Modified siRNA selectively suppresses the expression of mutant FUS

Abstract

[Problem] To provide a chemically-modified siRNA that has improved stability against RNase enzymes and selectively inhibits the expression of P525L mutant FUS which is a causative gene of ALS. [Solution] Provided is a chemically-modified siRNA comprising a sense strand and an antisense strand, or a salt thereof, wherein: the antisense strand includes a complementary or substantially complementary region in a portion of a mRNA that codes P525L mutant FUS protein; the complementary region has a nucleotide length of 19 to 21; and the siRNA contains at least one substitution selected from the group consisting of a 2'-F- nucleotide, a 2'-OMe-nucleotide, a nucleotide in which a 2'-O atom and a 4'-O atom are crosslinked by methylene, a 2'-deoxy-nucleotide, and a phosphorothioate bond that forms an internucleotide bond.

Description

[Technical Field]

[0001] The present invention relates to chemically modified siRNAs with improved stability against RNases, which selectively suppress the expression of P525L point-mutated FUS (fused in sarcoma), a gene that causes amyotrophic lateral sclerosis (ALS), and pharmaceutical compositions containing the chemically modified siRNAs. [Background technology]

[0002] Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, is a fatal, progressive neurodegenerative disease characterized by the predominant loss of motor neurons (MNs) in the primary motor cortex, brainstem, and spinal cord. The loss of motor neurons disrupts basic motor functions, such as breathing, and typically leads to death within two to five years of diagnosis. Progressive deterioration of patients' motor function severely reduces their ability to breathe, necessitating some form of respiratory support for survival. Other symptoms include weakness of the hands, arms, legs, or swallowing muscles. Some patients may also develop frontotemporal dementia (FTD). ALS typically develops between the ages of 50 and 70, making it a disease of the elderly.

[0003] ALS can be broadly classified into two types: sporadic ALS and familial ALS. Most cases are non-hereditary sporadic ALS, while familial ALS is a disease with a relatively small number of patients, accounting for approximately 5-10% of all ALS cases.

[0004] The cause of ALS is complex. It is generally believed to be a complex genetic disease caused by mutations in multiple genes coupled with environmental exposure. Factors related to the onset of ALS include SOD1 (Cu 2+ / Zn 2+More than a dozen causative genes have been identified, including TDP-43 (TAR DNA-binding protein-43kD), TDP-43 (superoxide dismutase), FUS (fused in sarcoma), ANG (angiogenin), ATXN2 (ataxin-2), VCP (valosin-containing protein), OPTN (optineurin), and C9orf72 (chromosome 9 open reading frame 72). However, the exact mechanism of motor neuron degeneration remains unclear.

[0005] FUS is known to be the causative gene for familial ALS, second only to SOD1. FUS, the causative gene for ALS6 linked to chromosome 16, is an RNA-binding protein identified in 2009 and is known to be a causative gene that is more common in relatively young people with familial ALS (Non-Patent Document 1).

[0006] FUS travels between the nucleus and cytoplasm, where it plays an important role in RNA metabolism, including DNA repair and splicing regulation. Mutations in FUS cause abnormal aggregation in the cytoplasm, and two hypotheses have been proposed as the cause of familial ALS. The first is a loss-of-function hypothesis, in which normal RNA metabolism, which should occur in the nucleus, is disrupted. The second is that the mutant protein aggregates in the cytoplasm, resulting in toxicity.

[0007] The FUS protein contains a nuclear localization signal at its C-terminus. Mutations in this region can reduce the affinity of FUS for the nuclear receptor transporter, transportin. This disrupts normal nuclear localization, leading to cytoplasmic accumulation of mutant FUS. Investigations of FUS mutation locations have revealed that the most common mutations in the nuclear localization signal are P495X, G507D, K510R / E, S513P, R514G / S, R514S, G515C, H517Q / P, R518G / K, R521G / C / H, R522G, R524W / T / S, and P525L. Furthermore, the P525L point mutation is common in early-onset ALS, which develops in people in their teens and twenties. Most patients die within two years of onset, and no therapeutic treatments are currently available.

[0008] As mentioned above, wild-type FUS plays an important role in RNA metabolism, and it has been reported that knockout of FUS in mouse forebrain cortical neurons results in decreased interaction with the RNA splicing factor SFPQ (splicing factor, proline and glutamine rich), resulting in changes in tau isoforms. Therefore, high selectivity for mutant FUS is essential for the development of therapeutics for ALS caused by FUS mutations.

[0009] Meanwhile, reports on siRNAs targeting genes with point mutations include, for example, siRNAs targeting G356D point-mutated epidermal growth factor receptor (EGFR) (Patent Document 1), siRNAs targeting V337M point-mutated amyloid precursor protein (APP) (Non-Patent Document 2), and siRNAs targeting G85R point-mutated SOD1 (Non-Patent Document 3). However, there are no teachings or suggestions regarding chemically modified siRNAs that selectively suppress the expression of P525L point-mutated FUS while improving stability against plasma RNases that nonspecifically cleave RNA strands. A method has been proposed for diagnosing ALS or a genetic predisposition to ALS using specific genetic markers and treating or preventing ALS using siRNA molecules that reduce the expression of mutant FUS, but no specific siRNA sequences are described (Patent Document 2). [Prior art documents] [Patent documents]

[0010] [Patent Document 1] WO2011 / 158924 Brochure [Patent Document 2] WO2010 / 011283 Pamphlet [Non-patent literature]

[0011] [Non-Patent Document 1] Kwiatkowski TJ, et al. Mutations in the FUS / TLS gene on chromosome 16 cause familial amyotrophic lateral sclerosis. Science, 2009;323: 1205-1208. [Non-patent document 2] Miller VM, et al. Targeting Alzheimer's disease genes with RNA interference: an efficient strategy for silencing mutant alleles. Nucleic Acids Res, 2004;32:661-668. [Non-patent document 3] Ding H, et al. Selective silencing by RNAi of a dominant allele that causes amyotrophic lateral sclerosis. Aging Cell, 2003;2:209-217. Summary of the Invention [Problem to be solved by the invention]

[0012] An object of the present invention is to provide a chemically modified siRNA that has improved stability against plasma RNases that nonspecifically cleave RNA strands and that selectively suppresses the expression of P525L point mutant FUS, the causative gene for ALS, and a pharmaceutical composition comprising the chemically modified siRNA. [Means for solving the problem]

[0013] To address the problem of low stability against RNases in plasma of siRNAs that selectively suppress the expression of P525L point-mutated FUS, the causative gene for ALS, the present inventors substituted the nucleotides constituting the siRNA with 2'-F-nucleotides and 2'-OMe-nucleotides. The 2'-F-nucleotides and 2'-OMe-nucleotides were arranged alternately in the RNA strand of the siRNA, but two or four consecutive 2'-F-nucleotides or 2'-OMe-nucleotides were placed at the site of cleavage of the RNA strand by Ago2, a component of the RNA-induced silencing complex (RISC) present in cells. The inventors discovered siRNAs that maintain high RNAi activity equivalent to that of natural siRNAs and high selectivity for the mRNA encoding P525L point-mutated FUS, while exhibiting dramatically improved stability against RNases, leading to the completion of the present invention.

[0014] The present disclosure includes the following features. [1] A chemically modified siRNA or a salt thereof, comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region complementary or substantially complementary to a portion of an mRNA encoding a P525L point-mutated FUS protein, the complementary region being 19 to 21 nucleotides in length, and the siRNA comprises at least one substitution selected from the group consisting of 2'-F-nucleotides, 2'-OMe-nucleotides, nucleotides in which the 2'-O atom and the 4'-C atom are bridged with a methylene, 2'-deoxy-nucleotides, and phosphorothioate bonds that form internucleotide bonds. [2] The chemically modified siRNA or salt thereof according to [1], which comprises a motif of 2 or 4 consecutive 2'-F-nucleotides or 2'-OMe-nucleotides at or adjacent to the cleavage site of the RNA strand by Ago2, and in the RNA strand other than the motif, 2'-F-nucleotides and 2'-OMe-nucleotides are alternately contained along the RNA strand. [3] The siRNA is represented by the following formula (I): Sense strand: 5'-(YX)a-(YY)b-(XY)c-(XX)d-(YX)e-(YY)f-(XY)g-(XYX)h-(YXY)i-3' Antisense strand: 3'-(YX)j-(XY)k-(XY)a-(XX)b-(YX)c-(YY)d-(XY)e-(XX)f-(YX)g-(Y)h-(X)i-5' (I) During the ceremony, X and Y are 2'-F-nucleotides and 2'-OMe-nucleotides, respectively; a, b, c, d, e, f, g, h, i, j, and k are independently integers from 0 to 4; (a, b, c, j, k) are (0, 0, 4, 1, 0), (0, 1, 3, 0, 1), (0, 2, 2, 0, 1), (1, 1, 2, 0, 1), (2, 1, 1, 0, 1), (1, 2, 1, 0, 1), (2, 2, 0, 0, 1) or (3, 1, 0, 0, 1); When d is 1, (e, f, g, h, i) are (4, 0, 0, 0, 1), (3, 1, 0, 1, 0), (2, 2, 0, 1, 0), (2, 1, 1, 1, 0), (1, 1, 2, 1, 0), (1, 2, 1, 1, 0), (0, 2, 2, 1, 0) or (0, 1, 3, 1, 0); When d is 2, (e, f, g, h, i) are (3, 0, 0, 0, 1), (2, 1, 0, 1, 0), (1, 2, 0, 1, 0), (1, 1, 1, 1, 0), (0, 2, 1, 1, 0) or (0, 1, 2, 1, 0); The chemically modified siRNA or a salt thereof according to [2], which is 21 nucleotides in length. [4] Double-stranded RNA consisting of the sense strand of SEQ ID NO: 5 and the antisense strand of SEQ ID NO: 6, double-stranded RNA consisting of the sense strand of SEQ ID NO: 13 and the antisense strand of SEQ ID NO: 14, double-stranded RNA consisting of the sense strand of SEQ ID NO: 15 and the antisense strand of SEQ ID NO: 16, double-stranded RNA consisting of the sense strand of SEQ ID NO: 17 and the antisense strand of SEQ ID NO: 18, double-stranded RNA consisting of the sense strand of SEQ ID NO: 19 and the antisense strand of SEQ ID NO: 20, double-stranded RNA consisting of the sense strand of SEQ ID NO: 21 and the antisense strand of SEQ ID NO: 22, and the sense strand of SEQ ID NO: 23 and double-stranded RNA consisting of the antisense strand of SEQ ID NO: 24, double-stranded RNA consisting of the sense strand of SEQ ID NO: 35 and the antisense strand of SEQ ID NO: 36, double-stranded RNA consisting of the sense strand of SEQ ID NO: 37 and the antisense strand of SEQ ID NO: 38, double-stranded RNA consisting of the sense strand of SEQ ID NO: 39 and the antisense strand of SEQ ID NO: 40, double-stranded RNA consisting of the sense strand of SEQ ID NO: 41 and the antisense strand of SEQ ID NO: 42, double-stranded RNA consisting of the sense strand of SEQ ID NO: 43 and the antisense strand of SEQ ID NO: 44, double-stranded RNA consisting of the sense strand of SEQ ID NO: 45 and the antisense strand of SEQ ID NO: 46 double-stranded RNA consisting of a sense strand of SEQ ID NO: 47 and an antisense strand of SEQ ID NO: 48; double-stranded RNA consisting of a sense strand of SEQ ID NO: 49 and an antisense strand of SEQ ID NO: 50; double-stranded RNA consisting of a sense strand of SEQ ID NO: 51 and an antisense strand of SEQ ID NO: 52; double-stranded RNA consisting of a sense strand of SEQ ID NO: 53 and an antisense strand of SEQ ID NO: 54; double-stranded RNA consisting of a sense strand of SEQ ID NO: 55 and an antisense strand of SEQ ID NO: 56; double-stranded RNA consisting of a sense strand of SEQ ID NO: 57 and an antisense strand of SEQ ID NO: 58 double-stranded RNA consisting of a sense strand of SEQ ID NO: 59 and an antisense strand of SEQ ID NO: 60; double-stranded RNA consisting of a sense strand of SEQ ID NO: 63 and an antisense strand of SEQ ID NO: 64; double-stranded RNA consisting of a sense strand of SEQ ID NO: 67 and an antisense strand of SEQ ID NO: 68; double-stranded RNA consisting of a sense strand of SEQ ID NO: 71 and an antisense strand of SEQ ID NO: 72; double-stranded RNA consisting of a sense strand of SEQ ID NO: 73 and an antisense strand of SEQ ID NO: 74; double-stranded RNA consisting of a sense strand of SEQ ID NO: 75 and an antisense strand of SEQ ID NO: 76;Double-stranded RNA consisting of the sense strand of SEQ ID NO:79 and the antisense strand of SEQ ID NO:80, double-stranded RNA consisting of the sense strand of SEQ ID NO:81 and the antisense strand of SEQ ID NO:82, double-stranded RNA consisting of the sense strand of SEQ ID NO:85 and the antisense strand of SEQ ID NO:86, double-stranded RNA consisting of the sense strand of SEQ ID NO:87 and the antisense strand of SEQ ID NO:88, double-stranded RNA consisting of the sense strand of SEQ ID NO:89 and the antisense strand of SEQ ID NO:90, double-stranded RNA consisting of the sense strand of SEQ ID NO:91 and the antisense strand of SEQ ID NO:92, double-stranded RNA consisting of the sense strand of SEQ ID NO:93 and the antisense strand of SEQ ID NO:94, double-stranded RNA consisting of the sense strand of SEQ ID NO:97 and the antisense strand of SEQ ID NO:98, double-stranded RNA consisting of the sense strand of SEQ ID NO:99 and the antisense strand of SEQ ID NO:100, double-stranded RNA consisting of the sense strand of SEQ ID NO:103 and the antisense strand of SEQ ID NO:104, double-stranded RNA consisting of the sense strand of SEQ ID NO:105 and the antisense strand of SEQ ID NO:106, double-stranded RNA consisting of the sense strand of SEQ ID NO:107 The chemically modified siRNA or salt thereof according to [2] or [3] is selected from the group consisting of a double-stranded RNA consisting of a sense strand of SEQ ID NO: 109 and an antisense strand of SEQ ID NO: 110, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 111 and an antisense strand of SEQ ID NO: 112, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 113 and an antisense strand of SEQ ID NO: 114, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 117 and an antisense strand of SEQ ID NO: 118, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 119 and an antisense strand of SEQ ID NO: 120, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 123 and an antisense strand of SEQ ID NO: 124, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 125 and an antisense strand of SEQ ID NO: 126, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 127 and an antisense strand of SEQ ID NO: 128, and a double-stranded RNA consisting of a sense strand of SEQ ID NO: 129 and an antisense strand of SEQ ID NO: 130. [5] Double-stranded RNA consisting of the sense strand of SEQ ID NO: 13 and the antisense strand of SEQ ID NO: 14, double-stranded RNA consisting of the sense strand of SEQ ID NO: 21 and the antisense strand of SEQ ID NO: 22, double-stranded RNA consisting of the sense strand of SEQ ID NO: 39 and the antisense strand of SEQ ID NO: 40, double-stranded RNA consisting of the sense strand of SEQ ID NO: 43 and the antisense strand of SEQ ID NO: 44, double-stranded RNA consisting of the sense strand of SEQ ID NO: 49 and the antisense strand of SEQ ID NO: 50, double-stranded RNA consisting of the sense strand of SEQ ID NO: 53 and the antisense strand of SEQ ID NO: 54, double-stranded RNA consisting of the sense strand of SEQ ID NO: 55 and the antisense strand of SEQ ID NO: 56, double-stranded RNA consisting of the sense strand of SEQ ID NO: 57 and the antisense strand of SEQ ID NO: 58, double-stranded RNA consisting of the sense strand of SEQ ID NO: 59 and the antisense strand of SEQ ID NO: 60, double-stranded RNA consisting of the sense strand of SEQ ID NO: 87 and the antisense strand of SEQ ID NO: 88, double-stranded RNA consisting of the sense strand of SEQ ID NO: 89 the double-stranded RNA consisting of a sense strand of SEQ ID NO: 111 and an antisense strand of SEQ ID NO: 112; the double-stranded RNA consisting of a sense strand of SEQ ID NO: 113 and an antisense strand of SEQ ID NO: 114; the double-stranded RNA consisting of a sense strand of SEQ ID NO: 123 and an antisense strand of SEQ ID NO: 124; the double-stranded RNA consisting of a sense strand of SEQ ID NO: 125 and an antisense strand of SEQ ID NO: 126; the double-stranded RNA consisting of a sense strand of SEQ ID NO: 127 and an antisense strand of SEQ ID NO: 128; and the double-stranded RNA consisting of a sense strand of SEQ ID NO: 129 and an antisense strand of SEQ ID NO: 130. [6] Double-stranded RNA consisting of the sense strand of SEQ ID NO: 3 and the antisense strand of SEQ ID NO: 4, double-stranded RNA consisting of the sense strand of SEQ ID NO: 7 and the antisense strand of SEQ ID NO: 8, double-stranded RNA consisting of the sense strand of SEQ ID NO: 9 and the antisense strand of SEQ ID NO: 10, double-stranded RNA consisting of the sense strand of SEQ ID NO: 11 and the antisense strand of SEQ ID NO: 12, double-stranded RNA consisting of the sense strand of SEQ ID NO: 25 and the antisense strand of SEQ ID NO: 26, double-stranded RNA consisting of the sense strand of SEQ ID NO: 27 and the antisense strand of SEQ ID NO: 28, double-stranded RNA consisting of the sense strand of SEQ ID NO: 29 and the antisense strand of SEQ ID NO: 30 The chemically modified siRNA or salt thereof according to [1], selected from the group consisting of a double-stranded RNA consisting of an antisense strand, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 31 and an antisense strand of SEQ ID NO: 32, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 33 and an antisense strand of SEQ ID NO: 34, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 65 and an antisense strand of SEQ ID NO: 66, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 83 and an antisense strand of SEQ ID NO: 84, and a double-stranded RNA consisting of a sense strand of SEQ ID NO: 121 and an antisense strand of SEQ ID NO: 122. [7] The chemically modified siRNA or a salt thereof according to any one of [1] to [6], wherein the internucleotide bonds between adjacent nucleotides in the three nucleotides located at the 5' and 3' ends of the sense strand and the antisense strand are phosphorothioate bonds. [8] The chemically modified siRNA or a salt thereof according to any one of [1] to [7], for suppressing the expression of P525L point mutation FUS. [9] The chemically modified siRNA or salt thereof according to any one of [1] to [7], for selectively suppressing the expression of P525L point mutant FUS without substantially suppressing the expression of wild-type FUS.

[10] An agent for suppressing the expression of P525L point mutant FUS, comprising the chemically modified siRNA or a salt thereof according to any one of [1] to [9].

[11] A pharmaceutical composition for preventing or treating ALS, comprising the chemically modified siRNA or a salt thereof according to any one of [1] to

[10] as an active ingredient.

[12] The pharmaceutical composition according to

[11] , wherein the ALS is ALS with a P525L point mutation in FUS. [Effects of the Invention]

[0015] The present invention provides chemically modified siRNAs or salts thereof that have dramatically improved stability against RNases while maintaining high RNAi activity equivalent to that of natural siRNAs and high selectivity for the mRNA encoding the P525L point mutant FUS.

[0016] By using the chemically modified siRNA or a salt thereof of the present disclosure, it is possible to selectively suppress the expression of the P525L point mutant FUS without substantially suppressing the expression of wild-type FUS. Furthermore, a pharmaceutical composition containing the chemically modified siRNA or a salt thereof of the present disclosure enables effective treatment of ALS or ALS with a P525L point mutant FUS. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 shows the effect of chemically modified siRNAs of the present disclosure on the mRNA expression rates of wild-type FUS and P525L point mutant FUS. [Figure 2] FIG. 1 shows the effect of chemically modified siRNAs of the present disclosure on the mRNA expression rates of wild-type FUS and P525L point mutant FUS. [Figure 3] FIG. 1 shows the effect of chemically modified siRNAs of the present disclosure on the mRNA expression rates of wild-type FUS and P525L point mutant FUS. [Figure 4] FIG. 1 shows the effect of chemically modified siRNAs of the present disclosure on the mRNA expression rates of wild-type FUS and P525L point mutant FUS. [Figure 5] FIG. 1 shows the effect of chemically modified siRNAs of the present disclosure on the mRNA expression rates of wild-type FUS and P525L point mutant FUS. [Figure 6] FIG. 1 shows the stability of natural siRNA in human serum. [Figure 7] FIG. 1 shows the stability of chemically modified siRNAs of the present disclosure in human serum. DETAILED DESCRIPTION OF THE INVENTION

[0018] The chemically modified siRNA (small interfering RNA) of the present disclosure is a double-stranded RNA consisting of an RNA (antisense strand) complementary to the mRNA transcribed from the P525L point-mutated FUS gene, which is a causative gene for ALS, and an RNA (sense strand) complementary to the antisense strand. The chemically modified siRNA can degrade the P525L point-mutated FUS mRNA by RNA interference (RNAi), thereby selectively suppressing the expression of P525L point-mutated FUS, which is involved in ALS.

[0019] The chemically modified siRNA of the present disclosure comprises a region complementary or substantially complementary to a portion of the mRNA encoding P525L point-mutated FUS, and the complementary region is 19 to 21 nucleotides in length. In some embodiments, the chemically modified siRNA of the present disclosure has a sense strand and an antisense strand each having a length of 19 to 26 nucleotides. In some embodiments, the chemically modified siRNA of the present disclosure has a length of 19 to 23 nucleotides.

[0020] In this specification, "complementary" means that the sense strand and antisense strand of siRNA, or the antisense strand of siRNA and target mRNA, are bound by hydrogen bonds formed by the complementary base portions of opposing nucleotides.In this specification, "substantially complementary" means that one or several opposing nucleotides are not complementary nucleotides, but the oligonucleotide as a whole forms a base pair and binds.

[0021] The chemically modified siRNA of the present disclosure contains at least one substitution selected from the group consisting of 2'-F-nucleotides, 2'-OMe-nucleotides, nucleotides in which the 2'-O atom and the 4'-C atom are methylene-bridged (LNA), 2'-deoxy-nucleotides, and phosphorothioate bonds forming the internucleotide bond.

[0022] In some embodiments, the chemically modified siRNA of the present disclosure comprises a motif of two or four consecutive 2'-F-nucleotides or 2'-OMe-nucleotides at or adjacent to the Ago2 RNA cleavage site, and the RNA strand other than the motif contains alternating 2'-F-nucleotides and 2'-OMe-nucleotides along the RNA strand. Ago2 (Argonaute 2) is one of the components of the RNA-induced silencing complex (RISC). The siRNA is incorporated into RISC, and after the sense strand is removed, the antisense strand recognizes the target mRNA, which is then cleaved by Ago2.

[0023] The chemically modified siRNA can be represented by the following formula (I): Sense strand: 5'-(YX)a-(YY)b-(XY)c-(XX)d-(YX)e-(YY)f-(XY)g-(XYX)h-(YXY)i-3' Antisense strand: 3'-(YX)j-(XY)k-(XY)a-(XX)b-(YX)c-(YY)d-(XY)e-(XX)f-(YX)g-(Y)h-(X)i-5' (I)

[0024] In the above formula (I), X and Y are 2'-F-nucleotide and 2'-OMe-nucleotide, respectively; a, b, c, d, e, f, g, h, i, j and k are independently integers of 0 to 4; (a, b, c, j, k) are, in this order, (0, 0, 4, 1, 0), (0, 1, 3, 0, 1), (0, 2, 2, 0, 1), (1, 1, 2, 0, 1), (2, 1, 1, 0, 1), (1, 2, 1, 0, 1), (2, 2, 0, 0, 1), or (3, 1, 0, 0, 1); when d is 1, (e, f, g, h, i) are, in this order, (4, 0, 0, 0, 1), (3, 1, 0, 1, 0), (2, 2, 0, 1, 0), (2, 1, 1, 1, 0), (1, 1, 2, 1, 1, 0), (1, 2, 1, 1, 0), (0, 2, 2, 1, 0), or (0, 1, 3, 1, 0); when d is 2, (e, f, g, h, i) are (3, 0, 0, 0, 1), (2, 1, 0, 1, 0), (1, 2, 0, 1, 0), (1, 1, 1, 1, 0), (0, 2, 1, 1, 0), or (0, 1, 2, 1, 0), respectively. Here, a, b, c, d, e, f, g, h, i, j, and k represent the number of repeats of the sequence. For example, (YX)a indicates a sequence consisting of four nucleotides, YXYX, when a=2; a=3 indicates a sequence consisting of six nucleotides, YXYXYX; and a=0 indicates that the sequence in parentheses does not exist.

[0025] The double-stranded siRNA is cleaved by Ago2. In some embodiments, in the case of the 21-nucleotide-long chemically modified siRNA of the present disclosure, the cleavage site by Ago2 is the bond between positions 9 and 10 or between positions 10 and 11 from the 5' end of the sense strand.

[0026] In some embodiments, the chemically modified siRNA of the present disclosure is a double-stranded RNA consisting of a sense strand of SEQ ID NO: 5 and an antisense strand of SEQ ID NO: 6, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 13 and an antisense strand of SEQ ID NO: 14, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 15 and an antisense strand of SEQ ID NO: 16, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 17 and an antisense strand of SEQ ID NO: 18, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 19 and an antisense strand of SEQ ID NO: 20, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 21 and an antisense strand of SEQ ID NO: 22, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 23 and an antisense strand of SEQ ID NO: 24, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 35 and an antisense strand of SEQ ID NO: 36, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 37 and an antisense strand of SEQ ID NO: 38, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 39 and an antisense strand of SEQ ID NO: 40, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 41 and an antisense strand of SEQ ID NO: 42, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 43 and an antisense strand of SEQ ID NO: 44, as shown in Table 1 below. double-stranded RNA consisting of a sense strand of SEQ ID NO: 45 and an antisense strand of SEQ ID NO: 46; double-stranded RNA consisting of a sense strand of SEQ ID NO: 47 and an antisense strand of SEQ ID NO: 48; double-stranded RNA consisting of a sense strand of SEQ ID NO: 49 and an antisense strand of SEQ ID NO: 50; double-stranded RNA consisting of a sense strand of SEQ ID NO: 51 and an antisense strand of SEQ ID NO: 52; double-stranded RNA consisting of a sense strand of SEQ ID NO: 53 and an antisense strand of SEQ ID NO: 54; double-stranded RNA consisting of a sense strand of SEQ ID NO: 55 and an antisense strand of SEQ ID NO: 56 double-stranded RNA consisting of a sense strand of SEQ ID NO: 57 and an antisense strand of SEQ ID NO: 58; double-stranded RNA consisting of a sense strand of SEQ ID NO: 59 and an antisense strand of SEQ ID NO: 60; double-stranded RNA consisting of a sense strand of SEQ ID NO: 63 and an antisense strand of SEQ ID NO: 64; double-stranded RNA consisting of a sense strand of SEQ ID NO: 67 and an antisense strand of SEQ ID NO: 68; double-stranded RNA consisting of a sense strand of SEQ ID NO: 71 and an antisense strand of SEQ ID NO: 72; double-stranded RNA consisting of a sense strand of SEQ ID NO: 73 and an antisense strand of SEQ ID NO: 74;Double-stranded RNA consisting of the sense strand of SEQ ID NO: 75 and the antisense strand of SEQ ID NO: 76, double-stranded RNA consisting of the sense strand of SEQ ID NO: 79 and the antisense strand of SEQ ID NO: 80, double-stranded RNA consisting of the sense strand of SEQ ID NO: 81 and the antisense strand of SEQ ID NO: 82, double-stranded RNA consisting of the sense strand of SEQ ID NO: 85 and the antisense strand of SEQ ID NO: 86, double-stranded RNA consisting of the sense strand of SEQ ID NO: 87 and the antisense strand of SEQ ID NO: 88, double-stranded RNA consisting of the sense strand of SEQ ID NO: 89 and the antisense strand of SEQ ID NO: 90, double-stranded RNA consisting of the sense strand of SEQ ID NO: 91 and the antisense strand of SEQ ID NO: 92, double-stranded RNA consisting of the sense strand of SEQ ID NO: 93 and the antisense strand of SEQ ID NO: 94, double-stranded RNA consisting of the sense strand of SEQ ID NO: 97 and the antisense strand of SEQ ID NO: 98, double-stranded RNA consisting of the sense strand of SEQ ID NO: 99 and the antisense strand of SEQ ID NO: 100, double-stranded RNA consisting of the sense strand of SEQ ID NO: 103 and the antisense strand of SEQ ID NO: 104, double-stranded RNA consisting of the sense strand of SEQ ID NO: 105 and the antisense strand of SEQ ID NO: 106 double-stranded RNA consisting of a sense strand, double-stranded RNA consisting of a sense strand of SEQ ID NO: 107 and an antisense strand of SEQ ID NO: 108, double-stranded RNA consisting of a sense strand of SEQ ID NO: 109 and an antisense strand of SEQ ID NO: 110, double-stranded RNA consisting of a sense strand of SEQ ID NO: 111 and an antisense strand of SEQ ID NO: 112, double-stranded RNA consisting of a sense strand of SEQ ID NO: 113 and an antisense strand of SEQ ID NO: 114, double-stranded RNA consisting of a sense strand of SEQ ID NO: 117 and an antisense strand of SEQ ID NO: 118, double-stranded RNA consisting of a sense strand of SEQ ID NO: 119 and an antisense strand of SEQ ID NO: 120, double-stranded RNA consisting of a sense strand of SEQ ID NO: 123 and an antisense strand of SEQ ID NO: 124, double-stranded RNA consisting of a sense strand of SEQ ID NO: 125 and an antisense strand of SEQ ID NO: 126, double-stranded RNA consisting of a sense strand of SEQ ID NO: 127 and an antisense strand of SEQ ID NO: 128, and double-stranded RNA consisting of the antisense strand of a double-stranded RNA consisting of a sense strand of SEQ ID NO: 129 and an antisense strand of SEQ ID NO: 130. ,

[0027] In some embodiments, the chemically modified siRNA of the present disclosure is a double-stranded RNA consisting of a sense strand of SEQ ID NO: 13 and an antisense strand of SEQ ID NO: 14, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 21 and an antisense strand of SEQ ID NO: 22, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 39 and an antisense strand of SEQ ID NO: 40, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 43 and an antisense strand of SEQ ID NO: 44, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 49 and an antisense strand of SEQ ID NO: 50, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 53 and an antisense strand of SEQ ID NO: 54, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 55 and an antisense strand of SEQ ID NO: 56, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 57 and an antisense strand of SEQ ID NO: 58, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 59 and an antisense strand of SEQ ID NO: 60, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 87 and an antisense strand of SEQ ID NO: 88, or a double-stranded RNA consisting of a sense strand of SEQ ID NO: 89 and an antisense strand of SEQ ID NO: 90. double-stranded RNA consisting of the antisense strand of SEQ ID NO: 88, double-stranded RNA consisting of the sense strand of SEQ ID NO: 89 and the antisense strand of SEQ ID NO: 90, double-stranded RNA consisting of the sense strand of SEQ ID NO: 91 and the antisense strand of SEQ ID NO: 92, double-stranded RNA consisting of the sense strand of SEQ ID NO: 93 and the antisense strand of SEQ ID NO: 94, double-stranded RNA consisting of the sense strand of SEQ ID NO: 111 and the antisense strand of SEQ ID NO: 112, double-stranded RNA consisting of the sense strand of SEQ ID NO: 113 and the antisense strand of SEQ ID NO: 114, double-stranded RNA consisting of the sense strand of SEQ ID NO: 123 and the antisense strand of SEQ ID NO: 124, double-stranded RNA consisting of the sense strand of SEQ ID NO: 125 and the antisense strand of SEQ ID NO: 126, double-stranded RNA consisting of the sense strand of SEQ ID NO: 127 and the antisense strand of SEQ ID NO: 128, and double-stranded RNA consisting of the sense strand of SEQ ID NO: 129 and the antisense strand of SEQ ID NO: 130.

[0028] In some embodiments, the chemically modified siRNA of the present disclosure is a double-stranded RNA consisting of a sense strand of SEQ ID NO: 3 and an antisense strand of SEQ ID NO: 4, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 7 and an antisense strand of SEQ ID NO: 8, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 9 and an antisense strand of SEQ ID NO: 10, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 11 and an antisense strand of SEQ ID NO: 12, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 25 and an antisense strand of SEQ ID NO: 26, or a double-stranded RNA consisting of a sense strand of SEQ ID NO: 27 and an antisense strand of SEQ ID NO: 28, as shown in Table 1 below. The double-stranded RNA is selected from the group consisting of double-stranded RNA, double-stranded RNA consisting of a sense strand of SEQ ID NO: 29 and an antisense strand of SEQ ID NO: 30, double-stranded RNA consisting of a sense strand of SEQ ID NO: 31 and an antisense strand of SEQ ID NO: 32, double-stranded RNA consisting of a sense strand of SEQ ID NO: 33 and an antisense strand of SEQ ID NO: 34, double-stranded RNA consisting of a sense strand of SEQ ID NO: 65 and an antisense strand of SEQ ID NO: 66, double-stranded RNA consisting of a sense strand of SEQ ID NO: 83 and an antisense strand of SEQ ID NO: 84, and double-stranded RNA consisting of a sense strand of SEQ ID NO: 121 and an antisense strand of SEQ ID NO: 122.

[0029] (Table 1) Strand (S = sense strand, AS = antisense strand) JPEG0007776924000001.jpg211148JPEG0007776924000002.jpg224148JPEG0007776924000003.jpg224148JPEG0007776924000004.jpg140148

[0030] siRNAs-010, -002, -003, -006, -008, -009, and -011 listed in Table 1 are all natural siRNAs, and were prepared and tested to compare their stability against RNases and RNAi activity with the chemically modified siRNAs disclosed herein.

[0031] The chemically modified nucleotides contained in the chemically modified siRNAs listed in Table 1 and their abbreviations are shown in Table 2 below. (Table 2) JPEG0007776924000005.jpg199167In Table 2, "LNA (locked nucleic acid) modification" refers to a chemically modified nucleotide in which the ribose constituting the nucleotide is bridged between the 2'-O atom and the 4'-C atom with methylene.

[0032] In some embodiments, the sense strand and antisense strand constituting the chemically modified siRNA of the present disclosure have the sequences set forth in Table 1, but may also have substantially identical sequences to those set forth in Table 1. "Substantially identical sequences" means that the antisense strand of the siRNA and the target mRNA may contain chemical modifications and mismatched bases in the sequences set forth in Table 1, as long as they retain the ability to form double-stranded RNA. In some embodiments, the number of mismatched bases is three or less. In some embodiments, the number of mismatched bases may be up to one.

[0033] The sense strand and antisense strand constituting the chemically modified siRNA of the present disclosure may comprise a dinucleotide overhang at the 3' end. In some embodiments, the chemically modified siRNA of the present disclosure comprises UU (U: uridine) as the overhang.

[0034] Normally, siRNA has phosphodiester bonds, but in the chemically modified siRNA of the present disclosure, in both the sense and antisense strands, the two phosphodiester bonds between adjacent nucleotides in the three nucleotides located at the 5' and 3' ends are all replaced with phosphorothioate bonds.

[0035] The chemically modified siRNA of the present disclosure can be produced by nucleic acid molecule synthesis methods well known to those skilled in the art, such as those described in "Creation and Application Development of Nucleic Acid Drugs" (CMC Publishing, 2016) and "Synthetic Techniques for Peptides, Nucleic Acids, and Sugar Chains Contributing to Medium-Molecule Drug Discovery" (CMC Publishing, 2018).

[0036] The chemically modified siRNA of the present disclosure can be double-stranded by associating a synthesized single-stranded oligonucleotide with another complementary single-stranded oligonucleotide.Specific examples of the association method include heating to a temperature at which the double-stranded oligonucleotide dissociates, and then gradually cooling to anneal the complementary oligonucleotides.

[0037] The oligonucleotides can be synthesized by solid-phase synthesis using commercially available amidites. Solid-phase synthesis is performed using a commercially available nucleic acid synthesizer and a solid support. The 3' end of a monomer nucleotide is attached to the surface of the solid support via an alkyl chain, and an amidite is then added thereto. That is, the desired oligonucleotide can be synthesized by repeating a cycle of extending the desired oligonucleotide sequence one nucleotide at a time from the 3' end toward the 5' end. After the synthesis cycle is completed, the oligonucleotide is cleaved from the solid support, and the base moiety and 2' position are deprotected to prepare the desired single-stranded RNA. However, the deprotection step is not necessary when synthesizing 2'-F-modified RNA, 2'-OMe-modified RNA, or RNA in which the 2'-O atom and the 4'-C atom are crosslinked with a methylene.

[0038] The chemically modified siRNA of the present disclosure can be synthesized by Gene Design using the phosphoramidite method described above. The obtained siRNA can be subjected to simple column purification and then quality confirmation by mass spectrometry and electrophoresis.

[0039] The chemically modified siRNA of the present disclosure can be prepared by selecting a contiguous base sequence that is the target of the P525L point mutation FUS mRNA. Specifically, the base sequence is selected from the mRNA sequence of 19 to 21 nucleotides in the region containing the P525L point mutation. The resulting siRNA sequence can be prepared by selecting a base sequence in which one or several nucleotides have been substituted, deleted, inserted, and / or added from the above sequence, as long as it can induce RNA interference and degrade the target P525L point mutation FUS mRNA.

[0040] In some embodiments, a chemically modified siRNA or a salt thereof comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region complementary or substantially complementary to a portion of an mRNA encoding a P525L point-mutated FUS protein, the complementary region being 19 to 21 nucleotides in length, and the siRNA comprising at least one substitution selected from the group consisting of 2'-F-nucleotides, 2'-OMe-nucleotides, nucleotides in which the 2'-O atom and the 4'-C atom are bridged with a methylene, 2'-deoxy-nucleotides, and phosphorothioate bonds forming internucleotide bonds, can be produced by a method for synthesizing nucleic acid molecules well known to those skilled in the art.

[0041] In some embodiments, a chemically modified siRNA or a salt thereof containing a motif of 2 or 4 consecutive 2'-F-nucleotides or 2'-OMe-nucleotides at or adjacent to the Ago2 cleavage site of the RNA strand, and containing alternating 2'-F-nucleotides and 2'-OMe-nucleotides along the RNA strand other than the motif, can be produced by a method for synthesizing nucleic acid molecules well known to those skilled in the art.

[0042] In some embodiments, the siRNA is represented by the following formula (I): Sense strand: 5'-(YX)a-(YY)b-(XY)c-(XX)d-(YX)e-(YY)f-(XY)g-(XYX)h-(YXY)i-3' Antisense strand: 3'-(YX)j-(XY)k-(XY)a-(XX)b-(YX)c-(YY)d-(XY)e-(XX)f-(YX)g-(Y)h-(X)i-5' (I) During the ceremony, X and Y are 2'-F-nucleotides and 2'-OMe-nucleotides, respectively; a, b, c, d, e, f, g, h, i, j, and k are independently integers from 0 to 4; (a, b, c, j, k) are (0, 0, 4, 1, 0), (0, 1, 3, 0, 1), (0, 2, 2, 0, 1), (1, 1, 2, 0, 1), (2, 1, 1, 0, 1), (1, 2, 1, 0, 1), (2, 2, 0, 0, 1) or (3, 1, 0, 0, 1); When d is 1, (e, f, g, h, i) are (4, 0, 0, 0, 1), (3, 1, 0, 1, 0), (2, 2, 0, 1, 0), (2, 1, 1, 1, 0), (1, 1, 2, 1, 0), (1, 2, 1, 1, 0), (0, 2, 2, 1, 0) or (0, 1, 3, 1, 0); When d is 2, (e, f, g, h, i) are (3, 0, 0, 0, 1), (2, 1, 0, 1, 0), (1, 2, 0, 1, 0), (1, 1, 1, 1, 0), (0, 2, 1, 1, 0) or (0, 1, 2, 1, 0); The chemically modified 21-nucleotide-long siRNA or a salt thereof can be produced by methods for synthesizing nucleic acid molecules well known to those skilled in the art.

[0043] Those skilled in the art can prepare the chemically modified siRNA of the present disclosure based on the base sequence disclosed herein. Specifically, double-stranded RNA can be prepared based on any of the base sequences of SEQ ID NOs: 1 to 130. Once one nucleotide strand is identified, those skilled in the art can easily understand the base sequence of the complementary nucleotide strand. The chemically modified siRNA of the present disclosure may be prepared using a commercially available nucleic acid synthesizer or may be obtained using a general synthesis service.

[0044] The chemically modified siRNA of the present disclosure can induce RNA interference, target and degrade P525L point mutant FUS mRNA, and selectively suppress the expression of P525L point mutant FUS, which is involved in the onset of ALS.

[0045] The chemically modified siRNA of the present disclosure suppresses the expression of P525L point mutant FUS, while having no substantial effect on the expression of wild-type FUS without mutation. That is, the chemically modified siRNA of the present disclosure selectively suppresses the expression of P525L point mutant FUS without substantially suppressing the expression of wild-type FUS. "Without substantially suppressing the expression of wild-type FUS" means that undesirable symptoms caused by the suppression of wild-type FUS expression in ALS do not substantially appear.

[0046] The inhibitory effect of the chemically modified siRNA of the present disclosure on the expression of P525L point mutant FUS can be expressed as an expression inhibition rate (%) using the calculation formula described below. In some embodiments, the chemically modified siRNA of the present disclosure inhibits the expression of P525L point mutant FUS by 30% or more. In some embodiments, the chemically modified siRNA of the present disclosure inhibits the expression of P525L point mutant FUS by 50% or more.

[0047] In the FUS expression suppression effect of the chemically modified siRNA of the present disclosure, the selectivity for the P525L point mutant type compared to the wild type is: Selectivity in expression rate = wild-type FUS expression rate / P525L point mutant FUS expression rate; or Selectivity in expression suppression rate = P525L point mutant FUS expression suppression rate (%) - wild-type FUS expression suppression rate (%); The expression rate and the expression inhibition rate are defined as follows. The higher the selectivity for both the expression rate and the expression inhibition rate, the higher the selectivity for inhibiting the expression of P525L point mutant FUS. The expression rate and expression inhibition rate of the siRNA of the present disclosure for wild-type FUS and P525L point mutant FUS can be calculated using the formula described below.

[0048] In some embodiments, the selectivity of the chemically modified siRNA of the present disclosure in terms of the expression rate is 1.5 or higher. In some embodiments, the selectivity of the chemically modified siRNA of the present disclosure in terms of the expression rate is 2 or higher. In some embodiments, the selectivity of the chemically modified siRNA of the present disclosure in terms of the expression suppression rate is 20% or higher. In some embodiments, the selectivity of the chemically modified siRNA of the present disclosure in terms of the expression suppression rate is 40% or higher. The selectivity of FUS expression suppression by the chemically modified siRNA may be evaluated based on either the selectivity of the expression rate or the selectivity of the expression suppression rate, or a combination of both.

[0049] The chemically modified siRNA of the present disclosure can be in the form of salt.In some embodiments, said salt is a pharmaceutically acceptable salt.In some embodiments, said salt includes but is not limited to alkali metal salt such as sodium salt, potassium salt, lithium salt, and alkaline earth metal salt such as calcium salt, magnesium salt.

[0050] The chemically modified siRNA or a salt thereof of the present disclosure is useful as a therapeutic agent for ALS, and its therapeutic effect can be evaluated using, for example, the methods described in the following documents or methods equivalent thereto. McCampbell A, et al. Antisense oligonucleotides extend survival and reverse decrement in muscle response in ALS models. J Clin Invest, 2018;128:3558-3567. Akiyama T, et al. Aberrant axon branching via Fos-B dysregulation in FUS-ALS motor neurons. EBioMedicine, 2019;45:362-378. Shiihashi G, Mislocated FUS is sufficient for gain-of-toxic-function amyotrophic lateral sclerosis phenotypes in mice. Brain, 2016;139:2380-94.

[0051] In some embodiments, the present disclosure provides a pharmaceutical composition for preventing or treating ALS, comprising the chemically modified siRNA or its salt and a pharmaceutically acceptable carrier.In some embodiments, the ALS is the ALS with P525L point mutation FUS.

[0052] In some embodiments, the pharmaceutical compositions of the present disclosure may be in a dosage form for oral use or a dosage form for parenteral use. These dosage forms can be formulated by those skilled in the art by appropriately combining pharmaceutically acceptable carriers and additives and blending them into a unit dosage form required for generally accepted pharmaceutical practice. In some embodiments, the pharmaceutical compositions of the present disclosure can be manufactured according to known methods, such as those described in the Japanese Pharmacopoeia or the United States Pharmacopoeia (USP).

[0053] In some embodiments, a method for preventing or treating ALS or ALS with P525L point mutation FUS is provided, comprising administering to a patient in need of treatment an effective amount of an expression inhibitor of P525L point mutation FUS containing the chemically modified siRNA or a salt thereof disclosed herein.

[0054] In some embodiments, the present invention provides an agent for suppressing the expression of P525L point mutant FUS, comprising a chemically modified siRNA or a salt thereof according to the present disclosure, for the prevention or treatment of ALS or ALS with P525L point mutant FUS.

[0055] In some embodiments, the present invention provides an agent for suppressing the expression of P525L point mutant FUS, comprising a chemically modified siRNA or a salt thereof according to the present disclosure, for producing an agent for preventing or treating ALS or ALS having P525L point mutant FUS.

[0056] In some embodiments, there is provided a method for preventing or treating ALS or ALS with P525L point mutation FUS, comprising administering to a patient in need of treatment an effective amount of a pharmaceutical composition for preventing or treating ALS, comprising the chemically modified siRNA disclosed herein or a salt thereof and a pharmaceutically acceptable carrier.

[0057] In some embodiments, there is provided a pharmaceutical composition comprising a chemically modified siRNA of the present disclosure or a salt thereof and a pharmaceutically acceptable carrier for the prevention or treatment of ALS or ALS with a P525L point mutation in FUS.

[0058] In some embodiments, a pharmaceutical composition is provided, comprising a chemically modified siRNA of the present disclosure or a salt thereof and a pharmaceutically acceptable carrier, for producing an agent for preventing or treating ALS or ALS with a P525L point mutation in FUS.

[0059] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples. [Example]

[0060] [Human FUS 野生型 and FUS P525L cDNA synthesis Human wild-type FUS (hereafter referred to as FUS 野生型 The cDNA sequence (SEQ ID NO: 1) of human P525L point mutant FUS (hereinafter referred to as FUS) is shown in SEQ ID NO: 131. P525L The cDNA sequence (Sequence 2) is shown in SEQ ID NO: 132. The artificial gene was obtained from GenScript Japan Co., Ltd. The synthetic gene was inserted into the BamHI / XhoI site in the multicloning site of the pcDNA3.1+ vector.

[0061] (Array 1)

[0062] (Array 2) [Example]

[0063] [GFP fusion FUS 野生型 and FP635 fusion FUS P525L Construction of gene expression vector] PCR was performed using the pTurboGFP vector, pTurboFP635 vector, and the artificial gene prepared in Example 1 with the primer set shown in Table 3. For PCR, 25 μL of PrimeSTAR Max Premix (Takara Bio Inc.), 4 μL each of 2.5 μM Primer (final concentration 0.2 μM), 1 μL (20 ng) of template, and 20 μL of water were mixed and incubated at 98°C for 10 seconds, followed by 35 temperature cycles of 98°C for 10 seconds, 55°C for 5 seconds, and 72°C for 10 seconds (25 seconds when vector was used as template). The vector amplified fragment and FUS gene amplified fragment were ligated by an In-Fusion reaction. Specifically, 2 μL of the insert fragment, 1 μL of the vector amplification product, 2 μL of 5x In-Fusion HD Enzyme Premix (Takara Bio Inc.), and 5 μL of water were mixed, reacted at 50°C for 15 minutes, and then transformed into NEB Turbo Competent E. coli (New England Biolabs Japan). Next, a plasmid vector was prepared from the transformant, and its DNA sequence confirmed that the target cDNA had been properly inserted. FUS 野生型 contains the Turbo GFP fluorescent protein at its N-terminus and the FUS P525L were cloned in-frame into the pTurboGFP vector (Evrogen) and the pTurboFP635 vector (Evrogen), respectively, so that the TurboFP635 fluorescent protein was attached to the N-terminus thereof.

[0064] (Table 3) JPEG0007776924000006.jpg56144 [Example]

[0065] [Cell culture] HEK293 cells were cultured in Advanced DMEM (Thermo Fisher Scientific) containing 10% FBS and 4 mM GlutaMAX® Supplement at 37°C in a 5% CO environment. HEK293 cells were obtained from the JCRB Cell Bank, Culture Resources Laboratory, National Institutes of Biomedical Innovation, Health and Nutrition (cell number JCRB9068). [Example]

[0066] [Generation of FUS knockout (KO) HEK293 cell lines] FUS KO HEK cell lines were generated by transfection with the FUS / TLS CRISPR / Cas9 KO (sc-400612) plasmid (Santa Cruz) and the FUS / TLS HDR plasmid (h) (sc-400612-HDR) (Santa Cruz) using TransIT®-293 Transfection Reagent (Mirus). FUS gene knockout was confirmed by RT-PCR (using the SuperScript® IV One-Step RT-PCR System with ezDNase®, Invitrogen, #12595100) to confirm FUS mRNA expression. The primer sets used are listed in Table 4. Total RNA was prepared using the RNeasy Plus Mini Kit (QIAGEN), and gDNA was digested by mixing 1 μL of 10x ezDNase Buffer, 1 μL of ezDNase Enzyme, 1 μL of template RNA (500 ng / μL), and 7 μL of water at 37°C for 5 minutes. For RT-PCR, 10 μL of template RNA (digested gDNA), 25 μL of 2x Platinum SuperFi RT-PCR Master Mix, 2.5 μL of Primer Set I Mixture (10 μM each), 2.5 μL of Primer Set V Mixture (10 μM each), 0.5 μL of SuperScript IV RT Mix, and 9.5 μL of water were mixed and incubated at 60°C for 10 minutes, 98°C for 2 minutes, and then 40 cycles of 98°C for 10 seconds, 62°C for 10 seconds, and 72°C for 1 minute, followed by a reaction at 72°C for 5 minutes.

[0067] (Table 4) JPEG0007776924000007.jpg32135 [Example]

[0068] [TurboGFP fusion FUS 野生型 and TurboFP635 fusion FUS P525L Preparation of co-expressing HEK293 cell lines TurboGFP fusion FUS 野生型 cDNA and TurboFP635 fused FUS P525L The cDNA was cloned into the multiple cloning site of pAAVS1-puro-DNR (Origene). pAAVS1-puro-DNR (Origene)_TurboGFP-FUS 野生型 , pAAVS1-puro-DNR(Origene)_TurboFP635-FUS P525L and pCas-Guide-AAVS1 (Origene) were transfected into pre-prepared FUS KO HEK293 cells, and TurboGFP-fused FUS was expressed. 野生型 and TurboFP635 fusion FUS P525L We generated cells co-expressing TurboGFP and TurboFP635. Cell lines were cloned by sorting double-positive cells for TurboGFP and TurboFP635 using On-chip Sort (On-chip Biotechnologies Co., Ltd.), then sorting single cells into 384-well plates using On-chip SPiS (On-chip Biotechnologies Co., Ltd.) and culturing them. [Example]

[0069] [TurboGFP fusion FUS 野生型 and TurboFP635 fusion FUS P525L Evaluation of RNA interference using co-expressing HEK293 cell line (imaging) A 25 μL mixture of 25 μL of Opti-MEM (Invitrogen) and 1.5 μL of Lipofectamine® RNAi MAX (Invitrogen) was mixed with a 25 μL mixture of 25 μL of Opti-MEM (Invitrogen) and 0.5 μL of 10 μM siRNA, and the mixture was incubated at room temperature for 15 to 20 minutes. 野生型 and TurboFP635 fusion FUS P525L The co-expressing HEK293 cell line was cultured at 3.0 × 10 in a medium (FluoroBrite® DMEM containing 5% FBS, hereinafter the same) pre-warmed to 37°C.5 The cells were suspended at 100 μL / mL. 100 μL of the cell suspension was mixed with 10 μL of the previously prepared Lipofectamine-siRNA complex and seeded onto a CellCarrier Ultra collagen-coated 96-well plate (PerkinElmer) and cultured at 37°C and 5% CO2 (the following cultures were performed under the same conditions). 24 hours after transfection, 100 μL of medium was added, and 48 hours after transfection, data were acquired using an Operetta CLS® High-Content Confocal Imaging System (PerkinElmer) (20x water immersion lens, confocal mode). The total number of cells (nuclei), the number of TurboGFP-positive cells, and the number of TurboFP635-positive cells were counted from the acquired image data, and the TurboGFP-positive cell rate (number of TurboGFP-positive cells / total number of cells) and the TurboFP635-positive cell rate (number of TurboFP635-positive cells / total number of cells) were calculated.

[0070] TurboGFP-positive cells and TurboFP635-positive cells were defined as follows. TurboGFP positive cells (FUS 野生型 Expressing cells): The sum of the TurboGFP fluorescence intensity within the nuclear region divided by the area of ​​the nuclear region (pixels) is 400 or greater. TurboFP635 positive cells (FUS P525L Expressing cells): The sum of the fluorescence intensity of TurboFP635 in the cytoplasmic region divided by the area (pixels) of the cytoplasmic region is 400 or more.

[0071] Each positive cell rate was substituted into the following formula to calculate the relative expression rate and expression inhibition rate. "Expression rate (%)" = 100 × {(rate of TurboGFP-positive cells after treatment with various siRNAs) - (rate of TurboGFP-positive cells after treatment with positive control siRNA)} / {(rate of TurboGFP-positive cells after treatment with negative control siRNA) - (rate of TurboGFP-positive cells after treatment with positive control siRNA)} "Expression suppression rate (%)" = 100 × {(rate of TurboGFP-positive cells after treatment with various siRNAs) - (rate of TurboGFP-positive cells after treatment with negative control siRNA)} / {(rate of TurboGFP-positive cells after treatment with positive control siRNA) - (rate of TurboGFP-positive cells after treatment with negative control siRNA)} The above formula is FUS 野生型 In the case of FUS P525L The percentage of positive cells can also be calculated in the same way from the TurboFP635-positive cell rate. In the above formula, the negative control siRNA is an siRNA with a sequence unrelated to known gene sequences in humans, mice, and rats, and is provided by Horizon Discovery. Its sequence is UAGCGACUAAACACAUCAA (SEQ ID NO: 145). On the other hand, the positive control siRNA is a mixture of four siRNAs (SEQ ID NOs: 146 to 149) designed to target specific regions of human FUS mRNA, and is provided by Horizon Discovery. The sequences of the four siRNAs are as follows: CCUACGGACAGCAGAGUUA (SEQ ID NO: 146) GAUUAUACCCAACAAGCAA (SEQ ID NO: 147) GAUCAAUCCUCCAUGAGUA (SEQ ID NO: 148) CGGGACAGCCCAUGAUUAA (SEQ ID NO: 149)

[0072] Tables 5 to 9 show the effects of the chemically modified siRNAs listed in Table 1 on the expression rates of wild-type FUS and P525L point mutant FUS, their expression-suppressing effects, and their respective selectivities. Tables 5 to 9 show the results of experiments performed independently of each other, so results for the same siRNA may be shown in each table. Examples of the same siRNA include siRNA-010 and siRNA-010-4. Here, the selectivity is Selectivity in expression rate = wild-type FUS expression rate (%) / P525L point mutant FUS expression rate (%); or Selectivity in expression suppression rate = P525L point mutant FUS expression suppression rate (%) - wild-type FUS expression suppression rate (%); is defined as:

[0073] (Table 5) JPEG0007776924000008.jpg44142

[0074] (Table 6) JPEG0007776924000009.jpg115142

[0075] (Table 7) JPEG0007776924000010.jpg134144

[0076] (Table 8) JPEG0007776924000011.jpg110142

[0077] (Table 9) JPEG0007776924000012.jpg145144

[0078] As shown in Tables 5 to 9, all chemically modified siRNAs had a selectivity for FUS expression of 1.5 times or more and / or a selectivity for FUS expression inhibition of 20% or more, and an inhibition rate of P525L point mutant FUS of 30% or more. Therefore, these chemically modified siRNAs are suggested to have the same inhibitory effect and high selectivity against P525L point mutant FUS compared to native siRNAs.

[0079] As shown in Tables 6, 8 and 9, siRNA-010-16, siRNA-010-16-12, siRNA-010-8, siRNA-010-4-13, siRNA-010-16-6, siRNA-010-16-13, siRNA-010-16-14, siRNA-010-16-15, siRNA-010-16-16, siRNA-006-16-13, siRNA-006-16-14, siRNA-006-16-15 , siRNA-006-16-16, siRNA-009-16-15, siRNA-009-16-16, siRNA-011-16-13, siRNA-011-16-14, siRNA-011-16-15, and siRNA-011-16-16 all showed ≥2-fold selectivity for FUS expression and / or ≥40% selectivity for FUS expression suppression, and ≥50% suppression of P525L point mutant FUS expression. These chemically modified siRNAs therefore demonstrate comparable suppressive activity and higher selectivity for P525L point mutant FUS compared to native siRNA. [Example]

[0080] [TurboGFP fusion FUS 野生型 and TurboFP635 fusion FUS P525L Evaluation of RNA interference using co-expressing HEK293 cell line (real-time PCR) Introduction of siRNA into cells was performed according to the method described in Example 6. 48 hours after transfection, the medium was completely removed, and 50 μL of cell lysate, prepared by mixing 0.5 μL of DNase I (Life Technologies Japan) and 49.5 μL of Lysis Solution (Life Technologies Japan), was added and incubated at room temperature for 5 minutes. 5 μL of Stop Solution (Life Technologies Japan) was then added and mixed, followed by incubation at room temperature for 2 minutes. This was then subjected to reverse transcription. 10 μL of the previously prepared cell lysate was added to a reverse transcription reaction solution prepared by mixing 25 μL of 2X Fast Advanced RT Buffer (Life Technologies Japan), 2.5 μL of 20X Fast Advanced RT Enzyme Mix (Life Technologies Japan), and 12.5 μL of nuclease-free water. The reaction was then incubated at 37°C for 30 minutes, followed by 95°C for 5 minutes to synthesize cDNA.

[0081] Real-time PCR of TurboGFP-fused FUS 野生型 gene, TurboFP635 fusion FUS P525LThe three genes, including the GFP gene and an endogenous control gene (GAPDH), were detected in the same reaction system. The reaction mixture was prepared by mixing 10 μL of TaqMan® Fast Advanced Master Mix (Life Technologies Japan), 0.06 μL each of 100 μM primers (GFP_X_F, GFP_X_R, FP635_X_F, FP635_X_R), 0.5 μL each of 10 μM TaqMan probes (TurboGFP (NED) and TurboFP635 (FAM)), 1.0 μL of 20X TaqMan Assay (GAPDH) (Life Technologies Japan), 3.76 μL of nuclease-free water, and 4 μL of pre-prepared cDNA. This reaction mixture was incubated in a real-time PCR device (QuantStudio 7 pro, Life Technologies Japan) at 50°C for 2 minutes, then at 95°C for 20 seconds, followed by 40 cycles of 95°C for 1 second and 60°C for 20 seconds. The primers and TaqMan probes used in the real-time PCR are listed in Tables 10 and 11, respectively. Gene expression levels were calculated as relative values ​​using the ΔΔCt method. Relative mRNA expression rates were calculated by setting the mRNA expression rate after treatment with the negative control siRNA at 100% and the mRNA expression rate after treatment with the positive control siRNA at 0%.

[0082] (Table 10) JPEG0007776924000013.jpg47168

[0083] (Table 11) JPEG0007776924000014.jpg29168

[0084] The effects of the chemically modified siRNAs described herein on the mRNA expression rates of wild-type FUS and P525L point mutant FUS are shown in Figures 1 to 5. Similar to the results of the quantitative analysis of FUS protein expression levels by imaging analysis in Example 6, the results of the quantitative analysis of mRNA expression levels by real-time PCR also suggest that each chemically modified siRNA maintains high selectivity for suppressing the expression of P525L point mutant FUS. [Example]

[0085] [Stability evaluation in human serum] The stability of native siRNA and the chemically modified siRNA described herein in human serum was evaluated. 10% (v / v) human serum was prepared by adding 100 μL of human serum (Cosmo Bio Co., Ltd.) to 900 μL of phosphate-buffered saline (PBS) and mixing. 5 μL of 100 μM siRNA was added to 95 μL of 10% (v / v) human serum preheated to 37°C and incubated at 37°C. After the start of incubation, 2 μL of the sample was sampled at predetermined times, mixed with 18 μL of 1x TBE Sample Buffer, and immediately frozen. The sampling times for each siRNA were 0, 15, 30, 45, 60, 75, and 90 minutes for native siRNA-006, siRNA-009, siRNA-010, and siRNA-011, and 0, 1, 3, 6, and 24 hours for the chemically modified siRNAs. Frozen samples were thawed, and 5 μL of the sample was electrophoresed in 20% TBE-PAGE and 1× TBE buffer (150 CV, 40 min). The gel was stained with SYBER® Gold (Thermo Fisher Scientific) and detected with an Amersham Imager 680 UV transilluminator at 312 nm (Cytiva).

[0086] The results of investigating the stability of the siRNA in human serum are shown in Figure 6 for the native siRNA and in Figure 7 for the chemically modified siRNA. Stability in human serum was confirmed by the presence or absence of band shifts and multiple bands due to incubation relative to the band observed without incubation (0 min). As a result, band shifts and multiple bands were observed with the native siRNA within 1 hour of the start of incubation, indicating that degradation by RNase proceeded rapidly. On the other hand, no clear band shift was observed with the chemically modified siRNA even 24 hours after the start of incubation, indicating that degradation by RNase did not occur. Therefore, the chemically modified siRNA of the present disclosure demonstrated dramatically improved stability against RNase.

Claims

1. A chemically modified siRNA or a salt thereof that selectively suppresses expression of P525L point mutant FUS compared to expression of wild-type FUS, the siRNA is a double-stranded RNA consisting of a sense strand of SEQ ID NO: 1 and an antisense strand of SEQ ID NO: 2, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 61 and an antisense strand of SEQ ID NO: 62, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 69 and an antisense strand of SEQ ID NO: 70, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 77 and an antisense strand of SEQ ID NO: 78, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 95 and an antisense strand of SEQ ID NO: 96, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 101 and an antisense strand of SEQ ID NO: 102, or a double-stranded RNA consisting of a sense strand of SEQ ID NO: 115 and an antisense strand of SEQ ID NO: 116, the chemical modification is at least one substitution selected from the group consisting of a 2'-F-nucleotide, a 2'-OMe-nucleotide, a nucleotide in which the 2'-O atom and the 4'-C atom are bridged with methylene, a 2'-deoxy-nucleotide, and a phosphorothioate bond forming an internucleotide bond in the siRNA; the siRNA comprises a motif of 2 or 4 consecutive 2'-F-nucleotides or 2'-OMe-nucleotides at or adjacent to the cleavage site of the RNA strand by Ago2, and the 2'-F-nucleotides and 2'-OMe-nucleotides are alternately contained along the RNA strand other than the motif; Chemically modified siRNA or a salt thereof.

2. The chemically modified siRNA is represented by the following formula (I): Sense strand: 5'-(YX)a-(YY)b-(XY)c-(XX)d-(YX)e-(YY)f-(XY)g-(XYX)h-(YXY)i-3' Antisense strand: 3'-(YX)j-(XY)k-(XY)a-(XX)b-(YX)c-(YY)d-(XY)e-(XX)f-(YX)g-(Y)h-(X)i-5' (I) “During the ceremony, X and Y are 2'-F-nucleotides and 2'-OMe-nucleotides, respectively; a, b, c, d, e, f, g, h, i, j and k are independently integers from 0 to 4; (a, b, c, j, k) are, in this order, (0, 0, 4, 1, 0), (0, 1, 3, 0, 1), (0, 2, 2, 0, 1), (1, 1, 2, 0, 1), (2, 1, 1, 0, 1), (1, 2, 1, 0, 1), (2, 2, 0, 0, 1) or (3, 1, 0, 0, 1); When d is 1, (e, f, g, h, i) are (4, 0, 0, 0, 1), (3, 1, 0, 1, 0), (2, 2, 0, 1, 0), (2, 1, 1, 1, 0), (1, 1, 2, 1, 0), (1, 2, 1, 1, 0), (0, 2, 2, 1, 0) or (0, 1, 3, 1, 0); When d is 2, (e, f, g, h, i) are (3, 0, 0, 0, 1), (2, 1, 0, 1, 0), (1, 2, 0, 1, 0), (1, 1, 1, 1, 0), (0, 2, 1, 1, 0) or (0, 1, 2, 1, 0) in that order. 21 nucleotides in length, The chemically modified siRNA or a salt thereof according to claim 1 .

3. Double-stranded RNA consisting of the sense strand of SEQ ID NO: 5 and the antisense strand of SEQ ID NO: 6, double-stranded RNA consisting of the sense strand of SEQ ID NO: 13 and the antisense strand of SEQ ID NO: 14, double-stranded RNA consisting of the sense strand of SEQ ID NO: 15 and the antisense strand of SEQ ID NO: 16, double-stranded RNA consisting of the sense strand of SEQ ID NO: 17 and the antisense strand of SEQ ID NO: 18, double-stranded RNA consisting of the sense strand of SEQ ID NO: 19 and the antisense strand of SEQ ID NO: 20, double-stranded RNA consisting of the sense strand of SEQ ID NO: 21 and the antisense strand of SEQ ID NO: 22, and the sense strand of SEQ ID NO: 23 and a double-stranded RNA consisting of the antisense strand of SEQ ID NO: 24, a double-stranded RNA consisting of the sense strand of SEQ ID NO: 35 and the antisense strand of SEQ ID NO: 36, a double-stranded RNA consisting of the sense strand of SEQ ID NO: 37 and the antisense strand of SEQ ID NO: 38, a double-stranded RNA consisting of the sense strand of SEQ ID NO: 39 and the antisense strand of SEQ ID NO: 40, a double-stranded RNA consisting of the sense strand of SEQ ID NO: 41 and the antisense strand of SEQ ID NO: 42, a double-stranded RNA consisting of the sense strand of SEQ ID NO: 43 and the antisense strand of SEQ ID NO: 44, a double-stranded RNA consisting of the sense strand of SEQ ID NO: 45 and the antisense strand of SEQ ID NO: 46 double-stranded RNA consisting of a sense strand of SEQ ID NO: 47 and an antisense strand of SEQ ID NO: 48; double-stranded RNA consisting of a sense strand of SEQ ID NO: 49 and an antisense strand of SEQ ID NO: 50; double-stranded RNA consisting of a sense strand of SEQ ID NO: 51 and an antisense strand of SEQ ID NO: 52; double-stranded RNA consisting of a sense strand of SEQ ID NO: 53 and an antisense strand of SEQ ID NO: 54; double-stranded RNA consisting of a sense strand of SEQ ID NO: 55 and an antisense strand of SEQ ID NO: 56; double-stranded RNA consisting of a sense strand of SEQ ID NO: 57 and an antisense strand of SEQ ID NO: 58 a double-stranded RNA consisting of a sense strand of SEQ ID NO: 59 and an antisense strand of SEQ ID NO: 60; a double-stranded RNA consisting of a sense strand of SEQ ID NO: 63 and an antisense strand of SEQ ID NO: 64; a double-stranded RNA consisting of a sense strand of SEQ ID NO: 67 and an antisense strand of SEQ ID NO: 68; a double-stranded RNA consisting of a sense strand of SEQ ID NO: 71 and an antisense strand of SEQ ID NO: 72; a double-stranded RNA consisting of a sense strand of SEQ ID NO: 73 and an antisense strand of SEQ ID NO: 74; a double-stranded RNA consisting of a sense strand of SEQ ID NO: 75 and an antisense strand of SEQ ID NO: 76;double-stranded RNA consisting of the sense strand of SEQ ID NO:79 and the antisense strand of SEQ ID NO:80, double-stranded RNA consisting of the sense strand of SEQ ID NO:81 and the antisense strand of SEQ ID NO:82, double-stranded RNA consisting of the sense strand of SEQ ID NO:85 and the antisense strand of SEQ ID NO:86, double-stranded RNA consisting of the sense strand of SEQ ID NO:87 and the antisense strand of SEQ ID NO:88, double-stranded RNA consisting of the sense strand of SEQ ID NO:89 and the antisense strand of SEQ ID NO:90, double-stranded RNA consisting of the sense strand of SEQ ID NO:91 and the antisense strand of SEQ ID NO:92, double-stranded RNA consisting of the sense strand of SEQ ID NO:93 and the antisense strand of SEQ ID NO:94, double-stranded RNA consisting of the sense strand of SEQ ID NO:97 and the antisense strand of SEQ ID NO:98, double-stranded RNA consisting of the sense strand of SEQ ID NO:99 and the antisense strand of SEQ ID NO:100, double-stranded RNA consisting of the sense strand of SEQ ID NO:103 and the antisense strand of SEQ ID NO:104, double-stranded RNA consisting of the sense strand of SEQ ID NO:105 and the antisense strand of SEQ ID NO:106, double-stranded RNA consisting of SEQ ID NO:10 3. The chemically modified siRNA or salt thereof according to claim 1 or 2, comprising a sequence selected from the group consisting of a double-stranded RNA consisting of a sense strand of SEQ ID NO: 7 and an antisense strand of SEQ ID NO: 108, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 109 and an antisense strand of SEQ ID NO: 110, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 111 and an antisense strand of SEQ ID NO: 112, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 113 and an antisense strand of SEQ ID NO: 114, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 117 and an antisense strand of SEQ ID NO: 118, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 119 and an antisense strand of SEQ ID NO: 120, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 123 and an antisense strand of SEQ ID NO: 124, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 125 and an antisense strand of SEQ ID NO: 126, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 127 and an antisense strand of SEQ ID NO: 128, and a double-stranded RNA consisting of a sense strand of SEQ ID NO: 129 and an antisense strand of SEQ ID NO:

130.

4. Double-stranded RNA consisting of the sense strand of SEQ ID NO: 13 and the antisense strand of SEQ ID NO: 14, double-stranded RNA consisting of the sense strand of SEQ ID NO: 21 and the antisense strand of SEQ ID NO: 22, double-stranded RNA consisting of the sense strand of SEQ ID NO: 39 and the antisense strand of SEQ ID NO: 40, double-stranded RNA consisting of the sense strand of SEQ ID NO: 43 and the antisense strand of SEQ ID NO: 44, double-stranded RNA consisting of the sense strand of SEQ ID NO: 49 and the antisense strand of SEQ ID NO: 50, double-stranded RNA consisting of the sense strand of SEQ ID NO: 53 and the antisense strand of SEQ ID NO: 54, double-stranded RNA consisting of the sense strand of SEQ ID NO: 55 and the antisense strand of SEQ ID NO: 56, double-stranded RNA consisting of the sense strand of SEQ ID NO: 57 and the antisense strand of SEQ ID NO: 58, double-stranded RNA consisting of the sense strand of SEQ ID NO: 59 and the antisense strand of SEQ ID NO: 60, double-stranded RNA consisting of the sense strand of SEQ ID NO: 87 and the antisense strand of SEQ ID NO: 88, double-stranded RNA consisting of the sense strand of SEQ ID NO: 8 3. The chemically modified siRNA or salt thereof according to claim 1 or 2, comprising a sequence selected from the group consisting of a double-stranded RNA consisting of a sense strand of SEQ ID NO: 9 and an antisense strand of SEQ ID NO: 90, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 91 and an antisense strand of SEQ ID NO: 92, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 93 and an antisense strand of SEQ ID NO: 94, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 111 and an antisense strand of SEQ ID NO: 112, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 113 and an antisense strand of SEQ ID NO: 114, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 123 and an antisense strand of SEQ ID NO: 124, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 125 and an antisense strand of SEQ ID NO: 126, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 127 and an antisense strand of SEQ ID NO: 128, and a double-stranded RNA consisting of a sense strand of SEQ ID NO: 129 and an antisense strand of SEQ ID NO:

130.

5. A chemically modified siRNA or a salt thereof that selectively suppresses expression of P525L point mutant FUS compared to expression of wild-type FUS, The siRNAs include double-stranded RNA consisting of a sense strand of SEQ ID NO: 3 and an antisense strand of SEQ ID NO: 4, double-stranded RNA consisting of a sense strand of SEQ ID NO: 7 and an antisense strand of SEQ ID NO: 8, double-stranded RNA consisting of a sense strand of SEQ ID NO: 9 and an antisense strand of SEQ ID NO: 10, double-stranded RNA consisting of a sense strand of SEQ ID NO: 11 and an antisense strand of SEQ ID NO: 12, double-stranded RNA consisting of a sense strand of SEQ ID NO: 25 and an antisense strand of SEQ ID NO: 26, double-stranded RNA consisting of a sense strand of SEQ ID NO: 27 and an antisense strand of SEQ ID NO: 28, double-stranded RNA consisting of a sense strand of SEQ ID NO: 29, and a double-stranded RNA consisting of an antisense strand of SEQ ID NO: 30, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 31 and an antisense strand of SEQ ID NO: 32, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 33 and an antisense strand of SEQ ID NO: 34, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 65 and an antisense strand of SEQ ID NO: 66, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 83 and an antisense strand of SEQ ID NO: 84, or a double-stranded RNA consisting of a sense strand of SEQ ID NO: 121 and an antisense strand of SEQ ID NO: 122, or a salt thereof.

6. The chemically modified siRNA or a salt thereof according to any one of claims 1 to 5, wherein the internucleotide bonds between adjacent 3 nucleotides located at the 5' and 3' ends of the sense strand and the antisense strand are phosphorothioate bonds.

7. An agent for suppressing the expression of P525L point-mutated FUS, comprising the chemically modified siRNA or a salt thereof according to any one of claims 1 to 6.

8. A pharmaceutical composition comprising the chemically modified siRNA or a salt thereof according to any one of claims 1 to 6.

9. The pharmaceutical composition according to claim 8, for selectively inhibiting the expression of P525L point mutant FUS compared to the expression of wild-type FUS.

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