WO2021172498A1 - エクソン51のスキッピングを誘導するアンチセンス核酸 - Google Patents
エクソン51のスキッピングを誘導するアンチセンス核酸 Download PDFInfo
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- WO2021172498A1 WO2021172498A1 PCT/JP2021/007286 JP2021007286W WO2021172498A1 WO 2021172498 A1 WO2021172498 A1 WO 2021172498A1 JP 2021007286 W JP2021007286 W JP 2021007286W WO 2021172498 A1 WO2021172498 A1 WO 2021172498A1
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- antisense oligomer
- exon
- antisense
- seq
- pharmaceutically acceptable
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Definitions
- the present invention relates to an antisense oligomer that induces skipping of the 51st exon of the human dystrophin gene and a pharmaceutical composition containing the antisense oligomer.
- DMD Duchenne muscular dystrophy
- Infants show almost the same motor function as healthy people, but muscle weakness is seen from around 4 to 5 years old. After that, muscle weakness of DMD patients progresses, and DMD patients become unable to walk by about 12 years old, and die from heart failure or respiratory failure in their 20s.
- the dystrophin gene is located on the X chromosome and is a huge gene consisting of 2.2 million base pairs of DNA. It is transcribed from DNA to the pre-mRNA, and the intron is further removed by splicing, and 79 exons are bound to make the mRNA corresponding to the translation region 11,058 bases. This mRNA is translated into 3,685 amino acids to produce the dystrophin protein.
- Dystrophin proteins are involved in maintaining muscle cell membrane stability and are required to make muscle cells less fragile. Since the dystrophin gene of DMD patients has a mutation, the dystrophin protein having a function in muscle cells is hardly expressed.
- the structure of the muscle cell cannot be maintained, and a large amount of calcium ions flow into the muscle cell. As a result, a reaction similar to inflammation occurs, and fibrosis progresses, making it difficult for muscle cells to regenerate.
- Becker-type muscular dystrophy is also caused by a mutation in the dystrophin gene, but its symptoms are generally milder than DMD, and the progression of muscle weakness is slower, often in adulthood, although it presents with muscle weakness due to muscle atrophy. Onset.
- the difference in clinical manifestations between DMD and BMD is believed to be due to whether mutations disrupt or maintain the amino acid reading frame when dystrophin mRNA is translated into dystrophin protein (non-patented). Document 1). That is, in DMD, the dystrophin protein having a function is hardly expressed due to the mutation that shifts the amino acid reading frame, but in BMD, a part of exons is deleted due to the mutation, but the amino acid reading frame is maintained. Therefore, an incomplete but functional dystrophin protein is produced.
- Exon skipping method is expected as a treatment method for DMD.
- This method is a method of repairing the amino acid reading frame of dystrophin mRNA by modifying splicing and inducing the expression of a partially restored dystrophin protein (Non-Patent Document 2).
- the amino acid sequence portion that is the target of exon skipping will be lost. Therefore, the dystrophin protein expressed by this treatment is shorter than that of the normal one, but the function of stabilizing muscle cells is partially retained because the amino acid reading frame is maintained. Therefore, exon skipping is expected to cause DMD to exhibit symptoms similar to milder BMD.
- the exon skipping method has been clinically tested in human DMD patients through animal experiments with mice and dogs.
- Exon skipping can be induced by binding of an antisense nucleic acid that targets either or both of the 5'or 3'splice sites, or the inside of the exon. Exons are included in the mRNA only if both splice sites are recognized by the spliceosome complex. Therefore, exon skipping can be induced by targeting the splice site with antisense nucleic acid. In addition, it is thought that SR protein binding to the exon splicing enhancer (ESE) is necessary for exons to be recognized by the splicing mechanism, and targeting the ESE can also induce exon skipping. can.
- ESE exon splicing enhancer
- Non-Patent Document 3 Since mutations in the dystrophin gene differ depending on the DMD patient, antisense nucleic acids are required according to the location and type of gene mutation. So far, Antisense nucleic acids that induce exon skipping for all 79 exons have been produced by Steve Wilton et al. Of the University of Western Australia (Non-Patent Document 3), and 39 types by Annemieek Artsma-Rus et al. In the Netherlands. An antisense nucleic acid that induces exon skipping for exons has been produced (Non-Patent Document 4).
- exon 51 About 13% of all DMD patients are considered to be treatable by skipping the 51st exon (hereinafter referred to as "exon 51").
- exon 51 a plurality of reports have been made on studies targeting exon 51 of the dystrophin gene for exon skipping (Patent Documents 1 to 10 and Non-Patent Documents 3 to 7).
- a novel antisense oligomer that induces skipping of exon 51 of the dystrophin gene with high efficiency is desired. Further, an antisense oligomer is desired, which maintains the activity of inducing the skipping of exon 51 of the dystrophin gene with high efficiency and has excellent properties (for example, solubility and safety) as a medicine.
- the present inventors administer an antisense oligomer having the base sequence shown in any of SEQ ID NOs: 1 to 89 and 91 to 93. As a result, it was found that the skipping of exon 51 of the human dystrophin gene is induced with high efficiency. In addition, as a result of research, we have found an antisense oligomer that induces skipping of exon 51 of the human dystrophin gene with high efficiency and has excellent solubility and safety. The present inventors have completed the present invention based on this finding.
- the present invention is as follows. [1] The following (a1) to (d1): (A1) An antisense oligomer containing any of the nucleotide sequences of SEQ ID NOs: 1 to 89 and 91 to 93; (B1) A human dystrophin gene containing a base sequence in which 1 to 5 bases are deleted, substituted, inserted, and / or added to any of the base sequences of SEQ ID NOs: 1 to 89 and 91 to 93.
- an antisense oligomer selected from the group consisting of antisense oligomers having an activity of inducing skipping of exxon 51 of the human dystrophin gene (provided that the antisense oligomer consists of any of the nucleotide sequences of SEQ ID NOs: 90 and 97 to 126). ) Or its pharmaceutically acceptable salts or their hydrates.
- Antisense oligomer with activity to induce (G) With respect to any of the nucleotide sequences of SEQ ID NOs: 1 to 89 and 91 to 93, it is composed of a nucleotide sequence having 80% or more sequence identity and has an activity of inducing skipping of exon 51 of the human distrophin gene. Antisense oligomer having; and (h) an antisense oligomer that hybridizes with an oligonucleotide having a base sequence complementary to any of the base sequences of SEQ ID NOs: 1 to 89 and 91 to 93 under high stringent conditions.
- An antisense oligomer selected from the group consisting of antisense oligomers having an activity of inducing skipping of exxon 51 of the human dystrophin gene (provided that the antisense oligomer consists of any of the nucleotide sequences of SEQ ID NOs: 90 and 97 to 126). (Excluding oligomers) or pharmaceutically acceptable salts thereof or hydrates thereof. [3]
- the antisense oligomer An anti that has a nucleotide sequence having 90% or more sequence identity to any of the nucleotide sequences of SEQ ID NOs: 1 to 89 and 91 to 93 and has an activity of inducing skipping of exon 51 of the human dystrophin gene.
- the antisense oligomer (A2) An antisense oligomer containing the base sequence of any of SEQ ID NOs: 7, 8, 10, 16, 21, 24, 31, 42, 67 and 76; (B2) 1 to 5 bases are deleted, substituted, inserted, and / or in any of the base sequences of SEQ ID NOs: 7, 8, 10, 16, 21, 24, 31, 42, 67 and 76.
- An antisense oligomer containing an added base sequence and having an activity of inducing skipping of exon 51 of the human dystrophin gene (C2) A human containing a base sequence having 80% or more sequence identity with respect to any of the base sequences of SEQ ID NO: 7, 8, 10, 16, 21, 24, 31, 42, 67 and 76, and human.
- antisense oligomers which are antisense oligomers that hybridize with oligonucleotides having a specific base sequence under stringent conditions and which have an activity of inducing skipping of exon 51 of the human distrophin gene.
- the sugar moiety of at least one nucleotide constituting the oligonucleotide has an -OH group at the 2'position of OR, R, R'OR, SH, SR, NH 2 , NHR, NR 2 , N 3 , CN, F.
- the phosphate binding moiety of at least one nucleotide constituting the oligonucleotide is selected from the group consisting of a phosphorothioate bond, a phosphorodithioate bond, an alkylphosphonate bond, a phosphoramidate bond, and a boranephosphate bond.
- the 5'end is the following chemical formulas (1) to (3): The antisense oligomer according to [9] or [10] above, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, which is the basis of any of the above.
- a pharmaceutical composition for treating muscular dystrophy which comprises the antisense oligomer according to any one of the above [1] to [11], a pharmaceutically acceptable salt thereof, or a hydrate thereof.
- the pharmaceutical composition according to the above [12] further comprising a pharmaceutically acceptable carrier.
- [18] Use of the antisense oligomer according to any one of the above [1] to [11], a pharmaceutically acceptable salt thereof, or a hydrate thereof in the manufacture of a pharmaceutical for treating muscular dystrophy.
- a method for treating muscular dystrophy which comprises the step of administering the pharmaceutical composition of the above to a patient with muscular dystrophy.
- the treatment method according to [19] above, wherein the patient is a human.
- the pharmaceutical composition according to any one of. [22] The antisense oligomer according to [21] above or a pharmaceutically acceptable salt thereof or a hydrate thereof, or a pharmaceutical composition, wherein the patient with muscular dystrophy is a human in the treatment.
- an antisense oligomer that induces skipping of exon 51 of the human dystrophin gene with high efficiency. Further, according to the present invention, it is possible to provide an antisense oligomer having excellent solubility while maintaining the activity of inducing the skipping of exon 51 of the human dystrophin gene with high efficiency. Furthermore, according to the present invention, while maintaining the activity of inducing the skipping of the exon 51 of the human dystrophin gene with high efficiency, and having excellent solubility and safety (for example, whether there is any effect on the function of the kidney and liver). , Very unlikely to affect) antisense oligomers can be provided.
- PMO No. The skipping efficiency of exon 51 of the human dystrophin gene in human rhabdomyosarcoma cells (RD cells) of antisense oligomers 43, 44, 45 and 46 is shown.
- PMO No. The skipping efficiency of exon 51 of the human dystrophin gene in RD cells of 42, 45, 47, 48, 49, 50 antisense oligomers is shown.
- PMO No. The skipping efficiency of exon 51 of the human dystrophin gene in RD cells of antisense oligomers 42, 62, 63 and 89 is shown.
- PMO No. The skipping efficiency of exon 51 of the human dystrophin gene in RD cells of 83 and 85 antisense oligomers is shown.
- the skipping efficiency of exon 51 of the human dystrophin gene in RD cells of 33, 34, 35, 36, 37, 38 antisense oligomers is shown.
- PMO No. The skipping efficiency of exon 51 of the human dystrophin gene in RD cells of 83, 85, 90 antisense oligomers is shown.
- PMO No. The skipping efficiency of exon 51 of the human dystrophin gene in RD cells of the antisense oligomers of 82, 84, 86 and 87 is shown.
- PMO No. The skipping efficiency of exon 51 of the human dystrophin gene in RD cells of antisense oligomers of 45, 51, 52, 56, 57, 58, 59, 60, 61 is shown.
- the skipping efficiency of exon 51 of the human dystrophin gene in RD cells of 42, 64, 65, 66, 85 antisense oligomers is shown.
- PMO No. The skipping efficiency of exon 51 of the human dystrophin gene in RD cells of antisense oligomers 1, 2, 42, 66, 67, 85, 88, 92, 93 is shown.
- PMO No. The skipping efficiency of exon 51 of the human dystrophin gene in RD cells of the antisense oligomers of 3, 4, 5, 6, 7, 8, 42, 68, 69, 70 and 85 is shown.
- the skipping efficiency of exon 51 of the human dystrophin gene in RD cells of the antisense oligomers of 8, 9, 10, 11, 12, 13, 14, 42, 71, 72, 73, 91 is shown.
- PMO No. The skipping efficiency of exon 51 of the human dystrophin gene in RD cells of the antisense oligomers of 8, 15, 16, 17, 42, 74, 75 and 76 is shown.
- PMO No. The skipping efficiency of exon 51 of the human dystrophin gene in RD cells of the antisense oligomers of 8, 18, 19, 20, 42, 63, 75, 76 and 77 is shown.
- the skipping efficiency of exon 51 of the human dystrophin gene in RD cells of the antisense oligomers of 8, 21, 22, 23, 24, 25, 26, 42, 77 and 78 is shown.
- PMO No. The skipping efficiency of exon 51 of the human dystrophin gene in RD cells of the antisense oligomers of 8, 21, 27, 28, 29, 30, 42, 79, 80 and 81 is shown.
- PMO No. The skipping efficiency of exon 51 of the human dystrophin gene in RD cells of the antisense oligomers of 8, 16, 21, 31, 32 and 67 is shown.
- PMO No. The skipping efficiency of exon 51 of the human dystrophin gene in RD cells of the antisense oligomers of 16, 21, and 94 is shown.
- mice of 42 antisense oligomers The results of the safety test in mice of 42 antisense oligomers are shown. From the left, the aspartate aminotransferase (AST) value, alanine aminotransferase (ALT) value, urea nitrogen amount (BUN) value, and creatinine value are shown by mean ⁇ standard deviation (Student's t-test significance level: p ⁇ 0. 05). PMO No. The results of safety tests on mice of 16 and 90 antisense oligomers are shown. From the left, the AST value, ALT value, BUN value, and creatinine value are shown by the mean ⁇ standard deviation, and the p-value is shown for the values that show a significant increase (significance level by Dunnett's test: p ⁇ 0.05).
- the present invention provides an antisense oligomer (hereinafter, referred to as “the antisense oligomer of the present invention”) that efficiently skips the 51st exon of the human dystrophin gene.
- the "gene” includes cDNA, pre-mRNA and mRNA in addition to the genomic gene.
- the gene is a pre-mRNA, i.e. pre-mRNA.
- the human dystrophin gene is located at locus Xp21.2.
- the human dystrophin gene has a size of 2.2 million base pairs and is the largest known human gene.
- the coding region of the human dystrophin gene is only 14 kb, and the coding region is dispersed in the dystrophin gene as 79 exons (Roberts, RG., Et al., Genomics, 16: 536-538 (Roberts, RG., Et al., Genomics, 16: 536-538). 1993); Koenig, M., et al., Cell 53: 219-228 (1988)).
- Pre-mRNA a transcript of the human dystrophin gene, is spliced to produce 14 kb of mature mRNA.
- the nucleotide sequence of the human wild-type dystrophin gene is known (GenBank Accession No. NM_004006).
- the nucleotide sequence of exon 51 of the human wild-type dystrophin gene is shown in SEQ ID NO: 127.
- the antisense oligomer of the present invention was prepared for the purpose of modifying a protein encoded by a DMD-type dystrophin gene into a BMD-type dystrophin protein by skipping the exon 51 of the human dystrophin gene. Therefore, the exon 51 of the dystrophin gene, which is the target of exon skipping of the antisense oligomer, includes not only the wild type but also the mutant type.
- the antisense oligomer of the present invention is the antisense oligomer according to any one selected from the group consisting of the following (a1) to (d1).
- A1 An antisense oligomer containing any of the nucleotide sequences of SEQ ID NOs: 1 to 89 and 91 to 93;
- B1 1 to 5, 1 to 4, 1 to 3, 1-2, or 1 base is deleted from any of the base sequences of SEQ ID NOs: 1 to 89 and 91 to 93.
- the antisense oligomer of the present invention is specifically the antisense oligomer according to any one selected from the group consisting of the following (e) to (h).
- E An antisense oligomer consisting of any of the nucleotide sequences of SEQ ID NOs: 1 to 89 and 91 to 93;
- F 1 to 5, 1 to 4, 1 to 3, 1-2, or 1 base is deleted from any of the base sequences of SEQ ID NOs: 1 to 89 and 91 to 93.
- an antisense oligomer consisting of a substituted nucleotide sequence and having an activity of inducing skipping of exon 51 of the human dystrophin gene; (G) 80% or more, 84% or more, 85% or more, 89% or more, 90% or more, 94% or more, or 95% or more with respect to any of the nucleotide sequences of SEQ ID NOs: 1 to 89 and 91 to 93.
- the antisense oligomer of the present invention the antisense oligomer according to any one selected from the group consisting of the following (a2) to (d2) is more preferable.
- (B2) 1 to 5 bases are deleted, substituted, inserted, and / or in any of the base sequences of SEQ ID NOs: 7, 8, 10, 16, 21, 24, 31, 42, 67 and 76.
- An antisense oligomer containing an added base sequence and having an activity of inducing skipping of exon 51 of the human dystrophin gene (C2) A human containing a base sequence having 80% or more sequence identity with respect to any of the base sequences of SEQ ID NO: 7, 8, 10, 16, 21, 24, 31, 42, 67 and 76, and human.
- antisense oligomers which are antisense oligomers that hybridize with oligonucleotides having a specific base sequence under stringent conditions and which have an activity of inducing skipping of exon 51 of the human distrophin gene. Antisense oligomer.
- the antisense oligomers (b1) to (d1), (f) to (h), and (b2) to (d2) are the antisense oligomers of (a1), (e), and (a2), respectively. It is a variant of the sense oligomer, with the intention of responding to mutations (eg, polymorphisms) in the dystrophin gene of patients.
- the antisense oligomer of the present invention excludes (does not include) the antisense oligomer consisting of the following base sequence described in International Publication No. 2015/137409.
- the "antisense oligomer that hybridizes under stringent conditions” refers to, for example, an oligonucleotide having a base sequence complementary to any of the base sequences of SEQ ID NOs: 1 to 89 and 91 to 93.
- An antisense oligomer obtained by using a colony hybridization method, a plaque hybridization method, a southern hybridization method, or the like, using all or part of the probe as a probe.
- Hybridization methods include, for example, “Sambrook & Russell, Molecular Cloning: A Laboratory Manual Vol. 3, Cold Spring Harbor, Laboratory Press 2001” and “Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987", etc. You can use the method described in.
- the "stringent condition” may be any of a low stringent condition, a medium stringent condition, and a high stringent condition.
- the "low stringent condition” is, for example, a condition of 5 ⁇ SSC, 5 ⁇ Denhardt solution, 0.5% SDS, 50% formamide, 32 ° C.
- the “medium stringent conditions” are, for example, 5 ⁇ SSC, 5 ⁇ Denhardt solution, 0.5% SDS, 50% formamide, 42 ° C. or 5 ⁇ SSC, 1% SDS, 50 mM Tris-HCl ( The conditions are pH 7.5), 50% formamide, and 42 ° C.
- “High stringent conditions” include, for example, (1) 5 ⁇ SSC, 5 ⁇ Denhardt solution, 0.5% SDS, 50% formamide, 50 ° C., (2) 0.2 ⁇ SSC, 0.1%. SDS, 60 ° C, (3) 0.2 x SSC, 0.1% SDS, 62 ° C, (4) 0.2 x SSC, 0.1% SDS, 65 ° C, or (5) 0.1 x SSC , 0.1% SDS, 65 ° C., but is not limited thereto. Under these conditions, it can be expected that the antisense oligomer having higher sequence identity can be efficiently obtained as the temperature is raised.
- sequence identity means the identity of a pair of two nucleic acids in the entire range of the base sequence to be compared, and is created by using a mathematical algorithm known in the technical field of the present invention. It is represented by the percentage of matching bases in the optimal alignment of the base sequences.
- an antisense oligomer having a base sequence of "80% sequence identity" with respect to an antisense oligomer having a base sequence of 20 bases is 16 with respect to the antisense oligomer having 20 bases. It means an antisense oligomer having the same base as more than a base.
- sequence identity is determined by FASTA (Science 227 (4693): 1435-1441, (1985)) and the algorithm BLAST (Basic Local Alignment Search Tool) by Carlin and Arthur (Proc. Natl. Acad. Sci. USA 872264-2268). , 1990; Proc Natl Acad Sci USA 90: 5873, 1993).
- Programs called blastn, blastx, tblastn and tblastx based on the BLAST algorithm have been developed (Altschul SF, et al: J Mol Biol 215: 403, 1990).
- BLAST and Gapped BLAST programs use the default parameters of each program.
- a commercially available kit for example, Alkphos Direct Labeling and Detection System (GE Healthcare) can be used. In this case, the membrane was incubated overnight with a primary wash buffer containing 0.1% (w / v) SDS under 55 ° C. conditions according to the protocol attached to the kit. After washing, hybridized antisense oligomers can be detected.
- a commercially available reagent for example, PCR labeling mix (Roche. When the probe is labeled with digoxigenin (DIG) using (Diagnostics), etc.), hybridization can be detected using a DIG nucleic acid detection kit (Roche Diagnostics).
- Examples of the antisense oligomer that can be hybridized other than the above include any of the nucleotide sequences of SEQ ID NOs: 1 to 89 and 91 to 93 when calculated using the default parameters by homology search software such as FASTA and BLAST. 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2 % Or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more sequence identity.
- the antisense oligomer having can be mentioned.
- “Inducing skipping of the 51st exon of the human dystrophin gene” means that the anti-exon 51 of the transcript of the human dystrophin gene (eg, pre-mRNA) and / or the site corresponding to the adjacent intron of the present invention.
- the binding of the sense oligomer causes the exclusion of exon 51 when the transcript is spliced, for example, in the case of DMD patients lacking exon 52, exon 53 is assigned to the base sequence corresponding to the 3'end of exon 50. It means that the base sequences corresponding to the 5'ends of the gene are ligated to form mature mRNAs in which no frame shift of codons has occurred.
- DMD patients with a mutation in the dystrophin gene that is the target of exon 51 skipping can be treated by exon 51 skipping.
- Examples of such DMD patients include DMD patients having a frameshift mutation due to a deletion of exon at least in the vicinity of exon 51 and having a dystrophin gene whose amino acid reading frame is modified by skipping exon 51. More specifically, for example, exons 13-50, 29-50, 40-50, 43-50, 45-50, 47-50, 48-50, 49-50, 50, 52, 52- of the dystrophin gene. DMD patients with frameshift mutations due to having a deletion such as 63 can be mentioned.
- binding means that when the antisense oligomer of the present invention and a transcript of the human dystrophin gene are mixed, they hybridize under physiological conditions to form a double strand.
- physiological conditions mean conditions adjusted to a pH, salt composition, and temperature similar to those in a living body.
- the condition is 25 to 40 ° C., preferably 37 ° C., pH 5 to 8, preferably pH 7.4, and the sodium chloride concentration is 150 mM.
- Whether or not skipping of the exon 51 of the human dystrophin gene has occurred is determined by introducing the antisense oligomer of the present invention into dystrophin-expressing cells (for example, human dystrophin myoma cells) and using the total RNA of the dystrophin-expressing cells to determine whether or not human dystrophin is skipped. It can be confirmed by RT-PCR amplification of the region around exon 51 of the gene mRNA and performing nested PCR or sequence analysis on the PCR amplification product.
- dystrophin-expressing cells for example, human dystrophin myoma cells
- the skipping efficiency ES (unit:%) was such that the mRNA of the human dystrophin gene was recovered from the test cells, and among the mRNA, the amount of polynucleotide "A" in the band skipped by exon 51 and the amount of polynucleotide "A” skipped by exon 51 were not skipped.
- the amount of polynucleotide "B” in the band can be measured and calculated according to the following formula (1) based on the measured values of these "A" and "B". For the calculation of skipping efficiency, International Publication No. 2012/029986 can be referred to.
- the antisense oligomer of the present invention exons with an efficiency of 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more.
- Skip 51 the antisense oligomer of the present invention exons with an efficiency of 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more.
- the antisense oligomer of the present invention preferably has high solubility in physiological saline. Unlike those with low solubility, antisense oligomers, which are highly soluble in physiological saline, are extremely unlikely to precipitate and become unusable during storage of the preparation, and precipitate when an infusion solution containing salt is used. It is extremely unlikely that it will become unusable. Furthermore, since antisense oligomers, which are highly soluble in physiological saline, are difficult to precipitate, they are extremely unlikely to be toxic at the time of administration (Bulletin of Osaka University of Pharmaceutical Sciences 1, 91-99 (2007)). Therefore, the antisense oligomer, which is highly soluble in physiological saline, has a very high utility value as an active ingredient of a pharmaceutical product.
- Solubility in physiological saline is preferably 20 mg / mL or more, more preferably 30 mg / mL or more, further preferably 40 mg / mL or more, and 50 mg / mL or more. Is particularly preferred.
- the solubility of the antisense oligomer in physiological saline can be evaluated by dissolving the antisense oligomer in physiological saline at a desired concentration and visually confirming the presence or absence of precipitation after a certain period of time.
- the antisense oligomer has high safety as an active ingredient of a drug.
- Safety can be evaluated, for example, by using aspartate aminotransferase (AST) value, alanine aminotransferase (ALT) value, urea nitrogen (BUN) value and creatinine level in blood after administration of antisense oligomer as an index. ..
- the AST value increases when the liver is damaged, the ALT value increases when there is a problem with the liver, the BUN value increases when the kidney function declines, and the creatinine level increases when the renal glomerular filtration function decreases. Therefore, it is possible to evaluate the effect of antisense oligomers on the function of kidney and liver using these values as an index.
- the AST value, ALT value, BUN value and creatinine value in the blood are measured to perform a statistically significant difference test, and a control group (solvent administration) is performed. If a significant increase is observed compared to the measured value (or no treatment), it is judged to be an abnormal value, and the administered antisense oligomer may or may affect the function of the kidney and liver. It can be judged to be expensive. Conversely, if no significant increase is seen, it can be determined that there is no or unlikely effect on kidney and liver function. Further, it may be determined as an abnormal value when a specific increase rate, specifically, for example, an increase rate of 30% or more is observed as compared with the measured value of the control group.
- Examples of the antisense oligomer of the present invention include 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35. Examples thereof include oligonucleotides, morpholino oligomers, and peptide nucleic acid (PNA) oligomers having a base length.
- the length of the antisense oligomer is preferably 20 to 30 bases, 20 to 29 bases, 22 to 30 bases, 22 to 29 bases or 25 to 29 bases, and 22 to 30 bases, 22 to 29 bases or 25 bases. A length of up to 29 bases is more preferred, with morpholino oligomers being preferred.
- oligonucleotide of the present invention is an antisense oligomer of the present invention having a nucleotide as a constituent unit, and such a nucleotide may be a ribonucleotide, a deoxyribonucleotide, or a modified nucleotide. good.
- a modified nucleotide means a ribonucleotide or a deoxyribonucleotide in which all or part of the nucleobase, sugar moiety, and phosphate binding moiety that composes the ribonucleotide or deoxyribonucleotide is modified.
- examples of the nucleobase include adenine, guanine, hypoxanthine, cytosine, thymine, uracil, and modified bases thereof.
- modified bases include, for example, pseudouracil, 3-methyluracil, dihydrouracil, 5-alkylcytosine (eg, 5-methylcytosine), 5-alkyluracil (eg, 5-ethyluracil), 5-halouracil (5).
- 6-azapyrimidine 6-alkylpyrimidine (6-methyluracil), 2-thiouracil, 4-thiouracil, 4-acetylcytosine, 5- (carboxyhydroxymethyl) uracil, 5'-carboxymethylaminomethyl -2-thiouracil, 5-carboxymethylaminomethyluracil, 1-methyladenine, 1-methylhypoxanthin, 2,2-dimethylguanine, 3-methylcytosine, 2-methyladenine, 2-methylguanine, N6-methyladenine , 7-Methylguanine, 5-methoxyaminomethyl-2-thiouracil, 5-methylaminomethyluracil, 5-methylcarbonylmethyluracil, 5-methyloxyuracil, 5-methyl-2-thiouracil, 2-methylthio-N6- Examples thereof include, but are not limited to, isopentenyladenine, uracil-5-oxyacetic acid, 2-thiocytosine, purine, 2,6-d
- Modifications of the sugar moiety include, for example, modification of the 2'position of ribose and modification of other moieties of sugar.
- the modification of the 2'position of ribose for example, the -OH group at the 2'position of ribose is OR, R, R'OR, SH, SR, NH 2 , NHR, NR 2 , N 3 , CN, F, Cl. , Br, I can be mentioned.
- R represents alkyl or aryl.
- Modifications of other portions of sugar include, for example, ribose or deoxyribose in which O at the 4'position is replaced with S, sugar in which the 2'position and the 4'position are crosslinked, for example, LNA (Locked Nucleic Acid). ) Or ENA (2'-O, 4'-C-Ethylene-bridged Nucleic Acids) and the like, but are not limited thereto.
- LNA Locked Nucleic Acid
- ENA 2'-O, 4'-C-Ethylene-bridged Nucleic Acids
- Modifications of the phosphate bond moiety include, for example, phosphodiester bonds, phosphorothioate bonds, phosphorodithioate bonds, alkylphosphonate bonds, phosphoromidate bonds, and borane phosphate bonds (Enya et al: Bioorganic & Medical Chemistry, 2008). , 18, 9154-9160) (see, for example, Patent Republishing Publication Nos. 2006/129594 and 2006/038608).
- alkyl a linear or branched alkyl having 1 to 6 carbon atoms is preferable. Specific examples thereof include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, n-hexyl and isohexyl. Be done.
- the alkyl may be substituted, and examples of such substituents include halogen, alkoxy, cyano, and nitro, and 1 to 3 of these may be substituted.
- the cycloalkyl is preferably a cycloalkyl having 5 to 12 carbon atoms. Specific examples thereof include cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl and cyclododecyl.
- examples of the halogen include fluorine, chlorine, bromine and iodine.
- Alkoxy includes linear or branched alkoxy having 1 to 6 carbon atoms, for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-. Examples thereof include pentyloxy, isopentyloxy, n-hexyloxy, isohexyloxy and the like. In particular, alkoxy having 1 to 3 carbon atoms is preferable.
- the aryl is preferably an aryl having 6 to 10 carbon atoms. Specifically, for example, phenyl, ⁇ -naphthyl, ⁇ -naphthyl can be mentioned. Phenyl is particularly preferable.
- the aryl may be substituted, and examples of such substituents include alkyl, halogen, alkoxy, cyano, and nitro, and 1 to 3 of these may be substituted.
- the alkylene a linear or branched alkylene having 1 to 6 carbon atoms is preferable.
- examples of the acyl include linear or branched alkanoyl or aloyl.
- alkanoyl include formyl, acetyl, 2-methylacetyl, 2,2-dimethylacetyl, propionyl, butyryl, isobutyryl, pentanoyl, 2,2-dimethylpropionyl, hexanoyl and the like.
- aloyl include benzoyl, toluoil, and naphthoyl. Such aloyl may be substituted at a substitutable position or may be substituted with an alkyl.
- the oligonucleotide of the present invention preferably has a group represented by the following general formula as a constituent unit in which the -OH group at the 2'position of ribose is replaced with methoxy and the phosphate binding moiety is a phosphorothioate bond.
- Antisense oligomer In the formula, Base represents a nucleobase.
- the oligonucleotide of the present invention can be easily synthesized using various automatic synthesizers (for example, AKTA oligopilot plus 10/100 (GE Healthcare)), or can be easily synthesized by a third party organization (for example, Promega, Inc.). It can also be manufactured by outsourcing to Takara Co., Ltd. or Japan Bioservice Co., Ltd.).
- automatic synthesizers for example, AKTA oligopilot plus 10/100 (GE Healthcare)
- a third party organization for example, Promega, Inc.
- It can also be manufactured by outsourcing to Takara Co., Ltd. or Japan Bioservice Co., Ltd.
- the morpholino oligomer of the present invention is an antisense oligomer of the present invention having a group represented by the following general formula as a constituent unit.
- Base is synonymous with the above; W represents a group represented by any of the following equations.
- X represents -CH 2 R 1 , -O-CH 2 R 1 , -S-CH 2 R 1 , -NR 2 R 3 or F;
- R 1 represents H, alkyl;
- R 2 and R 3 represent H, alkyl, cycloalkyl, or aryl, the same or different;
- Y 1 represents O, S, CH 2 or NR 1 ;
- Y 2 represents O, S or NR 1 ;
- Z represents O or S.
- Examples of the morpholino monomer compound used for the synthesis of the morpholino oligomer of the present invention include the morpholino monomer compound (A), the morpholino monomer compound (C), the morpholino monomer compound (T), and the morpholino monomer compound (G) shown in the table below. ), But is not limited to these.
- the morpholino oligomer is preferably an oligomer having a group represented by the following formula as a constituent unit (phosphologiamidate morpholino oligomer (hereinafter referred to as “PMO”)).
- PMO phosphologiamidate morpholino oligomer
- Base, R 2 and R 3 have the same meanings as described above.
- the morpholino oligomer of the present invention includes nucleobases, morpholino ring moieties, phosphate binding moieties, and all or part of the 3'end and / or 5'end that make up such oligomers.
- Modifications of the phosphate binding moiety include, for example, phosphorodiamidate bond, phosphorothioate bond, phosphorodithioate bond, alkylphosphonate bond, phosphoramidate bond, boranephosphate bond (Enya et al., Bioorganic & Medicinal). Chemistry, 2008, 18, 9154-9160) can be mentioned (see, for example, Patent Republishing Publications 2006/129594 and 2006/038608).
- Morpholine oligomers can be produced, for example, in accordance with WO 1991/090333 or WO 2009/064471.
- the PMO can be produced according to the method described in WO 2009/064471, or can be produced according to the method shown below.
- PMO manufacturing method As one aspect of the PMO, for example, a compound represented by the following general formula (I) (hereinafter referred to as PMO (I)) can be mentioned.
- PMO (I) a compound represented by the following general formula (I) (hereinafter referred to as PMO (I)) can be mentioned.
- n is any integer in the range 1-99, preferably any integer in the range 19-29, 19-28, 21-29, 21-28 or 24-28. More preferably, it is 21-29, 21-28 or 24-28.
- n is any integer in the range 1-99, preferably any integer in the range 19-29, 19-28, 21-29, 21-28 or 24-28. More preferably, it is 21-29, 21-28 or 24-28.
- PMO (I) can be produced according to a known method, and can be produced, for example, by carrying out the following steps.
- the compounds and reagents used in the following steps are not particularly limited as long as they are generally used in the production of PMO.
- all the following steps can be carried out by the liquid phase method or the solid phase method (manual or using a commercially available solid phase automatic synthesizer).
- a method using an automatic synthesizer is desirable from the viewpoint of simplification of the operation procedure and accuracy of synthesis.
- Step A By allowing an acid to act on the compound represented by the following general formula (II) (hereinafter referred to as compound (II)), the compound represented by the following general formula (III) (hereinafter referred to as compound (III)). .) The process of manufacturing.
- n, R 2 and R 3 are synonymous with the above; Each BP independently represents a nucleobase that may be protected; T represents a trityl group, a monomethoxytrityl group, or a dimethoxytrityl group; L represents hydrogen, an acyl, or a group represented by the following general formula (IV) (hereinafter referred to as a group (IV)).
- nucleobase related to BP
- the same “nucleobase” as Base can be mentioned.
- the amino group or hydroxyl group of the nucleobase related to BP may be protected.
- the protecting group for such an amino group is not particularly limited as long as it is used as a protecting group for a nucleic acid, and specifically, for example, benzoyl, 4-methoxybenzoyl, acetyl, propionyl, butyryl, isobutyryl, and phenylacetyl. , Phenoxyacetyl, 4-tert-butylphenoxyacetyl, 4-isopropylphenoxyacetyl, (dimethylamino) methylene.
- protecting group for the hydroxyl group for example, 2-cyanoethyl, 4-nitrophenethyl, phenylsulfonylethyl, methylsulfonylethyl, trimethylsilylethyl, and 1 to 5 electron-withdrawing groups are substituted at any substitutable position.
- the "solid phase carrier” is not particularly limited as long as it is a carrier that can be used for the solid phase reaction of nucleic acid, and for example, (i) reagents that can be used for the synthesis of morpholino nucleic acid derivatives (for example, dichloromethane, acetonitrile, tetrazole, etc. It is almost insoluble in N-methylimidazole, pyridine, acetic anhydride, lutidine, trifluoroacetic acid), is chemically stable to reagents that can be used in the synthesis of (ii) morpholino nucleic acid derivatives, and is (iii) chemically modified.
- morpholino nucleic acid derivatives for example, dichloromethane, acetonitrile, tetrazole, etc. It is almost insoluble in N-methylimidazole, pyridine, acetic anhydride, lutidine, trifluoroacetic acid
- swellable polystyrene for example, aminomethylpolystyrene resin 1% divinylbenzene crosslinked (200 to 400 mesh) (2.4 to 3.0 mmol / g) (manufactured by Tokyo Kasei Co., Ltd.), Aminomethylated Polystyrene Resin ⁇ HCl [ Divinylbenzene 1%, 100-200 mesh] (manufactured by Peptide Research Institute), non-swellable polystyrene (eg, Primer Support (manufactured by GE Healthcare)), PEG chain-linked polystyrene (eg, NH 2- PEG resin) Watanabe Kagaku Co., Ltd.), TentaGel resin), polyswellable polystyrene (for example, aminomethylpolystyrene resin 1% divinylbenzene crosslinked (200 to 400 mesh) (2.4 to 3.0 mmol / g) (manufactured by Tokyo Kasei
- This step can be carried out by allowing an acid to act on compound (II).
- Examples of the "acid” that can be used in this step include trifluoroacetic acid, dichloroacetic acid, and trichloroacetic acid.
- the amount of the acid used is, for example, preferably in the range of 0.1 molar equivalent to 1000 molar equivalent, preferably in the range of 1 molar equivalent to 100 molar equivalent, relative to 1 mol of compound (II).
- an organic amine can be used together with the acid.
- the organic amine is not particularly limited, and examples thereof include triethylamine.
- the amount of the organic amine used is, for example, preferably in the range of 0.01 molar equivalent to 10 molar equivalent, preferably in the range of 0.1 molar equivalent to 2 molar equivalent, relative to 1 mol of acid. ..
- a salt or mixture of an acid and an organic amine is used in this step, for example, a salt or a mixture of trifluoroacetic acid and triethylamine can be mentioned, and more specifically, with respect to 2 equivalents of trifluoroacetic acid.
- a mixture of 1 equivalent of triethylamine can be mentioned.
- the acid that can be used in this step can also be diluted with an appropriate solvent so as to have a concentration in the range of 0.1% to 30%.
- the solvent is not particularly limited as long as it does not participate in the reaction, and examples thereof include dichloromethane, acetonitrile, alcohols (ethanol, isopropanol, trifluoroethanol, etc.), water, or a mixture thereof.
- the reaction temperature in the above reaction is, for example, preferably in the range of 10 ° C. to 50 ° C., more preferably in the range of 20 ° C. to 40 ° C., and further preferably in the range of 25 ° C. to 35 ° C.
- the reaction time varies depending on the type of acid used and the reaction temperature, but is usually in the range of 0.1 minutes to 24 hours. Preferably, it is in the range of 1 minute to 5 hours.
- a base can be added to neutralize the acid present in the system, if necessary.
- the “base” is not particularly limited, and examples thereof include diisopropylethylamine.
- the base can also be used by diluting it with a suitable solvent so that the concentration is in the range of 0.1% (v / v) to 30% (v / v).
- the solvent used in this step is not particularly limited as long as it is not involved in the reaction, and examples thereof include dichloromethane, acetonitrile, alcohols (ethanol, isopropanol, trifluoroethanol, etc.), water, or a mixture thereof.
- the reaction temperature is, for example, preferably in the range of 10 ° C.
- reaction time varies depending on the type of base used and the reaction temperature, but is usually preferably in the range of 0.1 minutes to 24 hours, preferably in the range of 1 minute to 5 hours.
- Step 1 A step of producing a compound represented by the following general formula (VI) (hereinafter referred to as compound (VI)) by allowing an acylating agent to act on the compound represented by the following general formula (V).
- a compound represented by the following general formula (VI) hereinafter referred to as compound (VI)
- an acylating agent to act on the compound represented by the following general formula (V).
- This step can be carried out by introducing a known linker using compound (V) as a starting material.
- the compound represented by the following general formula (VIa) can be produced by carrying out a method known as an esterification reaction using compound (V) and succinic anhydride.
- BP and T are synonymous with the above.
- Step 2 A step of producing compound (IIa) by reacting compound (VI) with a solid-phase carrier by allowing a condensing agent or the like to act on the compound (VI).
- This step can be produced by a method known as a condensation reaction using compound (VI) and a solid phase carrier.
- n 1 to 99 (in certain embodiments, n is, for example, 2 to 29, 2 to 28, 2 to 27, 2 to 26, 2 to 25, 2 to 24, 2 to 23, 2).
- the compound represented by the following general formula (IIa2) which is (to 29, 21 to 28 or 24 to 28) and L is the group (IV), is based on the compound (IIa) as a starting material. It can be produced by repeating step A and step B according to the method for producing PMO described in the specification a desired number of times.
- BP , n, R 2 , R 3 , T, linker, solid phase carrier are synonymous with the above.
- Step B A step of producing a compound represented by the following general formula (VII) (hereinafter referred to as compound (VII)) by allowing a morpholino monomer compound to act on compound (III) in the presence of a base.
- VII general formula
- This step can be carried out by allowing the morpholino monomer compound to act on the compound (III) in the presence of a base.
- Examples of the morpholinomonomer compound include a compound represented by the following general formula (VIII). [In the formula, BP , R 2 , R 3 , and T are synonymous with the above. ]
- Examples of the "base” that can be used in this step include diisopropylethylamine, triethylamine, and N-ethylmorpholine. The amount of the base used is, for example, appropriately in the range of 1 molar equivalent to 1000 molar equivalents, preferably in the range of 10 molar equivalents to 100 molar equivalents, relative to 1 mol of compound (III).
- the morpholinomonomer compound and base that can be used in this step can also be diluted with an appropriate solvent so as to have a concentration of 0.1% to 30%.
- the solvent is not particularly limited as long as it does not participate in the reaction, and examples thereof include N, N-dimethylimidazolidone, N-methylpiperidone, DMF, dichloromethane, acetonitrile, terrorahydrofuran, or a mixture thereof.
- the reaction temperature is, for example, preferably in the range of 0 ° C. to 100 ° C., more preferably in the range of 10 ° C. to 50 ° C.
- the reaction time varies depending on the type of base used and the reaction temperature, but is usually preferably in the range of 1 minute to 48 hours, preferably in the range of 30 minutes to 24 hours.
- an acylating agent can be added as needed.
- the "acylating agent” include acetic anhydride, chloride acetate, and phenoxyacetic anhydride.
- the acylating agent can also be used, for example, diluted with an appropriate solvent so as to have a concentration in the range of 0.1% to 30%.
- the solvent is not particularly limited as long as it does not participate in the reaction, and examples thereof include dichloromethane, acetonitrile, tetrahydrofuran, alcohols (ethanol, isopropanol, trifluoroethanol, etc.), water, or a mixture thereof.
- bases such as pyridine, lutidine, colisine, triethylamine, diisopropylethylamine, and N-ethylmorpholine can be used together with the acylating agent.
- the amount of the acylating agent used is preferably in the range of 0.1 molar equivalent to 10000 molar equivalent, and more preferably in the range of 1 molar equivalent to 1000 molar equivalent.
- the amount of the base used is, for example, preferably in the range of 0.1 molar equivalent to 100 molar equivalent, preferably in the range of 1 molar equivalent to 10 molar equivalent, relative to 1 mol of the acylating agent.
- the reaction temperature of this reaction is preferably in the range of 10 ° C.
- reaction time varies depending on, for example, the type of acylating agent used and the reaction temperature, but is usually preferably in the range of 0.1 minutes to 24 hours, preferably in the range of 1 minute to 5 hours.
- Step C In the compound (VII) produced in step B, a step of removing a protecting group using a deprotecting agent to produce a compound represented by the general formula (IX).
- a step of removing a protecting group using a deprotecting agent to produce a compound represented by the general formula (IX).
- This step can be carried out by allowing a deprotecting agent to act on compound (VII).
- Examples of the "deprotecting agent” include concentrated aqueous ammonia and methylamine.
- the "deprotecting agent” that can be used in this step is, for example, diluted with water, methanol, ethanol, isopropyl alcohol, acetonitrile, tetrahydrofuran, DMF, N, N-dimethylimidazolidone, N-methylpiperidone or a mixed solvent thereof. It can also be used. Of these, ethanol is preferable.
- the amount of the deprotectant to be used is, for example, preferably in the range of 1 mol equivalent to 100,000 molar equivalents, preferably in the range of 10 molar equivalents to 1000 molar equivalents, relative to 1 mol of compound (VII). Is.
- the reaction temperature is, for example, appropriately in the range of 15 ° C. to 75 ° C., preferably in the range of 40 ° C. to 70 ° C., and more preferably in the range of 50 ° C. to 60 ° C.
- the deprotection reaction time varies depending on the type of compound (VII), reaction temperature, etc., but is appropriately in the range of 10 minutes to 30 hours, preferably in the range of 30 minutes to 24 hours, and more preferably 5 It is in the range of hours to 20 hours.
- Step D A step of producing PMO (I) by allowing an acid to act on the compound (IX) produced in step C.
- This step can be carried out by adding an acid to compound (IX).
- Examples of the "acid” that can be used in this step include trichloroacetic acid, dichloroacetic acid, acetic acid, phosphoric acid, hydrochloric acid and the like.
- the amount of the acid used for example, it is appropriate to use the solution so that the pH is in the range of 0.1 to 4.0, and more preferably in the range of 1.0 to 3.0.
- the solvent is not particularly limited as long as it does not participate in the reaction, and examples thereof include acetonitrile, water, and a mixed solvent thereof.
- the reaction temperature is preferably in the range of 10 ° C. to 50 ° C., more preferably in the range of 20 ° C. to 40 ° C., and even more preferably in the range of 25 ° C. to 35 ° C.
- the deprotection reaction time varies depending on the type of compound (IX), reaction temperature, etc., but is preferably in the range of 0.1 minutes to 5 hours, preferably in the range of 1 minute to 1 hour, and more preferably. Is in the range of 1 to 30 minutes.
- PMO (I) are conventional separation and purification means from the reaction mixture obtained in this step, for example, extraction, concentration, neutralization, filtration, centrifugation, recrystallization, reversed phase column chromatography from C 8 C 18, It can be obtained by using means such as cation exchange column chromatography, anion exchange column chromatography, gel filtration column chromatography, high-speed liquid chromatography, dialysis, and limit filtration alone or in combination, and a desired PMO (I) can be obtained. ) Can be isolated and purified (see, eg, WO 1991/09033).
- a mixed solution of, for example, 20 mM triethylamine / acetate buffer and acetonitrile can be used as the elution solvent.
- purifying PMO (I) by ion exchange chromatography for example, a mixed solution of 1 M saline solution and 10 mM sodium hydroxide aqueous solution can be used.
- the peptide nucleic acid is an antisense oligomer of the present invention having a group represented by the following general formula as a constituent unit. (In the formula, Base is synonymous with the above.)
- Peptide nucleic acids can be produced, for example, according to the following literature. 1) P. E. Nielsen, M. Egholm, R. H. Berg, O. Buchardt, Science, 254, 1497 (1991) 2) M. Egholm, O. Buchardt, P. E. Nielsen, R. H. Berg, Jacs., 114, 1895 (1992) 3) K. L. Dueholm, M. Egholm, C. Behrens, L. Christensen, H. F. Hansen, T. Vulpius, K. H. Petersen, R. H. Berg, P. E. Nielsen, O. Buchardt, J. Org. Chem., 59, 5767 (1994) 4) L.
- the 5'end may be a group according to any of the following chemical formulas (1) to (3). It is preferably (3) -OH.
- the groups represented by the above (1), (2) and (3) are referred to as “group (1)”, “group (2)” and “group (3)”, respectively.
- the stereochemistry of the phosphorus atom may contain a compound that is optically pure.
- a person skilled in the art can obtain a pure optically active substance from a mixture of isomers (International Publication No. 2017/024264).
- the antisense oligomer of the present invention may be synthesized as a pure optically active substance.
- a person skilled in the art can control the synthesis reaction to obtain a pure optically active substance (Publication Patent Publication No. 2018-537952).
- the antisense oligomer of the present invention is a complex with a functional peptide aimed at improving efficacy (for example, a membrane-permeable peptide aimed at improving transport efficiency to target cells). It may be formed (International Publication No. 2008/036127, International Publication No. 2009/005793, International Publication No. 2012/150960, International Publication No. 2016/187425, International Publication No. 2018/118662. , International Publication No. 2018/118599, International Publication No.
- the binding site is not particularly limited, but it is preferable that the 5'-terminal or 3'-terminal of the antisense oligomer is bound to the amino-terminal or carboxyl-terminal of the functional peptide.
- the antisense oligomer of the present invention and the functional peptide may form a complex via a linker.
- the linker is not particularly limited, but the 5'end or 3'end of the antisense oligomer is bound to one end of the linker, and the amino terminus or carboxyl terminus of the functional peptide is bound to the other end of the linker. Is preferable. In addition, additional amino acids may be present between the functional peptide and the linker.
- the antisense oligomer of the present invention can induce skipping of exon 51 with high efficiency even when its length is shorter than that of the antisense oligomer according to the prior art.
- the antisense oligomer of the present invention has excellent solubility while maintaining the activity of inducing skipping of exon 51 with high efficiency.
- the antisense oligomer of the present invention has excellent solubility and safety while maintaining the activity of inducing skipping of exon 51 with high efficiency. Therefore, any DMD patient with a mutation in the dystrophin gene that is subject to skipping exxon 51 (eg, frameshift mutation, missense mutation / nonsense mutation in exson 51, etc.) will have the antisense of the invention.
- oligomers can alleviate the symptoms of muscle dystrophy with high efficiency.
- the symptoms of muscular dystrophy can be alleviated with high efficiency by administering the antisense oligomer of the present invention to a DMD patient having a predetermined mutant dystrophin gene lacking at least an exon in the vicinity of exon 51. ..
- the predetermined mutant dystrophin gene is a dystrophin gene in which the reading frame of an amino acid is modified when at least an exon near the exon 51 is deleted to have a frameshift mutation and the exon 51 is omitted (when skipped). Means.
- DMD patients include, for example, deficiencies of exons 13-50, 29-50, 40-50, 43-50, 45-50, 47-50, 48-50, 49-50, 50, 52, 52-63 and the like. DMD patients with frameshift mutations due to having a loss. More specifically, the pharmaceutical composition containing the antisense oligomer of the present invention is subjected to DMD patients (patients having a mutation that in-frames with exon 51 skipping, for example, exxon 13-50 deletion patients, exxon 29-50).
- Exxon 40-50 deletion patients, Exxon 43-50 deletion patients, Exxon 45-50 deletion patients, Exxon 47-50 deletion patients, Exxon 48-50 deletion patients, Exxon 49-50 deletion patients It is predicted that administration to patients, exxon 50-deficient patients, exxon 52-deficient patients, exxon 52-63-deficient patients, etc.) can alleviate the symptoms of muscular dystrophy with high efficiency.
- administration to patients, exxon 50-deficient patients, exxon 52-deficient patients, exxon 52-63-deficient patients, etc. can alleviate the symptoms of muscular dystrophy with high efficiency.
- the pharmaceutical composition containing the antisense oligomer of the present invention when used, the same therapeutic effect can be obtained even with a small dose as compared with the oligomer according to the prior art, so that side effects can be reduced and it is economical. Is the target.
- the antisense oligomer of the present invention is useful in the preparation of pharmaceutical compositions because it maintains the activity of inducing skipping of exon 51 with high efficiency and has excellent solubility. Furthermore, the antisense oligomer of the present invention is useful as a pharmaceutical composition because it maintains the activity of inducing skipping of Exxon 51 with high efficiency and has excellent solubility and safety. Therefore, as another embodiment, a pharmaceutical composition for treating muscular dystrophy containing the antisense oligomer of the present invention, a pharmaceutically acceptable salt or hydrate thereof as an active ingredient (hereinafter referred to as "the composition of the present invention"). )I will provide a.
- the present invention also provides a method for treating muscular dystrophy, which comprises the step of administering the antisense oligomer of the present invention to a DMD patient.
- the antisense oligomer of the present invention may be administered as the pharmaceutical composition for treating muscular dystrophy.
- the present invention provides the use of the antisense oligomer of the present invention in the production of a pharmaceutical composition for treating muscular dystrophy, and the antisense oligomer of the present invention for use in the treatment of muscular dystrophy.
- Examples of pharmaceutically acceptable salts of the antisense oligomers of the invention contained in the compositions of the invention include alkali metal salts such as sodium salts, potassium salts and lithium salts, calcium salts and magnesium salts.
- Alkaline earth metal salts such as aluminum salts, iron salts, zinc salts, copper salts, nickel salts, cobalt salts; ammonium salts; t-octylamine salts, dibenzylamine salts, morpholin salts, glucosamine salts, phenylglycine Alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N, N'-dibenzylethylenediamine salt, chloroprocine salt, prokine salt, diethanolamine salt, N-benzyl -Organic amine salts such as phenethylamine salts, piperaz
- Inorganic acid salts such as nitrates, perchlorates, sulfates, phosphates; Lower alcan sulfonates such as methanesulfonates, trifluoromethanesulfonates, ethanesulfonates; benzene Alyl sulfonates such as sulfonates, p-toluene sulfonates; organics such as acetates, malates, fumarates, succinates, citrates, tartrates, oxalates, maleates.
- Acid salts examples include glycine salts, lysine salts, arginine salts, ornithine salts, glutamate salts, amino acid salts such as asparaginate salts, and the like. These salts can be produced by known methods. Alternatively, the antisense oligomer of the invention contained in the composition of the invention may be in the form of its hydrate.
- the administration form of the composition of the present invention is not particularly limited as long as it is a pharmaceutically acceptable administration form, and can be selected according to the treatment method, but from the viewpoint of ease of delivery to muscle tissue, intravenously.
- Intra-arterial administration, intra-arterial administration, intramuscular administration, subcutaneous administration, oral administration, intra-tissue administration, transdermal administration and the like are preferable.
- the dosage form that the composition of the present invention can take is not particularly limited, and examples thereof include various injections, oral preparations, drip infusions, inhalants, ointments, lotions, and the like.
- the composition of the invention can include a carrier that facilitates delivery of the oligomer to muscle tissue.
- a carrier is not particularly limited as long as it is pharmaceutically acceptable, and examples thereof include a cationic carrier such as a cationic liposome and a cationic polymer, or a carrier utilizing a viral envelope. ..
- cationic liposomes include liposomes formed containing 2-O- (2-diethylaminoethyl) carbamoyl-1,3-O-dioreoilglycerol and phospholipogen as essential constituents (hereinafter, "liposomes A").
- Examples of the cationic polymer include JetSI (registered trademark) (manufactured by Qbiogene) and Jet-PEI (registered trademark) (polyethyleneimine, manufactured by Qbiogene).
- Examples of the carrier using the virus envelope include GenomeOne (registered trademark) (HVJ-E liposome, manufactured by Ishihara Sangyo Co., Ltd.).
- the pharmaceutical device described in Japanese Patent No. 2924179, the cationic carrier described in Patent Republishing Publication No. 2006/129594 and Patent Republishing Publication No. 2008/096690 can also be used.
- US Pat. Nos. 4,235,871, 4,737,323, WO 96/14057 "New RRC, Liposomes: A practical approach, IRL Press, Oxford (1990) pages 33-" 104 ”etc. can be referred to.
- the concentration of the antisense oligomer of the present invention contained in the composition of the present invention varies depending on the type of carrier and the like, but in one embodiment, the range of 0.1 nM to 100 ⁇ M is appropriate, and the concentration is in the range of 100 nM to 10 ⁇ M. Is preferable.
- the weight ratio of the antisense oligomer of the present invention to the carrier (carrier / antisense oligomer of the present invention) contained in the composition of the present invention varies depending on the properties of the oligomer, the type of the carrier, and the like. The range of 1 to 100 is suitable, and the range of 0.1 to 10 is preferable.
- composition of the present invention may be in the form of an aqueous solution.
- the composition of the present invention contains the antisense oligomer of the present invention in an amount of 2.5 to 500 mg / mL, 5 to 450 mg / mL, 10 to 400 mg / mL, 15 to 350 mg / mL, 20 to 300 mg / mL.
- the composition of the present invention contains the antisense oligomer of the present invention in an amount of 10 to 100 mg / mL, 15 to 95 mg / mL, 20 to 80 mg / mL, 25 to 75 mg / mL, 30 to 70 mg / mL, 35 to 65 mg.
- the composition of the present invention may be in a dry form.
- the composition of the present invention in a dry form containing 125 mg or 250 mg of the antisense oligomer of the present invention in a dry form is added to 0.5 mL to 100 mL of water.
- the antisense oligomer concentration of the present invention of 1.25 mg / mL to 250 mg / mL or 2.5 mg / mL to 500 mg / mL
- preferably mixed with 1 mL to 50 mL of water 2.
- composition of the present invention may optionally contain a pharmaceutically acceptable additive.
- additives include, for example, emulsifying aids (eg, fatty acids having 6 to 22 carbon atoms and pharmaceutically acceptable salts thereof, albumin, dextran), stabilizers (eg, cholesterol, phosphatidic acid, sucrose, mannitol, etc.).
- Sorbitol, xylitol Sorbitol, xylitol
- isotonic agents eg sodium chloride, glucose, maltose, lactose, sucrose, trehalose, mannitol, sorbitol, xylitol
- pH regulators eg hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, sodium hydroxide
- Potassium hydroxide Triethanolamine
- the composition of the present invention can be prepared by adding the antisense oligomer of the present invention to the dispersion liquid of the carrier and stirring appropriately. Further, the additive can be added in an appropriate step before or after the addition of the antisense oligomer of the present invention.
- the aqueous solvent that can be used when adding the antisense oligomer of the present invention is not particularly limited as long as it is pharmaceutically acceptable, and for example, water for injection. , Distilled water for injection, electrolyte solution such as physiological saline, glucose solution, sugar solution such as maltose solution, and the like. Further, conditions such as pH and temperature in such a case can be appropriately selected by those skilled in the art.
- the composition of the present invention can be, for example, a liquid preparation or a lyophilized preparation thereof.
- the lyophilized preparation can be prepared by freeze-drying the composition of the present invention having the form of a liquid agent by a conventional method. For example, after appropriately sterilizing the composition of the present invention having the form of a liquid preparation, a predetermined amount is dispensed into a vial, and pre-freezing is performed under the condition of about -40 to -20 ° C for about 2 hours. Then, the primary drying can be performed at about 0 to 10 ° C. under reduced pressure, and then the secondary drying can be performed at about 15 to 25 ° C. under reduced pressure to freeze-dry. Then, in general, the inside of the vial can be replaced with nitrogen gas and stoppered to obtain a lyophilized preparation of the composition of the present invention.
- the lyophilized preparation of the composition of the present invention can generally be redissolved and used by adding an arbitrary suitable solution (redissolving solution).
- suitable solution redissolving solution
- examples of such a redissolved solution include water for injection, physiological saline, and other general infusion solutions.
- the amount of this redissolved solution varies depending on the intended use and is not particularly limited, but 0.5 to 2 times the amount of the solution before freeze-drying, or 500 mL or less is appropriate.
- the dose of the composition of the present invention should be determined in consideration of the type, dosage form, age, body weight, and other conditions of the patient, the route of administration, and the nature and degree of the disease contained in the antisense oligomer of the present invention.
- the amount of the antisense oligomer of the present invention for adults is generally in the range of 0.1 mg to 10 g / human, preferably in the range of 1 mg to 1 g / human per day. Is the target. This value may also vary depending on the type of target disease, dosage form, and target molecule. Therefore, in some cases, less than this may be sufficient, and conversely, higher doses may be required. It can also be administered once to several times a day or at intervals of one to several days.
- composition of the present invention a pharmaceutical composition containing a vector capable of expressing the oligonucleotide of the present invention and the above-mentioned carrier can be mentioned.
- Such an expression vector may express a plurality of oligonucleotides of the present invention.
- a pharmaceutically acceptable additive can be added to the composition.
- concentration of the expression vector contained in the composition varies depending on the type of carrier and the like, but in one embodiment, the range of 0.1 nM to 100 ⁇ M is appropriate, and the concentration of 100 nM to 10 ⁇ M is preferable. ..
- the weight ratio of the expression vector to the carrier (carrier / expression vector) contained in the composition varies depending on the properties of the expression vector, the type of carrier, etc., but is appropriately in the range of 0.1 to 100, and is 0. The range of 1 to 10 is preferable. Further, the content of the carrier contained in the composition is the same as that of the composition of the present invention containing the antisense oligomer of the present invention, and the preparation method thereof is also the case of the composition of the present invention. Is similar to.
- Example 1 Synthesis of antisense oligomer
- the table also shows the theoretical value of the molecular weight of each antisense oligomer and the measured value by ESI-TOF-MS.
- H51_67-81_131-142 is used when the base at the 5'end of exon 51 of the human dystrophin gene is the first base and the bases following the 3'side are numbered in order. It is shown that the antisense oligomer targets the sequences of the 67th to 81st bases and the 131st to 142nd bases. The sequence of the base before the -1st in the target base sequence is the base sequence in the intron 50. The nucleotide sequence containing the sequence of exon 51 of the human wild-type dystrophin gene and the sequence near the 3'end of intron 50 is shown in SEQ ID NO: 128.
- Example 2 Exon skipping activity test of antisense oligomer
- Test method Tables 1 and 2 for 3.5 ⁇ 10 5 RD cells human dystrophin myoma cell line, CCL-136, purchased from ATCC
- Each of the antisense oligomers of 0.3 to 120 ⁇ M was introduced by Nucleoctor II (Lonza) using Amaxa Cell Line Nucleofector Kit L. T-030 was used as the pulse program for introduction.
- the RD cells after introduction are mixed in 2 mL of Eagle's minimal essential medium (EMEM) medium (manufactured by Sigma, the same applies hereinafter) containing 10% fetal bovine serum (FBS) (manufactured by Invitrogen) at 37 ° C. and 5% CO.
- EMEM Eagle's minimal essential medium
- FBS fetal bovine serum
- the cells were cultured under two conditions for three nights.
- PBS manufactured by Nissui, the same applies hereinafter
- Buffer RA1 manufactured by Takara Bio Inc.
- 2-mercaptoethanol manufactured by Nacalai Tesque
- One-Step RT-PCR was performed on 400 ng of the extracted total RNA using a QIAGEN OneStep RT-PCR Kit (manufactured by Qiagen) and a thermal cycler. Reaction solutions were prepared according to the protocol attached to the kit. As the thermal cycler, TakaRa PCR Thermal Cycler Dice Touch (manufactured by Takara Bio Inc.) was used. The RT-PCR program used is as follows.
- the nucleotide sequences of the forward and reverse primers used for RT-PCR are as follows. Forward primer: 5'-CTGAGTGGAAGGCGGTAAAC -3'(SEQ ID NO: 95) Reverse primer: 5'-GAAGTTTCAGGGCCAAGTCA -3'(SEQ ID NO: 96)
- Example 3 Solubility test of antisense oligomer
- Solubility tests of 7, 8, 10, 16, 21, 24, 31, 42, 67, 76 and 90 antisense oligomers in saline were conducted to further verify their usefulness in pharmaceutical applications. ..
- Test method 57 ⁇ L of water for injection was added to a sample bottle containing 5.7 mg of each of the above antisense oligomers, dissolved using ultrasonic waves and vortex, and then 57 ⁇ L of double-concentration physiological saline was added to vortex. was stirred to prepare a 50 mg / mL aqueous saline solution. The mixture was allowed to stand at room temperature for 24 hours, and after standing, the presence or absence of precipitation was visually confirmed, and those in which no precipitation was observed were evaluated as highly soluble antisense oligomers.
- Example 4 Safety evaluation of antisense oligomer
- PMO No. Safety evaluations were performed on 16, 21, 42, and 90 in order to verify the safety in pharmaceutical applications.
- the antisense oligomer of the present invention has an activity of inducing skipping of exon 51 of the dystrophin gene with high efficiency, and has excellent physical properties and safety as a pharmaceutical.
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Abstract
Description
[1]
下記(a1)~(d1):
(a1)配列番号1~89及び91~93のいずれかの塩基配列を含むアンチセンスオリゴマー;
(b1)配列番号1~89及び91~93のいずれかの塩基配列に対して1~5個の塩基が欠失、置換、挿入、および/または付加された塩基配列を含み、かつヒトジストロフィン遺伝子のエクソン51のスキッピングを誘導する活性を有するアンチセンスオリゴマー;
(c1)配列番号1~89及び91~93のいずれかの塩基配列に対して、80%以上の配列同一性を有する塩基配列を含み、かつヒトジストロフィン遺伝子のエクソン51のスキッピングを誘導する活性を有するアンチセンスオリゴマー;および
(d1)配列番号1~89及び91~93のいずれかの塩基配列と相補的な塩基配列からなるオリゴヌクレオチドとストリンジェントな条件下でハイブリダイズするアンチセンスオリゴマーであって、かつヒトジストロフィン遺伝子のエクソン51のスキッピングを誘導する活性を有するアンチセンスオリゴマー
からなる群から選択されるアンチセンスオリゴマー(但し、配列番号90および97~126のいずれかの塩基配列からなるアンチセンスオリゴマーを除く)もしくはその医薬的に許容可能な塩またはそれらの水和物。
[2]
以下の(e)~(h):
(e)配列番号1~89及び91~93のいずれかの塩基配列からなるアンチセンスオリゴマー;
(f)配列番号1~89及び91~93のいずれかの塩基配列に対して1~5個の塩基が欠失および/または置換された塩基配列からなり、かつヒトジストロフィン遺伝子のエクソン51のスキッピングを誘導する活性を有するアンチセンスオリゴマー;
(g)配列番号1~89及び91~93のいずれかの塩基配列に対して、80%以上の配列同一性を有する塩基配列からなり、かつヒトジストロフィン遺伝子のエクソン51のスキッピングを誘導する活性を有するアンチセンスオリゴマー;および
(h)配列番号1~89及び91~93のいずれかの塩基配列と相補的な塩基配列からなるオリゴヌクレオチドと高ストリンジェントな条件下でハイブリダイズするアンチセンスオリゴマーであって、かつヒトジストロフィン遺伝子のエクソン51のスキッピングを誘導する活性を有するアンチセンスオリゴマー
からなる群から選択されるアンチセンスオリゴマー(但し、配列番号90および97~126のいずれかの塩基配列からなるアンチセンスオリゴマーを除く)もしくはその医薬的に許容可能な塩またはそれらの水和物。
[3]
前記アンチセンスオリゴマーが、
配列番号1~89及び91~93のいずれかの塩基配列に対して、90%以上の配列同一性を有するヌクレオチド配列を有し、かつヒトジストロフィン遺伝子のエクソン51のスキッピングを誘導する活性を有するアンチセンスオリゴマー
である、上記[1]又は[2]に記載のアンチセンスオリゴマーもしくはその医薬的に許容可能な塩またはそれらの水和物。
[4]
前記アンチセンスオリゴマーが、
(a2)配列番号7、8、10、16、21、24、31、42、67及び76のいずれかの塩基配列を含むアンチセンスオリゴマー;
(b2)配列番号7、8、10、16、21、24、31、42、67及び76のいずれかの塩基配列に対して1~5個の塩基が欠失、置換、挿入、および/または付加された塩基配列を含み、かつヒトジストロフィン遺伝子のエクソン51のスキッピングを誘導する活性を有するアンチセンスオリゴマー;
(c2)配列番号7、8、10、16、21、24、31、42、67及び76のいずれかの塩基配列に対して、80%以上の配列同一性を有する塩基配列を含み、かつヒトジストロフィン遺伝子のエクソン51のスキッピングを誘導する活性を有するアンチセンスオリゴマー;および
(d2)配列番号7、8、10、16、21、24、31、42、67及び76のいずれかの塩基配列と相補的な塩基配列からなるオリゴヌクレオチドとストリンジェントな条件下でハイブリダイズするアンチセンスオリゴマーであって、かつヒトジストロフィン遺伝子のエクソン51のスキッピングを誘導する活性を有するアンチセンスオリゴマー
からなる群から選択されるアンチセンスオリゴマー
である、上記[1]に記載のアンチセンスオリゴマーもしくはその医薬的に許容可能な塩またはそれらの水和物。
[5]
オリゴヌクレオチドである、上記[1]~[4]のいずれかに記載のアンチセンスオリゴマーもしくはその医薬的に許容可能な塩またはそれらの水和物。
[6]
前記オリゴヌクレオチドを構成する少なくとも1つのヌクレオチドの糖部分及び/又はリン酸結合部分が修飾されている、上記[5]に記載のアンチセンスオリゴマーもしくはその医薬的に許容可能な塩またはそれらの水和物。
[7]
前記オリゴヌクレオチドを構成する少なくとも1つのヌクレオチドの糖部分は、2’位の-OH基が、OR、R、R’OR、SH、SR、NH2、NHR、NR2、N3、CN、F、Cl、Br及びIからなる群より選択されるいずれかの基(上記Rは、アルキル又はアリールを示し、上記R’は、アルキレンを示す。)で置換されたリボースである、上記[5]又は[6]に記載のアンチセンスオリゴマーもしくはその医薬的に許容可能な塩またはそれらの水和物。
[8]
前記オリゴヌクレオチドを構成する少なくとも1つのヌクレオチドのリン酸結合部分が、ホスホロチオエート結合、ホスホロジチオエート結合、アルキルホスホネート結合、ホスホロアミデート結合、及びボラノフォスフェート結合からなる群から選択されるいずれか1つのものである、上記[5]~[7]のいずれかに記載のアンチセンスオリゴマーもしくはその医薬的に許容可能な塩またはそれらの水和物。
[9]
モルホリノオリゴマーである、上記[1]~[4]のいずれかに記載のアンチセンスオリゴマーもしくはその医薬的に許容可能な塩またはそれらの水和物。
[10]
ホスホロジアミデートモルホリノオリゴマーである、上記[9]に記載のアンチセンスオリゴマーもしくはその医薬的に許容可能な塩またはそれらの水和物。
[11]
5’末端が、下記化学式(1)~(3):
のいずれかの基である、上記[9]または[10]に記載のアンチセンスオリゴマーもしくはその医薬的に許容可能な塩またはそれらの水和物。
[12]
上記[1]~[11]のいずれかに記載のアンチセンスオリゴマーもしくはその医薬的に許容可能な塩またはそれらの水和物を含む、筋ジストロフィー治療用医薬組成物。
[13]
さらに医薬的に許容可能な担体を含む、上記[12]に記載の医薬組成物。
[14]
筋ジストロフィー患者に投与するための上記[12]または[13]に記載の医薬組成物であって、前記患者が、ジストロフィン遺伝子にエクソン51のスキッピングの対象となる変異を有する患者である、医薬組成物。
[15]
前記患者が、少なくともエクソン51近傍のエクソンの欠失によるフレームシフト突然変異を有するとともにエクソン51のスキッピングによりアミノ酸の読み取り枠が修正されるジストロフィン遺伝子を有する、上記[14]に記載の医薬組成物。
[16]
前記患者が、ジストロフィン遺伝子にエクソン13-50、29-50、40-50、43-50、45-50、47-50、48-50、49-50、50、52、52-63の欠失によるフレームシフト突然変異を有する、上記[14]または[15]に記載の医薬組成物。
[17]
前記患者がヒトである、上記[14]~[16]のいずれかに記載の医薬組成物。
[18]
筋ジストロフィー治療用医薬の製造における上記[1]~[11]のいずれかに記載のアンチセンスオリゴマーもしくはその医薬的に許容可能な塩またはそれらの水和物の使用。
[19]
上記[1]~[11]のいずれかに記載のアンチセンスオリゴマーもしくはその医薬的に許容可能な塩またはそれらの水和物の有効量、または上記[12]~[16]のいずれかに記載の医薬組成物を、筋ジストロフィー患者に投与する工程を含む、筋ジストロフィーの治療方法。
[20]
前記患者がヒトである、上記[19]に記載の治療方法。
[21]
筋ジストロフィーの治療に使用するための、上記[1]~[11]のいずれかに記載のアンチセンスオリゴマーもしくはその医薬的に許容可能な塩またはそれらの水和物、または上記[12]~[16]のいずれかに記載の医薬組成物。
[22]
前記治療において、筋ジストロフィー患者がヒトである、上記[21]に記載のアンチセンスオリゴマーもしくはその医薬的に許容可能な塩またはそれらの水和物、または医薬組成物。
本発明は、ヒトジストロフィン遺伝子の第51番目のエクソンを高効率にスキッピングするアンチセンスオリゴマー(以下、「本発明のアンチセンスオリゴマー」という)を提供する。
本発明において、「遺伝子」には、ゲノム遺伝子以外に、cDNA、mRNA前駆体及びmRNAも含まれる。好ましくは、遺伝子は、mRNA前駆体、即ち、pre-mRNAである。
ヒトゲノムにおいて、ヒトジストロフィン遺伝子は遺伝子座Xp21.2に存在する。ヒトジストロフィン遺伝子は、220万塩基対のサイズを有しており、既知のヒト遺伝子としては最大の遺伝子である。但し、ヒトジストロフィン遺伝子のコード領域はわずか14kbに過ぎず、該コード領域は79個のエクソンとしてジストロフィン遺伝子内に分散している(Roberts, RG., et al., Genomics, 16: 536-538 (1993); Koenig, M., et al., Cell 53: 219-228 (1988))。ヒトジストロフィン遺伝子の転写物であるpre-mRNAは、スプライシングを受けて14kbの成熟mRNAを生成する。ヒトの野生型ジストロフィン遺伝子の塩基配列は公知である(GenBank Accession No. NM_004006)。
ヒトの野生型ジストロフィン遺伝子のエクソン51の塩基配列を配列番号127に示す。
本発明のアンチセンスオリゴマーは、ヒトジストロフィン遺伝子のエクソン51のスキッピングにより、DMD型ジストロフィン遺伝子でコードされるタンパク質を、BMD型ジストロフィンタンパク質に改変することを目的として作製されたものである。従って、アンチセンスオリゴマーのエクソンスキッピングの対象となるジストロフィン遺伝子のエクソン51には、野生型だけではなく、変異型も含まれる。
(a1)配列番号1~89及び91~93のいずれかの塩基配列を含むアンチセンスオリゴマー;
(b1)配列番号1~89及び91~93のいずれかの塩基配列に対して1~5個、1~4個、1~3個、1~2個、または1個の塩基が欠失、置換、挿入、及び/又は付加された塩基配列を含み、かつヒトジストロフィン遺伝子のエクソン51のスキッピングを誘導する活性を有するアンチセンスオリゴマー;
(c1)配列番号1~89及び91~93のいずれかの塩基配列に対して、80%以上、84%以上、85%以上、89%以上、90%以上、94%以上、または95%以上の配列同一性を有する塩基配列を含み、かつヒトジストロフィン遺伝子のエクソン51のスキッピングを誘導する活性を有するアンチセンスオリゴマー;および
(d1)配列番号1~89及び91~93のいずれかの塩基配列と相補的な塩基配列からなるオリゴヌクレオチドとストリンジェントな条件下でハイブリダイズするアンチセンスオリゴマーであって、かつヒトジストロフィン遺伝子のエクソン51のスキッピングを誘導する活性を有するアンチセンスオリゴマー
(e)配列番号1~89及び91~93のいずれかの塩基配列からなるアンチセンスオリゴマー;
(f)配列番号1~89及び91~93のいずれかの塩基配列に対して1~5個、1~4個、1~3個、1~2個、または1個の塩基が欠失および/または置換された塩基配列からなり、かつヒトジストロフィン遺伝子のエクソン51のスキッピングを誘導する活性を有するアンチセンスオリゴマー;
(g)配列番号1~89及び91~93のいずれかの塩基配列に対して、80%以上、84%以上、85%以上、89%以上、90%以上、94%以上、または95%以上の配列同一性を有する塩基配列からなり、かつヒトジストロフィン遺伝子のエクソン51のスキッピングを誘導する活性を有するアンチセンスオリゴマー;および
(h)配列番号1~89及び91~93のいずれかの塩基配列と相補的な塩基配列からなるオリゴヌクレオチドと高ストリンジェントな条件下でハイブリダイズするアンチセンスオリゴマーであって、かつヒトジストロフィン遺伝子のエクソン51のスキッピングを誘導する活性を有するアンチセンスオリゴマー
(a2)配列番号7、8、10、16、21、24、31、42、67及び76のいずれかの塩基配列を含むアンチセンスオリゴマー;
(b2)配列番号7、8、10、16、21、24、31、42、67及び76のいずれかの塩基配列に対して1~5個の塩基が欠失、置換、挿入、および/または付加された塩基配列を含み、かつヒトジストロフィン遺伝子のエクソン51のスキッピングを誘導する活性を有するアンチセンスオリゴマー;
(c2)配列番号7、8、10、16、21、24、31、42、67及び76のいずれかの塩基配列に対して、80%以上の配列同一性を有する塩基配列を含み、かつヒトジストロフィン遺伝子のエクソン51のスキッピングを誘導する活性を有するアンチセンスオリゴマー;および
(d2)配列番号7、8、10、16、21、24、31、42、67及び76のいずれかの塩基配列と相補的な塩基配列からなるオリゴヌクレオチドとストリンジェントな条件下でハイブリダイズするアンチセンスオリゴマーであって、かつヒトジストロフィン遺伝子のエクソン51のスキッピングを誘導する活性を有するアンチセンスオリゴマー
からなる群から選択されるアンチセンスオリゴマー。
スキッピング効率ES(単位:%)は、ヒトジストロフィン遺伝子のmRNAを被検細胞から回収し、該mRNAのうち、エクソン51がスキップしたバンドのポリヌクレオチド量「A」と、エクソン51がスキップしなかったバンドのポリヌクレオチド量「B」を測定し、これら「A」及び「B」の測定値に基づき、以下の式(1)に従って計算することができる。スキッピング効率の計算については、国際公開第2012/029986号を参照することができる。
糖のその他の部分の修飾としては、例えば、リボース又はデオキシリボースの4’位のOをSに置換したもの、糖の2’位と4’位を架橋したもの、例えば、LNA(Locked Nucleic Acid)又はENA(2’-O,4’-C-Ethylene-bridged Nucleic Acids)などが挙げられるが、これらに限定されるものではない。
本発明において、シクロアルキルとしては、炭素数5~12のシクロアルキルが好ましい。具体的には、例えば、シクロペンチル、シクロヘキシル、シクロヘプチル、シクロオクチル、シクロデシル、シクロドデシルが挙げられる。
本発明において、ハロゲンとしては、フッ素、塩素、臭素、ヨウ素を挙げることができる。
アルコキシとしては、直鎖状または分枝鎖状の炭素数1~6のアルコキシ、例えば、メトキシ、エトキシ、n-プロポキシ、イソプロポキシ、n-ブトキシ、イソブトキシ、sec-ブトキシ、tert-ブトキシ、n-ペンチルオキシ、イソペンチルオキシ、n-ヘキシルオキシ、イソヘキシルオキシ等を挙げることができる。とりわけ、炭素数1~3のアルコキシが好ましい。
本発明において、アルキレンとしては、直鎖状または分枝鎖状の炭素数1~6のアルキレンが好ましい。具体的には、例えば、メチレン、エチレン、トリメチレン、テトラメチレン、ペンタメチレン、ヘキサメチレン、2-(エチル)トリメチレン、1-(メチル)テトラメチレンを挙げることができる。
本発明において、アシルとしては、直鎖状若しくは分枝鎖状のアルカノイル、又はアロイルを挙げることができる。アルカノイルとしては、例えば、ホルミル、アセチル、2-メチルアセチル、2,2-ジメチルアセチル、プロピオニル、ブチリル、イソブチリル、ペンタノイル、2,2-ジメチルプロピオニル、ヘキサノイル等が挙げられる。アロイルとしては、例えば、ベンゾイル、トルオイル、ナフトイルを挙げることができる。かかるアロイルは置換可能な位置において置換されていてもよく、アルキルで置換されていてもよい。
(式中、Baseは、核酸塩基を表す。)
(式中、Xは、-CH2R1、-O-CH2R1、-S-CH2R1、-NR2R3又はFを表し;
R1は、H、アルキルを表し;
R2及びR3は、同一又は異なって、H、アルキル、シクロアルキル、又は、アリールを表し;
Y1は、O、S、CH2又はNR1を表し;
Y2は、O、S又はNR1を表し;
Zは、O又はSを表す。))
(式中、Base、R2、R3は、前記と同義である。)
本発明モルホリノオリゴマーは、かかるオリゴマーを構成する核酸塩基、モルホリノ環部分、リン酸結合部分、3’末端及び/又は5’末端の全部又は一部が修飾されているものを含む。
モルホリノオリゴマーは、例えば、国際公開第1991/009033号、又は国際公開第2009/064471号に従って製造することができる。特に、PMOは、国際公開第2009/064471号に記載の方法に従って製造するか、又は以下に示す方法に従って製造することができる。
PMOの1つの態様として、例えば、次の一般式(I)で表される化合物(以下、PMO(I)という。)を挙げることができる。
[式中、各Base、R2、R3は、前記と同義であり;
nは、1~99の範囲内にある任意の整数であり、好ましくは、19~29、19~28、21~29、21~28または24~28の範囲内にある任意の整数であり、より好ましくは、21~29、21~28または24~28である。]
下記工程に使用されている化合物及び試薬は、PMOの製造に一般的に使用されているものであれば特に限定されない。
また、下記のすべての工程は、液相法又は固相法(マニュアル又は市販の固相自動合成機を用いる)で実施することができる。固相法でPMOを製造する場合、操作手順の簡便化及び合成の正確性の点から自動合成機を用いる方法が望ましい。
次の一般式(II)で表される化合物(以下、化合物(II)という。)に酸を作用させることによって、次の一般式(III)で表される化合物(以下、化合物(III)という。)を製造する工程。
[式中、n、R2、R3は、前記と同義であり;
各BPは,独立して、保護されていてもよい核酸塩基を表し;
Tは、トリチル基、モノメトキシトリチル基、又はジメトキシトリチル基を表し;
Lは、水素、アシル、又は次の一般式(IV)で表される基(以下、基(IV)という。)を表す。]
かかるアミノ基の保護基としては、核酸の保護基として使用されるものであれば特に制限されず、具体的には、例えば、ベンゾイル、4-メトキシベンゾイル、アセチル、プロピオニル、ブチリル、イソブチリル、フェニルアセチル、フェノキシアセチル、4-tert-ブチルフェノキシアセチル、4-イソプロピルフェノキシアセチル、(ジメチルアミノ)メチレンを挙げることができる。水酸基の保護基としては、例えば、2-シアノエチル、4-ニトロフェネチル、フェニルスルホニルエチル、メチルスルホニルエチル、トリメチルシリルエチル、置換可能な任意の位置で1~5個の電子吸引性基で置換されていてもよいフェニル、ジフェニルカルバモイル、ジメチルカルバモイル、ジエチルカルバモイル、メチルフェニルカルバモイル、1-ピロリジニルカルバモイル、モルホリノカルバモイル、4-(tert-ブチルカルボキシ)ベンジル、4-[(ジメチルアミノ)カルボキシ]ベンジル、4-(フェニルカルボキシ)ベンジルを挙げることができる(例えば、国際公開第2009/064471号公報参照)。
「リンカー」としては、通常核酸やモルホリノ核酸誘導体を連結するために使用される公知のものを用いることができるが、例えば、3-アミノプロピル、スクシニル、2,2’-ジエタノールスルホニル、ロングチェーンアルキルアミノ(LCAA)を挙げることができる。
また、前記酸と一緒に、有機アミンを使用することができる。有機アミンとしては、特に限定されるものではないが、例えば、トリエチルアミンを挙げることができる。有機アミンの使用量は、例えば、酸1モルに対して、0.01モル当量~10モル当量の範囲内が適当であり、好ましくは、0.1モル当量~2モル当量の範囲内である。
本工程において酸と有機アミンとの塩又は混合物を使用する場合には、例えば、トリフルオロ酢酸とトリエチルアミンの塩又は混合物を挙げることができ、より具体的には、トリフルオロ酢酸2当量に対してトリエチルアミン1当量を混合したものを挙げることができる。
本工程に使用しうる酸は、0.1%~30%の範囲内の濃度になるように適当な溶媒で希釈して使用することもできる。溶媒としては、反応に関与しなければ特に限定されないが、例えば、ジクロロメタン、アセトニトリル、アルコール類(エタノール、イソプロパノール、トリフルオロエタノールなど)、水又はこれらの混合物を挙げることができる。
反応時間は、使用する酸の種類、反応温度によって異なるが、通常0.1分~24時間の範囲内が適当である。好ましくは、1分~5時間の範囲内である。
本工程に用いる溶媒としては、反応に関与しなければ特に限定されないが、ジクロロメタン、アセトニトリル、アルコール類(エタノール、イソプロパノール、トリフルオロエタノールなど)、水又はこれらの混合物を挙げることができる。反応温度は、例えば、10℃~50℃の範囲内が好ましく、より好ましくは、20℃~40℃の範囲内であり、さらに好ましくは、25℃~35℃の範囲内である。
反応時間は、使用する塩基の種類、反応温度によって異なるが、通常0.1分~24時間の範囲内が適当であり、好ましくは、1分~5時間の範囲内である。
[式中、BP、T、リンカー、固相担体は、前記と同義である。]
次の一般式(V)で表される化合物にアシル化剤を作用させることによって、次の一般式(VI)で表される化合物(以下、化合物(VI)という。)を製造する工程。
[式中、BP、T、リンカーは、前記と同義であり;
R4は、水酸基、ハロゲン、カルボキシル基、又は、アミノを表す。]
特に、次の一般式(VIa)で表される化合物は、化合物(V)と無水コハク酸とを用いてエステル化反応として知られた方法を実施することにより製造することができる。
[式中、BP、Tは、前記と同義である。]
化合物(VI)に縮合剤等を作用させることによって、固相担体と反応させ、化合物(IIa)を製造する工程。
[式中、BP、R4、T、リンカー、固相担体は、前記と同義である。]
本工程は、化合物(VI)と固相担体とを用いて縮合反応として知られた方法により製造することができる。
化合物(II)において、n=1~99(特定の態様ではnは、例えば、2~29、2~28、2~27、2~26、2~25、2~24、2~23、2~22、2~21または2~20であり、好ましくは19~29、19~28、21~29、21~28または24~28の範囲内にある任意の整数であり、より好ましくは、21~29、21~28または24~28である)であって、Lが基(IV)である、次の一般式(IIa2)で表される化合物は、化合物(IIa)を出発原料とし、本明細書に記載のPMOの製法にかかる工程A及び工程Bを所望の回数繰り返し実施することにより製造することができる。
[式中、BP、n、R2、R3、T、リンカー、固相担体は、前記と同義である。]
化合物(III)に塩基存在下にモルホリノモノマー化合物を作用させることによって、次の一般式(VII)で表される化合物(以下、化合物(VII)という。)を製造する工程。
[式中、各BP、L、n、R2、R3、Tは、前記と同義である。]
[式中、BP、R2、R3、Tは前記と同義である。]
本工程に使用しうる「塩基」としては、例えば、ジイソプロピルエチルアミン、トリエチルアミン、又は、N-エチルモルホリンを挙げることができる。塩基の使用量としては、例えば、化合物(III)1モルに対して、1モル当量~1000モル当量の範囲内が適当であり、好ましくは10モル当量~100モル当量の範囲内である。
本工程に使用しうるモルホリノモノマー化合物および塩基は、0.1%~30%の濃度になるように適当な溶媒で希釈して使用することもできる。溶媒としては、反応に関与しなければ特に限定されないが、例えば、N,N-ジメチルイミダゾリドン、N-メチルピペリドン、DMF、ジクロロメタン、アセトニトリル、テロラヒドロフラン、又はこれらの混合物を挙げることができる。
反応時間は、使用する塩基の種類、反応温度によって異なるが、通常1分~48時間の範囲内が適当であり、好ましくは、30分~24時間の範囲内である。
また、必要であれば、アシル化剤と一緒に、例えば、ピリジン、ルチジン、コリジン、トリエチルアミン、ジイソプロピルエチルアミン、N-エチルモルホリン等の塩基を使用することができる。アシル化剤の使用量としては、0.1モル当量~10000モル当量の範囲内が好ましく、1モル当量~1000モル当量の範囲内がより好ましい。塩基の使用量としては、例えば、アシル化剤1モルに対して、0.1モル当量~100モル当量の範囲内が適当であり、好ましくは1モル当量~10モル当量の範囲内である。
本反応の反応温度は、10℃~50℃の範囲内が好ましく、より好ましくは、10℃~50℃の範囲内が好ましく、より好ましくは、20℃~40℃の範囲内であり、さらに好ましくは、25℃~35℃の範囲内である。反応時間は、例えば、使用するアシル化剤の種類、反応温度によって異なるが、通常0.1分~24時間の範囲内が適当であり、好ましくは、1分から5時間の範囲内である。
工程Bにおいて製造される化合物(VII)において、脱保護剤を用いて保護基を脱離し、一般式(IX)で表される化合物を製造する工程。
[式中、Base、BP、L、n、R2、R3、Tは、前記と同義である。]
逆相クロマトグラフィーを用いてPMO(I)を精製する場合には、溶出溶媒として、例えば20mMのトリエチルアミン/酢酸緩衝液とアセトニトリルの混合溶液を使用することができる。
また、イオン交換クロマトグラフィーを用いてPMO(I)を精製する場合には、例えば、1Mの食塩水と10mMの水酸化ナトリウム水溶液の混合溶液を使用することができる。
1)P. E. Nielsen, M. Egholm, R. H. Berg, O. Buchardt,Science, 254, 1497 (1991)
2)M. Egholm, O. Buchardt, P. E. Nielsen, R. H. Berg,Jacs., 114, 1895 (1992)
3)K. L. Dueholm, M. Egholm, C. Behrens, L. Christensen, H. F. Hansen, T. Vulpius, K. H. Petersen, R. H. Berg, P. E. Nielsen, O. Buchardt,J. Org. Chem., 59, 5767 (1994)
4)L. Christensen, R. Fitzpatrick, B. Gildea, K. H. Petersen, H. F. Hansen, T. Koch, M. Egholm,O. Buchardt, P. E. Nielsen, J. Coull, R. H. Berg, J. Pept. Sci., 1, 175 (1995)
5)T. Koch, H. F. Hansen, P. Andersen, T. Larsen, H. G. Batz, K. Otteson, H. Orum, J. Pept. Res., 49, 80 (1997)
以下、上記(1)、(2)及び(3)で示される基を、それぞれ「基(1)」、「基(2)」及び「基(3)」と呼ぶ。
本発明のアンチセンスオリゴマーは、有効性の向上を目的とした機能性ペプチド(例えば、標的細胞への輸送効率の向上を目的とした膜透過性ペプチド)との複合体を形成しているものであってもよい(国際公開第2008/036127号、国際公開第2009/005793号、国際公開第2012/150960号、国際公開第2016/187425号、国際公開第2018/118662号、国際公開第2018/118599号、国際公開第2018/118627号、J. D. Ramsey, N. H. Flynn, Pharmacology & Therapeutics 154, 78-86 (2015)、M. K. Tsoumpra et al., EBioMedicine, 45, 630-645 (2019))。結合部位は特に限定されないが、アンチセンスオリゴマーの5’端または3’端と機能性ペプチドのアミノ末端またはカルボキシル末端が結合していることが好ましい。
また、別の態様として、本発明のアンチセンスオリゴマーと機能性ペプチドは、リンカーを介して複合体を形成していてもよい。リンカーは特に限定されないが、アンチセンスオリゴマーの5’端または3’端とリンカーの一方の端が結合し、機能性ペプチドのアミノ末端またはカルボキシル末端とリンカーのもう一方の端が結合していることが好ましい。また、機能性ペプチドとリンカーの間には、付加的なアミノ酸が存在していてもよい。
本発明のアンチセンスオリゴマーは、従来技術に係るアンチセンスオリゴマーと比較してその長さが短い場合であっても、エクソン51のスキッピングを高効率に誘導することができる。また、本発明のアンチセンスオリゴマーは、エクソン51のスキッピングを高効率に誘導する活性を維持しつつ、かつ優れた溶解性を有する。さらに、本発明のアンチセンスオリゴマーは、エクソン51のスキッピングを高効率に誘導する活性を維持しつつ、かつ優れた溶解性および安全性を有する。したがって、ジストロフィン遺伝子にエクソン51のスキッピングの対象となる変異(例えば、フレームシフト突然変異、エクソン51の中でのミスセンス突然変異/ナンセンス突然変異など)を有するDMD患者であれば、本発明のアンチセンスオリゴマーを投与することによって、高効率に筋ジストロフィーの症状を緩和することができると予測される。例えば、少なくともエクソン51近傍のエクソンを欠失した所定の変異ジストロフィン遺伝子を有するDMD患者に本発明のアンチセンスオリゴマーを投与することによって、高効率に筋ジストロフィーの症状を緩和することができると予測される。なお、所定の変異ジストロフィン遺伝子とは、少なくともエクソン51近傍のエクソンを欠失してフレームシフト突然変異を有するとともにエクソン51を省いた場合(スキップした場合)にアミノ酸の読み取り枠が修正されるジストロフィン遺伝子を意味する。DMD患者としては、例えば、エクソン13-50、29-50、40-50、43-50、45-50、47-50、48-50、49-50、50、52、52-63等の欠失を有することによるフレームシフト突然変異を有するDMD患者が挙げられる。
より具体的に述べると、本発明のアンチセンスオリゴマーを含む医薬組成物をDMD患者(エクソン51スキッピングでin-frame化する変異を有する患者、例えば、エクソン13-50欠失患者、エクソン29-50欠失患者、エクソン40-50欠失患者、エクソン43-50欠失患者、エクソン45-50欠失患者、エクソン47-50欠失患者、エクソン48-50欠失患者、エクソン49-50欠失患者、エクソン50欠失患者、エクソン52欠失患者、エクソン52-63欠失患者等)に投与することにより、高効率に筋ジストロフィーの症状を緩和することができると予測される。例えば、本発明のアンチセンスオリゴマーを含む医薬組成物を用いる場合、従来技術に係るオリゴマーと比べて少量の投与量でも同程度の治療効果を得られるため、副作用を軽減することができ、かつ経済的である。
また、本発明のアンチセンスオリゴマーは、エクソン51のスキッピングを高効率に誘導する活性を維持しつつ、かつ優れた溶解性を有するため、医薬組成物の調製において有用である。さらに、本発明のアンチセンスオリゴマーは、エクソン51のスキッピングを高効率に誘導する活性を維持しつつ、かつ優れた溶解性および安全性を有するため、医薬組成物として有用である。
そこで、別の実施態様として、本発明のアンチセンスオリゴマー、その医薬的に許容可能な塩又は水和物を有効成分とする、筋ジストロフィー治療用医薬組成物(以下、「本発明の組成物」という)を提供する。
また、本発明は、本発明のアンチセンスオリゴマーを、DMD患者に投与する工程を含む、筋ジストロフィーの治療方法を提供する。
当該治療方法において、本発明のアンチセンスオリゴマーは、前記筋ジストロフィー治療用医薬組成物として投与してもよい。
さらに、本発明は、筋ジストロフィー治療用医薬組成物の製造における本発明のアンチセンスオリゴマーの使用、及び筋ジストロフィー治療に使用するための本発明のアンチセンスオリゴマーを提供する。
詳細については米国特許第4,235,871号、同第4,737,323号、国際公開第96/14057号、“New RRC, Liposomes: A practical approach, IRL Press, Oxford (1990) pages 33-104”等を参照することができる。
国際公開第2015/137409号に記載の方法に従い、ヒトジストロフィン遺伝子のエクソン51および/またはその5’側隣接イントロンであるイントロン50の一部の塩基配列を標的とする、表1に示すアンチセンスオリゴマー(PMO No.1~93(配列番号1~93))を合成した。表中には、各アンチセンスオリゴマーの分子量の理論値およびESI-TOF-MSによる実測値も示す。
表1中において、例えば「H51_67-81_131-142」は、ヒトジストロフィン遺伝子のエクソン51の5’末端の塩基を1番目の塩基とし、3’側に続く塩基に順番に番号を付した場合に、アンチセンスオリゴマーが67番目~81番目の塩基の配列および131番目~142番目の塩基の配列を標的とするものであることを示す。なお、標的塩基配列中の-1番目以前の塩基の配列は、イントロン50中の塩基配列である。ヒトの野生型ジストロフィン遺伝子のエクソン51の配列とイントロン50の3’端付近の配列とを含む塩基配列を配列番号128に示す。
ヒトジストロフィン遺伝子のエクソン51スキッピングのIn vitro試験
(1)試験方法
RD細胞(ヒト横紋筋肉腫細胞株、CCL-136、ATCCより購入)3.5×105個に対して、表1、2の各アンチセンスオリゴマー0.3~120 μMをAmaxa Cell Line Nucleofector Kit Lを用いてNucleofector II(Lonza)により導入した。導入のためのパルスプログラムはT-030を用いた。
培養後のRD細胞をPBS(ニッスイ社製、以下同じ)で1回洗浄した後、1%の2-メルカプトエタノール(ナカライテスク社製)を含むBuffer RA1(タカラバイオ社製)を350 μL細胞に添加し、数分間室温に放置して細胞を溶解させ、NucleoSpin(登録商標) Filter(タカラバイオ社製)上に回収した。11,000×gで1分間遠心し、ホモジネートを作製した。NucleoSpin(登録商標) RNA(タカラバイオ社製)に添付のプロトコルに従って全RNAを抽出した。抽出した全RNAの濃度はNanoDrop ONE(Thermo Fisher社製)を用いて測定した。
50℃、30分間:逆転写反応
95℃、15分間:ポリメラーゼ活性化、逆転写酵素不活性化、cDNA熱変性
[94℃、30秒間;60℃、30秒間;72℃、1分間]×35サイクル:PCR増幅
72℃、10分間:最終伸長反応
フォワードプライマー:5’- CTGAGTGGAAGGCGGTAAAC -3’ (配列番号95)
リバースプライマー:5’- GAAGTTTCAGGGCCAAGTCA -3’ (配列番号96)
エクソン51がスキップしたバンドのポリヌクレオチド量「A」と、エクソン51がスキップしなかったバンドのポリヌクレオチド量「B」を、バンドのシグナル強度として測定した。これら「A」及び「B」の測定値に基づき、上述の式(1)に従って、スキッピング効率を求めた。
各アンチセンスオリゴマーについて得られたエクソン51のスキッピング効率の結果を図1~18に示す。なお、直接的または間接的な比較対照として、エクソン51のスキッピング薬であるeteplirsen(WHO Drug Information 24, 2, 137-139 (2010), Proposed INN List 103)と同一の塩基配列を有し、かつ同一の5’末端修飾:すなわち5’末端が上記の基(1)を有しており、そのため全体の構造が同一である表2に示すアンチセンスオリゴマー(PMO No.94(配列番号94))を、特開2015-91229号公報に記載の方法に従い合成して用いた。
表1の本発明のアンチセンスオリゴマーは、eteplirsenと全体の構造が同一である表2のアンチセンスオリゴマーと比較してスキッピング効率が顕著に高く、エクソン51を極めて有効にスキッピングさせた。
アンチセンスオリゴマーの生理食塩水に対する溶解性試験
実施例2でスキッピング効率が非常に高かったPMO No.7、8、10、16、21、24、31、42、67、76および90の各アンチセンスオリゴマーについて、医薬用途への有用性をさらに検証するため、生理食塩水に対する溶解性試験を行った。
5.7 mgの上記各アンチセンスオリゴマーの入ったサンプル瓶に注射用水57μLを加え、超音波とボルテックスを使って溶解させた後、2倍濃度生理食塩水57μLを加え、ボルテックスを使って撹拌し50 mg/mLの生理食塩水溶液とした。室温で24時間静置し、静置後に沈澱の有無を目視によって確認し、沈澱が見られなかったものを溶解性が高いアンチセンスオリゴマーと評価した。
試験した各アンチセンスオリゴマーのうち、PMO No.7、8、10、16、21、24、31、42、67および76の各アンチセンスオリゴマーは生理食塩水に対して50 mg/mL以上の溶解性を示した。
この結果から、これらのアンチセンスオリゴマーは、エクソン51のスキッピング効率が顕著に高く、生理食塩水に対する溶解性も高いため、医薬として利用価値が高いアンチセンスオリゴマーであることがわかった。
実施例3で生理食塩水に対する溶解性試験を実施した各アンチセンスオリゴマーのうち、PMO No.16、21、42、90について、医薬用途での安全性を検証するため、安全性評価を行った。
各アンチセンスオリゴマーを生理食塩水に溶解し、C57BL/6N雄性6週齢マウスの尾静脈内へ投与した。翌日、マウスより血清を回収し、血中のアスパラギン酸アミノトランスフェラーゼ(AST)値、アラニンアミノトランスフェラーゼ(ALT)値、尿素窒素量(BUN)値およびクレアチニン値を測定した。媒体として使用した生理食塩水のみを投与したマウスもしくは無処置マウスの対照群での測定値を正常値とし、統計学的有意差検定(Studentのt検定またはDunnettの検定)を行って各アンチセンスオリゴマーの投与群でp<0.05の有意水準で値に有意に上昇が見られた場合、異常値と判定した。各検定には、統計解析システムSAS(登録商標、SAS Institute社)バージョン9.3を用いた。1000 mg/kgの用量においてAST値、ALT値、BUN値およびクレアチニン値のいずれにも異常値が見られない場合、安全性が高いアンチセンスオリゴマーであると判断した。
PMO No.16、21、42の各アンチセンスオリゴマーについては、1000 mg/kgの用量においてAST値、ALT値、BUN値およびクレアチニン値のいずれにも異常値が見られず、安全性が高い(具体的には、腎臓および肝臓の機能への影響がないか、影響を与える可能性が極めて低い)アンチセンスオリゴマーであることを確認した。それぞれの結果について、図19~21に示す。なお、p<0.05の有意水準で有意に上昇が見られた値(異常値)にはp値を示す。
Claims (22)
- 下記(a1)~(d1):
(a1)配列番号1~89及び91~93のいずれかの塩基配列を含むアンチセンスオリゴマー;
(b1)配列番号1~89及び91~93のいずれかの塩基配列に対して1~5個の塩基が欠失、置換、挿入、および/または付加された塩基配列を含み、かつヒトジストロフィン遺伝子のエクソン51のスキッピングを誘導する活性を有するアンチセンスオリゴマー;
(c1)配列番号1~89及び91~93のいずれかの塩基配列に対して、80%以上の配列同一性を有する塩基配列を含み、かつヒトジストロフィン遺伝子のエクソン51のスキッピングを誘導する活性を有するアンチセンスオリゴマー;および
(d1)配列番号1~89及び91~93のいずれかの塩基配列と相補的な塩基配列からなるオリゴヌクレオチドとストリンジェントな条件下でハイブリダイズするアンチセンスオリゴマーであって、かつヒトジストロフィン遺伝子のエクソン51のスキッピングを誘導する活性を有するアンチセンスオリゴマー
からなる群から選択されるアンチセンスオリゴマー(但し、配列番号90および97~126のいずれかの塩基配列からなるアンチセンスオリゴマーを除く)もしくはその医薬的に許容可能な塩またはそれらの水和物。 - 以下の(e)~(h):
(e)配列番号1~89及び91~93のいずれかの塩基配列からなるアンチセンスオリゴマー;
(f)配列番号1~89及び91~93のいずれかの塩基配列に対して1~5個の塩基が欠失および/または置換された塩基配列からなり、かつヒトジストロフィン遺伝子のエクソン51のスキッピングを誘導する活性を有するアンチセンスオリゴマー;
(g)配列番号1~89及び91~93のいずれかの塩基配列に対して、80%以上の配列同一性を有する塩基配列からなり、かつヒトジストロフィン遺伝子のエクソン51のスキッピングを誘導する活性を有するアンチセンスオリゴマー;および
(h)配列番号1~89及び91~93のいずれかの塩基配列と相補的な塩基配列からなるオリゴヌクレオチドと高ストリンジェントな条件下でハイブリダイズするアンチセンスオリゴマーであって、かつヒトジストロフィン遺伝子のエクソン51のスキッピングを誘導する活性を有するアンチセンスオリゴマー
からなる群から選択されるアンチセンスオリゴマー(但し、配列番号90および97~126のいずれかの塩基配列からなるアンチセンスオリゴマーを除く)もしくはその医薬的に許容可能な塩またはそれらの水和物。 - 前記アンチセンスオリゴマーが、
配列番号1~89及び91~93のいずれかの塩基配列に対して、90%以上の配列同一性を有するヌクレオチド配列を有し、かつヒトジストロフィン遺伝子のエクソン51のスキッピングを誘導する活性を有するアンチセンスオリゴマー
である、請求項1又は2に記載のアンチセンスオリゴマーもしくはその医薬的に許容可能な塩またはそれらの水和物。 - 前記アンチセンスオリゴマーが、
(a2)配列番号7、8、10、16、21、24、31、42、67及び76のいずれかの塩基配列を含むアンチセンスオリゴマー;
(b2)配列番号7、8、10、16、21、24、31、42、67及び76のいずれかの塩基配列に対して1~5個の塩基が欠失、置換、挿入、および/または付加された塩基配列を含み、かつヒトジストロフィン遺伝子のエクソン51のスキッピングを誘導する活性を有するアンチセンスオリゴマー;
(c2)配列番号7、8、10、16、21、24、31、42、67及び76のいずれかの塩基配列に対して、80%以上の配列同一性を有する塩基配列を含み、かつヒトジストロフィン遺伝子のエクソン51のスキッピングを誘導する活性を有するアンチセンスオリゴマー;および
(d2)配列番号7、8、10、16、21、24、31、42、67及び76のいずれかの塩基配列と相補的な塩基配列からなるオリゴヌクレオチドとストリンジェントな条件下でハイブリダイズするアンチセンスオリゴマーであって、かつヒトジストロフィン遺伝子のエクソン51のスキッピングを誘導する活性を有するアンチセンスオリゴマー
からなる群から選択されるアンチセンスオリゴマー
である、請求項1に記載のアンチセンスオリゴマーもしくはその医薬的に許容可能な塩またはそれらの水和物。 - オリゴヌクレオチドである、請求項1~4のいずれか1項に記載のアンチセンスオリゴマーもしくはその医薬的に許容可能な塩またはそれらの水和物。
- 前記オリゴヌクレオチドを構成する少なくとも1つのヌクレオチドの糖部分及び/又はリン酸結合部分が修飾されている、請求項5に記載のアンチセンスオリゴマーもしくはその医薬的に許容可能な塩またはそれらの水和物。
- 前記オリゴヌクレオチドを構成する少なくとも1つのヌクレオチドの糖部分は、2’位の-OH基が、OR、R、R’OR、SH、SR、NH2、NHR、NR2、N3、CN、F、Cl、Br及びIからなる群より選択されるいずれかの基(上記Rは、アルキル又はアリールを示し、上記R’は、アルキレンを示す。)で置換されたリボースである、請求項5又は6に記載のアンチセンスオリゴマーもしくはその医薬的に許容可能な塩またはそれらの水和物。
- 前記オリゴヌクレオチドを構成する少なくとも1つのヌクレオチドのリン酸結合部分が、ホスホロチオエート結合、ホスホロジチオエート結合、アルキルホスホネート結合、ホスホロアミデート結合、及びボラノフォスフェート結合からなる群から選択されるいずれか1つのものである、請求項5~7のいずれか1項に記載のアンチセンスオリゴマーもしくはその医薬的に許容可能な塩またはそれらの水和物。
- モルホリノオリゴマーである、請求項1~4のいずれか1項に記載のアンチセンスオリゴマーもしくはその医薬的に許容可能な塩またはそれらの水和物。
- ホスホロジアミデートモルホリノオリゴマーである、請求項9に記載のアンチセンスオリゴマーもしくはその医薬的に許容可能な塩またはそれらの水和物。
- 請求項1~11のいずれか1項に記載のアンチセンスオリゴマーもしくはその医薬的に許容可能な塩またはそれらの水和物を含む、筋ジストロフィー治療用医薬組成物。
- さらに医薬的に許容可能な担体を含む、請求項12に記載の医薬組成物。
- 筋ジストロフィー患者に投与するための請求項12または13に記載の医薬組成物であって、前記患者が、ジストロフィン遺伝子にエクソン51のスキッピングの対象となる変異を有する患者である、医薬組成物。
- 前記患者が、少なくともエクソン51近傍のエクソンの欠失によるフレームシフト突然変異を有するとともにエクソン51のスキッピングによりアミノ酸の読み取り枠が修正されるジストロフィン遺伝子を有する、請求項14に記載の医薬組成物。
- 前記患者が、ジストロフィン遺伝子にエクソン13-50、29-50、40-50、43-50、45-50、47-50、48-50、49-50、50、52、52-63の欠失によるフレームシフト突然変異を有する、請求項14または15に記載の医薬組成物。
- 前記患者がヒトである、請求項14~16のいずれか1項に記載の医薬組成物。
- 筋ジストロフィー治療用医薬の製造における請求項1~11のいずれか1項に記載のアンチセンスオリゴマーもしくはその医薬的に許容可能な塩またはそれらの水和物の使用。
- 請求項1~11のいずれか1項に記載のアンチセンスオリゴマーもしくはその医薬的に許容可能な塩またはそれらの水和物の有効量、または請求項12~16のいずれか1項に記載の医薬組成物を、筋ジストロフィー患者に投与する工程を含む、筋ジストロフィーの治療方法。
- 前記患者がヒトである、請求項19に記載の治療方法。
- 筋ジストロフィーの治療に使用するための、請求項1~11のいずれか1項に記載のアンチセンスオリゴマーもしくはその医薬的に許容可能な塩またはそれらの水和物、または請求項12~16のいずれか1項に記載の医薬組成物。
- 前記治療において、筋ジストロフィー患者がヒトである、請求項21に記載のアンチセンスオリゴマーもしくはその医薬的に許容可能な塩またはそれらの水和物、または医薬組成物。
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WO2023127918A1 (ja) | 2021-12-27 | 2023-07-06 | 日本新薬株式会社 | オリゴ核酸化合物の製造方法 |
WO2023168427A1 (en) | 2022-03-03 | 2023-09-07 | Yale University | Compositions and methods for delivering therapeutic polynucleotides for exon skipping |
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