Mutations in the CEP290 gene in amyotrophic lateral sclerosis

Abstract

To provide methods for diagnosing and stratifying an ALS patient, and a method for determining ALS contraction and / or the risk thereof.SOLUTION: The present invention discloses a mutation found in a gene encoding CEP290 of a patient having amyotrophic lateral sclerosis (ALS). The present invention also discloses methods for diagnosing and stratifying an ALS patient based on the mutation.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to mutations found in the gene encoding CEP290 in patients with amyotrophic lateral sclerosis (ALS). [Background technology]

[0002] Amyotrophic lateral sclerosis (ALS) is a disease that impairs the motor neurons that transmit signals from the brain to the muscles and their function. Genetic abnormalities, oxidative stress, and glutamate excess have been proposed as possible causes of ALS. Genetic factors such as gene abnormalities (e.g., mutations in superoxide dismutase (SOD1), TDP43, FUS, optineurin, C9ORF72, SQSTM1, and TUBA4A) are thought to be the most common causes of ALS (Non-Patent Documents 1 to 4).

[0003] CEP290 is a 290-kDa centrosome protein encoded by the CEP290 gene in humans (Non-Patent Document 5). CEP290 has been known to be the causative gene for Senior Loken syndrome (SLC), Joubert syndrome (JS) (Non-Patent Document 6), fatal Meckel-Gruber syndrome (MKS) (Non-Patent Document 7), and Bardet-Biedl syndrome (BBS) (Non-Patent Document 8). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Andersen PM, and Al-Chalabi A. Nat. Rev. Neurol., 2011; 7: 603-615 [Non-patent document 2] Oda M et al., Brain Nerve., 2011; 63: 165-170 [Non-patent document 3] Deng HX et al., Nature, 2011; 477: 211-215 [Non-patent document 4] Fecto F et al., Arch. Neurol., 2011; 68: 1440-1446 [Non-Patent Document 5] Coppieters F et al., Hum. Mutat., 2010;31: 1097-1108 [Non-patent document 6] Sayer JA et al., Nature Genet. 2006; 38: 674-681 [Non-Patent Document 7] Baala L et al.,Am. J. Hum. Genet. 2007; 80: 186-194 [Non-patent document 8] Leitch CC et al., Nature Genet., 2008; 40: 927 Summary of the Invention [Problem to be solved by the invention]

[0005] The present disclosure aims to provide methods for diagnosing and stratifying ALS patients, etc. [Means for solving the problem]

[0006] The present disclosure provides mutations found in the gene encoding CEP290 in patients with amyotrophic lateral sclerosis (ALS). In one aspect, the present disclosure provides methods for diagnosing and stratifying ALS patients based on the mutations.

[0007] The present inventors have examined the genomic DNA sequences of ALS patients and non-ALS patients and found that mutations in the gene encoding CEP290 are associated with ALS. The present disclosure is based on such findings.

[0008] The present disclosure may provide the following inventions. (1) A method for selecting a subject from subjects suffering from ALS, comprising: detecting a mutation in the gene encoding CEP290 in genomic DNA obtained from a subject suffering from ALS; selecting a subject having the mutation; A method comprising: (2) A method for assessing whether or not a subject has ALS or is at risk of having ALS, comprising: A method comprising detecting a mutation in the gene encoding CEP290 in genomic DNA obtained from the subject. (3) The method according to (1) or (2) above, wherein the mutation is a loss-of-function mutation. (4) The method according to (3), wherein the mutation is at least one mutation selected from the group consisting of p.Phe2421fs, p.Val2310fs, p.Lys1930*, p.Arg1926*, p.Glu1664*, p.Gln1283*, p.Gln1268*, p.Val683fs, p.Arg549*, and p.Arg205*. (5) A kit for use in the method according to any one of (1) to (4) above, comprising a means for detecting a mutation in the gene encoding CEP290. (6) The kit according to (5), wherein the means for detecting a mutation in the gene encoding CEP290 is a means for amplifying or concentrating a DNA fragment containing a mutation in the gene encoding CEP290. (7) The method according to any one of (1) to (4) above, further comprising administering a nucleic acid encoding CEP290 to a subject in which a mutation in the gene encoding CEP290 has been detected, thereby expressing the CEP290 protein in the subject. (8) A method of treating a subject suffering from ALS, comprising: A method comprising administering to the subject an effective amount of a pharmaceutical composition comprising a nucleic acid encoding CEP290, thereby expressing CEP290 protein in the subject, wherein the subject has a mutation in the gene encoding CEP290. (9) The method according to (8) above, wherein the mutation is a loss-of-function mutation. (10) A pharmaceutical composition for use in treating a subject suffering from ALS, A pharmaceutical composition comprising an effective amount of a gene encoding CEP290 operably linked to a regulatory sequence, wherein the subject has a mutation in the gene encoding CEP290. [Effects of the Invention]

[0009] According to the present disclosure, it is possible to provide methods for diagnosing and stratifying ALS patients based on mutations found in the gene encoding CEP290. [Brief explanation of the drawings]

[0010] [Figure 1] Examples of mutations found in the gene encoding CEP290 are shown. DETAILED DESCRIPTION OF THE INVENTION

[0011] <Definition> As used herein, a "subject" may be a mammal, including a human, and in particular a human.

[0012] As used herein, "treatment" can refer to both preventive and therapeutic treatment. Preventive treatment is used to mean preventing the onset of a disease, delaying its onset, or reducing the incidence of the disease. Therapeutic treatment is used to mean reducing the rate of disease progression, delaying its onset, preventing its onset, alleviating the symptoms of the disease, curing the disease, and ameliorating the disease.

[0013] As used herein, "amyotrophic lateral sclerosis" (ALS) refers to a disease that impairs the motor nerves, which transmit signals from the brain to muscles, and their function. Impaired motor nerves and their function make it difficult for the brain to execute commands, leading to muscle wasting. Muscle weakness is primarily a result of impaired neural signals and is not considered a muscle disorder. Most patients develop the disease between the ages of 50 and 70, with a higher incidence of men. ALS is thought to be caused by a variety of factors, including genetic abnormalities, oxidative stress, and glutamate excess. Genetic factors, such as genetic abnormalities (e.g., mutations in superoxide dismutase (SOD1), TDP43, FUS, optineurin, C9ORF72, SQSTM1, and TUBA4A), are believed to be the most common causes of the disease. Approximately 5–10% of ALS cases are familial. The causative genes for familial ALS have been identified.

[0014] ALS is broadly divided into two types based on the initial symptoms: limb type and bulbar type. In the limb type, the first symptom is loss of strength in the arms and legs. In the bulbar type, the first symptom is difficulty moving the tongue and mouth. Symptoms include slurred speech and impaired swallowing. In contrast, in ALS, the central nervous system, the sensory nerves controlling the five senses (seeing, hearing, smelling, tasting, and touch), and the autonomic nervous system controlling excretory functions are relatively undamaged. While the condition of ALS progresses rapidly in some patients, it progresses slowly over a period of several decades in others. A definitive diagnosis of ALS is usually made in Japan by a neurologist. Neurologists examine patients for abnormalities in their eyes, facial and limb movements, hammer tendon reflexes, balance, and sensation to determine the extent of motor nerve damage. Specifically, (i) motor neuron signs (hypertendon reflexes, spasticity, pathological reflexes) and neuromotor neuron signs (muscle atrophy, fasciculations) are present across multiple spinal cord segments, (ii) symptoms are progressive and spread from the initial site to other sites, and (iii) diseases causing similar symptoms are differentiated (diagnoses of exclusion, e.g., sensory disturbance, sphincter disturbance, autonomic neuropathy, visual disturbance, extrapyramidal symptoms, and Alzheimer's disease cognitive impairment are excluded). A definitive diagnosis of ALS is made when three or more domains of upper and lower motor neuron dysfunction, a progressive course, and all criteria for exclusion are met. However, attempts have been made to grade the diagnostic certainty (specifically, definite, probable, possible, and suspected) to allow for earlier therapeutic intervention. The motor control areas of the body are divided into four regions: the brainstem, cervical spinal cord, thoracic spinal cord, and lumbosacral spinal cord. Findings indicating upper and lower motor neuron damage in two regions are considered probable, and findings indicating upper and lower motor neuron damage in three regions are considered definite. Probable and definite cases are candidates for treatment. Table 1 shows the Awaji criteria for diagnosing ALS, and Table 2 shows the revised EL Escorial diagnostic criteria that incorporate Awaji's recommendations.

[0015] [Table 1]

[0016] [Table 2]

[0017] Riluzole is administered as a drug therapy. Riluzole has glutamate-selective inhibitory effects, noncompetitive inhibitory effects on excitatory amino acid receptors, and voltage-dependent Na+ channel inhibitory effects, and is thought to exert neuronal protective effects primarily by suppressing glutamate-induced excitotoxicity. To slow the progression of ALS, patients are orally administered 100 mg daily (e.g., 50 mg twice daily).

[0018] As used herein, "genomic DNA" refers to DNA that constitutes the entire genetic makeup (genome) of a subject. Genomic DNA is particularly present in the cell nucleus.

[0019] As used herein, "CEP290" refers to a 290 kDa centrosomal protein encoded by the CEP290 gene in humans. CEP290 is located on the Q arm of chromosome 12 (Chr12:88.05-88.14 Mb) in humans. CEP290 is also known as 3H11Ag, BBS14, CT87, JBTS5, LCA10, MKS4, NPHP6, POC3, SLSN6, rd16, or centrosomal protein 290. CEP290 is registered in the National Center for Biotechnology Information (NCBI) database, for example, under Gene ID: 80184. In one embodiment, the human CEP290 protein has the amino acid sequence set forth in SEQ ID NO: 1.

[0020] As used herein, a "loss-of-function mutation" (LoF) is a mutation that reduces or eliminates the function of a gene product. For genes present on autosomes, loss-of-function mutations are autosomal recessive or haploinsufficient. Typical examples of loss-of-function mutations include nonsense mutations and frameshift mutations. A nonsense mutation is a mutation in which a codon encoding an amino acid becomes a stop codon, resulting in the production of a shorter protein. A frameshift mutation is a mutation in which the reading frame of a codon is changed by the insertion or deletion of a base, resulting in the amino acid following the insertion or deletion being replaced by a different amino acid than the original amino acid. Loss of a stop codon is also a typical example of a loss-of-function mutation. It is known that loss of a stop codon causes ribosomes to stall at the poly(A) sequence, leading to premature degradation of mRNA by nonstop-mediated decay. Loss of a start codon is also a typical example of a loss-of-function mutation. Furthermore, splicing abnormalities can also be a typical example of a loss-of-function mutation. Mutations or deletions in splice donors or splice acceptors disrupt the normal intron excision process by splicing, resulting in the generation of erroneous mRNAs, which can lead to loss-of-function mutations such as frameshift and nonsense mutations.

[0021] As used herein, "targeted resequencing" refers to a technique in which DNA fragments containing a region to be decoded are amplified or enriched and then sequenced using a sequencing technique such as next-generation sequencing. A primer set can be designed to amplify a sufficient number and variety of DNA fragments to cover the region to be decoded. The primer set is then used for gene amplification (e.g., gene amplification by PCR) to obtain multiple amplicons. Gene amplification (e.g., gene amplification by PCR) can be performed multiplex (e.g., gene amplification by multiplex PCR). The resulting amplicons can be sequenced to determine the nucleic acid sequence of the region. In contrast, whole genome resequencing is a technique for decoding nucleic acid sequences without amplifying or enriching specific regions. Targeted resequencing is preferably used for decoding the genomic DNA of organisms with large genomes, such as mammals.

[0022] As used herein, "next-generation sequencing" (NGS) is a sequencing technology capable of simultaneously sequencing millions to billions of DNA fragments. Prior to next-generation sequencing, DNA sequences were determined by the Sanger method. In the Sanger method, a single-stranded DNA template is placed in a solution and a complementary copy of the template is created. In addition to deoxyribonucleotides (A, T, G, and C), dideoxyribonucleotides (A, T, G, and C) labeled with fluorescent dyes that emit different colors are added. The complementary copy then extends along the template, terminating upon incorporation of a dideoxyribonucleotide. The fluorescent dyes at the ends of the extended copy allow for identification of whether the terminal nucleotide is ATGC or ATGC. In this way, DNA sequencing using the Sanger method allows sequencing of a single DNA fragment at a time. In contrast, next-generation sequencing allows for the simultaneous sequencing of millions to billions of DNA fragments. In next-generation sequencing, a library is prepared by fragmenting sample DNA to readable sizes (tens to hundreds of base pairs) and ligating adapter sequences to both ends. The DNA fragments are then amplified and sequenced. In bridge PCR, polynucleotides capable of hybridizing to the adapters are immobilized on a substrate. When both ends bind to the immobilized polynucleotides on the substrate, a bridge structure is formed on the substrate. Amplification is then carried out in this state, synthesizing a complementary copy of the amplified template. The sequence is decoded by identifying each base incorporated into the complementary strand using the fluorescent color of the base label. Each base has a terminator cap, which stops the extension after each base extension. After decoding, the terminator cap is removed and the base is further extended while the base is decoded. In emulsion PCR, DNA fragments with adapters attached and beads with immobilized polynucleotides that can hybridize to the adapters are introduced into an emulsion in a 1:1 ratio, and then the nucleic acids are amplified by PCR to obtain beads with one type of amplified nucleic acid bound to each bead.The resulting beads are subjected to sequencing of the nucleic acids on the beads by pyrosequencing, ligation sequencing, or ion semiconductor sequencing.

[0023] As used herein, "deep sequencing" refers to sequencing with a coverage of 30 or more. Coverage refers to the number of times a specific base is sequenced. As used herein, "paired-end sequencing" refers to sequencing from both ends of a fragment and handling the resulting pair of data as a set. As used herein, "adapter" refers to a nucleic acid having a unique sequence that is linked to the end of a DNA fragment. Adapters can be used for purposes such as immobilizing unique nucleic acids on a surface or providing a site for priming a sequencing primer.

[0024] The term "operably linked," when used with respect to a polynucleotide (or gene, hereinafter the same), means that a regulatory sequence, such as a promoter, is positioned sufficiently close to the gene sequence that the regulatory sequence, such as a promoter, can affect the expression of the gene sequence. For example, a polynucleotide operably linked to a promoter means that the polynucleotide is linked such that its expression is under the control of the promoter.

[0025] The term "expressible state" refers to a state in which a polynucleotide can be transcribed in a cell into which the polynucleotide has been introduced. The term "expression vector" refers to a vector containing a polynucleotide of interest and equipped with a mechanism for expressing the polynucleotide of interest in a cell into which the vector has been introduced. For example, a "polynucleotide expression vector" refers to a vector capable of expressing a polynucleotide in a cell into which the vector has been introduced. In a polynucleotide expression vector, the polynucleotide is operably linked to, for example, a regulatory sequence.

[0026] In this specification, the sequence identity (or homology) between nucleotide sequences is determined as the ratio of matching nucleotides to the entire nucleotide sequence, excluding gaps in the obtained alignment, by juxtaposing two nucleotide sequences with gaps inserted in the portions corresponding to insertions and deletions so that the corresponding bases match as many as possible. The sequence identity between nucleotide sequences can be determined using various homology search software known in the art. For example, the value of the sequence identity of a nucleotide sequence can be obtained by calculation based on the alignment obtained by the known homology search software BLASTN.

[0027] <Method for diagnosing and stratifying ALS patients, and method for evaluating the risk of developing ALS> (A) According to the present disclosure, A method for selecting a subject from subjects suffering from ALS, The method comprising detecting a mutation in the gene encoding CEP290 in genomic DNA obtained from a subject suffering from ALS. is provided. This method of the present disclosure may further comprise selecting a subject in which a mutation in the gene encoding CEP290 has been detected. The patients thus selected may be subjects for gene therapy (or normalization of genes by gene editing techniques). Also, patients having a loss-of-function mutation thus selected may be subjects for replacement therapy of CEP290 protein.

[0028] (B-1) According to the present disclosure, A method for examining a subject suffering from ALS in a subject, The method comprising detecting a mutation in the gene encoding CEP290 in genomic DNA obtained from the subject. is provided. This method of the present disclosure may further comprise presuming that, in the subject, the cause of ALS may be a mutation in the gene encoding CEP290. In this sense, (B-2) According to the present disclosure, A method for presuming the cause of ALS in a subject suffering from ALS, detecting a mutation in the gene encoding CEP290 in genomic DNA obtained from a subject suffering from ALS. The method of the present disclosure may further comprise inferring that the cause of ALS in the subject may be a mutation in the gene encoding CEP290. By inferring the causative gene, a treatment strategy for the subject may be more clearly defined. Hereinafter, (B-1) and (B-2) will be collectively referred to simply as (B).

[0029] (C) According to the present disclosure, 1. A method for predicting the efficacy of CEP290 replacement therapy or CEP290 gene therapy in a subject suffering from ALS, comprising: detecting a mutation in a gene encoding CEP290 in genomic DNA obtained from the subject. The present invention provides a method for predicting whether a subject having a CEP290-encoding gene mutation is likely to benefit from CEP290 gene therapy. When a mutation in the gene encoding CEP290 is detected in a subject, the subject may be shown to benefit from CEP290 gene therapy. Therefore, this method of the present disclosure may further include predicting whether a subject having a CEP290-encoding gene mutation detected in the subject will benefit from CEP290 gene therapy. Furthermore, the presence of a loss-of-function mutation in the gene encoding CEP290 may indicate that replacement therapy with CEP290 protein will be effective. Therefore, this method of the present disclosure may further include predicting whether a subject having a loss-of-function mutation in the gene encoding CEP290 will benefit from CEP290 protein replacement therapy. Here, the term "prediction method" can be interpreted as "a method for predicting," "a method for obtaining preliminary information for prediction," "a method for diagnosing," "a method for diagnosing," or "a method for obtaining preliminary routines for diagnosis." In certain embodiments, the method does not involve medical intervention on humans. For example, the method may be useful for non-medical technicians at a genetic analysis center to make the prediction.

[0030] (D) According to the present disclosure, 1. A method for assessing whether a subject has or is at risk of having ALS, comprising: detecting a mutation in a gene encoding CEP290 in genomic DNA obtained from the subject. The method of the present disclosure may further include evaluating a subject in whom a mutation in the gene encoding CEP290 is detected as having or at risk of having ALS. When a mutation in the gene encoding CEP290 is detected in a subject, the subject may be identified as having or at risk of having ALS. A definitive diagnosis may be made by a physician. Here, the term "evaluation method" as used above may be interpreted as "method for evaluation," "method for obtaining preliminary information for evaluation," "method for determining," "method for diagnosing," "method for diagnosing," or "method for obtaining preliminary routines for diagnosis." In some embodiments, the method does not involve medical intervention on a human. For example, it may be useful for a technician who is not a medical professional to perform the evaluation at a genetic analysis center.

[0031] In each of the methods (A) to (D) according to the present disclosure, the mutation in the gene encoding CEP290 may be a mutation in the CEP290 protein, particularly a loss-of-function mutation. Examples of loss-of-function mutations include nonsense mutations, frameshift mutations, splice donor mutations, splice acceptor mutations, loss of the start codon, and loss of the stop codon.

[0032] In each of the methods (A) to (D) of the present disclosure, CEP290 is a protein of approximately 2,500 amino acids in humans. An example of human CEP290 is CEP290 having the amino acid sequence registered with NCBI under Gene ID: 80184. In the amino acid sequence registered with NCBI under Gene ID: 80184, amino acid numbers 1 to 695 may be a region involved in self-association. In the amino acid sequence, amino acid numbers 696 to 896 may be a region involved in association with other proteins. In the amino acid sequence, amino acid numbers 1,966 to 2,479 may be a region involved in self-association. Furthermore, in the amino acid sequence, the regions of amino acid numbers 59 to 565, 598 to 664, 697 to 931, 958 to 1,027, 1,071 to 1,498, 1,533 to 1,584, and 1,635 to 2,452 may be coiled-coil regions. These regions may play important roles in protein interactions, so their loss may result in loss-of-function mutations.

[0033] In some embodiments, CEP290 in ALS patients may have a nonsense mutation as a loss-of-function mutation. A CEP290 mutant having a nonsense mutation may lose function or be degraded by nonsense-mediated mRNA decay (NMD).

[0034] In each of the methods (A) to (D) according to the present disclosure, in one aspect, each of these mutations can be a frameshift mutation. That is, the frameshift mutation may be present in one or more regions selected from the group consisting of the regions of amino acid numbers 1 to 59, 59 to 565, 565 to 598, 598 to 664, 664 to 695, 696 to 896, 897 to 931, 932 to 957, 958 to 1027, 1027 to 1070, 1071 to 1498, 1499 to 1532, 1533 to 1584, 1585 to 1634, 1635 to 1965, 1966 to 2452, and 2453 to 2497 of the amino acid sequence, or in the region of CEP290 corresponding to said region.

[0035] In each of the above methods (A) to (D) according to the present disclosure, in one aspect, the CEP290 mutation may be any of the mutations listed in Table 3. [Table 3]

[0036] In one embodiment, the loss-of-function mutation can be one or more selected from the group consisting of p.Phe2421fs, p.Val2310fs, p.Lys1930*, p.Arg1926*, p.Glu1664*, p.Gln1283*, p.Gln1268*, p.Val683fs, p.Arg549*, and p.Arg205*. Here, "p." indicates protein, the three letters represent the three-letter code for the amino acid, and the number indicates the amino acid number counted from the amino-terminal methionine. "fs" indicates that a frameshift mutation has occurred at that position, and "*" indicates that a stop codon has occurred at that position. Thus, "p.Phe2421fs" means that the mutation has resulted in a frameshift mutation at phenylalanine 2421 of the protein. "p.Lys1930*" means that the mutation changed the 1930th lysine in the protein to a stop codon.

[0037] In each of the methods (A) to (D) above according to the present disclosure, in one aspect, the mutation may be a nonsense mutation, and the nonsense mutation may be, but is not limited to, one or more selected from the group consisting of p.Lys1930*, p.Arg1926*, p.Glu1664*, p.Gln1283*, p.Gln1268*, p.Arg549*, and p.Arg205*.

[0038] In one aspect, the loss-of-function mutations can be one or more selected from the group consisting of c.7257delA, c.6927delA, c.5788A>T, c.5776C>T, c.4990G>T, c.3847C>T, c.3802C>T, c.2047_2050delGTTA, c.1645C>T, and c.613C>T. Here, "c." means the coding region, the numbers indicate the base number where the mutation occurred with the A of the start codon ATG being the first, ATGC indicates the base, "del" indicates the deletion of the base at the corresponding base number, and ">" indicates the substitution of the base at the corresponding base number.

[0039] In each of the methods described in (A) to (D) above according to the present disclosure, in one aspect, the loss-of-function mutation of ALS can be, for example, the loss of the start codon. In one aspect, the loss-of-function mutation of ALS can be, for example, the loss of the stop codon. The loss of the stop codon can occur, for example, due to a frameshift mutation. The loss of the stop codon can occur, for example, by one or more selected from the group consisting of base insertion, deletion, and substitution. The loss of the start codon inhibits protein synthesis, and the loss of the stop codon induces mRNA degradation.

[0040] <Method for detecting mutations in CEP290> Mutations in CEP290 can be sufficient to cause ALS. Therefore, the detection of mutations in CEP290 can indicate that a person has ALS or has a risk of developing ALS. Also, the detection of mutations in CEP290 suggests that a treatment that replenishes CEP290 with normal function is effective for a subject suffering from ALS. Therefore, the detection of mutations in CEP290 is important.

[0041] CEP290 mutations can be detected by sequencing. CEP290 is a large protein. Therefore, in order to decode its full-length sequence, it is preferable to decode the full-length CEP290 by next-generation sequencing. Mammals have a huge sequence. Therefore, in a preferred embodiment, CEP290 mutations can be detected by targeted resequencing. In a preferred embodiment, CEP290 mutations can be detected by whole-genome sequencing or targeted resequencing in next-generation sequencing.

[0042] A biological sample containing genomic DNA can be obtained from a subject. The biological sample can be peripheral blood, saliva, buccal mucosa, nails, or hair. Genomic DNA can be extracted by conventional methods. For example, genomic DNA can be extracted by treating the biological sample with a cell lysis solution and recovering the genomic DNA dissolved in the lysis solution. DNA recovery can be performed by methods well known to those skilled in the art, such as ethanol precipitation. The obtained genomic DNA can be frozen and stored.

[0043] Genomic DNA can be subjected to sequencing. Sequencing can be performed by the Sanger method. In the Sanger method, the sequence-reading portion of CEP290 can be amplified before sequencing. Amplification can be performed, for example, by PCR. PCR can be performed using two primers designed to flank the region to be amplified. The amplified product can be sequenced by the Sanger method. Sequencing can be performed by next-generation sequencing. In next-generation sequencing (NGS), a library can be prepared before sequencing. In library preparation, genomic DNA can first be cleaved into fragments of readable length (e.g., tens to hundreds of base pairs). Cleavage can be performed using a DNA cleaving enzyme (endonuclease) such as a restriction enzyme. Various sequence-specific endonucleases are known that can be used for DNA cleavage, and they can be used for this cleavage. Adapters can be ligated to both ends of the obtained DNA fragments. In this way, a library of DNA fragments bearing adapters at both ends can be obtained.

[0044] The library may be subjected to gene amplification, which may be done, for example, by bridge PCR or emulsion PCR.

[0045] Targeted resequencing is preferably performed. In targeted resequencing, during the library preparation process, labeled oligonucleotide probes hybridizable to CEP290 are mixed with single-stranded DNA fragments, either before or preferably after adapter ligation, to hybridize with the DNA fragments. The DNA fragments are then adsorbed onto a surface (e.g., beads) on which a labeled binding substance is immobilized, thereby enriching fragments of the gene encoding CEP290. Single-stranded DNA can be easily achieved, for example, by heat denaturation. The library is subjected to gene amplification, and the sequence can then be decoded. The oligonucleotides can be labeled, for example, with biotin. In this case, a surface on which avidin is immobilized can be used to enrich the desired fragments via biotin on the surface. Therefore, a labeled nucleic acid (or a nucleic acid having a sequence complementary to the gene) capable of hybridizing to the gene encoding CEP290 and a support (e.g., beads) having a surface on which a binding compound for the label is immobilized can be prepared, and DNA fragments containing the gene encoding CEP290 can be enriched using this. The fragments range in length from several tens to several hundreds of base pairs. Therefore, to enrich DNA fragments to cover the gene encoding CEP290 (over approximately 7 kb), multiple labeled nucleic acids capable of hybridizing to each DNA fragment can be prepared. For example, in preparing DNA fragments from genomic DNA, restriction enzymes can be used to clarify the start and end points of the resulting DNA fragments. Therefore, for example, the DNA fragments obtained using restriction enzymes can be estimated, and multiple labeled nucleic acids capable of hybridizing separately to each individual fragment can be prepared. The labeled nucleic acid can include, for example, a nucleic acid selected from the group consisting of DNA, RNA, and modified nucleic acids. In terms of binding strength to DNA, RNA is preferred, and modified nucleic acids are more preferred. After enrichment, copies of the complementary strands can be synthesized, and then adapter sequences can be added to the fragments, allowing for focused sequencing of the fragments of the gene encoding CEP290.

[0046] In modified nucleic acids, bases may be modified with 2'-O-methyl, 2'-fluoro, or 2'-methoxyethyl (MOE) to enhance RNA stability, and the phosphodiester bonds in the nucleic acid backbone may be replaced with phosphorothioate bonds. Examples of modified nucleic acids include nucleic acids in which the oxygen atom at the 2' position is bridged to the carbon atom at the 4' position. Examples of such modified nucleic acids include locked nucleic acids, which are cross-linked DNA in which the oxygen atom at the 2' position is bridged to the carbon atom at the 4' position via a methylene bridge. bridged nucleic acids (BNA) such as LNA (Likely a nucleotide), ENA (where the oxygen atom at the 2' position and the carbon atom at the 4' position are bridged via ethylene), BNACOC (where the oxygen atom at the 2' position and the carbon atom at the 4' position are bridged via -CH2OCH2-), and BNANC (where the oxygen atom at the 2' position and the carbon atom at the 4' position are bridged via -NR-CH2- (where R is a methyl or hydrogen atom)); and cMO (where the oxygen atom at the 2' position and the carbon atom at the 4' position are bridged via -CH2(OCH3)-). Examples of modified nucleic acids include E, cEt, in which the 2' oxygen atom and the 4' carbon atom are bridged via -CH2(CH3)-, AmNA, in which the 2' and 4' carbon atoms are bridged via an amide, scpBNA, in which the 2' oxygen atom and the 4' carbon atom are bridged via a methylene, forming a cyclopropane at the 6' position, and peptide nucleic acids (PNA), in which the backbone is a polymer in which N-(2-aminoethyl)glycine is amide-linked instead of deoxyribose or ribose. Morpholino oligos can also be used as modified nucleic acids. In morpholino oligos, base-modified morpholine rings are linked by covalent chemical bonds such as -P(=O)(NH2)-.

[0047] Alternatively, in one embodiment, in targeted resequencing, during the library preparation process, fragments can be amplified by PCR using a primer set capable of amplifying gene fragments encoding CEP290 before adapter ligation. Primers can be designed so that multiple amplification products cover the entire length of CEP290. PCR can be performed in a multiplexed manner, thereby amplifying various DNA fragments covering CEP290. Adapters can be included in the primers so that they are contained in the amplified fragments. For example, a primer set capable of amplifying multiple DNA fragments covering the entire length of the gene region encoding CEP290 can be prepared as a targeted sequence panel. Preferably, the primer set may contain an adapter sequence. The resulting library can be subjected to gene amplification. Gene amplification can be performed, for example, by bridge PCR or emulsion PCR.

[0048] In one embodiment, samples from multiple subjects can be sequenced in a single sequencing run. In this case, in addition to the adapter sequence, an index sequence that distinguishes the origin of the sample can be added to distinguish each subject. The index sequence can be a sequence unique to the subject, and after the index sequence is added (e.g., ligated) to the sample of each subject, the samples from the multiple subjects are mixed and sequenced. The sequence information obtained by sequencing includes the index sequence. By referring to the index sequence, the subject from which the sequence information is derived can be identified.

[0049] In one aspect, the method of the present disclosure may further include determining (haplotyping) whether two or more mutations in a subject having two or more mutations in the gene encoding CEP290 are haplotypes. A haplotype means a genetic constitution on the same chromosome for two or more genotypes. Haplotyping means determining whether two or more genotypes are in the same genetic constitution on the same chromosome. Haplotyping can be performed, for example, by obtaining a gene fragment containing at least two of the two or more mutations when two or more mutations are detected on the gene encoding CEP290 and decoding the sequence with a long-read sequencer. The gene fragment may be amplified prior to the sequencing. Long-read sequencing can be achieved by long-read sequencing such as nanopore sequencing. Haplotyping can also be performed by cloning the gene encoding CEP290 onto a plasmid, transforming Escherichia coli with the plasmid, picking up single colonies and sequencing them.

[0050] <Method for treating a subject suffering from ALS with a mutation in the gene encoding CEP290> In ALS patients with mutations in the gene encoding CEP290, loss-of-function mutations in the CEP290 protein are strongly suggested to be the cause of ALS onset. Therefore, ALS can be treated by increasing CEP290 protein in the subject by administering CEP290 protein or a gene encoding CEP290 to the subject. In particular, a nucleic acid encoding CEP290 can be administered to a subject with ALS associated with a mutation in the gene encoding CEP290. Furthermore, gene editing techniques such as CRISPR-Cas9 can be used to normalize the mutated portion of the gene encoding CEP290 (Nature Communications, 2020, 11, Article number: 482). For example, gene editing techniques can be used to introduce a break in the region containing the mutation (target region) in genomic DNA. This allows the target region to be replaced with a nucleic acid having a normal sequence (a sequence for recombination). Specifically, when a break is introduced into the target region in the presence of a DNA template containing, in this order, an upstream sequence of the target region, an upstream homology arm capable of homologous recombination, a recombination sequence, a granule sequence of the target region, and a downstream homology arm capable of homologous recombination, eukaryotic cells induce homologous recombination with the recombination nucleic acid during genome repair. This replaces the target region with the recombination sequence. The mutation can be normalized by using a functional sequence for the recombination sequence (e.g., a normalized sequence, e.g., a sequence with a correct frame due to a corrected frameshift mutation, a sequence without a stop codon, or a wild-type sequence). The break into the target region can be introduced using gene editing technology (e.g., a DNA-cleaving enzyme that introduces a break at a single site in genomic DNA, such as the CRISPR / Cas system, preferably the CRISPR / Cas9 system, TALEN, zinc finger nucleases, or megabase nucleases). Introduction can be carried out, for example, using an adeno-associated virus (AAV).In one embodiment, an AAV may include a gene encoding a Cas9 nuclease, a gene encoding a guide RNA, a DNA template, and, optionally, a promoter to drive these (Nature Communications, 2020, 11, Article number: 482).

[0051] In one embodiment, the nucleic acid encoding CEP290 is operably linked to a regulatory sequence. As used herein, a "regulatory sequence" refers to a sequence that drives a gene operably linked to it and has the activity of transcribing RNA from that gene. A regulatory sequence is, for example, a promoter. Examples of promoters include class I promoters (which can be used to transcribe pre-rRNA), class II promoters (which contain a core promoter and upstream promoter elements and can be used to transcribe mRNA), and class III promoters (further classified into types I, II, and III). A regulatory sequence, also known as a regulatory sequence, may be any promoter that can transcribe mRNA in cells, such as animal cells or plant cells. For example, various Pol II promoters can be used as the first regulatory sequence. Pol II promoters include, but are not limited to, the CMV promoter, the EF1 promoter (EF1α promoter), the SV40 promoter, the MSCV promoter, the hTERT promoter, the β-actin promoter, the CAG promoter, and the CBh promoter. Promoters can also include promoters that drive bacteriophage-derived RNA polymerases, such as the T7 promoter, T3 promoter, and SP6 promoter, as well as Pol III promoters, such as the U6 promoter. The T7 promoter is preferably used for transcription from circular DNA, while the SP6 promoter is preferably used for transcription from linear DNA. The promoter may also be inducible. An inducible promoter is a promoter that can induce expression of a polynucleotide operably linked to it only in the presence of an inducer that drives the promoter. Some inducible promoters can induce expression of a polynucleotide operably linked to it only in the absence of an inhibitor that suppresses promoter activity. Inducible promoters include, but are not limited to, promoters that induce gene expression upon heating, such as heat shock promoters. Inducible promoters also include drug-activated promoters.Such drug-inducible promoters include, for example, cumate operator sequences, lambda operator sequences (e.g., 12xlambda Op), tetracycline-inducible promoters, etc. Tetracycline-inducible promoters include, for example, promoters that drive gene expression in the presence of tetracycline or its derivatives (e.g., doxycycline) or reverse tetracycline-controlled transactivator (rtTA). An example of a tetracycline-inducible promoter is the TRE3G promoter.

[0052] A gene encoding CEP290 operably linked to a regulatory sequence can be transcribed and translated in mammalian cells to produce a CEP290 protein, thereby reducing the effects of a mutation in CEP290. The CEP290 protein may be, but is not limited to, a wild-type CEP290, such as a CEP290 having an amino acid sequence registered in the National Center for Biotechnology Information (NCBI) database as, for example, Gene ID: 80184, or a sequence corresponding thereto.

[0053] In one embodiment, a gene encoding CEP290 operably linked to a regulatory sequence can be carried in a protein expression vector. Thus, the present disclosure provides a protein expression vector carrying a gene encoding CEP290 operably linked to a regulatory sequence in an expressible manner. The protein expression vector can be, for example, a viral vector.

[0054] Thus, the present disclosure provides, for example, a pharmaceutical composition comprising a gene encoding CEP290 operably linked to such a regulatory sequence. The present disclosure provides, for example, a pharmaceutical composition comprising a protein expression vector carrying a gene encoding CEP290 operably linked to a regulatory sequence.

[0055] In some embodiments, the nucleic acid encoding CEP290 may be messenger RNA (mRNA). In some embodiments, at least one uridine in the mRNA may be changed to pseudouridine. The pseudouridine may be 1-methylpseudouridine. The mRNA may be transcribed from cDNA, i.e., it may not have an intron. The mRNA may also have a cap structure at the 5' end (Furuichi Y & Miura K. Nature. 1975;253(5490):374-5). The cap structure can be added to the mRNA by the Anti-Reverse Cap Analogues (ARCA) method using a cap analog (Stepinski J et al. RNA. 2001 Oct;7(10):1486-95). The Cap structure of the mRNA can be converted to a Cap structure by further treatment with 2'-O-methyltransferase. These can be performed by conventional methods, for example, using commercially available kits such as the ScriptCap m7G Capping System and ScriptCap 2'-O-Methyltransferase Kit, or the T7 mScript Standard mRNA Production System (AR Brown CO., LTD). The mRNA can have a poly(A) tail. The addition of a poly(A) tail can be performed by conventional methods, for example, using the A-Plus Poly(A) Polymerase Tailing Kit (AR Brown CO., LTD). Thus, in one embodiment, the mRNA can have a Cap structure at the 5'-end and a poly(A) at the 3'-end, and preferably, at least a portion of the uridines is pseudouridine (preferably 1-methylpseudouridine). The mRNA can be isolated or synthesized.

[0056] mRNA can be encapsulated in lipid nanoparticles (LNPs). This prevents the degradation of mRNA in vivo and improves the efficiency of delivering mRNA into cells. Thus, in one aspect, the mRNA can be an mRNA having a Cap structure at the 5' end, a polyA at the 3' end, and preferably at least a portion of uridine is pseudouridine (preferably, 1-methylpseudouridine). Lipid nanoparticles encapsulating such mRNA are also provided. The lipid nanoparticles are not particularly limited, and for example, the lipid nanoparticles described in US9364435B, US8822668B, US8802644B, and US8058069B2 can be used. Alternatively, the mRNA may be encapsulated in a polyion complex micelle or a polyion complex type polymerosome (Miyata et al., Chem. Soc. Rev., 2012, 41, 2562-2574).

[0057] Thus, according to the present disclosure, a pharmaceutical composition containing mRNA encoding CEP290 is provided. According to the present disclosure, nanovesicles encapsulating mRNA encoding CEP290 are also provided. The nanovesicles can be vesicles having a sub-micrometer particle size, and preferably can be vesicles having a diameter of 100 nm or less. The nanovesicles can be lipid nanovesicles. The nanovesicles can be polyion complex type micelles.

[0058] <Kit containing means for amplifying or concentrating a DNA fragment containing a mutation of the gene encoding CEP290> According to the present disclosure, a kit for use in any of the methods described in (A) to (D) above can be provided. The kit can include means for detecting a mutation of the gene encoding CEP290, and the means can include means (a) for amplifying or means (b) for concentrating a DNA fragment.

[0059] Means (a) for amplifying a DNA fragment containing a mutation in the gene encoding CEP290 includes a primer set designed to amplify part or all of the gene encoding CEP290. In one embodiment, the primer set in means (a) can amplify multiple DNA fragments so as to cover the entire gene encoding CEP290. That is, the primer set is designed so that no region of the gene encoding CEP290 is unamplified (overlap of amplified fragments is allowed). Therefore, means (a) may include one or more primer sets designed so that no region of the gene encoding CEP290 is unamplified (overlap of amplified fragments is allowed). It may also include each primer set that detects or amplifies a mutation site listed in Table 3. Means (a) may be provided as a test panel containing the primer sets described above.

[0060] In one embodiment, the primer may contain an adapter sequence. By including an adapter sequence in the primer, the resulting amplification product will contain adapter sequences at both ends. In one embodiment, the primer may further contain an index sequence for purposes such as identifying the origin of the sample. Alternatively, means (a) may contain an adapter sequence for ligating the amplification product and / or DNA having an index sequence. The above means (a) may further contain a DNA polymerase, such as a thermostable DNA polymerase, that can be used in PCR. The above means (a) may further contain a reaction solution used in PCR. The reaction solution may contain components necessary for PCR, such as a primer set, DNA polymerase, dNTPs (e.g., dATP, dGTP, dCTP, and dTTP), and Mg ions.

[0061] Means (b) for enriching DNA fragments containing mutations in the gene encoding CEP290 include nucleic acids that can specifically hybridize to the gene encoding CEP290. If the gene encoding CEP290 has already been fragmented into smaller DNA fragments, labeled nucleic acids that hybridize to all fragments can be prepared. The nucleic acid can be a labeled nucleic acid (e.g., a biotin-labeled nucleic acid). The labeled nucleic acid can be adsorbed onto a surface (e.g., a support, e.g., beads) on which a substance that binds to the label (a labeled binding substance) has been immobilized, thereby enabling enrichment of the labeled nucleic acid. Therefore, means (b) above can include labeled nucleic acids and / or supports (e.g., beads) having a surface on which a labeled binding substance has been immobilized. When the label is biotin, the labeled binding substance can be avidin, streptavidin, or neuroavidin.

[0062] The above-mentioned means (b) may further comprise a restriction enzyme for preparing DNA fragments. Furthermore, the above-mentioned means (b) may comprise a plurality of labeled nucleic acids capable of specifically hybridizing to each of the fragments of the gene encoding CEP290 obtained by the restriction enzyme treatment. The above-mentioned means (b) may further comprise a support on which a labeled binding substance is immobilized. The support may preferably be beads. The beads may be, but are not limited to, magnetic beads.

[0063] Treatment Methods and Pharmaceutical Compositions According to the present disclosure, 1. A method of treating a subject suffering from ALS, comprising: a method comprising administering to the subject an effective amount of a pharmaceutical composition comprising a nucleic acid encoding CEP290, thereby expressing a CEP290 protein in the subject, wherein the subject has a mutation in the gene encoding CEP290. An effective amount can be an amount that produces a therapeutically beneficial effect.

[0064] Here, the nucleic acid encoding CEP290 may be an mRNA encoding CEP290 or a gene encoding CEP290 operably linked to a regulatory sequence. Details of these nucleic acids are as described above.

[0065] A subject suffering from ALS may have any diagnostic grade selected from the group consisting of definite, probable, and possible in the Awaji criteria. In some embodiments, a subject suffering from ALS may have a definite diagnostic grade in the Awaji criteria. In some embodiments, a subject suffering from ALS may have a probable diagnostic grade in the Awaji criteria. A subject suffering from ALS may have a possible diagnostic grade in the Awaji criteria.

[0066] In some embodiments, a subject suffering from ALS may have a diagnostic grade selected from the group consisting of Definite, Probable, Possible, and Suspected according to the EL Escorial diagnostic criteria. In some embodiments, a subject suffering from ALS may have a diagnostic grade of Definite according to the EL Escorial diagnostic criteria. In some embodiments, a subject suffering from ALS may have a diagnostic grade of Probable according to the EL Escorial diagnostic criteria. A subject suffering from ALS may have a diagnostic grade of Possible according to the EL Escorial diagnostic criteria. A subject suffering from ALS may have a diagnostic grade of Suspected according to the EL Escorial diagnostic criteria.

[0067] In a preferred embodiment, the subject suffering from ALS may be a subject determined by the method of the present disclosure to have a mutation in the gene encoding CEP290. The mutation may be any of the mutations described above.

[0068] The present disclosure provides a pharmaceutical composition comprising mRNA encoding CEP290. The present disclosure also provides nanovesicles encapsulating mRNA encoding CEP290. The nanovesicles may be vesicles having a submicrometer particle size, preferably vesicles having a diameter of 100 nm or less. The nanovesicles may be lipid nanovesicles. The nanovesicles may be polyion complex micelles. A subject suffering from ALS has a mutation in the gene encoding CEP290. The mutation may be any of the mutations described above.

[0069] According to the present disclosure, a pharmaceutical composition is provided comprising a gene encoding CEP290 operably linked to a regulatory sequence. According to the present disclosure, for example, a pharmaceutical composition is provided comprising a protein expression vector carrying a gene encoding CEP290 operably linked to a regulatory sequence. A subject suffering from ALS is a subject having a mutation in the gene encoding CEP290. The mutation may be any of the mutations described above.

[0070] According to the present disclosure, pharmaceutical compositions comprising nucleic acids encoding CEP290 can be used in methods for treating subjects suffering from ALS. Subjects suffering from ALS are subjects who have a mutation in the gene encoding CEP290. The mutation can be any of the mutations described above.

[0071] According to the present disclosure, the nucleic acid encoding CEP290 can be used in the manufacture of the medicament for the method of treating the subject suffering from ALS.The subject suffering from ALS is the subject that has mutation in the gene encoding CEP290.Mutation can be any of the mutations as described above. [Example]

[0072] Example 1: Genomic analysis of ALS patients Genomic DNA was extracted from the peripheral blood of 706 physician-diagnosed amyotrophic lateral sclerosis (ALS) patients. Sequencing libraries were constructed from the extracted DNA using the TruSeq DNA PCR-Free Sample Prep Kit according to the manufacturer's protocol. Libraries that met quality criteria were sequenced using an Illumina HiSeq X system.

[0073] Genomic DNA was extracted from 4,263 patients with diseases other than ALS (non-ALS patients), including those with myocardial infarction, drug rash, cancer, and dementia, and the genomes were sequenced in the same way.

[0074] Genomic analysis focused on mutations, particularly loss-of-function mutations (LoF mutations). Specifically, we focused on frameshifts, splicing aberrations (splice acceptors and splice donors), gene start and end mutations, and nonsense mutations. LoF was defined as a high impact mutation using SnpEff software version 4.3t with default parameters. LoF was compared between non-ALS and ALS patient groups to identify LoFs associated with ALS. LoF was then evaluated at the gene level. Specifically, LoFs were clustered at the gene level, and the association of these clusters with ALS was assessed.

[0075] The results are shown in Table 4.

[0076] [Table 4]

[0077] Table 4 shows the statistical analysis results for loss-of-function mutations of CEP290. For CEP290, p-value <10 6Therefore, the technical significance of this study is considered clear. Thus, this analysis enabled the genome-wide identification of loss-of-function mutations in ALS.

[0078] The LoFs observed in CEP290 are shown in Table 5.

[0079] [Table 5]

[0080] The mutations in Table 5 above are mapped to the CEP290 gene as shown in FIG.

[0081] CEP290, Centrosomal protein 290 (also known as KIAA0373, NEPHROCYSTIN6 (NPHP6), the antigen identified by the monoclonal antibody 3H11, or BBS14), is involved in cilia assembly and trafficking. CEP290 has been shown to be a causative gene for Senior Loken syndrome (SLC), medullary polycystic kidney disease (MPHP), Joubert syndrome (JS), Bardet-Biedl syndrome (BBS), and fatal Meckel-Gruber syndrome (MKS). Loss-of-function mutations in CEP290 may be amenable to CEP290 replacement therapy or gene therapy. [Industrial Applicability]

[0082] According to the present disclosure, it is possible to provide methods for diagnosing and stratifying ALS patients based on mutations found in the gene encoding CEP290.

[0083] Sequence Listing SEQ ID NO: 1: Amino acid sequence of human CEP290 protein

Claims

1. 1. A kit for use in a method for selecting a subject from subjects suffering from ALS, comprising: The method comprises: Detecting a mutation in the gene encoding CEP290 in genomic DNA obtained from a subject suffering from ALS; selecting a subject having the mutation; A method comprising: A kit comprising a means for detecting a mutation in the gene encoding CEP290.

2. A kit for use in a method for assessing whether or not a person has ALS or is at risk of having ALS, comprising: The method comprises:

1. A method comprising detecting a mutation in a gene encoding CEP290 in genomic DNA obtained from a subject, A kit comprising a means for detecting a mutation in the gene encoding CEP290.

3. The kit according to claim 1 or 2, wherein the mutation is a loss-of-function mutation.

4. The kit according to claim 3, wherein the loss-of-function mutation is at least one mutation selected from the group consisting of p.Phe2421fs, p.Val2310fs, p.Lys1930*, p.Arg1926*, p.Glu1664*, p.Gln1283*, p.Gln1268*, p.Val683fs, p.Arg549*, and p.Arg205*.

5. The kit according to any one of claims 1 to 4, wherein the method further comprises administering a nucleic acid encoding CEP290 to a subject in whom a mutation in a gene encoding CEP290 has been detected, thereby expressing a CEP290 protein in the subject.

6. A pharmaceutical composition for use in treating a subject suffering from ALS, the pharmaceutical composition comprising a nucleic acid encoding CEP290.

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