Peptide biomarkers for neurological diseases, especially motor neuron diseases.

Abstract

The present invention relates to splice variants of the CERT1 protein that act as biomarkers for TDP-43 pathology, particularly motor neuron diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), as well as other neurological diseases such as Alzheimer's disease. In particular, the present invention relates to a method for identifying splice variants of CERT1 comprising cryptic peptide sequences, as well as related methods for identifying TDP-43 pathology and / or TDP-43 dysfunction in a subject, and a method for predicting the likelihood of successful treatment. Antibodies that bind to CERT1 splice variants and kits comprising said antibodies are also claimed.

Description

Technical Field

[0001] Field of the Invention The present invention relates to splice variants of the CERT1 protein that act as biomarkers for TDP-43 pathology. In particular, the present invention relates to methods for identifying splice variants of CERT1 that contain cryptic peptide sequences, as well as related methods for identifying TDP-43 pathology and / or reduced TDP-43 function in a subject.

Background Art

[0002] Background of the Invention TAR DNA-binding protein 43 (TDP-43) is a versatile RNA / DNA-binding protein involved in RNA-related metabolism. Dysregulation of TDP-43 deposits acts as inclusions in the brains and spinal cords of patients with motor neuron diseases: amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) (Prasad et al., Front. Mol. Neurosci., 2019).

[0003] TDP-43 is mainly localized in the nucleus but also shuttles to the cytoplasm for some of its functions (Ayala et al., 2008). In several neurodegenerative diseases, TDP-43 is a common factor. In diseases such as ALS and FTLD, there is an increase in cytoplasmic TDP-43 concentration that leads to cytoplasmic inclusion formation (Neumann et al., 2006; Winton et al., 2008a). Cytoplasmic mislocalization can be associated with nuclear depletion and can lead to a decrease or loss of TDP-43 function. For example, over 95% of ALS patients show pathological mislocalization of TDP-43, and several mutations in its gene cause familial ALS. There are TDP-43 mutations that result in abnormal splicing of TDP-43 target RNAs, leading to widespread splicing abnormalities (see, for example, Arnold et al., PNAS 2013 110 E736-745 and Yang et al., PNAS. U.S.A. 111, E1121-E1129).

[0004] Klim et al. reported that STMN2 loss during TDP-43 dysfunction is due to changes in STMN2 splicing, suggesting that restoring STMN2 is a therapeutic strategy for ALS.

[0005] TDP-43 depletion is manifested in a variety of diseases known as TDP-43 pathology, including, for example, amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), progressive supranuclear palsy (PSP), primary lateral sclerosis, progressive muscular atrophy, Alzheimer's disease, Parkinson's disease, autism, hippocampal sclerotic dementia, Down syndrome, Huntington's disease, polyglutamine diseases, such as spinocerebellar ataxia type 3, myopathy, and chronic traumatic encephalopathy.

[0006] Tollervey et al., Nature Neuroscience 2010, 452-458, reported on the characterization of RNA targets and the position-dependent splicing regulation of TDP-43 in healthy brain tissue and brain tissue from FTLD patients. Most TDP-43 binding sites mapped to introns, long non-coding RNAs (lncRNAs), and intergenetic transcripts, and UG-rich motifs were enriched. Conserved RNP segments in TDP-43 are involved in binding to TAR DNA sequences and RNA sequences with UG repeats (Ayala et al., J.Mol.Biol.2005;348:575-588). TDP-43 depletion in cells, such as in TDP pathology, correlates with the loss of RNA binding of TDP-43 to TDP-43 RNA targets.

[0007] TDP-43 binding sites in human RNA are available online from a database of RNA-binding proteins and related motifs (see https: / / attract.cnic.es / results / e9f29380-8921-406e-84a8-27ce9b9398b4#). Specific characterized human RNA TDP-43 binding sites disclosed include the following RNA sequences: GUGAAUGA, GUUGUGC, UGUGUGUGUGUG, GAAUGG, UGUGUGUG, GAAUGA, UGUGUG, GUUGUUC, and GUUUUGC.

[0008] The role of TDP-43 as a causative agent of pathology can typically only be identified post-mortem.

[0009] Therefore, there is a need for biomarkers that can be used to diagnose patients with TDP-43 pathology. This would allow for the diagnosis and treatment of patients before disease progression. [Overview of the project]

[0010] Summary of the Invention To our surprise, we have found that splice variants of CERT1 containing cryptic peptide sequences act as biomarkers for loss of TDP43 expression and / or function, and can therefore be used as diagnostic tools for identifying TDP43-related diseases / pathologies.

[0011] The present invention provides a method for identifying TDP-43 pathology and / or TDP-43 dysfunction in a subject, comprising the step of identifying splice variants of CERT1 in a sample obtained from the subject.

[0012] The present invention provides a method for identifying subjects at risk of developing TDP-43 pathology and / or TDP-43 dysfunction, the method comprising the step of identifying CERT1 splice variants in a sample obtained from the subject, wherein the presence of CERT1 splice variants in the sample indicates that the subject is likely to develop TDP-43 pathology.

[0013] The present invention provides a method for determining whether a subject suffering from TDP-43 pathology and / or TDP-43 dysfunction is likely to respond to treatment, the method comprising the step of identifying a splice variant of CERT1 in a sample obtained from the subject, wherein the presence of the splice variant of CERT1 in the sample indicates that the subject is likely to respond to treatment.

[0014] The present invention provides a method for monitoring the therapeutic success of treatment for TDP-43 pathology and / or TDP-43 dysfunction in a subject, the method comprising the steps of identifying splice variants of CERT1 in a sample obtained from the subject, and further comprising the steps of predicting the therapeutic success of treatment for TDP-43 pathology and / or TDP-43 dysfunction based on the detection of splice variants of CERT1.

[0015] In some embodiments, the treatment may include antisense oligonucleotides that can restore the functional phenotype of one or more TDP-43 target RNAs in cells that are depleted of TDP-43 or expressing abnormal TDP-43 proteins.

[0016] The present invention provides the use of splice variants of CERT1 as biomarkers for TDP-43 pathology and / or TDP-43 dysfunction.

[0017] In some embodiments, the splice variant of CERT1 may include a cryptic peptide sequence.

[0018] In some embodiments, the splice variant of CERT1 may contain a cryptic peptide sequence of 1 to 30 amino acids.

[0019] In some embodiments, the splice variant of CERT1 may contain a cryptic peptide sequence of 10 to 20 amino acids.

[0020] In some embodiments, the splice variant of CERT1 may include a 13-amino acid cryptic peptide sequence.

[0021] In some embodiments, the cryptoptic peptide sequence may be inserted at a position corresponding to position 158 of the amino acid sequence by Sequence ID No. 1, which encodes wild-type CERT1.

[0022] In some embodiments, the cryptic peptide sequence may include or consist of the amino acid sequence VEITGSQLHFTWN (SEQ ID NO: 3), or a variant thereof having at least 80% sequence identity.

[0023] In some embodiments, the cryptic peptide sequence may include or consist of an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence VEITGSQLHFTWN (SEQ ID NO: 3).

[0024] In some embodiments, the cryptotic peptide sequence may include or consist of the amino acid sequence VEITGSQLHFTWN (SEQ ID NO: 3).

[0025] In some embodiments, the splice variant of CERT1 may include or consist of the amino acid sequence according to SEQ ID NO: 4 or a variant having at least 80% sequence identity thereto.

[0026] In some embodiments, the splice variant of CERT1 can comprise or consist of amino acids having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 4.

[0027] In some embodiments, the splice variant of CERT1 can comprise or consist of the amino acid sequence according to SEQ ID NO: 4.

[0028] In some embodiments, the method can be an ex vivo method.

[0029] In some embodiments, the step of identifying a splice variant of CERT1 can include proteomic analysis or an antibody-based assay.

[0030] In some embodiments, the step of identifying a splice variant of CERT1 can include measuring peptides in a sample using liquid chromatography (LC) and / or mass spectrometry (MS).

[0031] In some embodiments, the sample can be obtained from a body fluid or tissue selected from blood, serum, plasma, urine, saliva, brain tissue, and cerebrospinal fluid (CSF).

[0032] In some embodiments, the subject can be human. In some embodiments, the subject can be a human patient.

[0033] In some embodiments, the TDP-43 pathology can be a neuropathy.

[0034] In some embodiments, the TDP-43 pathology may be a neurological disorder selected from amyotrophic lateral sclerosis (ALS), frontotemporal dementia, frontotemporal lobar degeneration (FTLD), progressive supranuclear palsy (PSP), primary lateral sclerosis, progressive muscular atrophy, Alzheimer's disease, Parkinson's disease, autism, hippocampal sclerotic dementia, Down syndrome, Huntington's disease, polyglutamine diseases, such as spinocerebellar ataxia type 3, myopathy, and chronic traumatic encephalopathy.

[0035] In some embodiments, the TDP-43 pathology may be a neurological disorder selected from amyotrophic lateral sclerosis (ALS), frontotemporal dementia, and frontotemporal lobar degeneration (FTLD).

[0036] This invention provides an antibody that binds to a splice variant of CERT1.

[0037] In some embodiments, the splice variant of CERT1 may include the amino acid sequence VEITGSQLHFTWN (SEQ ID NO: 3), or an epitope comprising a variant thereof having at least 80% sequence identity.

[0038] In some embodiments, the epitope may or may consist of an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence VEITGSQLHFTWN (SEQ ID NO: 3).

[0039] In some embodiments, the splice variant of CERT1 may include or consist of the amino acid sequence according to SEQ ID NO: 4 or a variant having at least 80% sequence identity with SEQ ID NO: 4.

[0040] In some embodiments, the splice variant of CERT1 may or may consist of amino acids having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 4.

[0041] The present invention provides a kit comprising at least one reagent for detecting splice variants of CERT1, wherein at least one reagent is an antibody according to the present invention. [Brief explanation of the drawing]

[0042] [Figure 1] Bar plots showing the intensity of TDP-43 protein (A) or CERT1 peptide (VEITGSQLHFTWNETEK) (B) across different samples. n=3. Data are shown as mean ± standard deviation (SD). [Figure 2] Structure of the LNA gapmer used to knock down TDP-43. [Figure 3] (CA)n antisense oligonucleotide (ASO) structure (mimics the function of TDP-43 and is used to rescue misspliced ​​transcripts in the event of TDP-43 loss). [Figure 4] The location and arrangement of the cryptic exon showing the insertion at position 158. [Modes for carrying out the invention]

[0043] Detailed description of the invention TDP-43 TDP-43 is a TAR RNA / DNA binding protein encoded in humans on the sequential strand of human chromosome 1:11,012,653-11,022,858 (gene ENSG00000120948, Chr 1:11,012,344-11,025,739; example of a typical TDP-43 transcript = ENST00000439080.6), and is broadly involved in RNA splicing, stability, and metabolism.

[0044] In healthy cells, the TDP-43 protein is located in the nucleus, but in some neurodegenerative diseases, dysfunctional TDP-43 aggregates are formed in the cytoplasm (often associated with hyperphosphorylation and ubiquitination of TDP-43).

[0045] TDP-43 is an example of an RNA-binding protein that binds to GU repeats in multiple independent RNA transcripts. The interaction between RNA-binding proteins such as TDP-43 and a population of multiple RNA transcripts has a significant impact on the biology of RNA transcripts, including splicing against premRNA, RNA stability, and RNA accumulation, and therefore provides a mechanism for the expression of independent RNA populations within a cell.

[0046] This is particularly relevant in cases of TDP-43 depletion, where the loss of functional TDP-43 RNA binding is closely associated with neurodegeneration, and TDP-43 depletion in neuronal cells leads to significant changes in RNA processing of large populations of RNA transcripts in the cell.

[0047] TDP-43 pathology is a disorder associated with decreased or abnormal expression of TDP-43 and / or impaired TDP-43 function, often associated with increased cytoplasmic TDP-43, particularly hyperphosphorylated and ubiquitinated TDP-43.

[0048] TDP-43 depletion is observed in a variety of diseases known as TDP-43 pathology, including, for example, amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), progressive supranuclear palsy (PSP), primary lateral sclerosis, progressive muscular atrophy, Alzheimer's disease, Parkinson's disease, autism, hippocampal sclerotic dementia, Down syndrome, Huntington's disease, polyglutamine diseases, such as spinocerebellar ataxia type 3, myopathy, and chronic traumatic encephalopathy.

[0049] TDP-43-depleted cells refer to cells with reduced levels of TDP-43 function. In TDP-43 pathology, it is understood that abnormal TDP-43 expression can lead to the accumulation of dysfunctional cytoplasmic TDP-43 and a decrease in functional nuclear TDP-43 levels. Therefore, TDP-43-depleted cells may be characterized by reduced levels of TDP-43 function and, consequently, may be associated with increased levels of dysfunctional TDP-43.

[0050] For in vitro evaluation, TDP-43 depletion can be manipulated, for example, by a genetic engineering approach (e.g., CRISPR / CAS9) or by the use of antisense oligonucleotide inhibitors of TDP-43 (exemplified by gapmer oligonucleotides targeting human TDP-43 transcripts), as illustrated in the examples.

[0051] In some embodiments, the cells that are depleted of TDP-43 are neuronal cells.

[0052] CERT1 splice variant CERT1, also known as COL4A3BP or ceramide transport protein, is a protein that mediates the intracellular transport of ceramides and diacylglycerol lipids from the endoplasmic reticulum (ER) to the Golgi apparatus.

[0053] The exemplary CERT1 sequence is the human CERT1 sequence according to UniProt:Q9Y5P4. The amino acid sequence of human CERT1 (UniProt:Q9Y5P4-1) is provided below as Sequence ID No. 1. As used herein, the amino acid sequence of human CERT1 (Sequence ID No. 1) may be considered the wild-type sequence, i.e., the canonical sequence. Sequence ID 1 - Human CERT1 MSDNQSWNSSGSEEDPETESGPPVERCGVLSKWTNYIHGWQDRWVVLKNNALSYYKSEDETEYGCRGSICLSKAVITPHDFDECRFDISVNDSVWYLRAQDPDHRQQWIDAIEQHKTESGYGSESSLRRHGSMVSLVSGASGYSATSTSSFKKGHS LREKLAEMETFRDILCRQVDTLQKYFDACADAVSKDELQRDKVVEDDEDDFPTTRSDGDFLHSTNGNKEKLFPHVTPKGINGIDFKGEAITFKATTAGILATLSHCIELMVKREDSWQKRLDKETEKKRRTEEAYKNAMTELKKKSHFGGPDYEEG PNSLINEEEFFDAVEAALDRQDKIEEQSQSEKVRLHWPTSLPSGDAFSSVGTHRFVQKPYSRSSSMSSIDLVSASDDVHRFSSQVEEMVQNHMTYSLQDVGGDANWQLVVEEGEMKVYRREVEENGIVLDPLKATHAVKGVTGHEVCNYFWNVDVR NDWETTIENFHVVETLADNAIIIYQTHKRVWPASQRDVLYLSVIRKIPALTENDPETWIVCNFSVDHDSAPLNNRCVRAKINVAMICQTLVSPPEGNQEISRDNILCKITYVANVNPGGWAPASVLRAVAKREYPKFLKRFTSYVQEKTAGKPILF

[0054] This invention is based on the remarkable finding that splice variants of CERT1 act as biomarkers for loss of TDP43 expression and / or function, and can therefore be used as a diagnostic tool to identify diseases / pathologies associated with abnormal TDP-43 expression and / or TDP43 dysfunction.

[0055] The term "CERT1 splice variant" should be understood to refer to a CERT1 protein whose amino acid sequence differs from that of the wild-type / canonical CERT1 protein, such as a human CERT1 protein with the amino acid sequence of Sequence ID No. 1.

[0056] Therefore, a distinguishing feature between the wild-type CERT1 protein and splice variants of CERT1 is that splice variants of CERT1 may contain additional amino sequences, such as cryptic peptide sequences, that are not present in the wild-type CERT1 protein. In other words, the insertion of a cryptic peptide sequence into the amino acid sequence of the wild-type CERT1 protein can result in splice variants of CERT1. The event that leads to the formation of splice variants may be known as missplicing or latent splicing.

[0057] In some embodiments, the splice variant of CERT1 may include a cryptic peptide sequence. In other words, the amino acid sequence encoding the splice variant of CERT1 may include the amino acid sequence encoding the wild-type CERT1 protein and may further include a further amino acid sequence that is a cryptic peptide sequence not present in the amino acid sequence encoding the wild-type CERT1 protein.

[0058] As used herein, the term “cryptic peptide sequence” may be understood as an amino acid sequence introduced (e.g., inserted) into the amino acid sequence encoding the wild-type CERT1 protein. Therefore, a cryptic peptide sequence may also be referred to as an insertion.

[0059] In some embodiments, the cryptic peptide sequence may be 1 to 50 amino acids long. In some embodiments, the insertion may be 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, or 50 amino acids long.

[0060] In some embodiments, the cryptic peptide sequence may be 1 to 40 amino acids long.

[0061] In some embodiments, the cryptic peptide sequence may be 1 to 30 amino acids long.

[0062] In some embodiments, the cryptic peptide sequence may be 1 to 20 amino acids long.

[0063] In some embodiments, the cryptic peptide sequence may be 10 to 40 amino acids long.

[0064] In some embodiments, the cryptic peptide sequence may be 10 to 30 amino acids long.

[0065] In some embodiments, the cryptic peptide sequence may be 10 to 20 amino acids long.

[0066] In some embodiments, the cryptic peptide sequence may be 15 to 20 amino acids long.

[0067] In some embodiments, the cryptic peptide sequence may be 17 amino acids long.

[0068] In some embodiments, the cryptic peptide sequence may be 10 to 15 amino acids long.

[0069] In some embodiments, the cryptic peptide sequence may be 13 amino acids long.

[0070] In some embodiments, the cryptic peptide sequence can be located at any point within the splice variant of CERT1. In other words, the cryptic peptide sequence can be inserted at any position in the amino sequence of wild-type CERT1.

[0071] In some embodiments, the nucleic acid sequence encoding the cryptopeptide sequence may be inserted between exon 7 and exon 8 of the mRNA sequence encoding the splice variant of CERT1.

[0072] In some embodiments, the cryptoptic peptide sequence may be inserted at a position corresponding to position 158 of the amino acid sequence by Sequence ID No. 1, which encodes wild-type CERT1.

[0073] In some embodiments, the cryptic peptide sequence may include or consist of the amino acid sequence:VEITGSQLHFTWNETEK(SEQ ID NO: 2). In some embodiments, the cryptic peptide sequence may include the amino acid sequence:VEITGSQLHFTWNETEK(SEQ ID NO: 2). In some embodiments, the cryptic peptide sequence may consist of the amino acid sequence:VEITGSQLHFTWNETEK(SEQ ID NO: 2).

[0074] In some embodiments, the cryptic peptide sequence may comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence:VEITGSQLHFTWNETEK(SEQ ID NO: 2).

[0075] In some embodiments, the amino acid sequence VEITGSQLHFTWNETEK (SEQ ID NO: 2) can be encoded by a nucleic acid sequence having the sequence specified by SEQ ID NO: 5. Nucleic acid sequence encoding sequence number 5-VEITGSQLHFTWNETEK (sequence number 2): GTTGAAATAACTGGATCCCAGTTACATTTTACATGGAATGAAACTGAGAAG.

[0076] In some embodiments, the cryptic peptide sequence may include or consist of the amino acid sequence:VEITGSQLHFTWN(SEQ ID NO: 3). In some embodiments, the cryptic peptide sequence may include the amino acid sequence:VEITGSQLHFTWN(SEQ ID NO: 3). In some embodiments, the cryptic peptide sequence may consist of the amino acid sequence:VEITGSQLHFTWN(SEQ ID NO: 3).

[0077] In some embodiments, the cryptic peptide sequence may comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence:VEITGSQLHFTWN(SEQ ID NO: 3).

[0078] In some embodiments, the amino acid sequence VEITGSQLHFTWN (SEQ ID NO: 3) can be encoded by a nucleic acid sequence having the sequence specified by SEQ ID NO: 6. Nucleic acid sequence encoding sequence number 6-VEITGSQLHFTWN (sequence number 3): GTTGAAATAACTGGATCCCAGTTACATTTTACATGGAAT.

[0079] In some embodiments, it will be understood that the actual cryptic peptide sequence may be shorter in length than the cryptic peptide sequence described herein, for example, due to protease digestion during mass spectrometry and sample preparation. In some embodiments, the actual cryptic peptide sequence may be shorter by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids. In some embodiments, the actual cryptic peptide sequence may be shorter by 4 amino acids.

[0080] In some embodiments, for example, the actual cryptotic peptide sequence may be "VEITGSQLHFTWN" (SEQ ID NO: 3), but the peptide produced and detected may be "VEITGSQLHFTWNETEK" (SEQ ID NO: 2), where the four amino acids "ETEK" belong to the correctly spliced / canonical CERT1.

[0081] Therefore, in some embodiments, VEITGSQLHFTWNETEK (SEQ ID NO: 2) may be called a proteotypic peptide, but the actual cryptic peptide sequence may be VEITGSQLHFTWN (SEQ ID NO: 3).

[0082] In some embodiments, the splice variant of CERT1 may include or consist of the amino acid sequence given by SEQ ID NO: 4.

[0083] In some embodiments, the splice variant of CERT1 may consist of an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 4. Amino acid sequence of the splice variant of SEQ ID NO: 4-CERT1: .

[0084] In some embodiments, the amino acid sequence of the splice variant of CERT1 (SEQ ID NO: 4) can be encoded by a nucleic acid sequence having the sequence given by SEQ ID NO: 7. Nucleic acid sequence encoding a splice variant of sequence number 7-CERT1:

[0085] biomarkers The term "biomarker" is generally understood to refer to an indicator of a biological state. Biomarkers can be, for example, molecules, genes, mRNA, nucleic acid sequences, peptides, proteins, or amino acid sequences. It is also understood that certain biomarkers may be associated with specific diseases or pathological symptoms.

[0086] Biomarkers may be used predictively as a means of identifying individuals at risk of developing a disease or pathological condition.

[0087] Alternatively, biomarkers may be used diagnostically as a means of identifying subjects who have a disease or pathological symptoms.

[0088] Alternatively, biomarkers may be used prognostically as a means of monitoring disease or pathological symptoms in subjects, particularly those who have received treatment for a disease or pathological symptom and / or are in the process of receiving treatment for a disease or pathological symptom. In this case, the presence or absence of the biomarker may indicate whether or not the treatment for the disease or pathological symptom has been successful.

[0089] As used herein, the present invention may include a biomarker. The biomarker according to the present invention is a splice variant of CERT1 according to the present invention, wherein the CERT1 protein contains the cryptographic peptide sequence according to the present invention.

[0090] It will be understood that the presence of splice variants (i.e., complete proteins) and / or cryptic peptide sequences (i.e., isolated peptides) of CERT1 may be biomarkers.

[0091] In some embodiments, the biomarker is the splice variant of CERT1 according to the present invention (i.e., the CERT1 protein containing the cryptic peptide sequence).

[0092] In some embodiments, the biomarker is a cryptographic peptide sequence (i.e., an isolated peptide) according to the present invention.

[0093] In some embodiments, the biomarker may be in the form of a nucleic acid sequence such as DNA, RNA, and / or mRNA that encodes a splice variant or crypteptide sequence of CERT1.

[0094] In some embodiments, the biomarker may be an amino acid sequence, for example, an amino acid sequence of a splice variant of the CERT1 protein or an amino acid sequence of a cryptic peptide.

[0095] Biomarkers can also be called "signatures." For example, a biomarker in the form of a nucleic acid sequence can be called a "nucleic acid signature," and a biomarker in the form of an amino acid sequence can be called an "amino acid signature."

[0096] Biomarker expression refers to the amount of biomarker present and / or detectable in a sample obtained from a subject. Therefore, the terms “expression,” “amount,” or “level” may be considered interchangeable in this respect.

[0097] Suitable methods for detecting and / or measuring biomarkers are known in the art. Suitable methods for detecting and / or measuring biomarkers are disclosed herein.

[0098] Diagnostic methods It will be understood that the methods according to the present invention can typically be carried out outside the human body or the body of an animal. For example, the methods according to the present invention can be carried out using a sample obtained from a subject.

[0099] The presence of a cryptic peptide sequence according to the present invention and / or a splice variant of CERT1, such as a splice variant of CERT1 containing the cryptic peptide sequence according to the present invention, may be used as a biomarker for TDP-43 pathology. The presence of a cryptic peptide sequence according to the present invention and / or a splice variant of CERT1 may be further (or alternatively) used as a biomarker for identifying target cells, tissues, or organs having TDP-43 dysfunction.

[0100] Accordingly, the present invention provides a method for identifying TDP-43 pathology and / or TDP-43 dysfunction in a subject, comprising the step of identifying splice variants of CERT1 in a sample obtained from the subject. In some embodiments, the identification of splice variants of CERT1 includes the identification of cryptic peptide sequences. In some embodiments, the method is ex vivo. In some embodiments, the method is in vitro.

[0101] In some cases, individuals may have undiagnosed TDP-43 pathology, for example, reduced or abnormal expression of TDP-43 in their neuronal cells, but without showing symptoms of TDP-43 pathology at that stage. Therefore, detecting the presence of CERT1 splice variants in samples from such individuals may provide an indicator that the individual is at risk of developing TDP-43 pathology in the future.

[0102] Accordingly, the present invention provides a method for identifying subjects at risk of developing TDP-43 pathology and / or TDP-43 dysfunction, the method comprising the step of identifying a splice variant of CERT1 in a sample obtained from the subject, wherein the presence of the splice variant of CERT1 in the sample indicates that the subject is likely to develop TDP-43 pathology. In some embodiments, the identification of the splice variant of CERT1 includes the identification of a cryptic peptide sequence. In some embodiments, the method is ex vivo. In some embodiments, the method is in vitro.

[0103] In some cases, patients may be diagnosed with a TDP-43-related pathology (e.g., amyotrophic lateral sclerosis (ALS) or frontotemporal dementia), but the cause of the pathology (i.e., TDP-43 dysfunction and / or TDP-43 expression deficiency) may be unknown. Therefore, detecting the presence of CERT1 splice variants in samples from such patients may be used to determine whether the patient has TDP-43 dysfunction and / or TDP-43 expression deficiency, and thus whether the patient may benefit from treatments that partially or completely restore TDP-43 function and / or TDP-43 expression deficiency.

[0104] Accordingly, the present invention provides a method for determining whether a subject suffering from TDP-43 pathology and / or TDP-43 dysfunction is likely to respond to treatment, the method comprising the step of identifying a splice variant of CERT1 in a sample obtained from the subject, wherein the presence of the splice variant of CERT1 in the sample indicates that the subject is likely to respond to treatment. In some embodiments, the identification of the splice variant of CERT1 includes the identification of a cryptic peptide sequence. In some embodiments, the method is ex vivo. In some embodiments, the method is in vitro.

[0105] During or after administration of treatment to address TDP-43 pathology and / or TDP-43 dysfunction, the expression of CERT1 splice variants and / or cryptic peptide sequences may be reduced or undetectable. In this specification, “reduced” means that the amount or concentration of CERT1 and / or cryptic peptide sequences detected in a post-treatment patient sample is lower than the amount or concentration of CERT1 and / or cryptic peptide sequences detected in a pre-treatment patient sample.

[0106] Decreased or absent expression of the CERT1 splice variant and / or cryptic peptide sequence according to the present invention in patient-derived samples may indicate that the treatment was successful (or successful), for example, due to the rescue of misspliced ​​transcripts. This decrease can be compared to the expression or presence of the CERT1 splice variant before administration of the treatment.

[0107] The attached examples demonstrate that cells not expressing TDP-43 express a splice variant of CERT1, but treatment of the cells with (CA)n antisense oligonucleotides reduces the expression of the CERT1 splice variant. These results suggest that the expression of the CERT1 splice variant and / or cryptic peptide sequence may be used to monitor the therapeutic success (e.g., ongoing or previously administered) of disease treatment with therapies that improve TDP-43 expression and / or TDP-43 function.

[0108] Accordingly, the present invention provides a method for monitoring the therapeutic success of treatment for TDP-43 pathology and / or TDP-43 dysfunction in a subject, the method comprising the steps of identifying splice variants of CERT1 in a sample obtained from the subject, and further comprising the steps of predicting the therapeutic success of treatment for TDP-43 pathology and / or TDP-43 dysfunction based on the detection of splice variants of CERT1. In some embodiments, the identification of splice variants of CERT1 includes the identification of cryptic peptide sequences. In some embodiments, the method is ex vivo. In some embodiments, the method is in vitro.

[0109] In some embodiments, the absence of a detectable splice variant and / or cryptic peptide sequence in CERT1 may indicate therapeutic success (i.e., the treatment is successful in treating the disease). In some embodiments, a decrease in the detectable splice variant and / or cryptic peptide sequence in CERT1 may indicate therapeutic success (i.e., the treatment is successful in treating the disease). The decrease may be a 5-100% decrease in the expression of the splice variant and / or cryptic peptide sequence in CERT1 compared to the expression of the splice variant and / or cryptic peptide sequence in CERT1 before administration of the treatment. In some embodiments, the decrease may be 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, or 90-100%. In some embodiments, the reduction may be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0110] In some embodiments, the treatment may include a protein or nucleic acid molecule capable of restoring the functional phenotype of one or more TDP-43 target RNAs in cells that are depleted of TDP-43 or expressing abnormal TDP-43 proteins.

[0111] In some embodiments, the treatment may comprise antisense oligonucleotides capable of restoring the functional phenotype of one or more TDP-43 target RNAs in cells that are TDP-43 depleted or expressing abnormal TDP-43 proteins. In some embodiments, the treatment may mimic the function of TDP-43 and rescue misspliced ​​transcripts upon TDP-43 loss.

[0112] use As described herein, loss of TDP-43 function and / or TDP-43 expression may result in the expression of splice variants and / or cryptic peptide sequences of CERT1. Therefore, these splice variants and / or cryptic peptide sequences of CERT1 may be used as biomarkers to identify subjects who have (or are at risk of developing) TDP-43 pathology and / or TDP-43 dysfunction.

[0113] Therefore, the present invention provides the use of splice variants of CERT1 as biomarkers for TDP-43 pathology and / or TDP-43 dysfunction. The present invention also provides the use of cryptic peptide sequences as biomarkers for TDP-43 pathology and / or TDP-43 dysfunction.

[0114] In some embodiments, the presence of biomarkers in samples obtained from subjects may be used to identify subjects as having TDP-43 pathology and / or TDP-43 dysfunction.

[0115] In some embodiments, the presence of biomarkers in samples obtained from subjects may be used to identify subjects at risk of developing TDP-43 pathology and / or TDP-43 dysfunction.

[0116] In some embodiments, the presence or absence of biomarkers in a sample obtained from a subject may be used to determine whether a subject suffering from TDP-43 pathology and / or TDP-43 dysfunction is likely to respond to treatment.

[0117] In some embodiments, the presence or absence of a biomarker in a sample obtained from a subject may be used to monitor the progression of TDP-43 pathology and / or TDP-43 function decline in the subject.

[0118] In some embodiments, the presence or absence of a biomarker in a sample obtained from a subject may be used to monitor the treatment of TDP-43 pathology and / or TDP-43 dysfunction in subjects who have previously received treatment for TDP-43 pathology and / or TDP-43 dysfunction, and / or who are in the process of receiving treatment for TDP-43 pathology and / or TDP-43 dysfunction.

[0119] Identification of splice variants in CERT1 It will be understood that TDP-43 pathology and / or TDP-43 dysfunction may be identified by: (i) detecting splice variants of CERT1 according to the present invention; (ii) detecting cryptic peptide sequences contained within splice variants of CERT1 according to the present invention; and / or (iii) detecting the cryptic peptide sequences themselves, isolated from splice variants of CERT1 according to the present invention.

[0120] In some embodiments, the complete splice variant of the CERT1 protein (i.e., including the cryptic peptide sequence) may be detected. In some embodiments, the cryptic peptide sequence (i.e., isolated from the complete splice variant of the CERT1 protein) may be detected.

[0121] In some embodiments, the expression of the cryptic peptide sequence and / or splice variant of CERT1 according to the present invention can be measured or detected at the mRNA expression level (i.e., at the transcript level) by measuring or detecting one or more nucleic acid sequences encoding the cryptic peptide sequence and / or splice variant of CERT1 according to the present invention.

[0122] The expression of mRNA encoding the cryptic peptide sequence and / or the splice variant of CERT1 according to the present invention can be measured or detected using any method or technique known in the art that is suitable for measuring mRNA or transcript expression. Suitable techniques include quantitative PCR (including quantitative PCR) and RNA sequencing.

[0123] In some embodiments, the expression of the cryptic peptide sequence and / or splice variant of CERT1 according to the present invention can be measured or detected at the level of protein expression.

[0124] The protein expression of the cryptic peptide sequence and / or the splice variant of CERT1 according to the present invention may be measured or detected using any method or technique known in the art that is suitable for measuring protein expression. Suitable techniques include proteomic analysis and antibody-based assays. For example, the protein expression of the cryptic peptide sequence and / or the splice variant of CERT1 according to the present invention may be measured or detected using proteomics, mass spectrometry (MS), chromatography, enzyme-linked immunosorbent assay (ELISA), flow cytometry, Western blotting, or ultra-high sensitivity immunoassays (see, for example, https: / / www.quanterix.com / simoa-technology / ). Mass spectrometry may include any form of mass spectrometry, including (but not limited to) hyperreaction monitoring mass spectrometry (HRM-MS).

[0125] It will be understood that comprehensive proteomics profiling using mass spectrometry is suitable for use in the present invention to identify splice variants of CERT1 and / or cryptic peptide sequences according to the present invention. For example, after digesting a sample with Lys-C protease, hyperreaction monitoring mass spectrometry (HRM-MS) protein profiling may be used to identify splice variants of CERT1 and / or cryptic peptide sequences according to the present invention.

[0126] In some embodiments, the step of identifying splice variants and / or cryptic peptide sequences of CERT1 may include proteomic analysis. In some embodiments, the proteomic analysis is performed using a sample obtained from the subject.

[0127] In some embodiments, the step of identifying the splice variant and / or cryptic peptide sequence of CERT1 includes measuring the peptide in the sample using liquid chromatography (LC). In some embodiments, the step of identifying the splice variant and / or cryptic peptide sequence of CERT1 includes measuring the peptide in the sample using mass spectrometry (MS). In some embodiments, the mass spectrometry is hyperreaction monitoring mass spectrometry (HRM-MS).

[0128] In some embodiments, the step of identifying splice variants and / or cryptic peptide sequences of CERT1 may include an antibody-based assay. In some embodiments, the antibody-based assay may be ELISA, flow cytometry, or Western blotting.

[0129] sample As described herein, the cryptic peptide sequence and / or splice variant of CERT1 according to the present invention may be detected in a sample obtained from a subject.

[0130] In some embodiments, the sample is obtained from body fluids or tissues. In some embodiments, the sample is selected from blood, serum, plasma, urine, saliva, brain tissue, and cerebrospinal fluid (CSF).

[0131] In some embodiments, the sample is blood.

[0132] In some embodiments, the sample is brain tissue.

[0133] In some embodiments, the sample is cerebrospinal fluid (CSF).

[0134] Methods and techniques for obtaining these samples from the subject are well known in the art.

[0135] Any method or technique suitable for obtaining these samples from a subject is included in the present invention.

[0136] subject In some embodiments, the methods and applications of the present invention may be mammals.

[0137] In some embodiments, the subject may be human.

[0138] In other embodiments, the subject may alternatively be a non-human mammal, such as a dog, cat, horse, cow, or pig.

[0139] In some embodiments, the subject is a patient. In some embodiments, the subject is a human patient.

[0140] The subjects may be patients undergoing treatment (i.e., ongoing treatment) such as treatment to address TDP-43 pathology and / or TDP-43 dysfunction. The subjects may also be patients who have previously received treatment such as treatment to address TDP-43 pathology and / or TDP-43 dysfunction.

[0141] It will be understood that, as used herein, the terms “patient” and “subject” are considered interchangeable.

[0142] disease Where used herein, the term “treatment” refers to both the treatment of an existing disease (e.g., a disease or disorder as referred herein) and the prevention of a disease, i.e., prevention. Therefore, it will be recognized that in some embodiments, treatments referred herein may be preventative. In some embodiments, treatments may not be preventative; for example, treatments may be treatments for existing disease symptoms diagnosed in a patient.

[0143] It will be understood that the terms "pathology" and "disease" are interchangeable.

[0144] It will be understood that the terms "procedure" and "treatment" are interchangeable.

[0145] TDP-43 pathology is a disorder associated with decreased or abnormal expression of TDP-43, often related to increased cytoplasmic TDP-43, particularly hyperphosphorylated and ubiquitinated TDP-43.

[0146] In some embodiments, TDP-43 pathology and / or TDP-43 dysfunction may be caused by neuronal cells expressing disease-associated variants of TDP-43 and / or dysfunctional TDP-43 and / or neuronal cells depleted of normal TDP-43 protein.

[0147] TDP-43 depletion is manifested in various diseases known as TDP-43 pathology.

[0148] In some embodiments, TDP-43 pathology may be neurological. In some embodiments, TDP-43 dysfunction may be associated with neurological disorders.

[0149] In some embodiments, the neurological disorder may be selected from amyotrophic lateral sclerosis (ALS), frontotemporal dementia, frontotemporal lobar degeneration (FTLD), progressive supranuclear palsy (PSP), primary lateral sclerosis, progressive muscular atrophy, Alzheimer's disease, Parkinson's disease, autism, hippocampal sclerotic dementia, Down syndrome, Huntington's disease, polyglutamine diseases, such as spinocerebellar ataxia type 3, myopathy, and chronic traumatic encephalopathy.

[0150] In some embodiments, the neurological disorder may be selected from amyotrophic lateral sclerosis (ALS), frontotemporal dementia, and frontotemporal lobar degeneration (FTLD). In some embodiments, the neurological disorder may be amyotrophic lateral sclerosis (ALS). In some embodiments, the neurological disorder may be frontotemporal dementia. In some embodiments, the neurological disorder may be frontotemporal lobar degeneration (FTLD).

[0151] antibody The present invention provides an antibody that binds to a splice variant of CERT1 according to the present invention.

[0152] In some embodiments, antibodies may bind to the cryptic peptide sequence according to the present invention, which is contained within the amino acid sequence of a splice variant of CERT1 according to the present invention.

[0153] In some embodiments, an antibody may bind to a cryptic peptide sequence according to the present invention, which is isolated from the complete sequence of a splice variant of CERT1 according to the present invention.

[0154] In some embodiments, the antibody may exclusively bind to the cryptic peptide sequence according to the present invention.

[0155] In some embodiments, the epitope to which the antibody can bind may be the amino acid sequence:VEITGSQLHFTWNETEK (SEQ ID NO: 2), or a cryptic peptide sequence comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto.

[0156] In some embodiments, the epitope to which the antibody can bind may be the amino acid sequence:VEITGSQLHFTWN (SEQ ID NO: 3), or a cryptic peptide sequence comprising or having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto.

[0157] In some embodiments, an antibody may bind to a portion of the cryptic peptide sequence according to the present invention (for example, the epitope may overlap with the cryptic peptide sequence according to the present invention). In some embodiments, the epitope to which the antibody may bind may include the amino acid sequence:VEITGSQLHFTWN (SEQ ID NO: 3), or a portion of a cryptic peptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity therewith.

[0158] kit The present invention provides a kit comprising at least one reagent for detecting a splice variant of CERT1 according to the present invention, wherein the at least one reagent is an antibody according to the present invention.

[0159] In some embodiments, the kit may include instructions for using the kit.

[0160] In some embodiments, the kit may include additional reagents.

[0161] General terms and definitions The term "polypeptide" is used in its conventional sense to refer to a series of amino acids, typically L-amino acids, linked to one another by peptide bonds between the α-amino and carboxyl groups of adjacent amino acids. The term "polypeptide" is used interchangeably with the terms "amino acid sequence," "peptide," and / or "protein." The term "residue" is used to refer to an amino acid in an amino acid sequence.

[0162] The term "variant" refers to a polypeptide that has equivalent function to the amino acid sequence described herein but includes one or more amino acid substitutions, insertions, or deletions.

[0163] As used herein, “variant” is synonymous with “mutant” and refers to a polynucleotide or amino acid sequence that differs from the corresponding wild-type sequence. The term “wild-type” is used to mean a gene or protein that has the same polynucleotide or amino acid sequence as the naturally occurring gene or protein, respectively.

[0164] A nucleic acid sequence can be an RNA sequence or a DNA sequence or a variant thereof. The term "polynucleotide" includes RNA sequences or DNA sequences. It can be single-stranded or double-stranded. It can be, for example, a genome, recombinant mRNA, or cDNA.

[0165] This disclosure is not limited to the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of embodiments of this disclosure. Numerical ranges include the number defining the range. Unless otherwise indicated, any nucleic acid sequence is written from left to right in the 5' to 3' direction. Amino acid sequences are written from left to right, each in the amino to carboxy direction.

[0166] Where a range of values ​​is provided, each intervening value is understood to be between the upper and lower limits of that range, with values ​​specifically disclosed to tenths of a unit of the lower limit, unless the context explicitly indicates otherwise. Each smaller range between any stated value or intervening value within a stated range and any other stated value or intervening value within that stated range is included in this disclosure. The upper and lower limits of these smaller ranges may independently be included in or excluded from the range, and if a smaller range includes either the upper or lower limit, neither of them, or both, each range is also included in this disclosure, and any upper or lower limits specifically excluded within the stated range are subject to that exclusion. If a stated range includes one or both of the upper or lower limits, the range excluding one or both of the upper or lower limits that they include is also included in this disclosure.

[0167] When used herein and in the appended claims, the singular forms "a," "an," and "the" refer to multiple subjects unless the context explicitly indicates otherwise.

[0168] The terms “comprising,” “comprises,” and “comprised of,” as used herein, are synonymous with “including,” “includes,” or “containing,” and are comprehensive or open-ended, and do not exclude additional unlisted members, elements, or method steps. The terms “comprising,” “comprises,” and “comprised of” also include the term “consisting of.”

[0169] The publications described herein are provided solely for disclosure prior to the filing date of this patent application. Nothing in this specification should be construed as acknowledging that such publications constitute prior art of the attached claims.

[0170] The present invention will now be further illustrated by examples, which are intended to assist those skilled in the art in carrying out the invention and are not intended to limit the scope of the invention.

[0171] identity The terms “identity” and “sequence identity %” as used herein may refer to the percentage of nucleotides in a sequence of nucleic acid molecules (e.g., oligonucleotides) that are identical to a reference sequence (e.g., a sequence motif) across the sequence. Therefore, the percentage of identity is calculated by counting the number of identical (matching) aligned nucleic acid bases between the two sequences (in the sequence of the compound of the present invention and in the reference sequence), dividing that number by the total number of nucleotides in the oligonucleotide, and multiplying by 100. Thus, the percentage of identity = (number of matches × 100) / length of the aligned region (e.g., sequence of nucleotides). Insertions and deletions are not permitted in the calculation of the percentage of identity of a sequence of nucleotides. It will be understood that, in determining identity, chemical modifications of nucleic acid bases are ignored as long as the nucleic acid base retains its functional ability to form Watson-Crick base pairs (e.g., 5-methylcytosine is considered identical to cytosine for the purposes of calculating identity %).

[0172] Similarly, the terms “identity” and “sequence identity %” may, as used herein, refer to the percentage of amino acids in an amino acid sequence within a peptide or protein that are identical to a reference sequence across the amino acid sequence. Therefore, the percentage of identity is calculated by counting the number of identical (matching) aligned amino acids between the two sequences (in the amino acid sequence of the peptide or protein of the present invention and in the reference sequence), dividing that number by the total number of amino acids in the amino acid sequence, and multiplying by 100. Thus, the percentage of identity = (number of matches × 100) / length of aligned region. Insertions and deletions are not permitted in the calculation of the percentage of amino acid sequence identity. Array Overview [Table 1] [Examples]

[0173] Example 1 method HRM mass spectrometry Commercially available hiPSC-derived glutamatergic cortical neurons, iCell GlutaNeurons (FUJIFILM Cellular Dynamics, Cat.R1034), were co-cultured with iCell Astrocytes (FUJIFILM Cellular Dynamics, Cat.R1092) according to the manufacturer's guidelines. The ratio of astrocytes to neurons was 1:4. The cells were then cultured with the corresponding antisense LNA gapmer or (CA). n Antisense oligonucleotides (ASOs) were used for treatment on days 2 and 5, respectively. The culture medium was changed every 3-4 days, and LNA gapmers or (CA) were used. n One of the ASOs was used. For TDP-43 KD cells, only the LNA gapmer was added with every other medium change. Cells were harvested and pelletized on day 30. The cell pellets were frozen and kept at -80°C. Each sample contained approximately 2 million cells.

[0174] Comprehensive proteomics profiling using mass spectrometry (MS) was performed at Biognosys AG (Schlieren, Switzerland). Briefly, frozen cell pellets were lysed and then digested with Lys-C protease. Hyperreaction monitoring (HRM)-MS protein profiling was performed. Data were analyzed using Spectronaut 15 Pulsar software.

[0175] ASO used in the test 1. LNA gapmer (used for knockdown of TDP-43): 5'-TTCCGTTTTGAACATGCAA-3' The structure of the LNA gapmer is shown in Figure 2.

[0176] 2. (CA)nASO (mimics the function of TDP-43 and is used to rescue misspliced ​​transcripts in the event of TDP-43 loss): 5'-[CholTEG]-CACACACACACACACACACACACAC-3' The structure of (CA)nASO is shown in Figure 3.

[0177] result TDP-43 knockdown was performed using LNA gapmers in co-cultures of hiPSC-derived neurons and astrocytes. TDP-43 levels were reduced by approximately 80% (Figure 1A). A novel proteotypic peptide, VEITGSQLHFTWNETEK, was identified by mass spectrometry upon TDP-43 loss. Peptide strength was significantly reduced upon treatment with (CA)nASO (Figure 1B).

[0178] All publications referenced in the above specification are incorporated herein by reference. Various modifications and variations of the methods and systems described in the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention is described in relation to certain preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. In fact, various modifications of the described embodiments for carrying out the present invention, which will be apparent to those skilled in the art in molecular biology or related fields, are intended to be within the scope of the following claims.

Claims

1. A method for identifying TDP-43 pathology and / or TDP-43 dysfunction in a subject, comprising the step of identifying a splice variant of CERT1 in a sample obtained from the subject.

2. A method for identifying subjects at risk of developing TDP-43 pathology and / or TDP-43 dysfunction, the method comprising the step of identifying a splice variant of CERT1 in a sample obtained from the subject, wherein the presence of the splice variant of CERT1 in the sample indicates that the subject is likely to develop TDP-43 pathology.

3. A method for determining whether a subject suffering from TDP-43 pathology and / or TDP-43 dysfunction is likely to respond to treatment, the method comprising the step of identifying a splice variant of CERT1 in a sample obtained from the subject, wherein the presence of the splice variant of CERT1 in the sample indicates that the subject is likely to respond to treatment.

4. A method for monitoring the therapeutic success of treatment for TDP-43 pathology and / or TDP-43 dysfunction in a subject, the method comprising the step of identifying a splice variant of CERT1 in a sample obtained from the subject, and further comprising the step of predicting the therapeutic success of the treatment for TDP-43 pathology and / or TDP-43 dysfunction based on the detection of the splice variant of CERT1.

5. The method according to claim 3 or 4, wherein the treatment comprises an antisense oligonucleotide capable of restoring the functional phenotype of one or more TDP-43 target RNAs in cells that are depleted of TDP-43 or expressing an abnormal TDP-43 protein.

6. Use of CERT1 splice variants as biomarkers for TDP-43 pathology and / or TDP-43 dysfunction.

7. The method according to any one of claims 1 to 5, or the use according to claim 6, wherein the splice variant of CERT1 comprises a cryptic peptide sequence.

8. The method or use according to claim 7, wherein the splice variant of CERT1 comprises a cryptic peptide sequence of 1 to 30 amino acids.

9. The method or use according to claim 8, wherein the splice variant of CERT1 comprises a cryptic peptide sequence of 10 to 20 amino acids.

10. The method or use according to claim 8 or 9, wherein the splice variant of CERT1 comprises a 13-amino acid cryptic peptide sequence.

11. The method or use according to any one of claims 7 to 10, wherein the cryptic peptide sequence is inserted at a position corresponding to position 158 of the amino acid sequence described in SEQ ID NO: 1, which encodes wild-type CERT1.

12. The method or use according to any one of claims 7 to 11, wherein the cryptic peptide sequence comprises or consists of the amino acid sequence VEITGSQLHFTWN (SEQ ID NO: 3), or a variant thereof having at least 80% sequence identity.

13. The method or use of claim 12, wherein the cryptic peptide sequence comprises or consists of an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence VEITGSQLHFTWN (SEQ ID NO: 3).

14. The method or use according to claim 12 or claim 13, wherein the cryptic peptide sequence comprises or consists of the amino acid sequence VEITGSQLHFTWN of SEQ ID NO:

3.

15. The method according to any one of claims 1 to 5 or 7 to 14, or the use according to any one of claims 6 to 14, wherein the splice variant of CERT1 comprises or consists of the amino acid sequence described in SEQ ID NO: 4 or a variant having at least 80% sequence identity therewith.

16. The method or use of claim 15, wherein the splice variant of CERT1 comprises or consists of an amino acid having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

4.

17. The method or use according to claim 15 or claim 16, wherein the splice variant of CERT1 comprises or consists of the amino acid sequence described in SEQ ID NO:

4.

18. The method according to any one of claims 1 to 5 or 7 to 17, wherein the method is an ex vivo method.

19. The method according to any one of claims 1 to 5 or 7 to 18, wherein the step of identifying the splice variant of CERT1 comprises proteomic analysis or an antibody-based assay.

20. The method according to any one of claims 1 to 5 or 7 to 19, wherein the step of identifying the splice variant of CERT1 comprises measuring the peptide in the sample using liquid chromatography (LC) and / or mass spectrometry (MS).

21. The method according to any one of claims 1 to 5 or 7 to 20, wherein the sample is obtained from a body fluid or tissue selected from blood, serum, plasma, urine, saliva, brain tissue, and cerebrospinal fluid (CSF).

22. The method according to any one of claims 1 to 5 or 7 to 21, wherein the subject is a human, preferably a human patient.

23. The method according to any one of claims 1 to 5 or 7 to 22, or the use according to any one of claims 6 to 17, wherein the TDP-43 pathology is a neurological disorder.

24. The method or use according to claim 23, wherein the TDP-43 pathology is a neurological disorder selected from amyotrophic lateral sclerosis (ALS), frontotemporal dementia, frontotemporal lobar degeneration (FTLD), progressive supranuclear palsy (PSP), primary lateral sclerosis, progressive muscular atrophy, Alzheimer's disease, Parkinson's disease, autism, hippocampal sclerotic dementia, Down syndrome, Huntington's disease, polyglutamine diseases, such as spinocerebellar ataxia type 3, myopathy, and chronic traumatic encephalopathy.

25. The method or use according to claim 23 or claim 24, wherein the TDP-43 pathology is a neurological disorder selected from amyotrophic lateral sclerosis (ALS), frontotemporal dementia, and frontotemporal lobar degeneration (FTLD).

26. An antibody that binds to the splice variant of CERT1.

27. The antibody according to claim 26, wherein the splice variant of CERT1 comprises the amino acid sequence VEITGSQLHFTWN (SEQ ID NO: 3), or a variant thereof having at least 80% sequence identity, or an epitope comprising the same.

28. The antibody according to claim 26 or claim 27, wherein the splice variant of CERT1 comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence VEITGSQLHFTWN (SEQ ID NO: 3), or comprises an epitope consisting thereof.

29. The antibody according to any one of claims 26 to 28, wherein the splice variant of CERT1 includes or consists of the amino acid sequence described in SEQ ID NO: 4 or a variant having at least 80% sequence identity with SEQ ID NO:

4.

30. The antibody according to any one of claims 26 to 29, wherein the splice variant of CERT1 comprises or consists of an amino acid having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

4.

31. A kit comprising at least one reagent for detecting a splice variant of CERT1, wherein the at least one reagent is the antibody described in any one of claims 26 to 30.

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