound medical judgment.The specific therapeutically effective dose level for any specific subject or organism will depend on a variety of factors, including: the severity of the disease and disorder being treated; the activity of the specific active ingredient used; the specific composition used; the age, weight, general health, sex and diet of the subject; the time of administration, route of administration and the rate of excretion of the specific active ingredient used; the duration of treatment; the drugs used in combination with or simultaneously with the specific active ingredient used; and similar factors well known in the medical field.

[0231] The therapeutic agent can be administered by any route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as powder, ointment, cream and / or drop), mucosal, nasal, buccal, sublingual; by intratracheal instillation, bronchial instillation, and / or inhalation; and / or as oral spray, nasal spray and / or aerosol. Particularly 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 site. In general, the most appropriate route of administration will depend on a variety of 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.

[0232] The exact amount of therapeutic agent required to achieve an effective dose varies from subject to subject, and depends, for example, on the subject's species, age, general condition, severity of side effects or disorders, identity of the specific compound, mode of administration, etc. An effective amount may be contained in a single dose (e.g., a single oral dose) or in multiple doses (e.g., multiple oral doses). In some embodiments, when multiple doses are administered to a subject or applied to a biological sample, tissue or cell, any two doses of the multiple doses contain different or substantially the same amount of the compound described herein. In some embodiments, when multiple doses are administered to a subject or applied to a biological sample, tissue or cell, the frequency of administering multiple doses to a subject or applying multiple doses to a biological sample, tissue or cell is 3 doses per day, 2 doses per day, 1 dose per day, 1 dose every 2 days, 1 dose every 3 days, 1 dose every week, 1 dose every 2 weeks, 1 dose every 3 weeks, or 1 dose every 4 weeks. In some embodiments, the frequency of administering multiple doses to a subject or applying multiple doses to a biological sample, tissue or cell is 1 dose per day.In some embodiments, the frequency of administering multiple doses to a subject or applying multiple doses to a biological sample, tissue or cell is 2 doses per day.In some embodiments, the frequency of administering multiple doses to a subject or applying multiple doses to a biological sample, tissue or cell is 3 doses per day.In some embodiments, when multiple doses are administered to a subject or applied to a biological sample, tissue or cell, the period between the first dose and the last dose of the multiple doses is 1 day, 2 days, 4 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 6 months, 8 months, 9 months, 1 year, 2 years, 3 years, 4 years, 5 years, 7 years, 10 years, 15 years, 20 years, or the lifespan of a subject, tissue or cell.In some embodiments, the period between the first dose and the last dose of the multiple doses is 3 months, 6 months or 1 year. In certain embodiments, the period between the first and last dose of the multiple doses is the lifespan of the subject, tissue, or cell.In some embodiments, the doses described herein (e.g., a single dose, or any of the multiple doses) independently comprise 0.1 μg to 1 μg, 0.001 mg to 0.01 mg, 0.01 mg to 0.1 mg, 0.1 mg to 1 mg, 1 mg to 3 mg, 3 mg to 10 mg, 10 mg to 30 mg, 30 mg to 100 mg, 100 mg to 300 mg, 300 mg to 1,000 mg, or 1 g to 10 g (inclusive) of a compound described herein. In some embodiments, the doses described herein independently comprise 1 mg to 3 mg (inclusive) of a compound described herein. In some embodiments, the doses described herein independently comprise 3 mg to 10 mg (inclusive) of a compound described herein. In some embodiments, the doses described herein independently comprise 10 mg to 30 mg (inclusive) of a compound described herein. In certain embodiments, the doses described herein independently comprise between 30 mg and 100 mg (inclusive) of a compound described herein.

[0233] Routes of administration include, but are not limited to, oral, parenteral, intravenous, intramuscular, intraperitoneal, intranasal, sublingual, intratracheal, inhalation, subcutaneous, ocular, vaginal, and rectal. Systemic routes include oral and parenteral. Several types of devices are regularly used for administration by inhalation. These types of devices include metered dose inhalers (MDIs), breath-actuated MDIs, dry powder inhalers (DPIs), spacer / holding chambers combined with MDIs, and nebulizers.

[0234] In some embodiments, the treatment for the disease associated with the expression of RAN protein is administered to the central nervous system (CNS) of the 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 neuronal cells, glial cells, astrocytes, cerebrospinal fluid, etc.The modality of administering therapeutic agent to the CNS of a subject includes 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.

[0235] In some embodiments, the treatment as described by the present disclosure is administered systemically to the subject, for example, by intravenous injection. The systemically administered therapeutic molecule may, in some embodiments, be modified to improve delivery of the molecule to the CNS of the subject. Examples of modifications that improve CNS delivery of therapeutic molecules include, but are not limited to, co-administration or conjugation to blood-brain barrier targeting agents (e.g., transferrin, melanotransferrin, low-density lipoprotein (LDL), angiopep, 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).

[0236] The following examples are intended to illustrate the advantages of the present invention and to describe certain embodiments, but are not intended to exemplify the complete scope of the invention. Thus, it will be understood that the examples are not intended to limit the scope of the invention.

[0237] example Microsatellite repeat expansions cause over 40 inherited neurodegenerative and neuromuscular diseases. The length of the repeat tract is typically <30 in the general population, about 30-40 in premutation carriers, and can range from about 40 to several thousand repeats in affected individuals, depending on the disease. Expansion mutations often undergo bidirectional transcription, resulting in sense and antisense transcripts, which can form RNA aggregates. Repeat-expanded RNAs can be translated by repeat-associated non-AUG translation (RAN) to generate polymeric proteins. These repeat expansions are generally difficult to detect by high-throughput sequencing.

[0238] Example 1 Alzheimer's disease (AD) is a progressive dementia that affects approximately 10% of the general population aged 65 years or older. While mutations in certain genes, including apolipoprotein (APP) and presenilin genes (PSEN1 and PSEN2), have been observed in a subset of AD cases, the cause of the majority of AD cases is currently unknown. AD is characterized by the accumulation of β-amyloid (Aβ) peptides and hyperphosphorylated tau protein throughout the brains of patients at autopsy. However, the weak correlation between Aβ deposition and cognitive decline, as well as the limited efficacy of approaches targeting Aβ and tau to date, indicate that other factors play important roles in AD.

[0239] This example describes the identification of repeat expansions that contribute to Alzheimer's disease, dementia, and other neurodegenerative diseases. These putative expansions may contribute to disease from a single unidentified repeat expansion mutation or from the combined effect of multiple genes, each with a smaller premutation length. The data show that the repeat RNAs and / or RNA proteins generated from these expansion mutations contribute to disease by disrupting protein homeostasis, proteasome function, and autophagy.

[0240] Identification of RAN protein translation in AD patient samples To screen AD patient samples for RAN protein expression, an antibody-based screening tool was developed. Figures 1A and 1B show a schematic of the screening protocol. First, to determine whether RAN protein aggregates are present in the sample, the sample is contacted with a panel of antibodies that bind to diverse repetitive peptide motifs (Figure 1A). This antibody-based approach identified positive RAN staining in 21 of 120 tested human AD autopsy brains. Briefly, both soluble and insoluble fractions of protein lysates extracted from frozen AD autopsy brain tissue were contacted with antibodies against diverse RAN proteins (e.g., α-polySer, α-polyGP, α-polyGA, α-polyGR, α-polyPR). Positive signals from these antibodies compared to age-matched healthy controls were found by dot blot screening of early insoluble proteins from 21 of 120 AD cases.

[0241] Dot blots and signal quantification are shown for a subset of samples positive for α-polyGR and polySer staining (Figures 2A and 2B). Immunohistochemical (IHC) staining of hippocampal and frontal cortex sections from 20 candidate cases and approximately 15 healthy and disease controls showed strong RAN positive signals in AD cases, with no similar staining in age-matched healthy or disease controls. Example staining with α-polyGR and α-polyPR is shown in Figure 2C. Staining for α-polyGR and α-polyPR did not resemble staining for phosphorylated TDP43 (e.g., Figure 2C). In addition, dual IHC staining for RAN-GR protein and tau (3R) in the AD hippocampus showed distinct patterns with overlapping and non-overlapping signals, represented by arrows (Figure 3A). Further control IF experiments in cells transiently expressing 3R tau and / or GR or PR RAN proteins show that α-polyGR and α-polyPR recognize their corresponding tagged RAN proteins (Figure 3C) but do not cross-react with 3R tau (Figure 3B). Together, these data indicate that RAN staining is present in a subset of AD cases and that these proteins accumulate in patterns that are distinct from pTDP43 and 3R tau.

[0242] To further characterize these candidate RAN-positive AD cases, we examined the distribution of RAN staining by IHC in various regions of AD autopsy brains. We compared the distribution of RAN protein with additional forms of tau protein (e.g., 4-repeat (4R) tau, phosphorylated tau) and Aβ. In addition, we compared RAN staining and distribution with Braak score to determine whether and how RAN protein accumulation correlates with disease stage. In addition, we screened all RAN-positive candidate cases to remove cases with known coexisting repeat expansions. All poly(GR) and poly(PR) RAN-positive samples were shown to be negative for C9ORF72 expansion mutations.

[0243] We then used an RNA aggregate screening approach to identify repeat expansion motifs encoding candidate RAN proteins, which we combined with biotin-tagged nuclease-deficient Cas9 (dCas9) to pull down candidate repeat expansion mutations and corresponding flanking sequences from genomic DNA isolated from AD tissue samples positive for both RAN protein aggregates and RNA aggregates (Figure 1B). Sequencing of the upstream and downstream regions flanking the repeats was used to identify the specific locations of the repeat expansions.

[0244] The putative AD RAN protein repeat motifs were used to identify all possible DNA sequences that could code for RAN proteins, for example, all possible GR, PR and polySer coding repeat motifs are shown below in Tables 1, 2 and 3. One of skill in the art will know how to construct a similar table of all possible nucleic acid sequences encoding other RAN repeats identified herein, including poly(CP), poly(GP), poly(G), poly(GA), poly(GD), poly(GE), poly(GQ), poly(GT), poly(L), poly(LP), poly(LPAC) (SEQ ID NO:260), poly(LS), poly(P), poly(PA), poly(QAGR) (SEQ ID NO:261), poly(RE), poly(SP), poly(VP), poly(FP), poly(GK), poly(FTPLSLPV) (SEQ ID NO:262), poly(LLPSPSRC) (SEQ ID NO:263), poly(YSPLPPGV) (SEQ ID NO:264), poly(HREGEGSK) (SEQ ID NO:255), poly(TGRERGVN) (SEQ ID NO:265), or poly(PGGRGE) (SEQ ID NO:258), using the standard vertebrate DNA translation code.

[0245] Table 1: All possible nucleic acid sequences encoding GR [Table 1]

[0246] Table 2: All possible nucleic acid sequences encoding PR. [Table 2]

[0247] Table 3: All possible nucleic acid sequences encoding polySer [Table 3]

[0248] The sequences were used to design fluorophore-conjugated DNA probes targeting all possible repeat motifs that may code for a given candidate RAN AD protein. Fluorescence in situ hybridization (FISH) screening of frozen AD brains with RAN protein nucleic acid probes showed punctate RNA aggregates characteristic of repeat expansion disease (Figure 4). No similar RNA aggregates were found in controls or RAN-negative AD cases. Detection of RNA aggregates and staining for RAN protein in candidate AD brain tissue indicates the presence of one or more novel AD repeat expansion mutations.

[0249] To identify specific loci containing repeat expansion mutations in RNA- and RNA aggregate-positive candidate AD cases, we use a biotin-tagged nuclease-deficient Cas9 (dCas9) approach to enrich for specific repeat expansion mutations and corresponding flanking sequences using a pull-down assay (Figure 1B). This dCas9-based enrichment tool takes advantage of the rapid kinetics and high stability of the single-guide RNA / dCas9 (sgRNA-dCas9) complex to pull down and enrich specific DNA sequences without the need to denaturate the target DNA. The extended repeats provide multiple binding sites for the sgRNA, thus increasing the likelihood of interaction between the sgRNA-dCas9 complex and the extended repeats compared to shorter repeat sequences (Figure 1B).

[0250] The data show that enrichment of C9ORF72 G4C2 expansion mutations can be detected using this method. PCR data show enrichment for 5' and 3' sequences adjacent to the C9ORF72 G4C2 repeat in some of the C9(+) cases compared to the C9(-) cases (Figure 1C). For candidate AD cases, DNA samples containing genomic repeat expansions are enriched using a set of sgRNAs targeting putative RAN expansion mutations predicted by IHC and RNA FISH experiments. The enriched and non-enriched samples are then sequenced using next-generation sequencing technology to identify the repeat expansion locus that produces the RAN protein in AD.

[0251] Example 2 Molecular pathways affected by RAN protein translation This example provides data showing that RAN protein translation modifies protein homeostasis in neurons and glia of subjects with AD and other CNS disorders. Repeat expansion patient iPSC-derived neurons and glia are used to study the disruption of proteasome and autophagy pathways through proteasome activity, autophagy flux, proteasome and autophagy markers (e.g., p62, LC3, proteasome subunits) and transcriptome analysis. To determine whether repeat expansion accelerates AD disease progression, short and long repeats encoding RAN protein are differentially expressed in AD iPSC-derived cells and proteasome, autophagy function, β-amyloid and tau pathology are measured.

[0252] Proteasome activity in iPSC-derived cells is measured using a fluorescence-based assay that determines the rate of peptide cleavage by the proteasome complex in protein lysates. Proteasome activity in live cells can also be studied by infecting cells with a vector expressing GFP and monitoring the GFP signal over time. A decrease in the 7-methylcoumarin fluorescence signal or a higher GFP signal in live cells compared to healthy control cells indicates a decrease in proteasome function.

[0253] To measure autophagy activity, dyes that stain autophagosomes are used to monitor autophagy flux. DALGreen, a small molecule-based dye, can also be used to monitor late autophagy. The subcellular location and levels of proteasome and autophagy markers are assayed using immunofluorescence and Western blot (e.g., p62, LC3 I / II, proteasome subunits). Increased levels and / or accumulation of p62 have been observed to be associated with inhibition of autophagy and proteasome, while the ratio of LC3 I / II has been observed to be associated with autophagy activity, and accumulation of proteasome subunits has been observed to be associated with proteasome stall and reduced ubiquitin-proteasome activity.

[0254] Proteasome and autophagy activity will be measured in specific induced cells (e.g., iNeuron, iAstrocyte, iMGL cells, etc.) to examine cell type variations. Proteasome and autophagy function will be tracked over time as cells differentiate and mature, providing information on when these pathways are affected. Since stress is a risk for neurodegenerative disease and has been associated with increased expression of RAN protein in C9 ALS / FTD, we will test how proteasome and autophagy are further affected under various stress conditions. To understand the global effects in cells associated with changes in the proteasome and autophagy systems, we will perform RNAseq experiments.

[0255] The data show that in cultured glioblastoma cells, C9 poly(GA)RAN protein aggregates colocalize with the autophagy marker LC3B and the 26S proteasome subunit (Figure 5A). Expression of poly(GA)RAN protein was observed to result in reduced proteasome activity in HEK293T cells. These abnormalities are rescued by targeting poly(GA) protein with α-polyGA antibody (Figure 5B). These results indicate that GA-RAN protein aggregates sequester key proteins involved in the proteasome and autophagy systems, thereby disrupting the function of these systems.

[0256] Toll-like receptor 3 (TLR3), a member of the Toll-like receptor family that can activate immunological autophagy, has been observed to target double-stranded RNA (dsRNA). Increased eIF2α phosphorylation can result from expanded RNAs themselves, which trigger a dsRNA-mediated PKR response, or from RAN aggregates, which trigger ER stress. Monitoring the flux of PKR, eIF2α, TLR3, and autophagy in the presence of repeat RNA and RAN protein provides mechanistic insight into autophagy dysfunction and disease progression in repeat expansion disorders (Figure 6). TLR3-mediated autophagy can also be studied by knocking down or knocking out TLR3 using siRNA and CRISPR technology.

[0257] Translation of RAN protein leads to dysfunction of proteasome and autophagy pathway in cells in some embodiments, such as reduced activity or inappropriate complex formation.Dysfunction of proteasome and autophagy can be detected in certain cell types, while co-culturing neurons with glial cells can accelerate destruction.Since dsRNA is known to be recognized by TLR3 and activate eIF2α-PKR pathway, autophagy may be altered early in AD, which may cause negative feedback that worsens disease over time.Based on the finding that poly(GA) aggregates sequester proteasome and autophagy markers, RAN protein may inhibit proteasome and autophagy complexes by sequestration in RAN protein aggregates.In addition, ER stress response caused by accumulation of RAN protein may lead to activation of autophagy through eIF2α-PERK pathway in some embodiments.

[0258] Example 3 This example describes a method that allows the isolation of repeat expansion mutations from a single DNA sample and the identification of locus-specific unique adjacent sequences. This then allows direct testing of whether they contribute to disease in a larger group of patients. The method described herein that utilizes inactivated clustered regularly interspaced short palindromic repeat associated protein 9 (dCas9) was observed to pull down microsatellite expansion mutations with repeat motifs containing AGG, TGG, CGG or GGG (NGG) sequences in the protospacer adjacent motif (PAM). This method is referred to herein as Cas9-based repeat enrichment and detection (dCas9READ).

[0259] Repeat expansion mutations are difficult to detect using conventional sequencing techniques. Presented herein is a novel assay (dCas9READ) that utilizes sgRNA and dCas9 to enrich and detect repeat expansions containing NGG protospacer adjacent motifs (PAMs) (e.g., GGGGCC in ALS / FTD and CCTG in DM2) and their unique adjacent sequences as well as non-NGG PAMs (Figure 8). Repeats containing non-NGG PAMs include CAG and CTG repeats. The assay as disclosed herein simultaneously identifies multiple repeat expansions containing sequences with non-NGG PAMs, allowing for the identification of repeat expansions that are 40-50 repeats longer than the corresponding normal allele. In contrast to the very long non-coding expansions found in DM1, DM2 and ALS / FTD, CAG expansions in spinocerebellar degeneration and Huntington's disease are often only slightly longer (10-100 repeats) than the normal repeat range. These novel repeat pull-down techniques therefore facilitate the identification of novel extension mutations, thereby aiding in the diagnosis and eventual treatment of RAN protein-associated diseases. The basis of dCas9READ works on the principle that repeat extension mutations provide more binding sites for single guide RNA (sgRNA)-dCas9 complexes to associate with compared to shorter repeats (Figure 7). Next-generation sequencing (NGS) of extension-enriched genomic DNA (gDNA) was used to identify both the repeat extensions and the corresponding flanking sequences after biotin-streptavidin pull-down of DNA containing the extensions. Identification of unique flanking sequences and candidate repeat extension mutations allows PCR and Southern blot testing of specific repeats as putative disease-causing mutations. Compared to conventional pull-down methods, dCas9READ offers rapid binding kinetics and high stability of the sgRNA-dCas9-DNA complex without the need to denature the target DNA.

[0260] The dCas9READ protocol was performed using human genomic DNA from C9orf72 ALS / FTD and myotonic dystrophy type 2 (DM2) patients. Using Streptococcus pyogenes dCas9 (spdCas9), dCas9READ successfully enriched both C9ALS / FTD G4C2 and DM2 CCTG repeat expansion DNA by 4-6 fold compared to expansion-negative controls (Figure 8A and Figure 8B). This enrichment combined with bioinformatics allowed unambiguous identification of these expansion mutations and their corresponding unique flanking DNA sequences.

[0261] Further control experiments performed without the G4C2 sgRNA did not show a similar enrichment of the C9 ALS / FTD locus, indicating that the specificity of the pulldown is determined by the repeat-containing guide RNA (Figure 8A). Next-generation sequencing of enriched GGGGCC and CCTG repeats from C9 and DM2 DNA samples showed a significant increase in the number of total reads in samples with expanded C9orf72 and DM2 repeats compared to negative controls (Figure 8C and Figure 8D). Importantly, NGS data reporting the localization of enrichment as the ratio of total reads in a 2-4 kb region surrounding a selected repeat region (e.g., G4C2) in enriched samples compared to non-enriched samples showed that the C9orf72 locus had the highest enrichment score. These data indicate that dCas9READ can identify repeat expansion loci and unique flanking sequences surrounding individual repeat expansions without prior knowledge of the location of the expansion mutation in the human genome.

[0262] Example 4 This example describes the application of dCas9READ to isolate repeat expansion mutations directly from genomic DNA of patients with a neurodegenerative disease of unknown genetic etiology. Identifying flanking sequences in the repeat expansion locus allows direct testing of the role of specific expansion mutations in the disease. A subset of anti-RAN protein antibodies was used to identify RAN protein accumulation in AD brains (Figure 9A). For these experiments, fixed and frozen brain tissue from 120 AD cases that had onset of clinical features after age 60 (late-onset) and 30 age-matched controls was screened. Initial immunoblot screening showed higher signals in AD cases compared to control cases using antibodies against glycine-arginine (α-GR) (i.e., anti-GR), proline-arginine (α-PR) (i.e., anti-PR), glycine-alanine (α-GA) (i.e., anti-GA), glycine-proline (α-GP) (i.e., anti-GP), and serine (α-Ser) (i.e., anti-Ser) antibodies. Initial dot blot screening of insoluble proteins showed that positive signals from at least one of these antibodies were found more frequently in AD cases (21 of 120 or 17.5%) compared to age-matched controls without neurological disease (1 of 30 or 3.3%). Representative dot blot images showing α-GR staining and signal quantification are shown in Figure 9B.

[0263] Immunohistochemical (IHC) studies performed on hippocampal sections from AD cases that were positive for α-GR or α-PR staining by dot blot showed strong perinuclear RAN positive staining for GR or PR aggregates (Figure 9C), whereas staining was absent or minimal in age-matched healthy or disease controls (e.g., Figure 9D). Intracellular GR and PR aggregates do not resemble the extracellular Aβ plaques or intracellular p-tau tangles or pTDP43 staining typically found in AD (Figure 9C). Double-labeling IHC with p-tau and PR or GR further indicates that the patterns of intracellular accumulation of these RAN proteins and p-tau are distinct. Control IF experiments on cells overexpressing GFP-tau and / or GR60 or PR60 proteins show that α-GR and α-PR antibodies recognize their intracellular RAN protein targets (Figure 9C), but not GFP-tau (Figures 9E-9G). These data indicate that RAN protein staining is present in 17.5% of AD cases in this initial cohort, and that RAN protein accumulates in a pattern distinct from staining typically associated with AD, including Aβ plaques, p-tau tangles, and p-TDP43.

[0264] Repeat-expanded RNA was detected using probes specific for α-GR, α-GA, or α-GP, GC-rich DNA. Fluorescence in situ hybridization (FISH) with repeat-containing CCCCGG (SEQ ID NO: 71) or CCCCGT (SEQ ID NO: 59) probes detected punctate RNA aggregates (FIG. 10A) in a subset of AD cases that showed high signals for α-GR, α-GA, or α-GP antibodies by dot blot screening, but not in AD cases that were negative for these antibodies.

[0265] Repeat-containing transcripts generated from repeat expansion mutations can also form secondary structures composed of RNA G-quadruplexes and hairpin structures containing mismatches. To examine whether dsRNA accumulates in RAN-positive AD cases, fixed brain tissue was stained with an α-dsRNA antibody. Initial data show increased dsRNA signal in the hippocampus of RAN-positive AD cases compared to non-neuronal disease controls and disease controls including a panel of SCAs caused by small CAG repeat expansion mutations (Figure 10B). RNAse A treatment significantly reduced the staining of α-dsRNA, supporting the notion that this antibody specifically stains double-stranded RNA (Figure 10C).

[0266] The data described in this example highlights a method for identifying repeat expansions that lead to the accumulation of RAN protein aggregates in AD brains (Figure 11). Antibodies against RAN protein repeat motifs are also used to screen human AD autopsy brains for RAN aggregates. The sequence encoding the RAN protein aggregates is used to design fluorescent in situ hybridization (FISH) probes that identify specific RNA aggregate signals that will provide sequence information for designing sgRNAs for enrichment and identification of repeats using dCas9READ.

[0267] Example 5 This example describes the generation of anti-RAN antibodies. Antibodies against the RAN protein regions listed in Table 9 were generated by injecting subjects with peptide repeat-containing antigens. Immunofluorescence data validating the antibodies in transfected cells expressing recombinant proteins were obtained as shown in Figures 13A-13E.

[0268] Table 9 - Antibodies raised against target RAN protein regions [Table F-1] [Table F-2]

[0269] Equivalent While several inventive embodiments have been described and illustrated herein, those skilled in the art will readily envision a variety of other means and / or structures for performing the functions and / or obtaining one or more of the results and / or advantages described herein. Each such variation and / or modification is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials and configurations described herein are intended to be exemplary, and that the actual parameters, dimensions, materials and / or configurations will depend on the particular application for which the inventive techniques are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. Thus, it should be understood that the foregoing embodiments are presented by way of example only, and that within the scope of the appended claims and equivalents thereto, the inventive embodiments may be practiced otherwise than as specifically described and claimed. The inventive aspects of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods is within the scope of the present disclosure, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0270] All definitions, as defined and used herein, take precedence over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meaning of the defined term. All references, patents, and patent applications disclosed herein are each incorporated by reference with respect to the subject matter for which it is cited, which in some cases may include the entirety of that document. The indefinite articles "a" and "an," as used herein and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

[0271] The phrase "and / or" as used herein and in the claims should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctive in some cases and disjunctive in other cases. Multiple elements listed with "and / or" should be interpreted in the same manner, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present in addition to the elements specifically identified by the "and / or" clause, whether related or unrelated to those specifically identified elements. Thus, as a non-limiting example, a reference to "A and / or B", when used in conjunction with open-ended language such as "comprising", refers in one embodiment to A only (optionally including elements other than B); in another embodiment to B only (optionally including elements other than A); in yet another embodiment to both A and B (optionally including other elements); and so forth.

[0272] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as being inclusive, i.e., the inclusion of at least one, but also as including more than one of a number or list of elements, and optionally, further unlisted items. Only terms clearly indicated to the contrary, such as "only one of" or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one of a number or list of elements. In general, the term "or," when used herein, should only be construed as indicating exclusive alternatives when preceded by terms of exclusivity such as "either," "one of," "only one of," or "exactly one of" (i.e., "one or the other but not both"). "Consisting essentially of," when used in the claims, should have its ordinary meaning as used in the field of patent law.

[0273] As used herein in the specification and in the claims, the phrase "at least one" in reference to a list of one or more elements should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows for elements, whether related or unrelated to those elements specifically identified, optionally may be present in addition to the elements specifically identified in the list of elements to which the phrase "at least one" refers. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently, "at least one of A and / or B") can refer, in one embodiment, to at least one, optionally including more than one, A (in which B is absent) (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B (in which A is absent) (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and including any other elements); etc.

[0274] Unless expressly indicated to the contrary, in any method claimed herein that includes more than one step or act, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are described.

[0275] In the claims, as well as in the above specification, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like, should be understood to be open-ended, i.e., meaning including but not limited to. As described in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03, only the transitional phrases "consisting of" and "consisting essentially of" should be closed or semi-closed transitional phrases, respectively. It should be understood that embodiments described in this document with open-ended transitional phrases (e.g., "comprising") are also contemplated in alternative embodiments to "consisting of" and "consisting essentially of" the features described by the open-ended transitional phrases. For example, if the disclosure describes "a composition comprising A and B," the disclosure also contemplates "a composition consisting of A and B" and "a composition consisting essentially of A and B."

Claims

1. An anti-RAN protein antibody or antigen-binding fragment thereof that specifically binds to a RAN protein, (a) the antibody or antigen-binding fragment thereof (i) a CDR1 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 117, 119, 121, and 123; (ii) a CDR2 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 125, 127, 129, and 131; and / or (iii) a CDR3 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 133, 135, 137, and 139. and / or a heavy chain variable region (VH) comprising: (b) the antibody or antigen-binding fragment is (i) a CDR1 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 118, 120, 122, and 124; (ii) a CDR2 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 126, 128, 130, and 132; and / or (iii) a CDR3 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 134, 136, 138, and 140. a light chain variable region (VL) comprising: The antibody or antigen-binding fragment.

2. 2. The antibody or antigen-binding fragment of claim 1, comprising a framework region amino acid sequence set forth in any one of SEQ ID NOs: 155-181 and 183-184.

3. 3. The antibody or antigen-binding fragment of claim 1 or 2, a variable heavy chain amino acid sequence set forth in any one of SEQ ID NOs: 109, 111, 113 and 115; and / or The variable light chain amino acid sequence set forth in any one of SEQ ID NOs: 110, 112, 114 and 116. The antibody or antigen-binding fragment comprising:

4. 4. The antibody or antigen-binding fragment of any one of claims 1 to 3, comprising a VH, wherein the VH comprises a CDR1 region comprising the amino acid sequence of SEQ ID NO: 117; a CDR2 region comprising the amino acid sequence of SEQ ID NO: 125; and a CDR3 region comprising the amino acid sequence of SEQ ID NO:

133.

5. 5. The antibody or antigen-binding fragment of any one of claims 1 to 4, comprising the framework region amino acid sequence set forth in SEQ ID NO: 155, the framework region amino acid sequence set forth in SEQ ID NO: 163, the framework region amino acid sequence set forth in SEQ ID NO: 171, and / or the framework region amino acid sequence set forth in SEQ ID NO:

179.

6. 6. The antibody or antigen-binding fragment of any one of claims 1 to 5, comprising a VL, wherein the VL comprises a CDR1 region comprising the amino acid sequence of SEQ ID NO: 118; a CDR2 region comprising the amino acid sequence of SEQ ID NO: 126; and a CDR3 region comprising the amino acid sequence of SEQ ID NO:

134.

7. 7. The antibody or antigen-binding fragment of any one of claims 1 to 6, comprising the framework region amino acid sequence set forth in SEQ ID NO: 156, the framework region amino acid sequence set forth in SEQ ID NO: 164, the framework region amino acid sequence set forth in SEQ ID NO: 172, and / or the framework region amino acid sequence set forth in SEQ ID NO:

180.

8. 8. The antibody or antigen-binding fragment of any one of claims 1 to 7, the variable heavy chain amino acid sequence set forth in SEQ ID NO: 109; and / or Variable light chain amino acid sequence set forth in SEQ ID NO: 110 The antibody or antigen-binding fragment comprising:

9. The antibody or antigen-binding fragment of any one of claims 1 to 8, wherein the antibody binds to polyGA.

10. The antibody or antigen-binding fragment of any one of claims 1 to 3, wherein the antibody binds to polySer.

11. The antibody or antigen-binding fragment of any one of claims 1 to 3, wherein the antibody binds to polyPR.

12. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment of any one of claims 1 to 11.

13. An isolated cell transformed with the nucleic acid molecule of claim 12.

14. The antibody or antigen-binding fragment of any one of claims 1 to 11; and Pharmaceutically acceptable carrier and / or pharmaceutically acceptable buffer A composition comprising:

15. 1. A composition for use in a method of treating a RAN protein-associated disease in a subject, the composition comprising: An anti-RAN protein antibody or antigen-binding fragment thereof that specifically binds to poly-GA RAN protein, poly-Ser RAN protein, poly-PR RAN protein, or poly-GR RAN protein; and Pharmaceutically acceptable carrier and / or pharmaceutically acceptable buffer Including, wherein the method comprises administering to a subject a therapeutic agent for treating a RAN protein-associated disease, wherein the subject has been characterized as having the RAN protein-associated disease by detection of at least one RAN protein in a biological sample obtained from the subject. The composition.

16. 16. The composition of claim 15, wherein the subject is a mammalian subject.

17. 17. The composition of claim 15 or 16, wherein the subject is a human subject.

18. RAN protein-related diseases include Alzheimer's disease (AD); amyotrophic lateral sclerosis (ALS); or frontotemporal dementia; myotonic dystrophy type 1 (DM1); myotonic dystrophy type 2 (DM2); spinocerebellar degeneration 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); fragile X tremor ataxia syndrome (FXTAS); Fuchs endothelial corneal dystrophy (FECD); Huntington's disease type 2 (HDL2); fragile X syndrome (FXS); 2ql 1. The composition of any one of claims 15 to 17, wherein the folate-sensitive fragile site is selected from disorders involving the folate-sensitive fragile site FRA7A; disorders involving the folate-sensitive fragile site 2ql 1 FRA2A; and fragile XE syndrome (FRAXE).

19. the method further comprises administering to the subject a second therapeutic agent; Optionally, wherein the second therapeutic agent is selected from donepezil, galantamine, memantine, rivastigmine, or a combination thereof. The composition according to any one of claims 15 to 18.

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