Method for predicting prognosis and therapeutic effect on motor neuron disease

Abstract

As a result of performing an analysis exclusively on lower limb type and upper limb type ALS, excluding the bulbar palsy type ALS, among ALS for which it had been considered in existing reports that a reduction in Crtac1B was not observed, a significant correlation between the prognosis and the Crtac1B concentration in the cerebrospinal fluid was observed. Thus, it was discovered that Crtac1B is useful as a biomarker for predicting the prognosis of lower limb type and upper limb type ALS. As a result of analyzing the association between the motor function and the Crtac1B concentration in the cerebrospinal fluid before and after treatment in SMA patients treated with an SMA therapeutic agent, it was found that the Crtac1B concentration in the cerebrospinal fluid reflects the disease progression of SMA, and that Crtac1B is useful as a biomarker for diagnosing the therapeutic effect by the SMA therapeutic agent.

Description

Method for predicting prognosis and therapeutic effect of motor neuron disease

[0001] The present invention relates to a method for predicting the prognosis and therapeutic effect of motor neuron disease, and more particularly to a method for predicting the prognosis of patients with lower limb or upper limb amyotrophic lateral sclerosis, and a method for assessing the therapeutic effect in patients administered a therapeutic drug for spinal muscular atrophy.

[0002] Amyotrophic lateral sclerosis (ALS) is an intractable neurological disease in which motor neurons in the central nervous system undergo degeneration and cell death, leading to paralysis of the respiratory muscles and death within a few years. Although various molecular pathologies have been proposed, they have not yet been fully elucidated, and no fundamental treatment is available. One of the reasons for the lack of progress in treatment development is the delay in the development of biomarkers. Biomarkers that can predict the progression and prognosis of ALS are particularly essential for developing new treatments. Therefore, the development of novel biomarkers that reflect ALS pathology is an urgent issue for overcoming the disease.

[0003] Recently, it has been reported that neurofilament light chain (NfL), a cytoskeletal component abundant in neuronal axons, leaks into the cerebrospinal fluid (CSF) and blood, reflecting neuronal damage, and its increased concentration has been shown to correlate with prognosis in ALS patients (Non-Patent Document 1). However, the changes are far from clear, raising doubts about its practical utility in clinical practice. Furthermore, transactive response DNA-binding protein-43 (TDP-43), an important pathological molecule that accumulates in ALS neurons, has been shown to be mildly elevated in the CSF of ALS patients, but the difference is small, and no correlation with patient prognosis has been demonstrated (Non-Patent Document 1).

[0004] Spinal muscular atrophy (SMA), an autosomal recessive hereditary neuromuscular disease, causes degeneration of spinal anterior horn motor neurons, resulting in progressive muscle atrophy and weakness (Non-Patent Documents 2, 3). The clinical spectrum of the disease ranges from the most severe form, which leads to early death in infancy, to forms that allow patients to live a normal life. According to the Rare Diseases Information Center, the incidence of the most severe form is approximately 1 in 20,000 (as of December 2016), making SMA a leading cause of death from genetic diseases in infants.

[0005] The pathology and cause of SMA has been shown to be due to a lack of SMN protein caused by deletion or mutation of the SMN1 gene (Gene ID: 6606), resulting in degeneration of lower motor neurons in the anterior horn of the spinal cord (Non-Patent Documents 2, 4, 5). SMN1 has a duplicate gene, SMN2 (Gene ID: 6607). While the SMN1 gene expresses full-length functional SMN protein, the SMN2 gene primarily expresses a truncated, non-functional SMN protein due to exon 7 skipping, resulting in approximately 10% full-length SMN protein expression. In SMA patients, deletion or mutation of the SMN1 gene results in the loss or severe reduction of full-length functional SMN protein expression from the SMN1 gene, while the SMN2 gene cannot adequately supply full-length functional SMN protein, resulting in SMN protein deficiency. Therefore, SMA can be improved by supplementing functional SMN protein in neurons.

[0006] Methods for replenishing functional SMN protein in neurons include increasing endogenous functional SMN protein by inhibiting exon 7 skipping of the SMN2 pre-mRNA, or producing exogenous functional SMN protein by administering a vector capable of expressing the SMN protein gene. All currently available SMA treatments slow SMA progression and improve motor function through one of two mechanisms. Spinraza (trade name), which contains the sodium salt of the antisense oligonucleotide nusinersen as its active ingredient, and Evrisdi (trade name), which contains the small molecule compound risdiplam as its active ingredient, are splicing modulators that suppress exon 7 skipping of the SMN2 pre-mRNA. Zongelsma (trade name), which contains the non-replicating recombinant adeno-associated virus onasemnogene abeparvovec that expresses functional SMN protein, as its active ingredient, falls into the latter category. It is likely that various SMA treatments that slow SMA progression using similar mechanisms of action will be developed in the future.

[0007] As mentioned above, SMA therapeutic drugs are already in practical use, but their effectiveness is assessed using a rating scale that scores motor function, which is subjective to the evaluator's subjectivity. If biochemical biomarkers existed that could predict disease progression and reflect treatment efficacy, treatment efficacy could be objectively evaluated, but such biomarkers currently do not exist. NfL, which has been suggested to be related to prognosis in ALS, is sometimes used as a reference for assessing the effectiveness of SMA therapeutic drugs. However, because it is not a molecule directly related to the pathology, it is not sufficient to predict disease progression or treatment efficacy, as is the case with ALS. Although SMN protein expression has been shown to be a promising biomarker (Non-Patent Document 6), because it is not neurospecific, blood levels do not necessarily correlate with expression levels in motor neurons or clinical symptoms, making it insufficient for assessing disease progression or treatment efficacy.

[0008] Previously, the present inventors' group identified a novel endogenous molecule, Lateral Olfactory Tract Usher Substance (LOTUS), which promotes nerve regeneration by antagonizing the nerve regeneration inhibitor Nogo receptor type 1 (NgR1) and inhibiting NgR1 signaling (Non-Patent Document 7). LOTUS is a splicing variant of Crtac1, known as a marker of chondrocyte precursor cells, called Crtac1B, and is primarily expressed in the nervous system. Subsequently, the present inventors' group found that in neurodegenerative diseases accompanied by inflammation and / or demyelination, such as multiple sclerosis, LOTUS concentrations in cerebrospinal fluid (CSF) are reduced in response to disease progression, reflecting axonal degeneration (Non-Patent Documents 8-10). They then developed a technique for detecting neurodegenerative diseases accompanied by inflammation and / or demyelination using low LOTUS levels in samples such as CSF (Patent Document 1). According to Patent Document 1 and Non-Patent Documents 8-10, LOTUS levels are not reduced in ALS, a neurodegenerative disease without inflammation or demyelination, and thus ALS was previously considered undetectable by LOTUS levels. Like ALS, SMA is a neurodegenerative disease that does not involve inflammation or demyelination.

[0009] WO 2014 / 080979 A1

[0010] Kasai T, et al., Ann Clin Transl Neurol. 2019;6:2489-502Lunn MR et al., Lancet. 2008;371(9630):2120-2133Darras BT et al., Pediatr Clin North Am. 2015;62(3):743-766Kolb SJ et al., Arch Neurol. 2011;68(8):979-984Lefebvre S et al., Cell. 1995;80(1):155-165Alves CRR, et al., Mol Ther Methods Clin Dev. 2021; 23: 524-538Sato Y et al. Science 2011; 333: 769-773Takahashi K et al. JAMA Neurol. 2015; 72(2):176-9.Takahashi K et al. Neurodegener Dis Manag 2015; 5(6):469-72Takahashi K et al. Clin Exp Neuroimmunol 2015; 6(S1):64-69

[0011] One object of the present invention is to provide a biomarker for predicting the prognosis of ALS. Another object of the present invention is to provide a biomarker for objectively evaluating the therapeutic effect of a therapeutic drug for SMA.

[0012] The present inventors analyzed only lower limb and upper limb ALS, excluding bulbar paralysis, among the ALS types for which a decrease in LOTUS / Crtac1B was not observed in Patent Document 1 and Non-Patent Documents 8 to 10. As a result, they found a significant correlation between prognosis and LOTUS / Crtac1B concentration in cerebrospinal fluid, and discovered that LOTUS / Crtac1B is useful as a biomarker for predicting the prognosis of lower limb and upper limb ALS. They have thus completed an invention of a method for predicting the prognosis of patients with lower limb or upper limb ALS, which includes the following aspects.

[0013] Furthermore, the inventors analyzed the relationship between motor function and cerebrospinal fluid LOTUS / Crtac1B concentration before and after treatment in SMA patients receiving treatment with an SMA therapeutic drug, and found that the cerebrospinal fluid LOTUS / Crtac1B concentration reflects the progression of SMA disease and that LOTUS / Crtac1B is useful as a biomarker for diagnosing the therapeutic effect of an SMA therapeutic drug. They then completed an invention of a method for assessing the effectiveness of an SMA therapeutic drug, which includes the following aspects.

[0014] (Method for predicting prognosis of a patient with lower limb or upper limb amyotrophic lateral sclerosis) [1] A method for predicting the prognosis of a patient with lower limb or upper limb amyotrophic lateral sclerosis, the method comprising measuring Crtac1B protein in a sample derived from the patient, wherein a higher concentration of Crtac1B protein in the sample indicates a higher likelihood of a better prognosis. [2] The method described in [1], wherein the sample is blood or cerebrospinal fluid.

[0015] (Method for assessing the effectiveness of a therapeutic drug for SMA) [3] A method for assessing the therapeutic effect in a patient administered a therapeutic drug for spinal muscular atrophy, comprising: measuring, as a current measurement, the Crtac1B protein concentration in a sample collected from the patient after administration of the therapeutic drug; and comparing the current measurement with a pre-treatment measurement, which is the Crtac1B protein concentration measured in a sample collected from the patient before administration of the therapeutic drug, or with a previous measurement of the patient; wherein a therapeutic effect is indicated when the current measurement is at the same level or higher as the pre-treatment measurement or the previous measurement. [4] The method of [3], wherein the therapeutic effect is improvement or maintenance of motor function. [5] The method of [3] or [4], wherein the sample is blood or cerebrospinal fluid. [6] The method of any one of [3] to [5], wherein the spinal muscular atrophy is spinal muscular atrophy caused by biallelic deletion or mutation of the SMN1 gene.

[0016] According to the prognosis prediction method for patients with lower limb or upper limb ALS of the present invention, the patient's prognosis can be predicted based on the Crtac1B protein concentration in a patient-derived sample. The prognosis prediction by the method of the present invention is possible early after onset. Therefore, for patients predicted to have a high probability of a poor prognosis, intensive treatment with a combination of multiple therapeutic agents from early onset can slow the progression of the disease. Furthermore, if respiratory support with a ventilator has not yet been initiated, prompt preparation for ventilator use can prevent death from rapidly progressing respiratory failure, providing important benefits in treatment. On the other hand, for patients predicted to have a high probability of a good prognosis, important benefits in treatment selection can be provided, such as avoiding highly invasive treatments and improving quality of life. This provides a new biomarker that reflects the ALS pathology and contributes to the development of ALS therapies. According to the method for assessing the effectiveness of an SMA therapeutic drug of the present invention, the therapeutic effect of an SMA therapeutic drug can be determined using an objective indicator, i.e., the Crtac1B protein concentration in a sample collected from the patient.

[0017] Analysis of the relationship between CSF LOTUS concentrations and survival time in patients with upper and lower limb ALS. The top row shows data from 31 patients, and the bottom row shows data from 28 patients who also met the condition that CSF was collected within 24 months of symptom onset. Analysis of the relationship between CSF LOTUS concentrations and survival time in patients with bulbar ALS. The top row shows data from 20 patients, and the bottom row shows data from 18 patients who also met the condition that CSF was collected within 24 months of symptom onset. SOD1-Tg mice were overexpressed with mouse LOTUS, and survival time (top row) and motor function (bottom row) were analyzed. CSF LOTUS concentrations and motor function assessment scales were measured immediately before treatment administration (before treatment initiation) and after three loading doses (after treatment initiation) in two SMA patients who showed significant improvements in functional assessment scales after loading doses of Spinraza. These are the LOTUS concentrations in cerebrospinal fluid and motor function assessment scale scores (total values ​​for both left and right) in two patients receiving maintenance administration of Spinraza.

[0018] Hereinafter, the present invention will be described, starting with the points common to the method for predicting the prognosis of a patient with lower limb type or upper limb type ALS (hereinafter, sometimes referred to as the ALS prognosis prediction method) and the method for assessing the effectiveness of a therapeutic drug for SMA (hereinafter, sometimes referred to as the SMA therapeutic effectiveness assessment method).

[0019] I. Factors Common to the ALS Prognosis Prediction Method and the SMA Treatment Efficacy Assessment Method The cartilage acidic protein 1 (Crtac1) gene (NCBI Gene ID: 55118) encodes the Crtac1B protein (hereinafter, Crtac1B may be referred to as LOTUS) and has two known transcript variants, Crtac1A and Crtac1B, which are registered in NCBI GenBank under accession numbers NM_018058.7 (protein: NP_060528.3) and NM_001206528.3 (protein: NP_001193457.1), respectively. These sequences are shown in SEQ ID NOS: 1 to 4 in the Sequence Listing. The Crtac1A and Crtac1B proteins share the same region from amino acid 1 to amino acid 606, with the regions thereafter being characteristic of each variant. However, the above-mentioned sequences registered with GenBank are examples of wild-type sequences, and naturally occurring Crtac1B proteins may have amino acid sequences in which a small number of amino acids (e.g., one to several) have been substituted, deleted, inserted, or added as a result of polymorphisms in the gene sequence. The Crtac1B protein referred to in the present invention also encompasses such naturally occurring variants. SEQ ID NOs: 5 and 6 show the sequences of mouse LOTUS / Crtac1B (NM_145123.5, NP_660105.3). SEQ ID NOs: 7 and 8 show the sequences of rat LOTUS / Crtac1B (NM_134401.2, NP_599228.2).

[0020] As samples, blood samples (whole blood, serum, plasma) and cerebrospinal fluid (cerebrospinal fluid) samples can be preferably used. Although cerebrospinal fluid samples are used in the following examples, the method of the present invention can also be carried out using blood samples because cerebrospinal fluid flows into a vein.

[0021] The method for analyzing the sample is not particularly limited, and any method that can quantitatively measure the Crtac1B protein present in the sample may be used. For example, various techniques have been established, such as mass spectrometry, chromatography (liquid chromatography, gas chromatography, etc.), electrophoresis (capillary electrophoresis, etc.), and immunoassays using antibodies, and methods that use aptamers instead of antibodies may also be used, but are not limited to these.

[0022] Examples of mass spectrometry include laser-desorption / ionization time-of-flight mass spectrometry (LDI-TOF / MS) and electrospray ionization mass spectrometry (ESI-MS). Analytical equipment for both methods is commercially available, and those skilled in the art can easily perform these methods.

[0023] Immunoassays can be classified based on the reaction format, including sandwich, competitive, agglutination, and Western blotting. A blot-free Western assay, an improved version of Western blotting, uses capillary electrophoresis to separate target protein molecules and detect them with specific antibodies. Immunoassays can also be classified based on labeling, including radioimmunoassays, fluorescent immunoassays, enzyme immunoassays (EIA), and biotin immunoassays. All of these methods are included in the "immunoassay" category and can be used to measure Crtac1B protein. Non-limiting examples include ELISA, turbidimetric immunoassay (TIA), latex immunoagglutination (LATEX), electrochemiluminescence immunoassay (ECLIA), and immunochromatography. Protein chips, which immobilize antibodies on glass or polymer substrates to detect target proteins, can also be used.

[0024] Either polyclonal or monoclonal antibodies can be used for immunoassay, but monoclonal antibodies or antigen-binding fragments thereof are preferred from the standpoint of reproducibility, etc. When immunoassaying Crtac1B protein in the present invention, a commercially available anti-Crtac1 antibody may be used, or an antibody or antigen-binding fragment thereof that binds to Crtac1B protein may be prepared and used.

[0025] Methods for producing antibodies and their antigen-binding fragments are also well known. For example, antibodies can be produced by the hybridoma method. Specifically, for example, Crtac1B protein or a fragment thereof prepared by chemical synthesis or genetic engineering techniques is used as an immunogen to immunize an animal (excluding humans) together with an appropriate adjuvant, thereby inducing antibodies against Crtac1B in the animal. Antibody-producing cells, such as splenocytes or lymphocytes, are collected from the animal and fused with immortalized cells, such as myeloma cells, to produce hybridomas. Using the Crtac1B protein to be detected as a screening antigen, hybridomas that specifically bind to the Crtac1B protein can be selected from the hybridomas, which can then be grown and cultured to obtain monoclonal antibodies against the Crtac1B protein from the culture supernatant. Since the amino acid sequence of the Crtac1B protein and the nucleotide sequence encoding it are publicly known, as described above, those skilled in the art can easily prepare the Crtac1B protein or its fragments to be used as immunogens using standard methods. In the present invention, Crtac1B protein is primarily measured in samples derived from human patients, and therefore, when preparing antibodies for the immunoassay, an immunogen prepared based on the amino acid sequence of human Crtac1B is usually used. However, as long as an antibody capable of binding to the Crtac1B protein to be measured can be obtained, an immunogen prepared based on the amino acid sequence of Crtac1B from an animal species other than humans, such as mouse or rat, may also be used.

[0026] An "antigen-binding fragment" may be any antibody fragment as long as it maintains the binding ability of the original antibody to its corresponding antigen (antigen-antibody reactivity). Specific examples include, but are not limited to, Fab, F(ab')2, and scFv. As is well known, Fab and F(ab')2 can be obtained by treating a monoclonal antibody with a protease such as papain or pepsin. Methods for producing scFv (single-chain fragment of variable region, single-chain antibody) are also well known. For example, mRNA from a hybridoma prepared as described above is extracted, single-stranded cDNA is prepared, and PCR is performed using primers specific to the immunoglobulin heavy and light chains to amplify the immunoglobulin heavy and light chain genes. These genes are then linked with a linker, inserted into a plasmid vector after providing appropriate restriction enzyme sites, and transformed into Escherichia coli with the vector to express the scFv, which can then be recovered from the Escherichia coli to obtain the scFv.

[0027] When using immunoassays (e.g., sandwich assays, competitive assays, agglutination assays) that require specific detection of Crtac1B protein in distinction from Crtac1A, antibodies or antigen-binding fragments thereof that are highly specific for Crtac1B protein but do not substantially bind to Crtac1A protein are used. Such antibodies highly specific for Crtac1B protein (anti-Crtac1B antibodies) can be obtained, for example, by generating antibodies using the C-terminal region from amino acid 607 onwards, or a portion thereof, which is a region that is not present in human Crtac1A but is characteristic of human Crtac1B, as an immunogen, and then using Crtac1A protein and Crtac1B protein as screening antigens to select antibodies that bind to Crtac1B but not Crtac1A. In mouse LOTUS / Crtac1B (SEQ ID NO: 6), the region corresponding to the region characteristic of human Crtac1B is the C-terminal region from amino acid 608 onwards. In rat LOTUS / Crtac1B (SEQ ID NO: 8), the region corresponding to the above-mentioned region characteristic of human Crtac1B is the C-terminal region from amino acid 608 onwards.

[0028] When using immunoassays that can separate and analyze target proteins based on molecular size, such as Western blotting and blot-free Western blots, antibodies with high specificity for Crtac1B protein are not the only options. Anti-Crtac1 antibodies specific for Crtac1 that bind to both Crtac1A and Crtac1B proteins can also be used. Anti-Crtac1 antibodies that bind to both Crtac1A and Crtac1B proteins can be produced using the region of amino acids 1 to 606 common to human Crtac1A and human Crtac1B, or a portion thereof, as an immunogen. The region of amino acids 1 to 607 in mouse LOTUS / Crtac1B (SEQ ID NO: 6) and rat LOTUS / Crtac1B (SEQ ID NO: 8) corresponds to the aforementioned human consensus region. In the Examples below, an anti-Crtac1 antibody was used that was produced using as an immunogen the region of residues 42 to 56 of rat LOTUS / Crtac1B, which corresponds to the position of the β-turn between the α-helix and the β-sheet (this structure is thought to be suitable as an antibody recognition site). A specific example of an anti-Crtac1 antibody is an antibody produced using as an immunogen a peptide containing a rat LOTUS / Crtac1B partial region that encompasses the region of residues 42 to 56 of rat LOTUS / Crtac1B. When using a short peptide as an immunogen, immunogenicity can be enhanced by immunizing non-human animals with a peptide with a Cys residue added to the N-terminus (which can be easily prepared by chemical synthesis).

[0029] Measurements using the sandwich method with anti-Crtac1B antibodies can be performed, for example, as follows: Anti-Crtac1B antibodies are immobilized on a solid-phase support such as a plate or particles and used as solid-phase antibodies. Alternatively, anti-Crtac1 antibodies that bind to both Crtac1A and Crtac1B are conjugated to a labeling substance (enzyme, fluorescent substance, chemiluminescent substance, radioactive substance, etc.) and used as labeled antibodies. By contacting the solid-phase antibody with a sample, Crtac1B in the sample specifically binds to the solid-phase antibody, and Crtac1B is captured on the support via the solid-phase antibody. The support is washed and then reacted with the labeled antibody. After washing the support to remove unreacted labeled antibody, the signal from the labeled substance is measured by an appropriate method. When an enzyme is used as a labeling substance, a substrate such as a chromogenic, fluorescent, or luminescent substrate corresponding to the enzyme is reacted with the enzyme, and the resulting signal is measured.

[0030] The method for detecting the signal is selected appropriately depending on the type of labeling substance. For example, if the signal is colorimetric, a colorimeter or absorptiometer can be used; if it is fluorescent, a fluorometer can be used; if it is luminescent, a photon counter can be used; and if it is radioactive, a radiation measuring device can be used. Standard samples containing various concentrations of Crtac1B with known concentrations are subjected to immunoassay to measure the amount of Crtac1B. A calibration curve is then prepared by plotting the correlation between the amount of signal from the labeling substance and the Crtac1B concentration in the standard samples. The same measurement procedure is then performed on samples with unknown Crtac1B concentrations to measure the amount of signal from the label. The measured values ​​can then be applied to this calibration curve to quantify Crtac1B in the sample.

[0031] When measuring Crtac1B levels by Western blotting, Crtac1B is detected as a band with a molecular weight of 70 kD. Therefore, as described above, an anti-Crtac1 antibody that also binds to Crtac1A can be used as the primary antibody. A label-conjugated anti-immunoglobulin antibody, typically an enzyme-labeled antibody, is used as the secondary antibody. Standard samples containing known concentrations of Crtac1B are electrophoretically separated together with the test sample. After transfer to a membrane, the sample is reacted sequentially with the primary antibody and a labeled secondary antibody. After washing, if the label is an enzyme, the membrane is further reacted with an appropriate substrate to detect the bands. The band intensity on the membrane can be quantified using a commercially available image analyzer. A calibration curve can be created from the band intensities of the standard samples, and the band intensities of the test sample can be applied to this calibration curve to quantify Crtac1B in the test sample.

[0032] For the blot-free Western method, an automated system that integrates the entire protein separation and detection process is commercially available (the automated capillary electrophoresis and analysis device used in the Examples below), and Crtac1B in the test sample can be quantified using an anti-Crtac1 antibody that also binds to Crtac1A.

[0033] II. ALS Prognosis Prediction Method The method for predicting the prognosis of a patient with lower limb or upper limb ALS according to the present invention comprises measuring Crtac1B protein in a sample derived from a patient with lower limb or upper limb ALS. The patient is a patient diagnosed with lower limb or upper limb ALS, typically a human patient. Samples derived from a patient with lower limb or upper limb ALS include samples collected from patients already diagnosed with ALS and samples collected from patients at a pre-diagnosis stage of suspected ALS. In accordance with the purpose of the present invention of predicting prognosis, it is desirable to perform the ALS prognosis prediction method of the present invention by collecting a sample from a patient at a suspected stage of ALS, at the time of diagnosis, or early after diagnosis (if a sample is collected at a suspected stage of ALS, the ALS prognosis prediction method of the present invention is performed after a diagnosis of ALS), thereby providing prognostic information to patients and their families at an early stage.

[0034] A higher Crtac1B protein concentration in a sample indicates a higher probability of a good prognosis. Conversely, a lower Crtac1B protein concentration in a sample indicates a higher probability of a poor prognosis. A good prognosis for ALS means that the disease progresses slowly, physical functions are maintained, and the survival period or remaining lifespan is long. A long remaining lifespan means, for example, a remaining lifespan of more than about 30 months, more than about 40 months, or more than about 50 months. A poor prognosis for ALS means that the disease progresses rapidly, physical functions are significantly deteriorated, and the survival period or remaining lifespan is short. A short remaining lifespan means, for example, a remaining lifespan of about 30 months or less.

[0035] When setting a cutoff value or reference value for the Crtac1B protein concentration in a sample to determine whether the prognosis is good or poor, it can be set depending on the type of sample. Examples of such values ​​include: (1) a value that distinguishes between good and poor prognosis, i.e., a value below which a measured value indicates a high probability of a poor prognosis and a value above which a measured value indicates a high probability of a good prognosis; (2) a value above which a measured value indicates a high probability of a good prognosis; and (3) a value below which a measured value indicates a high probability of a poor prognosis. Hereinafter, (1) may be referred to as the good / poor reference value, (2) as the good reference value, and (3) as the poor reference value. Simply referring to reference values ​​refers to all of (1) to (3). In implementing the ALS prognosis prediction method, only one of (1) to (3) may be set, or both (2) and (3) may be set. When both (2) and (3) are set, (2) is a higher value than (3).

[0036] When the sample is cerebrospinal fluid, the reference value can be set, for example, within the range of 150 to 250 ng / ml, 180 to 230 ng / ml, 150 to 200 ng / ml, 200 to 280 ng / ml, 200 to 250 ng / ml, 230 to 280 ng / ml, or 250 to 300 ng / ml. When any one of (1), (2), and (3) is set, the reference value can be set within the range of 150 to 250 ng / ml, 180 to 230 ng / ml, 150 to 200 ng / ml, 200 to 280 ng / ml, 200 to 250 ng / ml, 230 to 280 ng / ml, or 250 to 300 ng / ml. When (2) and (3) are set, two reference values ​​(where (2) > (3)) can be set within the range of 150 to 250 ng / ml, 180 to 230 ng / ml, 150 to 200 ng / ml, 200 to 280 ng / ml, 200 to 250 ng / ml, 230 to 280 ng / ml, or 250 to 300 ng / ml. As an example of an embodiment in which only a poor reference value is used as the reference value, the poor reference value can be set at 180 ng / ml or 200 ng / ml, and if the Crtac1B protein concentration in the cerebrospinal fluid is less than 180 ng / ml or less than 200 ng / ml, it can be predicted that the prognosis is likely to be poor, for example, that the patient's life expectancy is likely to be approximately 30 months or less.

[0037] The above-mentioned reference value examples are examples of measuring Crtac1B protein in cerebrospinal fluid samples by blot-free Western blotting. It has been confirmed that when Crtac1B protein in cerebrospinal fluid samples is measured by ELISA, the measured value is approximately 1 / 7 of that by blot-free Western blotting (data from the examples omitted). Therefore, when measuring Crtac1B protein in cerebrospinal fluid samples by ELISA, the reference value can be set, for example, within the range of 20-35 ng / ml, 25-33 ng / ml, 20-30 ng / ml, 30-40 ng / ml, 30-35 ng / ml, 33-40 ng / ml, or 35-43 ng / ml. When setting any one of (1), (2), and (3), the reference value can be set within the range of 20 to 35 ng / ml, 25 to 33 ng / ml, 20 to 30 ng / ml, 30 to 40 ng / ml, 30 to 35 ng / ml, 33 to 40 ng / ml, or 35 to 43 ng / ml. When setting (2) and (3), two reference values ​​(provided that (2) > (3)) can be set within the range of 20 to 35 ng / ml, 25 to 33 ng / ml, 20 to 30 ng / ml, 30 to 40 ng / ml, 30 to 35 ng / ml, 33 to 40 ng / ml, or 35 to 43 ng / ml. As an example of an embodiment in which only the poor standard value is used as the standard value, the poor standard value can be set at 25 ng / ml or 30 ng / ml, and if the Crtac1B protein concentration in the cerebrospinal fluid is less than 25 ng / ml or less than 30 ng / ml, it can be predicted that the prognosis is likely to be poor, for example, that the patient's life expectancy is likely to be approximately 30 months or less.

[0038] For patients predicted to have a high probability of a poor prognosis, it is possible to implement at least one of the following measures: intensive treatment combining multiple therapeutic drugs, or prompt preparation for the use of a ventilator (if respiratory support with a ventilator has not yet been initiated).For patients predicted to have a high probability of a good prognosis, it is possible to prioritize improving quality of life by reviewing their treatment method, specifically, switching to a less invasive treatment if they were receiving a highly invasive treatment, or selecting a less invasive treatment if they have not yet started ALS treatment and were considering a highly invasive treatment.

[0039] In embodiments in which only a good reference value and a bad reference value, or both, are set, the ALS prognosis prediction method of the present invention may not provide a clear prediction result. For example, in embodiments in which only a good reference value is set, the patient's measured value is below the good reference value; in embodiments in which only a bad reference value is set, the patient's measured value is above the bad reference value; and in embodiments in which both a good reference value and a bad reference value are set, the patient's measured value is below the good reference value and above the bad reference value. In such cases, for patients who have already started ALS treatment, the current treatment can be continued and a treatment method can be selected according to the progression of symptoms. For patients who have not started treatment, standard treatment can be selected and, after a period of time, the ALS prognosis prediction method of the present invention can be used again to reconsider the treatment method.

[0040] Furthermore, since increasing Crtac1B levels is expected to improve patient prognosis, the prognosis prediction method of the present invention can also be applied to the development of therapeutic drugs for lower limb or upper limb ALS. For example, samples such as cerebrospinal fluid are collected from a model animal of lower limb or upper limb ALS, or from a patient with lower limb or upper limb ALS, before and after administration of a candidate therapeutic substance, and the Crtac1B protein concentrations in the samples are compared. If the Crtac1B protein concentration after administration is higher than before administration, this indicates that the candidate substance is likely to have a therapeutic effect on lower limb or upper limb ALS.

[0041] III. Method for assessing the effectiveness of SMA treatment A method for assessing the effectiveness of treatment in a patient administered a therapeutic drug for SMA according to the present invention includes the steps of measuring, as a current measurement, the Crtac1B protein concentration in a sample collected from the patient after administration of the SMA therapeutic drug, and comparing the current measurement with a pre-treatment measurement, which is the Crtac1B protein concentration measured in a sample collected from the patient before administration of the SMA therapeutic drug, or with a previous measurement of the patient.

[0042] Patients who are the target of the method for assessing the effectiveness of a treatment for SMA are patients who are receiving treatment with a therapeutic drug for SMA, and include patients who have developed clinical symptoms (patients who have developed SMA), as well as patients who have not yet developed clinical symptoms but who are predicted to develop SMA by genetic testing (mainly young patients under the age of 2). In one embodiment, the patient is a patient who has developed SMA.

[0043] The SMA targeted by the present invention is typically SMA caused by deletion or mutation of the biallelic SMN1 gene.

[0044] SMA treatments are broadly divided into splicing modulators that suppress the skipping of exon 7 of the SMN2 mRNA precursor, and gene therapy drugs containing vectors capable of expressing the gene encoding the SMN protein. Splicing modulators include Spinraza (trade name), whose active ingredient is the sodium salt of the antisense oligonucleotide nusinersen, and Evrisdi (trade name), whose active ingredient is the small molecule compound risdiplam. Gene therapy drugs include Zongelsma (trade name), whose active ingredient is onasemnogene abeparvovec, a non-replicating recombinant adeno-associated virus that expresses functional SMN protein.

[0045] Splicing modulators increase the production of functional SMN protein from the SMN2 gene. Therefore, all of the known splicing modulators listed above are intended for administration to patients who have been confirmed by genetic testing to have a deletion or mutation in the SMN1 gene and one or more copies of the SMN2 gene (see package inserts for Spinraza and Evrisdy). Therefore, when a therapeutic drug for SMA is a splicing modulator, SMA patients may have one or more copies of the SMN2 gene. Gene therapy drugs supplement exogenous functional SMN protein, regardless of the number of copies of the SMN2 gene in SMA patients. The nucleotide sequence of human SMN1 gene mRNA (NM_000344.4) and the amino acid sequence encoded thereby are shown in SEQ ID NOs: 9 and 10, and the human SMN2 gene mRNA (NM_017411.4) and the amino acid sequence encoded thereby are shown in SEQ ID NOs: 11 and 12, respectively. However, the SMN2 gene mainly expresses a truncated, non-functional SMN protein due to skipping of exon 7, and expression of the full-length SMN protein is approximately 10%, so the amount of SMN protein with the amino acid sequence shown in SEQ ID NO: 12 expressed from the SMN2 gene is low.

[0046] In one embodiment, the SMA therapeutic agent is a splicing modulator that suppresses skipping of exon 7 of the SMN2 pre-mRNA, the SMA is caused by a deletion or mutation in the biallelic SMN1 gene, and the patient has one or more copies of the SMN2 gene. In another embodiment, the SMA therapeutic agent is nusinersen or a pharmaceutically acceptable salt thereof, risdiplam, or onasemnogene abeparvovec.

[0047] Splicing modulators are typically substances that bind to a specific region of the SMN2 pre-mRNA and suppress the skipping of exon 7, and include antisense oligonucleotides, antibody drugs, peptide compounds, and small molecule compounds. In one embodiment, the splicing modulator is an antisense oligonucleotide or a small molecule compound.

[0048] The specific region of the SMN2 pre-mRNA is a region in the SMN2 pre-mRNA that is associated with exon 7 skipping, and the following regions have been reported to date:(1) The ISS motif (CCAGCATTATGAAAG, SEQ ID NO: 13) located at +10 to +24 in SMN2 intron 7 (Singh NK et al. MOLECULAR AND CELLULAR BIOLOGY, February 2006, pp. 1333-1346; Hua Y, et al. The American Journal of Human Genetics 82, 834-848, April 2008) (nusinersen binding site). (2) The regions known to function similarly to the ISS motif in SMN2 intron 7 are +4 to +21 (TTTAGACAAAATCAAAAA, region 855-872 in SEQ ID NO: 11) and +34 to +51 (TCACATTCCTTAAATTAA, region 885-902 in SEQ ID NO: 11) in SMN2 exon 7 (Hua Y, et al. (2007) PLoS Biol 5(4): e73. https: / / doi.org / 10.1371 / journal.pbio.0050073) (3) ESE2 (aaaaagaaggaa, region 868-879 in SEQ ID NO: 11), which is present in exon 7 of the SMN2 pre-mRNA and is one of the sequences that controls the incorporation of exon 7 into mRNA (Sivaramakrishnan, M., McCarthy, KD, Campagne, S. et al. Binding to SMN2 pre-mRNA-protein complex elicits specificity for small molecule splicing modifiers. Nat Commun 8, 1476 (2017). https: / / doi.org / 10.1038 / s41467-017-01559-4) (one of the sites where risdiplam binds) (4) SMN2 with U1 snRNA (U1 small nuclear RNA) bound The 5's sequence (GA--AAGT) present at the exon 7-intron 7 boundary of the pre-mRNA (Sivaramakrishnan, M., McCarthy, KD, Campagne, S. et al. (2017) cited above) (another site where risdiplam binds).

[0049] A substance that binds to at least one of the above-mentioned regions can suppress exon 7 skipping, promote incorporation of exon 7 into SMN2 mRNA, and increase the production of endogenous functional SMN protein. When the splicing regulator is an antisense oligonucleotide, a substance that binds to at least one of these regions is an oligonucleotide that contains a base sequence complementary to one of these regions.

[0050] Antisense oligonucleotide drugs themselves are a well-known technology that has already been put to practical use, and generally, oligonucleotides that have been chemically modified to improve in vivo stability (conferring nuclease resistance) or binding affinity to target regions are used. Examples of chemical modifications include techniques such as changing some or all of the internucleotide phosphodiester bonds to phosphorothioate bonds, or replacing some or all of the nucleotides with artificial nucleic acids such as LNA, AmNA, or 2'-MOE. Nusinersen is an antisense oligonucleotide that employs such techniques, and other antisense oligonucleotides used to treat SMA also typically employ such techniques.

[0051] When the splicing modulator is a low molecular weight compound, risdiplam is a representative example, and in addition to related compounds with a similar structure to risdiplam, various low molecular weight compounds that bind to at least one of the regions related to exon 7 skipping in the SMN2 pre-mRNA are included.

[0052] Gene therapy drugs themselves are well-known technologies that have already been put to practical use. Examples of vectors used in gene therapy drugs for SMA include viral vectors such as adeno-associated viruses (AAVs), which are also used in onasemnogene abeparvovec, adenoviruses, and retroviruses. However, viral vectors with high central nervous system delivery (e.g., AAV9) are generally preferred for SMA gene therapy drugs. Examples of the SMN gene incorporated into viral vectors include the sequence of the coding region of the SMN1 gene mRNA shown in SEQ ID NO: 9 (positions 18 to 902 of SEQ ID NO: 9) and nucleotides of a base sequence having 90% or more, e.g., 95% or more, or 98% or more identity to said sequence.

[0053] In the method for assessing the efficacy of SMA treatment, the Crtac1B protein concentration in a sample collected from a patient after administration of an SMA therapeutic drug is measured as a current measurement value, and this current measurement value is compared with a pre-treatment measurement value or a previous measurement value. A pre-treatment measurement value is a Crtac1B protein concentration measured in a sample collected from the patient before administration of the SMA therapeutic drug. In patients who have received approximately three or more doses, in addition to the current measurement value, comparisons may also be made with measurements from the time before last and any time before that, and comparisons with pre-treatment measurements may be made as desired.

[0054] If the current measurement is at the same level as or higher than the pre-treatment or previous measurement, it indicates that a therapeutic effect has been achieved. The therapeutic effect here refers to the improvement or maintenance of motor function. If the current measurement is higher than the pre-treatment or previous measurement, it indicates that motor function has improved. If the current measurement is at the same level as the pre-treatment or previous measurement, it indicates that motor function has been maintained (not deteriorated). This same level also includes a temporary slight decrease. If the current measurement is compared with the previous, previous-previous, and previous measurements, and if it appears to maintain a generally constant level, with slight decreases or increases, it can be determined that there is no change in Crtac1B protein concentration (motor function is maintained), and if the current measurement is lower than the pre-treatment or previous measurement, it indicates that a therapeutic effect has not been achieved. In this case, it is desirable to further compare the current measurement with the previous-previous and previous measurements (for patients who have been receiving the drug three or more times) to determine whether the Crtac1B protein concentration has clearly decreased rather than being a temporary decrease.

[0055] Patients who are shown to be benefiting from treatment may continue to receive the same SMA drug, while patients who are shown not to be benefiting from treatment may be discontinued or switched to a different SMA drug.

[0056] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to the following examples.

[0057] 1. Prognosis prediction for patients with amyotrophic lateral sclerosis (lower limb or upper limb type) 1-1. Methods Cerebrospinal fluid LOTUS concentrations were measured using an automated capillary electrophoresis / analysis device (trade name WES), which separates and analyzes target molecules based on molecular size, to detect the target protein using a specific antibody. The specific antibody used was a synthetic peptide (SEQ ID NO: 14) produced by adding Cys to the N-terminus of a partial peptide of rat LOTUS (residues 42 to 56 of rat LOTUS) to enhance immunogenicity, using the immunogen as the immunogen.

[0058] The samples used were cerebrospinal fluid from a total of 51 ALS patients, including 46 patients who met the following criteria: "death from the disease and the date of death could be confirmed" and "cerebrospinal fluid was collected within 24 months of the onset of symptoms." A correlation analysis was conducted between LOTUS concentration and survival time.

[0059] Human mutant SOD1 transgenic mice overexpressing LOTUS (SOD1-Tg) were generated by crossbreeding mouse LOTUS-overexpressing transgenic mice (LOTUS-Tg) with SOD1-Tg mice. The motor function of the mice was evaluated using a wire hang test. The mice were asked to hold onto a wire mesh, which was then inverted, and the time it took for the mice to fall off the mesh was measured.

[0060] 1-2. Results As with the results of the analysis conducted on ALS patients in 2015 (Patent Document 1, Non-Patent Documents 8-10), no significant results were observed in the analysis combining bulbar, upper limb, and lower limb types (data omitted). However, when the clinical disease types of bulbar, upper limb, and lower limb were analyzed separately, a strong correlation was found between LOTUS concentration and survival time in the upper limb and lower limb types (Figure 1-1). No correlation was observed in the bulbar type (Figure 1-2).

[0061] To clarify the molecular basis, LOTUS was overexpressed in SOD1-Tg mice, a model mouse of ALS, and motor function and survival time were analyzed. As a result, motor function and survival time were significantly improved in LOTUS-overexpressing SOD1-Tg mice compared with SOD1-Tg mice (Figure 2), suggesting a relationship between the expression level of LOTUS in the central nervous system and the progression and prognosis of ALS pathology.

[0062] 1-3. Discussion: High LOTUS concentrations in the cerebrospinal fluid of ALS patients may improve disease progression and prognosis. Furthermore, it has been shown that ALS model mice overexpressing LOTUS exhibit improved motor function and prognosis. Therefore, LOTUS is considered to be a highly promising biomarker for reflecting the pathology of ALS and predicting prognosis.

[0063] The bottom row of Figure 1-1 shows the results of an analysis limited to CSF ​​samples collected within 24 months of onset from 28 patients. In the top row of the analysis results for CSF from 31 patients, the three samples enclosed by dashed lines were collected more than 24 months after onset, but these three samples are plotted relatively close to the regression line. The analysis was conducted under the assumption that the condition of measuring LOTUS concentrations in samples collected long after onset, such as "sample collection within 24 months of symptom onset," may be important, but it was confirmed that such a condition is not important for predicting the prognosis of ALS based on LOTUS concentrations in samples.

[0064] 2. Evaluation of therapeutic efficacy of spinal muscular atrophy medications 2-1. Methods We analyzed the Revised Upper Limb Module (RULM) scores immediately before and after multiple doses of the drug from SMA patients whose cerebrospinal fluid was collected and stored before and after treatment with the SMA medication Spinraza at Yokohama City University Hospital, as well as the LOTUS concentration in cerebrospinal fluid collected at the same time as functional assessment. Concentration measurements were performed using an automated capillary electrophoresis analyzer (product name WES) as in 1. above.

[0065] 2-2. Results After the initial dose, Spinraza is administered at 4 and 12 weeks (loading dose), and then at six-month intervals (maintenance dose). Patients receiving Spinraza treatment generally see a significant improvement in motor function with the loading dose, and while further improvement in motor function is unlikely during maintenance doses, there is no worsening of symptoms and the improved motor function after the loading dose is maintained.

[0066] Figure 3 shows the LOTUS concentration in cerebrospinal fluid and the motor function assessment scale immediately before the administration of the drug (before the start of treatment) and after three loading doses (after the start of treatment) in two SMA patients who showed a significant improvement in the functional assessment scale after the loading dose. The LOTUS concentration after the start of treatment tended to increase in both cases, which coincided with the improvement in the functional assessment scale.

[0067] Figure 4 shows the cerebrospinal fluid LOTUS concentration and motor function assessment scale for two patients undergoing maintenance administration. Neither motor function nor cerebrospinal fluid LOTUS concentration remained significant, remaining stable. It was confirmed that in patients whose motor function deterioration was prevented by SPINRAZA administration, cerebrospinal fluid LOTUS concentration was also stable.

[0068] 2-3. Discussion LOTUS concentrations reflect the progression of SMA disease, increasing in patients whose symptoms improved, and remaining stable in patients whose symptoms stabilized and whose motor function was prevented from worsening. Therefore, LOTUS concentrations are useful as a biomarker for diagnosing the progression of SMA disease and the effectiveness of treatment.

Claims

1. A method for predicting the prognosis of a patient with lower limb or upper limb amyotrophic lateral sclerosis, comprising measuring Crtac1B protein in a sample derived from the patient, wherein a higher concentration of Crtac1B protein in the sample indicates a higher likelihood of a better prognosis.

2. The method of claim 1, wherein the sample is blood or cerebrospinal fluid.

3. A method for assessing the effectiveness of treatment in a patient administered a therapeutic drug for spinal muscular atrophy, comprising: measuring, as a current measurement, the Crtac1B protein concentration in a sample taken from the patient after administration of the therapeutic drug; and comparing the current measurement with a pre-treatment measurement, which is the Crtac1B protein concentration measured in a sample taken from the patient before administration of the therapeutic drug, or with a previous measurement of the patient; wherein if the current measurement is at the same level or higher than the pre-treatment measurement or the previous measurement, it is indicated that a therapeutic effect has been obtained.

4. The method according to claim 3, wherein the therapeutic effect is improvement or maintenance of motor function.

5. The method of claim 3 or 4, wherein the sample is blood or cerebrospinal fluid.

6. The method according to any one of claims 3 to 5, wherein the spinal muscular atrophy is spinal muscular atrophy caused by a deletion or mutation in the biallelic SMN1 gene.