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Definitions

• the present invention is a chemically modified siRNA with improved stability against RNA degrading enzymes, which is used to express the P525L point mutation FUS (Fused in Sarcoma), which is a causative gene for amyotrophic lateral sclerosis (ALS).
• the present invention relates to a chemically modified siRNA that selectively inhibits siRNA, and a pharmaceutical composition containing the chemically modified siRNA.
• ALS Amyotrophic lateral sclerosis
• MNs motor neurons
• FTD frontotemporal dementia
• ALS can be broadly classified into two types: sporadic ALS and familial ALS. Most of these are sporadic ALS, which is non-genetic. Familial ALS has a relatively large number of patients, accounting for 5-10% of all ALS. It is a disease with few cases.
• ALS a complex genetic disease due to mutations in multiple genes coupled with environmental exposures.
• Factors related to the onset of ALS include SOD1 (Cu 2+ /Zn 2+ superoxide dismutase), TDP-43 (TAR DNA binding protein-43kD), FUS (Fused in sarcoma), ANG (angiogenin), and ATXN2 (ataxin- More than a dozen causative genes have been identified, including 2), VCP (valosin-containing protein), OPTN (optineurin), and C9orf72 (chromosome 9 open reading frame 72).
• SOD1 Cu 2+ /Zn 2+ superoxide dismutase
• TDP-43 TAR DNA binding protein-43kD
• FUS Feused in sarcoma
• ANG angiogenin
• ATXN2 angiogenin
• ATXN2 angiogenin
• ATXN2 angiogenin
• ATXN2 ataxin- More
• FUS is known to be the second most frequent causative gene for familial ALS after SOD1.
• FUS the causative gene of ALS6 linked to chromosome 16
• RNA-binding protein identified in 2009
• Non-patent Document 1 the causative gene that is more common in young people among familial ALS.
• FUS travels between the nucleus and the cytoplasm and plays important RNA metabolic functions such as DNA repair and splicing regulation. It is known that mutations in this FUS cause abnormal aggregation in the cytoplasm, and the following two hypotheses have been proposed as a factor in the onset of familial ALS. The first is a loss-of-function theory in which RNA metabolism, which should take place in the nucleus, cannot be carried out normally, and the second is the theory that toxicity is acquired due to the mutant protein aggregating in the cytoplasm.
• mutation positions of FUS we found that P495X, G507D, K510R/E, S513P, R514G/S, R514S, G515C, H517Q/P, R518G/K, R521G/C/H, R522G, R524W/T/S It has been found that there are many mutations in nuclear export signal sites such as , P525L.
• the P525L point mutation is common in young-onset ALS that develops between the ages of 10 and 20, and it is known that most of these patients die within 2 years after onset. , so far no therapeutic drug has been developed.
• wild-type FUS has an important RNA metabolic function, and in fact, knockout of FUS in mouse forebrain cortical neurons reduced the interaction with RNA splicing factor, proline and glutamine rich (SFPQ). It has been reported that it causes changes in tau isoforms. Therefore, high selectivity for mutant FUS is essential for the development of therapeutic agents for ALS caused by FUS mutations.
• siRNA targeting genes with point mutations include, for example, siRNA targeting G356D point mutation EGFR (epidermal growth factor receptor) (Patent Document 1), and V337M point mutation APP (amyloid precursor protein).
• siRNA targeting P525L point mutation FUS Non-patent document 2
• siRNA targeting G85R point mutation SOD1 Non-patent document 3
• chemically modified siRNAs that have improved stability against RNAases in plasma that non-specifically cleave siRNAs. Methods have been proposed to diagnose ALS or a genetic predisposition to ALS using specific genetic markers and to treat or prevent ALS using siRNA molecules that reduce the expression of mutant FUS. There is no description (Patent Document 2).
• the purpose of the present invention is to provide chemically modified siRNA that has improved stability against plasma RNA degrading enzymes that nonspecifically cleave RNA strands, and that selectively inhibits the expression of the P525L point mutation FUS, which is the causative gene of ALS.
• the purpose of the present invention is to provide chemically modified siRNA that suppresses
• a further object of the present invention is to provide a pharmaceutical composition comprising the chemically modified siRNA.
• the present inventors constructed the siRNA to solve the problem that siRNA that selectively suppresses the expression of the P525L point mutation FUS, which is the causative gene of ALS, has low stability against RNA degrading enzymes in plasma. Nucleotides were replaced 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, and were determined by Ago2, which is one of the constituent proteins of the RNA-induced silencing complex (RISC) present in cells.
• RISC RNA-induced silencing complex
• RNAi activity equivalent to that of natural siRNA and the P525L point mutation FUS can be achieved.
• the present inventors have discovered siRNA that dramatically improves stability against RNA degrading enzymes while maintaining high selectivity for the encoded mRNA, and have completed the present invention.
• a chemically modified siRNA or a salt thereof consisting of a sense strand and an antisense strand, the antisense strand comprising a region complementary or substantially complementary to a part of mRNA encoding the P525L point mutant FUS protein,
• the complementary region is 19 to 21 nucleotides long, and the siRNA contains a 2'-F-nucleotide, a 2'-OMe-nucleotide, a nucleotide in which a 2'-O atom and a 4'-C atom are bridged with methylene.
• the cleavage site of the RNA strand by Ago2 or adjacent to the cleavage site contains a motif with 2 or 4 consecutive residues of 2'-F-nucleotides or 2'-OMe-nucleotides, and in the RNA strand other than the motif, 2
• the chemically modified siRNA or a salt thereof according to [1] which contains '-F-nucleotides and 2'-OMe-nucleotides alternately along the RNA strand.
• 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-nucleotide and 2'-OMe-nucleotide, 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)
• Double-stranded RNA consisting of the sense strand of SEQ ID NO: 5 and 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, the sense strand and sequence of SEQ ID NO: 15
• a double-stranded RNA consisting of the antisense strand of SEQ ID NO: 16 a double-stranded RNA consisting of the sense strand of SEQ ID NO: 17 and an antisense strand of SEQ ID NO: 18, a sense strand of SEQ ID NO: 19 and an antisense strand of SEQ ID NO: 20.
• double-stranded RNA double-stranded RNA consisting of the sense strand of SEQ ID NO: 21 and antisense strand of SEQ ID NO: 22
• double-stranded RNA consisting of the sense strand of SEQ ID NO: 23 and the antisense strand of SEQ ID NO: 24
• SEQ ID NO: 35 A double-stranded RNA consisting of a sense strand of SEQ ID NO: 36 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 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: 36 and an antisense strand of SEQ ID NO: 36
• a double-stranded RNA consisting of a sense strand of SEQ ID NO: 37
• a double-stranded RNA consisting of an antisense strand 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.
• RNA double-stranded RNA consisting of the sense strand of SEQ ID NO: 45 and antisense strand of SEQ ID NO: 46
• double-stranded RNA consisting of the sense strand of SEQ ID NO: 47 and the antisense strand of SEQ ID NO: 48
• sense strand of SEQ ID NO: 49 and double-stranded RNA consisting of the antisense strand of SEQ ID NO: 50
• double-stranded RNA consisting of the sense strand of SEQ ID NO: 51 and the antisense strand of SEQ ID NO: 52
• 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: 45 and antisense strand of SEQ ID NO: 46
• double-stranded RNA consisting of the sense strand of SEQ ID NO: 47 and the antisense strand of SEQ ID NO: 48
• double-stranded RNA consisting of the sense strand of SEQ ID NO: 75 and 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: 79 and the antisense strand of SEQ ID NO: 80; A double-stranded RNA consisting of a sense strand and an antisense strand of SEQ ID NO: 82, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 85 and an antisense strand of SEQ ID NO: 86, a sense strand of SEQ ID NO: 87 and an antisense strand of SEQ ID NO: 88.
• 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: 97 and an antisense strand of SEQ ID NO: 98
• a sense strand of SEQ ID NO: 99 and Double-stranded RNA consisting of 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
• the sense strand of SEQ ID NO: 105 and the antisense strand of SEQ ID NO: 106 the antisense strand of SEQ ID NO: 106.
• RNA 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: 127 and an antisense strand of SEQ ID NO: 128;
• Double-stranded RNA consisting of the sense strand of SEQ ID NO: 13 and 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, the sense strand and sequence of SEQ ID NO: 39
• Double-stranded RNA consisting of 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 sense strand of SEQ ID NO: 49 and an antisense strand of SEQ ID NO: 50 Double-stranded RNA consisting of the sense strand of SEQ ID NO: 13 and 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 the sense strand and sequence of SEQ ID
• double-stranded RNA double-stranded RNA, double-stranded RNA consisting of the sense strand of SEQ ID NO: 53 and 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, SEQ ID NO: 57
• a double-stranded RNA consisting of a sense strand of SEQ ID NO: 59 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 sense strand of SEQ ID NO: 87 and an antisense strand of SEQ ID NO: 88.
• a double-stranded RNA consisting of an antisense strand a double-stranded RNA consisting of a sense strand of SEQ ID NO: 89 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.
• RNA double-stranded RNA consisting of the sense strand of SEQ ID NO: 93 and 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, the sense strand of SEQ ID NO: 113 and double-stranded RNA consisting of 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, the sense strand of SEQ ID NO: 125 and the antisense strand of SEQ ID NO: 126.
• double-stranded RNA double-stranded RNA, double-stranded RNA consisting of the sense strand of SEQ ID NO: 27 and 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, SEQ ID NO: 31
• a double-stranded RNA consisting of a sense strand of SEQ ID NO: 32 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 sense strand of SEQ ID NO: 65 and an antisense strand of SEQ ID NO: 66.
• a double-stranded RNA consisting of an antisense strand 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.
• the present invention provides chemically modified siRNA or a salt thereof that has dramatically improved stability against RNA degrading enzymes while maintaining high RNAi activity equivalent to that of natural siRNA and high selectivity for mRNA encoding the P525L point mutation FUS. can be provided.
• the expression of the P525L point mutant FUS can be selectively suppressed without substantially suppressing the expression of wild type FUS.
• the pharmaceutical composition containing the chemically modified siRNA or a salt thereof of the present disclosure enables effective treatment of ALS or ALS having a P525L point mutation FUS.
• FIG. 3 is a diagram showing the effect of chemically modified siRNA of the present disclosure on the mRNA expression rate of wild-type FUS and P525L point mutation FUS.
• FIG. 3 is a diagram showing the effect of chemically modified siRNA of the present disclosure on the mRNA expression rate of wild-type FUS and P525L point mutation FUS.
• FIG. 3 is a diagram showing the effect of chemically modified siRNA of the present disclosure on the mRNA expression rate of wild-type FUS and P525L point mutation FUS.
• FIG. 3 is a diagram showing the effect of chemically modified siRNA of the present disclosure on the mRNA expression rate of wild-type FUS and P525L point mutation FUS.
• FIG. 3 is a diagram showing the effect of chemically modified siRNA of the present disclosure on the mRNA expression rate of wild-type FUS and P525L point mutation FUS.
• FIG. 3 is a diagram showing the effect of chemically modified siRNA of the present disclosure on the mRNA expression rate of wild-type FUS and P525L point mutation FUS.
• FIG. 2 is a diagram showing the stability of natural siRNA in human serum.
• FIG. 3 shows the stability of chemically modified siRNA of the present disclosure in human serum.
• the chemically modified siRNA (small interfering RNA) of the present disclosure includes an RNA complementary to the mRNA (antisense strand) transcribed from the P525L point mutation FUS, which is a causative gene of ALS, and an RNA complementary to the antisense strand (sense strand). It is a double-stranded RNA consisting of strands).
• the chemically modified siRNA can degrade the mRNA of P525L point mutant FUS by RNA interference (RNAi) and selectively suppress the expression of P525L point mutant FUS involved in ALS.
• RNAi RNA interference
• the chemically modified siRNA of the present disclosure includes a region that is complementary or substantially complementary to a portion of the mRNA encoding the P525L point mutation FUS, and the complementary region is 19 to 21 nucleotides long. Additionally, in some embodiments, the chemically modified siRNAs of the present disclosure have each of the sense and antisense strands between 19 and 26 nucleotides in length. In some embodiments, chemically modified siRNAs of the present disclosure are 19-23 nucleotides in length.
• complementary means that the sense strand and antisense strand of siRNA, or the antisense strand of siRNA and target mRNA, are bonded by hydrogen bonds formed when the base portions of opposing nucleotides are complementary. That's true.
• substantially complementary means that one or more of the opposing nucleotides are not complementary nucleotides, but the oligonucleotide as a whole binds by forming base pairs.
• the chemically modified siRNA of the present disclosure includes a 2'-F-nucleotide, a 2'-OMe-nucleotide, a nucleotide in which a 2'-O atom and a 4'-C atom are bridged with methylene (LNA), a 2'-deoxy- It contains at least one substitution selected from the group consisting of nucleotides and phosphorothioate bonds forming internucleotide bonds.
• the chemically modified siRNA of the present disclosure comprises 2 or 4 2'-F-nucleotide or 2'-OMe-nucleotide residues at or adjacent to the cleavage site of the RNA strand by Ago2. It contains a continuous motif, and in RNA strands other than said motif, it contains alternating 2'-F-nucleotides and 2'-OMe-nucleotides along said RNA strand.
• Ago2 (Argonaute2) is one of the constituent proteins of the RNA-induced silencing complex (RISC). siRNA is taken into RISC, and after the sense strand is removed, the antisense strand recognizes the target mRNA, and Ago2 recognizes the target mRNA. mRNA is cleaved.
• 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)
• 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 from 0 to 4; (a, b, c, j, k) are (0, 0, 4, 1, 0), (0, 1, 3, 0, 1) in this order ), (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) is (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,
• a 21 nucleotide long chemically modified siRNA or a salt thereof represents.
• a, b, c, d, e, f, g, h, i, j, and k indicate the number of repeats in the array.
• Double-stranded siRNA is cleaved by Ago2.
• the cleavage site by Ago2 is between positions 9 and 10 or between positions 10 and 11 from the 5' end of the sense strand. It is a combination of
• the chemically modified siRNA of the present disclosure is a double-stranded RNA consisting of the sense strand of SEQ ID NO: 5 and the antisense strand of SEQ ID NO: 6 as set forth in Table 1 below, the sense strand of SEQ ID NO: 13, and Double-stranded RNA consisting of 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, the sense strand of SEQ ID NO: 17 and the antisense strand of SEQ ID NO: 18.
• RNA 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
• the sense strand of SEQ ID NO: 47 double-stranded RNA consisting of a sense strand of SEQ ID NO: 49 and an antisense strand of SEQ ID NO: 50
• 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: 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
• the sense strand of SEQ ID NO: 47 double-stranded RNA consisting of a sense strand of S
• double-stranded RNA double-stranded RNA, double-stranded RNA consisting of the sense strand of SEQ ID NO: 73 and 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, SEQ ID NO: 79
• a double-stranded RNA consisting of a sense strand of SEQ ID NO: 80 and an antisense strand of SEQ ID NO: 80 a double-stranded RNA consisting of a sense strand of SEQ ID NO: 81 and an antisense strand of SEQ ID NO: 82, a sense strand of SEQ ID NO: 85 and an antisense strand of SEQ ID NO: 86.
• a double-stranded RNA consisting of an antisense strand a double-stranded RNA consisting of a sense strand of SEQ ID NO: 87 and an antisense strand of SEQ ID NO: 88, a double-stranded RNA consisting of a sense strand of SEQ ID NO: 89 and an antisense strand of SEQ ID NO: 90.
• RNA double-stranded RNA consisting of the sense strand of SEQ ID NO: 91 and 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, sense strand of SEQ ID NO: 97 and double-stranded RNA consisting of 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, 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: 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 a sense strand and an antisense strand of SEQ ID NO: 128, and a double-stranded RNA consisting of an 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. selected from the group.
• the chemically modified siRNA of the present disclosure comprises a double-stranded RNA consisting of a sense strand of SEQ ID NO: 13 and an antisense strand of SEQ ID NO: 14 as set forth in Table 1 below, a sense strand of SEQ ID NO: 21, and an antisense strand of SEQ ID NO: 14.
• Double-stranded RNA consisting of 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, the sense strand of SEQ ID NO: 43 and the antisense strand of SEQ ID NO: 44.
• RNA 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, the sense strand of SEQ ID NO: 111 double-stranded RNA consisting of a sense strand of SEQ ID NO: 113 and an antisense strand of SEQ ID NO: 114, 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
• 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 the sense strand of SEQ ID NO: 129 and the antisense strand of SEQ ID NO: 130.
• the chemically modified siRNA of the present disclosure comprises a double-stranded RNA consisting of a sense strand of SEQ ID NO: 3 and an antisense strand of SEQ ID NO: 4 as set forth in Table 1 below, a sense strand of SEQ ID NO: 7, and an antisense strand of SEQ ID NO: 4.
• Double-stranded RNA consisting of 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
• a double-stranded RNA consisting of a sense strand of SEQ ID NO: 31 and an antisense strand of SEQ ID NO: 32, a sense strand of SEQ ID NO: 33 and an antisense strand of SEQ ID NO: 34
• siRNA-010, -002, -003, -006, -008, -009, and -011 listed in Table 1 are all natural siRNAs, and their stability against RNA degrading enzymes and RNAi activity were evaluated according to the present disclosure. was prepared and used in the test for comparison with chemically modified siRNA.
• LNA locked nucleic acid
• the sense and antisense strands that make up the chemically modified siRNA of the present disclosure have the sequences listed in Table 1, but have sequences that are substantially identical to the sequences listed in Table 1. It may be a sense strand or an antisense strand with "Substantially the same sequence” refers to chemical modifications and mismatched bases in the sequences listed in Table 1, as long as the antisense strand of the siRNA and the target mRNA retain the ability to form double-stranded RNA. This means that it may contain. In some embodiments, the mismatched bases are 3 or less. In some embodiments, the mismatched base may include up to one.
• the sense strand and antisense strand that constitute the chemically modified siRNA of the present disclosure may include a dinucleotide overhang at the 3' end.
• the chemically modified siRNAs of the present disclosure include UU (U: uridine) as an overhang.
• siRNA has a phosphodiester bond
• the chemically modified siRNA of the present disclosure has two phosphodiester bonds between adjacent nucleotides in the three nucleotides located at the 5' and 3' ends of both the sense strand and the antisense strand. Both bonds are replaced with phosphorothioate bonds.
• the chemically modified siRNA of the present disclosure can be produced by a nucleic acid molecule synthesis method well known to those skilled in the art.
• the synthesis method is described, for example, in “Creation and Application of Nucleic Acid Medicines” (CMC Publishing, 2016) and “Synthesis Technology of Peptides, Nucleic Acids, and Sugar Chains Contributing to Middle Molecule Drug Discovery” (CMC Publishing, 2018). Examples include the method described in .
• the chemically modified siRNA of the present disclosure can be made double-stranded by associating a synthesized single-stranded oligonucleotide with another complementary single-stranded oligonucleotide.
• the association method include a method in which complementary oligonucleotides are annealed by heating to a temperature at which double-stranded oligonucleotides dissociate, and then gradually cooling.
• the oligonucleotide can be synthesized by a solid phase synthesis method using commercially available amidites. Solid phase synthesis is performed using a commercially available nucleic acid synthesizer and solid phase carrier. The 3' end of the nucleotide of the monomer is bonded to the surface of the solid support via an alkyl chain, and an amidite is added thereto. That is, the oligonucleotide of interest can be synthesized by repeating a cycle of elongating the oligonucleotide sequence of interest one nucleotide at a time from the 3' end to the 5' end.
• the oligonucleotide is excised from the solid support and the base portion and 2' position are deprotected to prepare the desired single-stranded RNA.
• the above deprotection step is not necessary. .
• the chemically modified siRNA of the present disclosure can be synthesized at Gene Design using the phosphoramidite method described above.
• the quality of the obtained siRNA can be confirmed by mass spectrometry and electrophoresis after simple column purification.
• the chemically modified siRNA of the present disclosure can be prepared by selecting a continuous base sequence that targets P525L point mutation FUS mRNA. Specifically, the mRNA sequence is selected from 19 to 21 nucleotides of the region containing the P525L point mutation.
• the sequence of the obtained siRNA can be modified by substitutions, deletions, insertions and/or additions of one or several nucleotides in said sequence, if it is capable of inducing RNA interference and degrading the targeted P525L point mutant FUS mRNA. nucleotide sequences can be selected and prepared.
• the chemically modified siRNA or salt thereof consists of a sense strand and an antisense strand, wherein the antisense strand is complementary to or substantially complementary to a portion of mRNA encoding the P525L point mutant FUS protein.
• the siRNA comprises a complementary region, the complementary region is 19-21 nucleotides long, and the siRNA contains 2'-F-nucleotides, 2'-OMe-nucleotides, 2'-O atoms and 4'-C atoms.
• a chemically modified siRNA or a salt thereof containing at least one substitution selected from the group consisting of methylene-bridged nucleotides, 2'-deoxy-nucleotides, and phosphorothioate bonds forming internucleotide bonds can be used as a nucleic acid molecule well known to those skilled in the art. It can be produced by the following synthesis method.
• the site of cleavage of the RNA strand by Ago2 or adjacent to the cleavage site comprises a motif of 2 or 4 consecutive residues of 2'-F-nucleotides or 2'-OMe-nucleotides, and the motif A chemically modified siRNA or a salt thereof containing alternating 2'-F-nucleotides and 2'-OMe-nucleotides along the RNA strand other than the RNA strand is produced by a nucleic acid molecule synthesis method well known to those skilled in the art. be able to.
• the siRNA is represented by 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-nucleotide and 2'-OMe-nucleotide, 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)
• the chemically modified siRNA of the present disclosure can be appropriately produced by those skilled in the art based on the base sequence disclosed in this specification. Specifically, double-stranded RNA can be produced based on any of the base sequences of SEQ ID NOs: 1 to 130. Once one nucleotide strand is known, those skilled in the art can easily understand the base sequence of the other complementary nucleotide strand.
• the chemically modified siRNA of the present disclosure may be produced using a commercially available nucleic acid synthesizer or the like, or may be obtained using a general synthesis contract service.
• 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.
• 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 the 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 in ALS, undesirable symptoms due to suppressing the expression of wild-type FUS do not substantially appear.
• the expression suppression effect of the P525L point mutant FUS by the chemically modified siRNA of the present disclosure can be expressed as an expression suppression rate (%) using the calculation formula described below.
• the expression inhibition rate of the P525L point mutation FUS of the chemically modified siRNA of the present disclosure is 30% or more.
• the expression inhibition rate of the P525L point mutation FUS of the chemically modified siRNA of the present disclosure is 50% or more.
• the expression rate and expression suppression rate shown by the siRNA of the present disclosure with respect to wild-type FUS and P525L point mutation FUS are obtained by the calculation formulas described below.
• the chemically modified siRNA of the present disclosure has a selectivity at the expression rate of 1.5 or more. In some embodiments, the chemically modified siRNA of the present disclosure has a selectivity of 2 or more in the expression rate. In some embodiments, the chemically modified siRNA of the present disclosure has a selectivity of 20% or more in the expression suppression rate. In some embodiments, the chemically modified siRNA of the present disclosure has a selectivity of 40% or more in the expression suppression rate.
• the selectivity of suppressing FUS expression by the chemically modified siRNA may be evaluated based on either the selectivity in expression rate or the selectivity in expression suppression rate, or may be evaluated in combination of both.
• the chemically modified siRNA of the present disclosure may be in the form of a salt.
• the salt is a pharmaceutically acceptable salt.
• the salts include, but are not limited to, alkali metal salts such as sodium salts, potassium salts, lithium salts, and alkaline earth metal salts such as calcium salts and magnesium salts.
• 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, for example, using the method described in the following literature or a method analogous 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.
• a pharmaceutical composition for preventing or treating ALS comprising the chemically modified siRNA of the present disclosure or a salt thereof and a pharmaceutically acceptable carrier.
• the ALS is an ALS with a P525L point mutation FUS.
• the pharmaceutical composition of the present disclosure may be in a dosage form that is used orally or parenterally. These dosage forms can be formulated by those skilled in the art by incorporating appropriate combinations of pharmaceutically acceptable carriers and excipients into unit dosage forms as required by generally accepted pharmaceutical practice. can. 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 Pharmacopeia (USP).
• USP United States Pharmacopeia
• the method comprises administering to a patient in need of treatment an effective amount of an expression inhibitor of P525L point mutation FUS containing a chemically modified siRNA of the present disclosure or a salt thereof.
• an effective amount of an expression inhibitor of P525L point mutation FUS containing a chemically modified siRNA of the present disclosure or a salt thereof is provided.
• a P525L point mutation FUS expression inhibitor containing the chemically modified siRNA of the present disclosure or a salt thereof for the prevention or treatment of ALS or ALS having a P525L point mutation FUS is provided.
• an agent for suppressing the expression of P525L point mutation FUS containing the chemically modified siRNA of the present disclosure or a salt thereof for producing a prophylactic or therapeutic agent for ALS or ALS having P525L point mutation FUS is provided. .
• an effective amount of a pharmaceutical composition for preventing or treating ALS comprising a chemically modified siRNA of the present disclosure or a salt thereof and a pharmaceutically acceptable carrier is administered to a patient in need of treatment.
• a method for preventing or treating ALS or ALS having a P525L point mutation FUS comprising:
• 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 having a P525L point mutation FUS.
• a pharmaceutical composition comprising a chemically modified siRNA of the present disclosure or a salt thereof and a pharmaceutically acceptable carrier for producing a prophylactic or therapeutic agent for ALS or ALS having a P525L point mutation FUS. provide.
• FUS wild type The human wild type FUS (hereinafter referred to as FUS wild type ) cDNA sequence (sequence 1) is SEQ ID NO: 131, and the human P525L point mutation FUS (hereinafter referred to as FUS P525L ) cDNA sequence (sequence 2) It 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 within the multiple cloning site of the pcDNA3.1+ vector.
• PCR For PCR, 25 ⁇ L of PrimeSTAR Max Premix (Takara Bio Inc.), 4 ⁇ L each of 2.5 ⁇ M Primer (final concentration 0.2 ⁇ M), 1 ⁇ L (20 ng) of template, and 20 ⁇ L of water were mixed, and after incubation at 98°C for 10 seconds, A temperature cycle of 98°C for 10 seconds, 55°C for 5 seconds, and 72°C for 10 seconds (25 seconds when the vector was used as a template) was performed for 35 cycles. The vector amplified fragment and the FUS gene amplified fragment were ligated by In-Fusion reaction.
• FUS wild type was cloned in frame into pTurboGFP vector (Evrogen) and pTurboFP635 vector (Evrogen), respectively, with Turbo GFP fluorescent protein added to its N-terminus and FUS P525L with TurboFP635 fluorescent protein added to its N-terminus. did.
• HEK293 cells were cultured at 37° C. in a 5% CO 2 environment using Advanced DMEM (Thermo Fisher Scientific Co., Ltd.) containing 10% FBS and 4 mM GlutaMAX (registered trademark) Supplement.
• HEK293 cells were obtained from the JCRB Cell Bank, Culture Resource Laboratory, National Institute of Biomedical Innovation, Health and Nutrition (cell number JCRB9068).
• FUS knockout (KO) HEK293 cell line FUS/TLS CRISPR/Cas9 KO (sc-400612) plasmid (Santa Cruz) and FUS/TLS HDR plasmid (h) (sc-400612-HDR) (Santa Cruz) were transfected with TransIT®-293 Transfection Reagent (Mirus ) was used to generate FUS KO HEK cell lines.
• FUS gene knockout was confirmed by RT-PCR (using SuperScript (registered trademark) IV One-Step RT-PCR System with ezDNase (registered trademark), invitrogen, #12595100) to confirm the presence or absence of FUS mRNA expression. Table 4 shows the primer sets used.
• Total RNA was prepared using the RNeasy Plus Mini Kit (QIAGEN), and gDNA was digested by mixing 1 ⁇ L of 10x ezDNase Buffer, 1 ⁇ L of ezDNase Enzyme, 1 ⁇ L of Template RNA (500 ng/ ⁇ L), and 7 ⁇ L of water, and incubating at 37°C for 5 minutes. I went for a minute.
• Template RNA (Digested gDNA) 10 ⁇ L, 2x Platinum SuperFi RT-PCR Master Mix 25 ⁇ L, Primer Set I Mixture (each 10 ⁇ M) 2.5 ⁇ L, Primer Set V Mixture (each 10 ⁇ M) 2.5 ⁇ L, SuperScript IV RT Mix 0.5 ⁇ L of Mix and 9.5 ⁇ L of water, and repeat 40 cycles of 10 minutes at 60°C, 2 minutes at 98°C, 10 seconds at 98°C, 10 seconds at 62°C, and 1 minute at 72°C. , and reacted at 72°C for 5 minutes.
• pAAVS1-puro-DNR (Origene)_TurboGFP-FUS wild type pAAVS1-puro-DNR (Origene)_TurboFP635-FUS P525L and pCas-Guide-AAVS1 (Origene) were introduced into pre-prepared FUS KO HEK293 cells to generate TurboGFP fusion.
• Cells co-expressing FUS wild type and TurboFP635-fused FUS P525L were generated.
• Cell line cloning was performed by sorting TurboGFP and TurboFP635 double-positive cells using On-chip Sort (On-chip Biotechnologies Co., Ltd.) and then using On-chip SPiS (On-chip Biotechnologies Co., Ltd.). ), single cells were sorted into 384 plates and cultured.
• RNA interference evaluation of RNA interference (imaging) using TurboGFP-fused FUS wild type and TurboFP635-fused FUS P525L co-expressing HEK293 cell line
• the HEK293 cell line co-expressing TurboGFP-fused FUS wild type and TurboFP635-fused FUS P525L has a density of 3.0 ⁇ 10 5 cells/mL in a medium (FluoroBrite® DMEM containing 5% FBS, hereinafter the same) heated to 37°C. It was suspended like this. 100 ⁇ L of the cell suspension and 10 ⁇ L of the previously prepared Lipofectamine-siRNA complex were mixed, seeded on a CellCarrier Ultra, collagen-coated 96-well plate (PerkinElmer), and cultured at 37°C and 5% CO 2 ( The following culturing was performed under the same conditions).
• TurboGFP-positive cells and TurboFP635-positive cells were defined as follows.
• TurboGFP-positive cells FUS wild-type expressing cells: Cells in which the total fluorescence intensity of TurboGFP in the nuclear region divided by the area (pixels) of the nuclear region is 400 or more.
• TurboFP635-positive cells FUS P525L- expressing cells: Cells in which the total fluorescence intensity of TurboFP635 in the cytoplasmic region divided by the area (pixel) of the cytoplasmic region is 400 or more.
• Each positive cell rate was substituted into the following formula to calculate the relative values of the expression rate and expression suppression rate.
• “Expression rate (%)” 100 ⁇ ⁇ (TurboGFP-positive cell rate after various siRNA treatments) - (TurboGFP-positive cell rate after positive control siRNA treatment) ⁇ / ⁇ (TurboGFP-positive cell rate after negative control siRNA treatment) - (TurboGFP positive cell rate after positive control siRNA treatment) ⁇
• “Expression suppression rate (%)” 100 ⁇ ⁇ (TurboGFP-positive cell rate after various siRNA treatments) - (TurboGFP-positive cell rate after negative control siRNA treatment) ⁇ / ⁇ (TurboGFP-positive cell rate after positive control siRNA treatment) ) - (TurboGFP positive cell rate after negative control siRNA treatment) ⁇
• the above calculation formula is for FUS wild type , but it can be similarly calculated from the TurboFP635 positive cell rate for FUS P525L .
• the negative control siRNA is an siRNA with a sequence that is not similar to known human, mouse, and rat gene sequences, and is provided by Horizon Discovery. Its sequence is UAGCGACUAAACACAUCAA (SEQ ID NO: 145).
• the positive control siRNA is a mixture of four types of siRNA (SEQ ID NOs: 146 to 149) designed to target any region of human FUS mRNA, and is provided by Horizon Discovery. The four types of arrangement are as follows.
• 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, as well as their expression suppression effects, as well as their respective selectivities. Since Tables 5 to 9 show the results of experiments performed independently of each other, the results for the same siRNA may be shown in each table. Examples of the same siRNA include siRNA-010 and siRNA-010-4.
• RNA interference using TurboGFP-fused FUS wild type and TurboFP635-fused FUS P525L co-expressing HEK293 cell line [Evaluation of RNA interference using TurboGFP-fused FUS wild type and TurboFP635-fused FUS P525L co-expressing HEK293 cell line (real-time PCR)] Introduction of siRNA into cells was performed according to the method shown in Example 6. 48 hours after transfection, the medium was completely removed, and 50 ⁇ L of a cell lysate containing 0.5 ⁇ L of DNase I (Life Technologies Japan) and 49.5 ⁇ L of Lysis Solution (Life Technologies Japan) was added thereto, followed by incubation at room temperature for 5 minutes. Thereafter, 5 ⁇ L of Stop Solution (Life Technologies Japan) was added, mixed, and incubated at room temperature for 2 minutes, which was then subjected to reverse transcription reaction.
• DNase I Life Technologies Japan
• Stop Solution Stop Solution
• cDNA was synthesized by reacting at 37°C for 30 minutes and then at 95°C for 5 minutes.
• Real-time PCR detected three genes: TurboGFP-fused FUS wild-type gene, TurboFP635-fused FUS P525L gene, and endogenous control gene (using GAPDH) in the same reaction system.
• the reaction solution was 10 ⁇ L of TaqMan (registered trademark) Fast Advanced Master Mix (Life Technologies Japan), 0.06 ⁇ L each of 100 ⁇ M primers (GFP_X_F, GFP_X_R, FP635_X_F, FP635_X_R), and 10 ⁇ M TaqMan probes (TurboGFP (NED) and TurboFP635 (FAM)).
• the sampling time for each siRNA was 0, 15, 30, 45, 60, 75, and 90 minutes for natural siRNA-006, siRNA-009, siRNA-010, and siRNA-011, and for the chemically modified siRNA, The time periods were 0, 1, 3, 6 and 24 hours.
• the frozen sampling sample was thawed, and 5 ⁇ L thereof was subjected to electrophoresis on 20% TBE-PAGE and 1 ⁇ TBE Buffer (150 CV, 40 minutes). Gels were stained with SYBER® Gold (Thermo Fisher Scientific) and detected with an Amersham Imager 680 UV transilluminator 312 nm (cytiva).
• FIG. 6 The results of examining the stability of the siRNA in human serum are shown in FIG. 6 for the natural siRNA and in FIG. 7 for the chemically modified siRNA. Stability in human serum was confirmed by band shift due to incubation and the presence or absence of multiple bands compared to the band observed without incubation (0 min). As a result, a band shift and multiple bands were observed for the natural siRNA within 1 hour after the start of incubation, indicating that degradation by RNase proceeded rapidly. On the other hand, with the chemically modified siRNA, no clear band shift was observed even after 24 hours had passed after the start of incubation, indicating that it was not degraded by RNAase. Therefore, the chemically modified siRNA of the present disclosure was shown to have dramatically improved stability against RNA degrading enzymes.

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