Patent Application
20240301416
The present invention relates to a pharmaceutical composition or a pharmaceutical combination for use in treatment of muscular dystrophy, a method for treatment of muscular dystrophy, and the like.
In recent years, exon skipping therapy has received attention which involves causing exon skipping of a gene having a mutation that causes a disease so that a protein having partial functions arises, thereby treating the disease. Examples of the disease that may be treated by such exon skipping therapy include Duchenne muscular dystrophy (DMD).
DMD is the most frequent form of hereditary progressive muscular disease that affects one in about 3,500 newborn boys. Although DMD patients exhibit motor functions rarely different from healthy humans in their infancy and childhood, muscle weakness is observed in children from around 4 to 5 years old. Then, muscle weakness in DMD patients progresses with age to the loss of ambulation by about 12 years old and death due to cardiac or respiratory insufficiency in the twenties. Therefore, it has been strongly desired to develop an effective therapeutic agent.
DMD is known to be caused by a mutation in the dystrophin gene. The dystrophin gene is located on X chromosome and is a huge gene consisting of 2.2 million DNA base pairs. DNA is transcribed into pre-mRNA, and introns are removed by splicing to synthesize mRNA of 13, 993 bases in which 79 exons are joined together. This mRNA is translated into 3,685 amino acids to produce dystrophin protein. The dystrophin protein is associated with the maintenance of membrane stability in muscle cells and necessary to make muscle cells less fragile. Patients with DMD have a mutation in the dystrophin gene and hence, the functional dystrophin protein is rarely expressed in muscle cells of the patients. Therefore, the structure of muscle cells cannot be maintained at the time of muscle contraction in the body of the patients with DMD, leading to a large influx of calcium ions into muscle cells. Consequently, muscle cell necrosis and fibrosis progress so that muscle cells can be eventually regenerated only with difficulty.
Becker muscular dystrophy (BMD) is also caused by a mutation in the dystrophin gene. The symptoms involve muscle weakness but are typically mild and slow in the progress of muscle weakness, when compared to DMD. In many cases, its onset is in adulthood. Differences in clinical symptoms between DMD and BMD are considered to reside in whether the reading frame for amino acids on the translation of dystrophin mRNA into the dystrophin protein is disrupted by the mutation or not (Non Patent Literature 1). More specifically, in DMD, the presence of mutation shifts the amino acid reading frame so that the expression of functional dystrophin protein is abolished, whereas in BMD the dystrophin protein that is capable of functioning, though imperfectly, is produced because the amino acid reading frame is preserved, while a part of the exons are deleted by the mutation.
Exon skipping is expected to serve as a method for treating DMD. This method involves modifying splicing to restore the amino acid reading frame of dystrophin mRNA and induce expression of the dystrophin protein having the function partially restored (Non Patent Literature 2). The amino acid sequence part to be translated from an exon, which is a target for exon skipping, will be lost. For this reason, the dystrophin protein expressed by this treatment becomes shorter than normal one but since the amino acid reading frame is maintained, the function to stabilize muscle cells is partially retained. Consequently, it is expected that exon skipping will lead DMD to the similar symptoms to that of BMD which is milder. The exon skipping approach has passed the animal tests using mice or dogs and now is currently assessed in clinical trials on human DMD patients.
The skipping of an exon can be induced by binding of antisense nucleic acids targeting site (s) surrounding either 5′ or 3′ splice site or both sites, or exon-internal sites. An exon will only be included in the mRNA when both splice sites thereof are recognized by the spliceosome complex. Thus, exon skipping can be induced by targeting the sites surrounding the splice sites with antisense nucleic acids. Furthermore, the binding of an SR protein rich in serine and arginine to an exonic splicing enhancer (ESE) is considered necessary for an exon to be recognized by the splicing mechanism. Accordingly, exon skipping can also be induced by targeting ESE.
Since a mutation of the dystrophin gene may vary depending on DMD patients, antisense nucleic acids need to be designed based on the site or type of respective genetic mutation. There are a plurality of reports on an antisense nucleic acid that induces exon skipping targeting one sequence of consecutive bases for a single exon in the dystrophin gene (Patent Literatures 1 to 6 and Non Patent Literatures 1 and 2). It has also been reported that when two types of antisense nucleic acids that target the same exon in the dystrophin gene are mixed and allowed to act (dual targeting), skipping activity may be enhanced as compared to use of each antisense nucleic acid alone (Patent Literature 7).
A method called multi-exon skipping has received attention which involves causing skipping of a plurality of exons (exon group), not one exon as described above. This method enables a wide range of mutations in the dystrophin gene to be treated by exon skipping. For example, exons 45 to 55 in the dystrophin gene are known as hot spots of genetic mutation, and it has been reported that skipping of these 11 exons enables about 60% of DMD patients having a deletion mutation to be treated (Non Patent Literature 3). Most of patients congenitally lacking exons 45 to 55 are known to manifest no or mild symptoms, though developing BMD (Non Patent Literature 4). Thus, it is expected that drugs capable of inducing exon 45 to 55 skipping are promising as therapeutic agents for DMD.
For example, a method using antisense nucleic acids respectively targeting all exons in a region which is the target of exon skipping (Non Patent Literatures 5, 7, 8, and 10), a method using antisense nucleic acids respectively targeting two different exons on the 3′ side and 5′ side of a region which is the target of exon skipping (Non Patent Literatures 6 and 9 and Patent Literatures 8, 9, and 11), and a method using an antisense nucleic acid targeting only an exon on the 5′ side of a region which is the target of exon skipping (Patent Literature 10) have been reported as methods for inducing multi-exon skipping.
The effects of drugs causing simultaneous skipping a plurality of exons (exon group) in objective pre-mRNA are not always sufficient. Under the foregoing circumstances, medicaments for treating patients having various mutations by causing simultaneous skipping of a plurality of exons (exon group) in objective pre-mRNA have been desired.
The present invention provides a combination of antisense oligomers or pharmaceutically acceptable salts thereof, or hydrates thereof, a pharmaceutical composition, a pharmaceutical combination, a method for treatment of muscular dystrophy, and the like as follows:
(1)
A combination of antisense oligomers or pharmaceutically acceptable salts thereof, or hydrates thereof which cause simultaneous skipping of any two or more numerically consecutive exons selected from the group consisting of the 45th exon to the 55th exon in human dystrophin pre-mRNA, the combination comprising:
The combination according to (1), wherein
The combination according to (1) or (2), wherein
The combination according to any one of (1) to (3),
The combination according to any one of (1) to (4), wherein the first antisense oligomer comprises the first unit oligomer and the second unit oligomer from the 5′ ends in this order, the first unit oligomer comprises any one base sequence selected from SEQ ID NOs: 907 to 1602, the second unit oligomer comprises any one base sequence selected from SEQ ID NOs: 106 to 210, and the second antisense oligomer comprises any one base sequence selected from SEQ ID NOs: 4299 to 5090.
(6)
The combination according to any one of (1) to (5), wherein the first unit oligomer comprises any one base sequence selected from the group consisting of SEQ ID NOs: 1180, 1190, 1201, 1212, 1222, 1224, and 1239.
(7)
The combination according to any one of (1) to (6), wherein the second unit oligomer comprises any one base sequence selected from the group consisting of SEQ ID NOs: 114, 124, 151, 201, 203, and 205.
(8)
The combination according to (6) or (7), wherein
The combination according to any one of (1) to (8), wherein the second antisense oligomer comprises a base sequence selected from the group consisting of SEQ ID NOs: 4698, 4702, 4752, 4923, 4926, 4936, and 4977.
(10)
The combination according to any one of (1) to (9), wherein
The combination according to any one of (5) to (10), wherein the first unit oligomer comprises a base sequence of SEQ ID NO: 1201, the second unit oligomer comprises a base sequence of SEQ ID NO: 151, and the second antisense oligomer comprises a base sequence of SEQ ID NO: 4950 or 4880.
(12)
The combination according to any one of (1) to (11), further comprising:
The combination according to (12), wherein the third antisense oligomer comprises a base sequence complementary to consecutive 15 to 30 bases of a base sequence consisting of a base sequence of 23 bases in the upstream direction from the 3′ end of the 45th exon and a base sequence of 73 bases in the downstream direction from the 5′ end of the 45th intron in the human dystrophin pre-mRNA.
(14)
The combination according to (12) or (13), wherein
The combination according to (14), wherein the third antisense oligomer comprises a base sequence complementary to:
The combination according to (14), wherein the third antisense oligomer comprises a base sequence complementary to:
The combination according to (14), wherein the third antisense oligomer comprises a base sequence complementary to:
The combination according to any one of (1) to (14), wherein the third antisense oligomer comprises a base sequence selected from the group consisting of SEQ ID NOs: 3060, 3065, 3077, 3082, 3087, 3090, 3096, 3108, 3119, and 3320.
(17-2)
The combination according to any one of (1) to (14), wherein the third antisense oligomer comprises a base sequence selected from the group consisting of SEQ ID NOs: 3077, 3082, 3087, 3090, 3096, 3108, and 3119.
(17-3)
The combination according to any one of (1) to (14), wherein the third antisense oligomer comprises a base sequence selected from the group consisting of SEQ ID NOs: 3082, 3087, 3090, 3096, 3108, and 3119.
(18)
The combination according to any one of (12) to (17), wherein the first antisense oligomer comprises the first unit oligomer and the second unit oligomer from the 5′ ends in this order, the first unit oligomer comprises any one base sequence selected from SEQ ID NOs: 907 to 1602, the second unit oligomer comprises any one base sequence selected from SEQ ID NOS: 106 to 210, the second antisense oligomer comprises any one base sequence selected from SEQ ID NOs: 4299 to 5090, and the third antisense oligomer comprises any one base sequence selected from SEQ ID NOs: 2555 to 3506.
(19)
The combination according to any one of (1) to (18), wherein
The combination according to (18) or (19), wherein the first unit oligomer comprises a base sequence of SEQ ID NO: 1201, the second unit oligomer comprises a base sequence of SEQ ID NO: 151, the second antisense oligomer comprises a base sequence of SEQ ID NO: 4950 or 4880, and the third antisense oligomer comprises a base sequence of SEQ ID NO: 3082, 3090, or 3096.
(21)
The combination according to any one of (1) to (20), the combination causing skipping of all exons from the 45th exon to the 55th exon in the human dystrophin pre-mRNA.
(22)
The combination according to any one of (1) to (11), wherein the first and second antisense oligomers are oligonucleotides, or the combination according to any one of (12) to (21), wherein the first to third antisense oligomers are oligonucleotides.
(23)
The combination according to (22), wherein a sugar moiety and/or a phosphate-binding region of at least one nucleotide constituting the oligonucleotide is modified.
(24)
The combination according to (22) or (23), wherein the sugar moiety of at least one nucleotide constituting the oligonucleotide is a ribose in which the 2′-OH group is replaced by any one group selected from the group consisting of —OR, —R, —R′OR, —SH, —SR, —NH2, —NHR, —NR2, —N3, —CN, —F, —Cl, —Br, and —I (wherein R is an alkyl or an aryl and R′ is an alkylene).
(25)
The combination according to any one of (22) to (24), wherein the phosphate-binding region of at least one nucleotide constituting the oligonucleotide is any one selected from the group consisting of a phosphorothioate bond, a phosphorodithioate bond, an alkylphosphonate bond, a phosphoramidate bond and a boranophosphate bond.
(26)
The combination according to any one of (1) to (11), wherein the first and second antisense oligomers are morpholino oligomers, or the combination according to any one of (12) to (21), wherein the first to third antisense oligomers are oligonucleotides.
(27)
The combination according to (26), wherein the first to third antisense oligomers are phosphorodiamidate morpholino oligomers.
(28)
The combination according to (26) or (27), wherein the 5′ end of each of the first to third antisense oligomers is a group represented by any one of the following chemical formulae (1) to (3):
The pharmaceutical composition or the pharmaceutical combination according to (29) or (30), wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
(32)
The pharmaceutical composition or the pharmaceutical combination according to any one of (29) to (31), for treatment of muscular dystrophy.
(33)
The pharmaceutical composition or the pharmaceutical combination according to any one of (29) to (32), for being administered to a human patient.
(34)
A method for treatment of muscular dystrophy, comprising administering to a patient with muscular dystrophy (i) the first and second antisense oligomers according to any one of (1) to (28), or pharmaceutically acceptable salts thereof, or hydrates thereof, (ii) the first to third antisense oligomers according to any one of (12) to (28), or pharmaceutically acceptable salts thereof, or hydrates thereof, or (iii) the pharmaceutical composition or the pharmaceutical combination according to any one of (29) to (33).
(35)
The method for treatment according to (34), wherein the muscular dystrophy patient is a patient with a mutation that is a target of exon 45 to 55 skipping in dystrophin gene.
(36)
The method for treatment according to (34) or (35), wherein the patient is a human.
The present invention provides a combination of antisense oligomers that cause simultaneous skipping of a plurality of exons in a target. Another aspect of the present invention provides a pharmaceutical composition or combination for treating muscular dystrophy patients having various mutations by causing simultaneous skipping of a plurality of exons in objective pre-mRNA. An alternative aspect of the present invention enables simultaneous skipping of exons 45 to 55 in human dystrophin pre-mRNA to be caused with a high efficiency.
Hereinafter, the present invention is described in detail. The embodiments described below are intended to be presented by way of example merely to describe the invention but not to limit the invention only to the following embodiments. The present invention may be implemented in various ways without departing from the gist of the invention.
The present invention provides a combination of antisense oligomers or pharmaceutically acceptable salts thereof, or hydrates thereof which cause simultaneous skipping of two or more numerically consecutive exons selected from the group consisting of the 45th exon to the 55th exon in human dystrophin pre-mRNA, the combination comprising:
As used herein, the term “combination” means a substance combination, a pharmaceutical combination, an agent combination, and the like. In one embodiment, respective antisense oligomers in the combination of the present invention are comprised in one pharmaceutical composition, and simultaneously administered. In another embodiment, respective antisense oligomers in the combination of the present invention are comprised in a plurality of pharmaceutical compositions, and separately (simultaneously or sequentially) administered. As used herein, the term “simultaneously” administering a plurality of pharmaceutical compositions means that a plurality of pharmaceutical compositions are administered at the same time. As used herein, the term “sequentially” administering a plurality of pharmaceutical compositions means that these are administered at different times. Specifically, one pharmaceutical composition may be administered before or after another pharmaceutical composition, and an administration interval in this case is not limited, but may be, for example, a few minutes, a few hours, or a few days.
Hereinafter, a first antisense oligomer or a pharmaceutically acceptable salt thereof, or a hydrate thereof, and a second antisense oligomer or a pharmaceutically acceptable salt thereof, or a hydrate thereof (and optionally a third antisense oligomer or a pharmaceutically acceptable salt thereof, or a hydrate thereof described herein) may be collectively referred to as the “antisense oligomer of the present invention”. The antisense oligomer of the present invention may refer to each of antisense oligomers or pharmaceutically acceptable salts thereof, or hydrates thereof. A first antisense oligomer or a pharmaceutically acceptable salt thereof, or a hydrate thereof described above as (i) may be referred to as the “first antisense oligomer of the present invention”, and a second antisense oligomer or a pharmaceutically acceptable salt thereof, or a hydrate thereof described above as (ii) may be referred to as the “second antisense oligomer of the present invention”.
As used herein, the term “gene” is intended to mean a genomic gene and also include cDNA, pre-mRNA and mRNA. Preferably, the gene is pre-mRNA. As used herein, the term “pre-mRNA” is an RNA molecule comprising an exon and an intron transcribed from a target gene on the genome and is a mRNA precursor.
The human dystrophin pre-mRNA is an RNA molecule comprising an exon and an intron transcribed from the human dystrophin gene on the genome and is a mRNA precursor. Those skilled in the art can obtain information on the base sequence of the human dystrophin pre-mRNA by analogy from the genomic sequence of the human dystrophin gene (GenBank Accession Nos. NG_012232.1).
In the human genome, the human dystrophin gene locates at locus Xp21.2. The human dystrophin gene has a size of about 3.0 Mbp and is the largest gene among known human genes. However, the coding regions of the human dystrophin gene are only about 14 kb, distributed as 79 exons throughout the human dystrophin gene (Roberts, R G, et al., Genomics, 16: 536-538 (1993)). The pre-mRNA, which is the transcript of the human dystrophin gene, undergoes splicing to generate mature mRNA of about 14 kb. The base sequence of mature mRNA of human wild-type dystrophin gene is known (GenBank Accession Nos. NM_004006).
The first antisense oligomer of the present invention comprises the first unit oligomer and the second unit oligomer, or consists of the first unit oligomer and the second unit oligomer.
The first unit oligomer targets a base sequence of 11 bases in the upstream direction from the 3′ end of the 44th intron and a base sequence of 69 bases in the downstream direction from the 5′ end of the 45th exon in the human dystrophin pre-mRNA. As used herein, the term “targeting” means that an intended base sequence is a base sequence complementary to the base sequence of a target region or a partial base sequence of the target sequence.
A target sequence of the first unit oligomer can be indicated by the range of −11 bases to +69 bases when the boundary between the 3′ end of intron 44 and the 5′ end of exon 45 is defined as basing point 0, a base sequence region on the 5′ side (upstream) from the basing point in the dystrophin gene is indicated by “−” (minus), and a base sequence region on the 3′ side (downstream) therefrom is indicated by “+”. In this respect, the region indicated by the range of −11 bases to −1 base belongs to intron 44, and the region indicated by the range of +1 base to +69 bases belongs to exon 45.
The first unit oligomer comprises a base sequence complementary to a base sequence consisting of a base sequence of 11 bases in the upstream direction from the 3′ end of the 44th intron and a base sequence of 69 bases in the downstream direction from the 5′ end of the 45th exon in the human dystrophin pre-mRNA, or a partial base sequence thereof.
The second unit oligomer targets a base sequence of from the 52nd to 75th bases in the upstream direction from the 3′ end of the 44th intron in the human dystrophin pre-mRNA.
A target sequence of the second unit oligomer can be indicated by the range of −75 bases to −52 bases when the boundary between the 3′ end of intron 44 and the 5′ end of exon 45 is defined as basing point 0, a base sequence region on the 5′ side (upstream) from the basing point in the dystrophin gene is indicated by “−” (minus), and a base sequence region on the 3′ side (downstream) therefrom is indicated by “+”. In this respect, the region indicated by the range of −75 bases to −52 bases belongs to intron 44.
The second unit oligomer comprises a base sequence complementary to a base sequence of from the 52nd to 75th bases in the upstream direction from the 3′ end of the 44th intron in the human dystrophin pre-mRNA, or a partial base sequence thereof.
The second antisense oligomer of the present invention targets a base sequence consisting of a base sequence of 33 bases in the upstream direction from the 3′ end of the 54th intron and a base sequence of 53 bases in the downstream direction from the 5′ end of the 55th exon in the human dystrophin pre-mRNA.
A target sequence of the second antisense oligomer can be indicated by the range of −33 bases to +53 bases when the boundary between the 3′ end of intron 54 and the 5′ end of exon 55 is defined as basing point 0, a base sequence region on the 5′ side (upstream) from the basing point in the dystrophin gene is indicated by “−” (minus), and a base sequence region on the 3′ side (downstream) therefrom is indicated by “+”. In this respect, the region indicated by the range of −33 bases to −1 base belongs to intron 54, and the region indicated by the range of +1 base to +53 bases belongs to exon 55.
The second antisense oligomer comprises a base sequence complementary to a base sequence of 33 bases in the upstream direction from the 3′ end of the 54th intron and a base sequence of 53 bases in the downstream direction from the 5′ end of the 55th exon in the human dystrophin pre-mRNA, or a partial base sequence thereof.
The combination of the present invention may further comprise, in addition to the first antisense oligomer and the second antisense oligomer of the present invention, a third antisense oligomer or a pharmaceutically acceptable salt thereof, or a hydrate thereof, comprising a base sequence complementary to a base sequence consisting of a base sequence of 23 bases in the upstream direction from the 3′ end of the 45th exon, and a base sequence of 73 bases in the downstream direction from the 5′ end of the 45th intron in the human dystrophin pre-mRNA, or a partial base sequence thereof. Hereinafter, a third antisense oligomer or a pharmaceutically acceptable salt thereof, or a hydrate thereof is referred to also as the “third antisense oligomer of the present invention”.
The third antisense oligomer of the present invention targets a base sequence consisting of a base sequence of 23 bases in the upstream direction from the 3′ end of the 45th exon and a base sequence of 73 bases in the downstream direction from the 5′ end of the 45th intron in the human dystrophin pre-mRNA.
A target sequence of the third antisense oligomer can be indicated by the range of −23 bases to +73 bases when the boundary between the 3′ end of exon 45 and the 5′ end of intron 46 is defined as basing point 0, a base sequence region on the 5′ side (upstream) from the basing point in the dystrophin gene is indicated by “−” (minus), and a base sequence region on the 3′ side (downstream) therefrom is indicated by “+”. In this respect, the region indicated by the range of −23 bases to −1 base belongs to exon 45, and the region indicated by the range of +1 base to +73 bases belongs to intron 46.
The third antisense oligomer comprises a base sequence complementary to a base sequence consisting of a base sequence of 23 bases in the upstream direction from the 3′ end of the 45th exon and a base sequence of 73 bases in the downstream direction from the 5′ end of the 45th intron in the human dystrophin pre-mRNA, or a partial base sequence thereof.
Specific examples of surrounding sequences of the target sequences of the first unit oligomer and the second unit oligomer comprised in the first antisense oligomer, the second antisense oligomer, and the third antisense oligomer of the present invention include those shown in Table 1 below.
ACTCCAGGATGGCATTGGGCAGCGGCAAACTGTTGTCAGAACATTGAATGCAACTGGGGAAGAAATA
CAGCTGTCAGACAGAAAAAAGAGGTAGGGCGACAGATCTAATAGGAATGAAAACATTTTAGCAGACTTTTTAAGCTTTCTTTAGAA
GAATATTTCATGAGAGATTATAAGCAGGGTGAAAGGCACTAACATTAAAGAACCTATCAACCATTAATCAACAGCAGTAAAGAAAT
AACTCATAGATTACTGCAACAGT
Specific examples of the target sequences of the first unit oligomer and the second unit oligomer comprised in the first antisense oligomer, the second antisense oligomer, and the third antisense oligomer of the present invention include those shown in Table 2 below.
As used herein, thymine “T” and uracil “U” are interchangeable with each other. Neither “T” nor “U” essentially influences the exon skipping activity of the antisense oligomer of the present invention. Therefore, as used herein, identical base sequences except for “T” or “U” are represented by the same SEQ ID NO. In the tables below, “U” may be described as “T” even in the base sequence of pre-mRNA. Those skilled in the art can understand an RNA sequence by appropriately replacing “T” with “U”.
Herein, a target base sequence is described as “Ha_b-c”.
“Ha” represents the ath exon of the human dystrophin gene, “b” represents the 5′-terminal base of the target base sequence, and “c” represents the 3′-terminal base of the target base sequence.
When “b” and “c” are positive integers, “b” and “c” each represent a base number in the downstream direction when the 5′-terminal base of the ath exon is counted as the 1st base. On the other hand, when “b” and “c” are negative integers, “b” and “c” each represent a base number in the upstream direction when the 3′-terminal base of the (a-1) th intron is counted as the 1st base.
For example, “H55_(-75)-(-52)” means a base sequence in which the 5′ end of the target base sequence is the 75th base in the upstream direction from the 3′ end of the 54th intron and the 3′ end of the target base sequence is the 52nd base in the upstream direction from the 3′ end of the 54th intron.
The surrounding sequence of the target region or the target sequence of the antisense oligomer of the present invention includes both wild (e.g., the base sequences represented by SEQ ID NOs: 5021 to 5027) and mutant types in relation to the human dystrophin pre-mRNA. Such a mutant type has, for example, any one base sequence selected from the group consisting of base sequences (B0) and (B1) to (B16) below:
As used herein, the term “base sequence that hybridizes under stringent conditions” refers to, for example, a base sequence obtained by colony hybridization, plaque hybridization, Southern hybridization or the like, using as a probe all or part of a base sequence complementary to, e.g., any one base sequence selected from the group consisting of SEQ ID NOs: 5021 to 5027. The hybridization method which may be used includes methods described in, for example, “Sambrook & Russell, Molecular Cloning: A Laboratory Manual Vol. 3, Cold Spring Harbor, Laboratory Press, 2001,” “Ausubel, Current Protocols in Molecular Biology, John Wiley & Sons, 1987-1997,” etc.
As used herein, the term “complementary base sequence” is not limited to a base sequence that forms Watson-Crick pairs with an intended base sequence, and also includes a base sequence that forms wobble base pairs therewith. Herein, the Watson-Crick pair means a base pair that forms a hydrogen bond between adenine and thymine, between adenine and uracil, or between guanine and cytosine, and the wobble base pair means a base pair that forms a hydrogen bond between guanine and uracil, between inosine and uracil, between inosine and adenine, or between inosine and cytosine. The term “complementary base sequence” does not have to have 100% complementarity with the intended base sequence and may contain, for example, 1, 2, 3, 4, or 5 noncomplementary bases based on the intended base sequence or may be a base sequence shorter by 1 base, 2 bases, 3 bases, 4 bases, or 5 bases than the intended base sequence.
As used herein, the term “stringent conditions” may be any of low stringent conditions, moderate stringent conditions or high stringent conditions. The term “low stringent condition” is, for example, 5×SSC, 5×Denhardt's solution, 0.5% SDS, 50% formamide at 32° C. The term “moderate stringent condition” is, for example, 5×SSC, 5×Denhardt's solution, 0.5% SDS, 50% formamide at 42° C., or 5×SSC, 1% SDS, 50 mM Tris-HCl (pH 7.5), 50% formamide at 42° C. The term “high stringent condition” is, for example, 5×SSC, 5×Denhardt's solution, 0.5% SDS, 50% formamide at 50° C., or 0.2×SSC, 0.1% SDS at 65° C. Under these conditions, base sequences with higher homology are expected to be obtained efficiently at higher temperatures, although multiple factors are involved in hybridization stringency including temperature, probe concentration, probe length, ionic strength, time, salt concentration and others, and those skilled in the art may approximately select these factors to achieve similar stringency.
When commercially available kits are used for hybridization, for example, an Alkphos Direct Labelling and Detection System (GE Healthcare) may be used. In this case, according to the attached protocol, after cultivation with a labeled probe overnight, the membrane can be washed with a primary wash buffer containing 0.1% (w/v) SDS at 55° C., thereby detecting hybridization. Alternatively, when the probe is labeled with digoxigenin (DIG) using a commercially available reagent (e.g., a PCR Labelling Mix (Roche Diagnostics), etc.) in producing a probe based on all or part of the complementary sequence to any one base sequence selected from the group consisting of SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389, hybridization can be detected with a DIG Nucleic Acid Detection Kit (Roche Diagnostics) or the like.
The identity between base sequences may be determined using algorithm BLAST (Basic Local Alignment Search Tool) by Karlin and Altschul (Proc. Natl. Acad. Sci. U.S. Pat. No. 872,264-2268, 1990; Proc. Natl. Acad. Sci. USA 90: 5873, 1993). Programs called BLASTN and BLASTX based on the BLAST algorithm have been developed (Altschul S F, et al: J. Mol. Biol. 215: 403, 1990). When a base sequence is sequenced using BLASTN, the parameters are, for example, score=100 and wordlength=12. When BLAST and Gapped BLAST programs are used, the default parameters for each program are employed.
The antisense oligomer of the present invention comprises a base sequence complementary to a base sequence of the target regions of the present invention, or a partial base sequence thereof. The term “partial” means a region, except for the full length, of the target regions, i.e., a partial region of the target regions. The partial region may be 10 to 60 bases long, 10 to 55 bases long, 10 to 50 bases long, 10 to 45 bases long, 10 to 40 bases long, 10 to 35 bases long, 10 to 30 bases long, 10 to 25 bases long, 15 to 60 bases long, 15 to 55 bases long, 15 to 50 bases long, 15 to 45 bases long, 15 to 40 bases long, 15 to 35 bases long, 15 to 30 bases long, 15 to 25 bases long, 16 to 60 bases long, 16 to 55 bases long, 16 to 50 bases long, 16 to 45 bases long, 16 to 40 bases long, 16 to 35 bases long, 16 to 30 bases long, 16 to 25 bases long, 17 to 60 bases long, 17 to 55 bases long, 17 to 50 bases long, 17 to 45 bases long, 17 to 40 bases long, 17 to 35 bases long, 17 to 30 bases long, 17 to 25 bases long, 18 to 60 bases long, 18 to 55 bases long, 18 to 50 bases long, 18 to 45 bases long, 18 to 40 bases long, 18 to 35 bases long, 18 to 30 bases long, 18 to 25 bases long, 19 to 60 bases long, 19 to 55 bases long, 19 to 50 bases long, 19 to 45 bases long, 19 to 40 bases long, 19 to 35 bases long, 19 to 30 bases long, 19 to 25 bases long, 20 to 60 bases long, 20 to 55 bases long, 20 to 50 bases long, 20 to 45 bases long, 20 to 40 bases long, 20 to 35 bases long, 20 to 30 bases long, 20 to 25 bases long, 15 to 30 bases long, 15 to 29 bases long, 15 to 28 bases long, 15 to 27 bases long, 15 to 26 bases long, 15 to 25 bases long, 15 to 24 bases long, 15 to 23 bases long, 15 to 22 bases long, 15 to 21 bases long, 15 to 20 bases long, 15 to 19 bases long, 15 to 18 bases long, 16 to 30 bases long, 16 to 29 bases long, 16 to 28 bases long, 16 to 27 bases long, 16 to 26 bases long, 16 to 25 bases long, 16 to 24 bases long, 16 to 23 bases long, 16 to 22 bases long, 16 to 21 bases long, 16 to 20 bases long, 16 to 19 bases long, 16 to 18 bases long, 17 to 30 bases long, 17 to 29 bases long, 17 to 28 bases long, 17 to 27 bases long, 17 to 26 bases long, 17 to 25 bases long, 17 to 24 bases long, 17 to 23 bases long, 17 to 22 bases long, 17 to 21 bases long, 17 to 20 bases long, 17 to 19 bases long, 17 to 18 bases long, 18 to 30 bases long, 18 to 29 bases long, 18 to 28 bases long, 18 to 27 bases long, 18 to 26 bases long, 18 to 25 bases long, 18 to 24 bases long, 18 to 23 bases long, 18 to 22 bases long, 18 to 21 bases long, 18 to 20 bases long, 18 to 19 bases long, 19 to 30 bases long, 19 to 29 bases long, 19 to 28 bases long, 19 to 27 bases long, 19 to 26 bases long, 19 to 25 bases long, 19 to 24 bases long, 19 to 23 bases long, 19 to 22 bases long, 19 to 21 bases long, 19 to 20 bases long, 20 to 30 bases long, 20 to 29 bases long, 20 to 28 bases long, 20 to 27 bases long, 20 to 26 bases long, 20 to 25 bases long, 20 to 24 bases long, 20 to 23 bases long, 20 to 22 bases long, 20 to 21 bases long, 5 to 25 bases long, 5 to 24 bases long, 5 to 23 bases long, 5 to 22 bases long, 5 to 21 bases long, 5 to 20 bases long, 5 to 19 bases long, 5 to 18 bases long, 5 to 17 bases long, 5 to 16 bases long, 5 to 15 bases long, 5 to 14 bases long, 5 to 13 bases long, 5 to 12 bases long, 7 to 25 bases long, 7 to 24 bases long, 7 to 23 bases long, 7 to 22 bases long, 7 to 21 bases long, 7 to 20 bases long, 7 to 19 bases long, 7 to 18 bases long, 7 to 17 bases long, 7 to 16 bases long, 7 to 15 bases long, 7 to 14 bases long, 7 to 13 bases long, 7 to 12 bases long, 9 to 25 bases long, 9 to 24 bases long, 9 to 23 bases long, 9 to 22 bases long, 9 to 21 bases long, 9 to 20 bases long, 9 to 19 bases long, 9 to 18 bases long, 9 to 17 bases long, 9 to 16 bases long, 9 to 15 bases long, 9 to 14 bases long, 9 to 13 bases long, 9 to 12 bases long, 10 to 25 bases long, 10 to 24 bases long, 10 to 23 bases long, 10 to 22 bases long, 10 to 21 bases long, 10 to 20 bases long, 10 to 19 bases long, 10 to 18 bases long, 10 to 17 bases long, 10 to 16 bases long, 10 to 15 bases long, 10 to 14 bases long, 10 to 13 bases long, 10 to 12 bases long, 60 bases long, 59 bases long, 58 bases long, 57 bases long, 56 bases long, 55 bases long, 54 bases long, 53 bases long, 52 bases long, 51 bases long, 50 bases long, 49 bases long, 48 bases long, 47 bases long, 46 bases long, 45 bases long, 44 bases long, 43 bases long, 42 bases long, 41 bases long, 40 bases long, 39 bases long, 38 bases long, 37 bases long, 36 bases long, 35 bases long, 34 bases long, 33 bases long, 32 bases long, 31 bases long, 30 bases long, 29 bases long, 28 bases long, 27 bases long, 26 bases long, 25 bases long, 24 bases long, 23 bases long, 22 bases long, 21 bases long, 20 bases long, 19 bases long, 18 bases long, 17 bases long, 16 bases long, 15 bases long, 14 bases long, 13 bases long, 12 bases long, 11 bases long, 10 bases long, 9 bases long, 8 bases long, 7 bases long, 6 bases long, or 5 bases long, but not limited thereto. These lengths may be increased or decreased by 1, 2, or 3 bases.
The antisense oligomer of the present invention has an activity to cause simultaneous skipping of any two or more numerically consecutive exons selected from the group consisting of the 45th exon to the 55th exon in human dystrophin pre-mRNA. As used herein, such skipping of two or more numerically consecutive exons from objective pre-mRNA is referred to as “multi-exon skipping” or “multi-skipping”, and this activity is referred to as “multi-exon skipping activity” or “multi-skipping activity”.
As used herein, the term “cause simultaneous skipping” of two or more numerically consecutive exons includes not only removal of the respective exons from pre-mRNA at completely the same timings but also sequential removal of the respective exons within a period from pre-mRNA to mature mRNA. Specifically, the term “cause simultaneous skipping” of two or more numerically consecutive exons refers to removal of a plurality of (two or more) numerically consecutive exons from pre-mRNA.
As used herein, the term “two or more numerically consecutive exons” means a plurality of exons that increase one by one in exon number among exons (the total number of exons is referred to as Texon) contained in objective pre-mRNA. The exon number means a number assigned to exons in order from the 5′ end to the 3′ end with an exon at the most upstream position of pre-mRNA defined as the first exon, followed by the second, the third, . . . In the case of skipping of two or more numerically consecutive exons in a certain gene, its exon numbers a1, . . . , aj can be represented by the sequence {aj}. The general term aj in the sequence {aj} is represented by the expression below:
wherein m is a given natural number that satisfies 1≤m≤(Texon-1), and j is a natural number that satisfies 2≤(m+j)≤Texon+1.
When the objective pre-mRNA is, for example, human dystrophin pre-mRNA, Texon is 79.
In a certain aspect, j is a given natural number selected from 1 to 11. In another aspect, j is 11, j is 10, j is 9, j is 8, j is 7, j is 6, j is 5, j is 4, j is 3, j is 2, or j is 1.
Herein, the any two or more numerically consecutive exons selected from the group consisting of the 45th exon to the 55th exon mean a plurality of exons that increase one by one in exon number among 11 exons from the 45th exon to the 55th exon contained in pre-mRNA. The exon number means a number assigned to exons in order from the 5′ end to the 3′ end with an exon at the most upstream position of pre-mRNA defined as the first exon, followed by the second, the third, . . . , and the 79th exons among 79 exons contained in human dystrophin pre-mRNA. An intron is numbered as the same number as that of an exon positioned on the 5′ side thereof. Specifically, the 45th intron is flanked by the 45th exon positioned on the 5′ side thereof and the 46th exon positioned on the 3′ side thereof. As used herein, the “nth” exon or intron means the nth exon or intron counted from the 5′ end toward the 3′ end in pre-mRNA.
Table 3 shows combinations of exons included in the any two or more numerically consecutive exons selected from the group consisting of the 45th exon to the 55th exon.
Among the combinations of exons described in Table 3, for example, the combination 1, 2, 3, 4, 6, 8, 10, 18, 20, 21, 23, 25, 27, 28, 30, 32, 34, 36, 38, 40, 41, 43, 45, 46, 50, 52, or 55 is a skipping pattern expected to exert higher therapeutic effects on DMD. Multi-exon skipping in such a combination is expected to exert therapeutic effects on more patients with DMD. In one embodiment, the combination of the present invention causes skipping of all exons from the 45th exon to the 55th exon in human dystrophin pre-mRNA.
The any two or more numerically consecutive exons selected from the group consisting of the 45th exon to the 55th exon may include a plurality of groups of consecutive exons and may be, for example, but not limited to, (example 1) exons 45 and 46 (first exon group) and exons 48 to 53 (second exon group), or (example 2) exons 46 and 47 (first exon group), exons 49 and 50 (second exon group), and exons 52 to 54 (third exon group).
In the present invention, the term “activity to cause skipping” (i.e., multi-skipping activity) means, when human dystrophin pre-mRNA is taken as an example, an activity to produce human dystrophin mRNA having deletion of any two or more numerically consecutive exons selected from the group consisting of the 45th exon to the 55th exon in the human dystrophin pre-mRNA.
In other words, this activity means that by binding of the antisense oligomer of the present invention to a target site in human dystrophin pre-mRNA, the 5′-terminal nucleotide of an exon immediately downstream of the exons to be deleted is linked to the 3′-terminal nucleotide of an exon immediately upstream of the exons to be deleted when the pre-mRNA undergoes splicing, thus resulting in formation of mature mRNA which is free of codon frame shift (i.e., mature mRNA having deletion of the exons without frame shift).
The antisense oligomer of the present invention exhibits a multi-skipping activity under physiological conditions. The term “under physiological conditions” refers to conditions set to mimic the in vivo environment in terms of pH, salt composition and temperature. The conditions are, for example, 25 to 40° C., preferably 37° C., pH 5 to 8, preferably pH 7.4 and 150 mM of sodium chloride concentration.
Whether multi-skipping is caused or not can be confirmed by introducing the combination of the present invention into a dystrophin expression cell (e.g., human rhabdomyosarcoma cells), amplifying the region surrounding exons 45 to 55 of mRNA of the human dystrophin gene from the total RNA of the dystrophin expression cell by RT-PCR, and performing nested PCR or sequence analysis on the PCR amplified product. The multi-skipping efficiency can be determined as follows. The mRNA for the human dystrophin gene is collected from test cells; in the mRNA, the polynucleotide level “A” of the band where any two or more numerically consecutive exons among exons 45 to 55 are skipped, the polynucleotide level “B” of the band where any one exon among exons 45 to 55 is skipped, and the polynucleotide level “C” of the band where no skipping is caused are measured. Using these measurement values of “A”, “B”, and “C”, the efficiency is calculated by the following equation.
For example, the multi-skipping efficiency of exons 45 to 55 can be determined by using a forward primer for exon 44 and a reverse primer for exon 56 to measure the polynucleotide level “A” of the band where exons 45 to 55 are multi-skipped, using the forward primer for exon 44 and a reverse primer for exon 46 to measure the polynucleotide level “B” of the band where exon 45 is single-skipped, and using the forward primer for exon 44 and the reverse primer for exon 46 to measure the polynucleotide level “C” of the band where no skipping is caused, followed by calculation by the equation using these measurement values of “A”, “B”, and “C”.
The number of exons to be deleted in human dystrophin mRNA by the antisense oligomer of the present invention is 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. This is referred to as a deletion pattern, and various deletion patterns may exist in admixture in results obtained in one skipping experiment or skipping treatment. For example, mRNA admixture having deletion of 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 exons is obtained by introducing the antisense oligomer of the present invention to cells expressing human dystrophin pre-mRNA, and collecting its mRNA.
In a certain aspect, the term “activity to cause skipping” can be defined as (C1) to (C10) below.
(C1) Any two numerically consecutive exons selected from the group consisting of the 45th exon to the 55th exon in human dystrophin pre-mRNA are skipped with the efficiency of 5% or higher, 10% or higher, 15% or higher, 20% or higher, 25% or higher, 30% or higher, 35% or higher, 40% or higher, 45% or higher, 50% or higher, 55% or higher, 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher.
Herein, the two numerically consecutive exons may be the 45th and the 46th exons, the 46th and the 47th exons, the 47th and the 48th exons, the 48th and the 49th exons, the 49th and the 50th exons, the 50th and the 51st exons, the 51st and the 52nd exons, the 52nd and the 53rd exons, the 53rd and the 54th exons, or the 54th and the 55th exons.
(C2) Any three numerically consecutive exons selected from the group consisting of the 45th exon to the 55th exon in human dystrophin pre-mRNA are skipped with the efficiency of 5% or higher, 10% or higher, 15% or higher, 20% or higher, 25% or higher, 30% or higher, 35% or higher, 40% or higher, 45% or higher, 50% or higher, 55% or higher, 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher.
Herein, the three numerically consecutive exons may be the 45th to the 47th exons, the 46th to the 48th exons, the 47th to the 49th exons, the 48th to the 50th exons, the 49th to the 51st exons, the 50th to the 52nd exons, the 51st to the 53rd exons, the 52nd to the 54th exons, or the 53rd to the 55th exons.
(C3) Any four numerically consecutive exons selected from the group consisting of the 45th exon to the 55th exon in human dystrophin pre-mRNA are skipped with the efficiency of 5% or higher, 10% or higher, 15% or higher, 20% or higher, 25% or higher, 30% or higher, 35% or higher, 40% or higher, 45% or higher, 50% or higher, 55% or higher, 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher.
Herein, the four numerically consecutive exons may be the 45th to the 48th exons, the 46th to the 49th exons, the 47th to the 50th exons, the 48th to the 51st exons, the 49th to the 52nd exons, the 50th to the 53rd exons, the 51st to the 54th exons, or the 52nd to the 55th exons.
(C4) Any five numerically consecutive exons selected from the group consisting of the 45th exon to the 55th exon in human dystrophin pre-mRNA are skipped with the efficiency of 5% or higher, 10% or higher, 15% or higher, 20% or higher, 25% or higher, 30% or higher, 35% or higher, 40% or higher, 45% or higher, 50% or higher, 55% or higher, 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher.
Herein, the five numerically consecutive exons may be the 45th to the 49th exons, the 46th to the 50th exons, the 47th to the 51st exons, the 48th to the 52nd exons, the 49th to the 53rd exons, the 50th to the 54th exons, or the 51st to the 55th exons.
(C5) Any six numerically consecutive exons selected from the group consisting of the 45th exon to the 55th exon in human dystrophin pre-mRNA are skipped with the efficiency of 5% or higher, 10% or higher, 15% or higher, 20% or higher, 25% or higher, 30% or higher, 35% or higher, 40% or higher, 45% or higher, 50% or higher, 55% or higher, 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher.
Herein, the six numerically consecutive exons may be the 45th to the 50th exons, the 46th to the 51st exons, the 47th to the 52nd exons, the 48th to the 53rd exons, the 49th to the 54th exons, or the 50th to the 55th exons.
(C6) Any seven numerically consecutive exons selected from the group consisting of the 45th exon to the 55th exon in human dystrophin pre-mRNA are skipped with the efficiency of 5% or higher, 10% or higher, 15% or higher, 20% or higher, 25% or higher, 30% or higher, 35% or higher, 40% or higher, 45% or higher, 50% or higher, 55% or higher, 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher.
Herein, the seven numerically consecutive exons may be the 45th to the 51st exons, the 46th to the 52nd exons, the 47th to the 53rd exons, the 48th to the 54th exons, or the 49th to the 55th exons.
(C7) Any eight numerically consecutive exons selected from the group consisting of the 45th exon to the 55th exon in human dystrophin pre-mRNA are skipped with the efficiency of 5% or higher, 10% or higher, 15% or higher, 20% or higher, 25% or higher, 30% or higher, 35% or higher, 40% or higher, 45% or higher, 50% or higher, 55% or higher, 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher.
Herein, the eight numerically consecutive exons may be the 45th to the 52nd exons, the 46th to the 53rd exons, the 47th to the 54th exons, or the 48th to the 55th exons.
(C8) Any nine numerically consecutive exons selected from the group consisting of the 45th exon to the 55th exon in human dystrophin pre-mRNA are skipped with the efficiency of 5% or higher, 10% or higher, 15% or higher, 20% or higher, 25% or higher, 30% or higher, 35% or higher, 40% or higher, 45% or higher, 50% or higher, 55% or higher, 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher.
Herein, the nine numerically consecutive exons may be the 45th to the 53rd exons, the 46th to the 54th exons, or the 47th to the 55th exons.
(C9) Any ten numerically consecutive exons selected from the group consisting of the 45th exon to the 55th exon in human dystrophin pre-mRNA are skipped with the efficiency of 5% or higher, 10% or higher, 15% or higher, 20% or higher, 25% or higher, 30% or higher, 35% or higher, 40% or higher, 45% or higher, 50% or higher, 55% or higher, 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher.
Herein, the ten numerically consecutive exons may be the 45th to the 54th exons, or the 46th to the 55th exons.
(C10) Eleven numerically consecutive exons selected from the group consisting of the 45th exon to the 55th exon in human dystrophin pre-mRNA are skipped with the efficiency of 5% or higher, 10% or higher, 15% or higher, 20% or higher, 25% or higher, 30% or higher, 35% or higher, 40% or higher, 45% or higher, 50% or higher, 55% or higher, 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher.
Herein, the eleven numerically consecutive exons may be the 45th to the 55th exons.
The antisense oligomer of the present invention may be 10 to 60 bases long, 10 to 55 bases long, 10 to 50 bases long, 10 to 45 bases long, 10 to 40 bases long, 10 to 35 bases long, 10 to 30 bases long, 10 to 25 bases long, 15 to 60 bases long, 15 to 55 bases long, 15 to 50 bases long, 15 to 45 bases long, 15 to 40 bases long, 15 to 35 bases long, 15 to 30 bases long, 15 to 25 bases long, 16 to 60 bases long, 16 to 55 bases long, 16 to 50 bases long, 16 to 45 bases long, 16 to 40 bases long, 16 to 35 bases long, 16 to 30 bases long, 16 to 25 bases long, 17 to 60 bases long, 17 to 55 bases long, 17 to 50 bases long, 17 to 45 bases long, 17 to 40 bases long, 17 to 35 bases long, 17 to 30 bases long, 17 to 25 bases long, 18 to 60 bases long, 18 to 55 bases long, 18 to 50 bases long, 18 to 45 bases long, 18 to 40 bases long, 18 to 35 bases long, 18 to 30 bases long, 18 to 25 bases long, 19 to 60 bases long, 19 to 55 bases long, 19 to 50 bases long, 19 to 45 bases long, 19 to 40 bases long, 19 to 35 bases long, 19 to 30 bases long, 19 to 25 bases long, 20 to 60 bases long, 20 to 55 bases long, 20 to 50 bases long, 20 to 45 bases long, 20 to 40 bases long, 20 to 35 bases long, 20 to 30 bases long, 20 to 25 bases long, 15 to 30 bases long, 15 to 29 bases long, 15 to 28 bases long, 15 to 27 bases long, 15 to 26 bases long, 15 to 25 bases long, 15 to 24 bases long, 15 to 23 bases long, 15 to 22 bases long, 15 to 21 bases long, 15 to 20 bases long, 15 to 19 bases long, 15 to 18 bases long, 16 to 30 bases long, 16 to 29 bases long, 16 to 28 bases long, 16 to 27 bases long, 16 to 26 bases long, 16 to 25 bases long, 16 to 24 bases long, 16 to 23 bases long, 16 to 22 bases long, 16 to 21 bases long, 16 to 20 bases long, 16 to 19 bases long, 16 to 18 bases long, 17 to 30 bases long, 17 to 29 bases long, 17 to 28 bases long, 17 to 27 bases long, 17 to 26 bases long, 17 to 25 bases long, 17 to 24 bases long, 17 to 23 bases long, 17 to 22 bases long, 17 to 21 bases long, 17 to 20 bases long, 17 to 19 bases long, 17 to 18 bases long, 18 to 30 bases long, 18 to 29 bases long, 18 to 28 bases long, 18 to 27 bases long, 18 to 26 bases long, 18 to 25 bases long, 18 to 24 bases long, 18 to 23 bases long, 18 to 22 bases long, 18 to 21 bases long, 18 to 20 bases long, 18 to 19 bases long, 19 to 30 bases long, 19 to 29 bases long, 19 to 28 bases long, 19 to 27 bases long, 19 to 26 bases long, 19 to 25 bases long, 19 to 24 bases long, 19 to 23 bases long, 19 to 22 bases long, 19 to 21 bases long, 19 to 20 bases long, 20 to 30 bases long, 20 to 29 bases long, 20 to 28 bases long, 20 to 27 bases long, 20 to 26 bases long, 20 to 25 bases long, 20 to 24 bases long, 20 to 23 bases long, 20 to 22 bases long, 20 to 21 bases long, 60 bases long, 59 bases long, 58 bases long, 57 bases long, 56 bases long, 55 bases long, 54 bases long, 53 bases long, 52 bases long, 51 bases long, 50 bases long, 49 bases long, 48 bases long, 47 bases long, 46 bases long, 45 bases long, 44 bases long, 43 bases long, 42 bases long, 41 bases long, 40 bases long, 39 bases long, 38 bases long, 37 bases long, 36 bases long, 35 bases long, 34 bases long, 33 bases long, 32 bases long, 31 bases long, 30 bases long, 29 bases long, 28 bases long, 27 bases long, 26 bases long, 25 bases long, 24 bases long, 23 bases long, 22 bases long, 21 bases long, 20 bases long, 19 bases long, 18 bases long, 17 bases long, 16 bases long, 15 bases long, 14 bases long, 13 bases long, 12 bases long, 11 bases long, or 10 bases long, but not limited thereto. These lengths may be increased or decreased by 1, 2, or 3 bases.
The first antisense oligomer of the present invention is a linked-type antisense oligomer configured to comprise a plurality of unit oligomers linked to each other, a pharmaceutically acceptable salt thereof, or a hydrate thereof (hereinafter, also referred to as the “linked-type antisense oligomer of the present invention”). The unit oligomers mean respective oligomers constituting the linked-type antisense oligomer of the present invention. Specifically, the unit oligomers mean moieties (units) comprising base sequences that hybridize with target base sequences having consecutive base sequences when the linked-type antisense oligomer of the present invention binds to the target base sequences in human dystrophin pre-mRNA.
The unit oligomers may be linked via a linker that does not contribute to hybridization, or may be linked directly without the mediation of a linker. When the unit oligomers are linked directly to each other, the 3′ end of the unit positioned on the 5′ side and the 5′ end of the unit positioned on the 3′ side form a phosphate bond or any one of the following groups.
The first unit oligomer constituting the linked-type antisense oligomer of the present invention may comprise a base sequence complementary to a base sequence consisting of a base sequence of 11 bases in the upstream direction from the 3′ end of the 44th intron and a base sequence of 69 bases in the downstream direction from the 5′ end of the 45th exon in human dystrophin pre-mRNA, or a partial base sequence thereof. The second unit oligomer constituting the linked-type antisense oligomer of the present invention may comprise a base sequence complementary to a base sequence of from the 52nd to 75th bases in the upstream direction from the 3′ end of the 44th intron in human dystrophin pre-mRNA, or a partial base sequence thereof.
In relation to the target sequence of the unit oligomer, the term “partial” means a partial region of consecutive bases, except for the full length, of the target sequence. The partial region may be 5 to 30 bases long, 5 to 29 bases long, 5 to 28 bases long, 5 to 27 bases long, 5 to 26 bases long, 5 to 25 bases long, 5 to 24 bases long, 5 to 23 bases long, 5 to 22 bases long, 5 to 21 bases long, 5 to 20 bases long, 5 to 19 bases long, 5 to 18 bases long, 5 to 17 bases long, 5 to 16 bases long, 5 to 15 base long, 5 to 14 bases long, 5 to 13 bases long, 5 to 12 bases long, 7 to 30 bases long, 7 to 29 bases long, 7 to 28 bases long, 7 to 27 bases long, 7 to 26 bases long, 7 to 25 bases long, 7 to 24 bases long, 7 to 23 bases long, 7 to 22 bases long, 7 to 21 bases long, 7 to 20 bases long, 7 to 19 bases long, 7 to 18 bases long, 7 to 17 bases long, 7 to 16 bases long, 7 to 15 bases long, 7 to 14 bases long, 7 to 13 bases long, 7 to 12 bases long, 9 to 30 bases long, 9 to 29 bases long, 9 to 28 bases long, 9 to 27 bases long, 9 to 26 bases long, 9 to 25 bases long, 9 to 24 bases long, 9 to 23 bases long, 9 to 22 bases long, 9 to 21 bases long, 9 to 20 bases long, 9 to 19 bases long, 9 to 18 bases long, 9 to 17 bases long, 9 to 16 bases long, 9 to 15 bases long, 9 to 14 bases long, 9 to 13 bases long, 9 to 12 bases long, 10 to 30 bases long, 10 to 29 bases long, 10 to 28 bases long, 10 to 27 bases long, 10 to 26 bases long, 10 to 25 bases long, 10 to 24 bases long, 10 to 23 bases long, 10 to 22 bases long, 10 to 21 bases long, 10 to 20 bases long, 10 to 19 bases long, 10 to 18 bases long, 10 to 17 bases long, 10 to 16 bases long, 10 to 15 bases long, 10 to 14 bases long, 10 to 13 bases long, 10 to 12 bases long, 30 bases long, 29 bases long, 28 bases long, 27 bases long, 26 bases long, 25 bases long, 24 bases long, 23 bases long, 22 bases long, 21 bases long, 20 bases long, 19 bases long, 18 bases long, 17 bases long, 16 bases long, 15 bases long, 14 bases long, 13 bases long, 12 bases long, 11 bases long, 10 bases long, 9 bases long, 8 bases long, 7 bases long, 6 bases long, or 5 bases long, but is not limited thereto. These lengths may be increased or decreased by 1, 2, or 3 bases.
The size of each unit oligomer may be 5 to 30 bases long, 5 to 29 bases long, 5 to 28 bases long, 5 to 27 bases long, 5 to 26 bases long, 5 to 25 bases long, 5 to 24 bases long, 5 to 23 bases long, 5 to 22 bases long, 5 to 21 bases long, 5 to 20 bases long, 5 to 19 bases long, 5 to 18 bases long, 5 to 17 bases long, 5 to 16 bases long, 5 to 15 bases long, 5 to 14 bases long, 5 to 13 bases long, 5 to 12 bases long, 7 to 30 bases long, 7 to 29 bases long, 7 to 28 bases long, 7 to 27 bases long, 7 to 26 bases long, 7 to 25 bases long, 7 to 24 bases long, 7 to 23 bases long, 7 to 22 bases long, 7 to 21 bases long, 7 to 20 bases long, 7 to 19 bases long, 7 to 18 bases long, 7 to 17 bases long, 7 to 16 bases long, 7 to 15 bases long, 7 to 14 bases long, 7 to 13 bases long, 7 to 12 bases long, 9 to 30 bases long, 9 to 29 bases long, 9 to 28 bases long, 9 to 27 bases long, 9 to 26 bases long, 9 to 25 bases long, 9 to 24 bases long, 9 to 23 bases long, 9 to 22 bases long, 9 to 21 bases long, 9 to 20 bases long, 9 to 19 bases long, 9 to 18 bases long, 9 to 17 bases long, 9 to 16 bases long, 9 to 15 bases long, 9 to 14 bases long, 9 to 13 bases long, 9 to 12 bases long, 10 to 30 bases long, 10 to 29 bases long, 10 to 28 bases long, 10 to 27 bases long, 10 to 26 bases long, 10 to 25 bases long, 10 to 24 bases long, 10 to 23 bases long, 10 to 22 bases long, 10 to 21 bases long, 10 to 20 bases long, 10 to 19 bases long, 10 to 18 bases long, 10 to 17 bases long, 10 to 16 bases long, 10 to 15 bases long, 10 to 14 bases long, 10 to 13 bases long, 10 to 12 bases long, 30 bases long, 29 bases long, 28 bases long, 27 bases long, 26 bases long, 25 bases long, 24 bases long, 23 bases long, 22 bases long, 21 bases long, 20 bases long, 19 bases long, 18 bases long, 17 bases long, 16 bases long, 15 bases long, 14 bases long, 13 bases long, 12 bases long, 11 bases long, 10 bases long, 9 bases long, 8 bases long, 7 bases long, 6 bases long, 5 bases long, but not limited thereto. These lengths may be increased or decreased by 1, 2, or 3 bases. The unit oligomers may have the same size or different sizes.
In the first antisense oligomer, the order of the first unit oligomer and the second unit oligomer is not limited. The first antisense oligomer may comprise the first unit oligomer and the second unit oligomer from the 5′ ends in this order, or may comprise the second unit oligomer and the first unit oligomer from the 5′ ends in this order.
In one embodiment, the first unit oligomer comprises or consists of a base sequence complementary to consecutive 15 to 30 bases of a base sequence consisting of a base sequence 11 bases in the upstream direction from the 3′ end of the 44th intron and a base sequence of 69 bases in the downstream direction from the 5′ end of the 45th exon in human dystrophin pre-mRNA. In one embodiment, the second unit oligomer comprises or consists of a base sequence complementary to consecutive 1 to 10 bases of a base sequence of from the 52nd to 75th bases in the upstream direction from the 3′ end of the 44th intron in human dystrophin pre-mRNA. In one embodiment, the second antisense oligomer comprises a base sequence complementary to consecutive 15 to 30 bases of a base sequence consisting of a base sequence of 33 bases in the upstream direction from the 3′ end of the 54th intron and a base sequence of 53 bases in the downstream direction from the 5′ end of the 55th exon in human dystrophin pre-mRNA. In one embodiment, the third antisense oligomer comprises or consists of a base sequence complementary to consecutive 15 to 30 bases of a base sequence consisting of a base sequence of 23 bases in the upstream direction from the 3′ end of the 45th exon and a base sequence of 73 bases in the downstream direction from the 5′ end of the 45th intron in the human dystrophin pre-mRNA.
Table 4 below shows examples of the target sequence of the first unit oligomer, and the complementary sequence (antisense sequence) thereof.
In one embodiment, the first unit oligomer comprises a base sequence complementary to:
Herein, the base sequence (c) is a mutant type of the base sequence (a), and examples of such a mutant type also include
In one embodiment, the first unit oligomer comprises or consists of:
Herein, the base sequence (b) is a mutant type of the base sequence (a), and examples of such a mutant type also include:
In one embodiment, the first unit oligomer comprises or consists of any one base sequence selected from the group consisting of SEQ ID NOs: 907 to 1602.
In one embodiment, the first unit oligomer comprises or consists of any one base sequence selected from the group consisting of SEQ ID NOs: 1180, 1190, 1201, 1212, 1222, 1224, and 1239.
Table 5 below shows examples of the target sequence of the second unit oligomer, and a complementary sequence (antisense sequence) thereof.
In one embodiment, the second unit oligomer comprises a base sequence complementary to:
Herein, the base sequence (c) is a mutant type of the base sequence (a), and examples of such a mutant type also include:
In one embodiment, the second unit oligomer comprises or consists of:
Herein, the base sequence (b) is a mutant type of the base sequence (a), and examples of such a mutant type also include
In one embodiment, the second unit oligomer comprises or consists of any one base sequence selected from the group consisting of SEQ ID NOS: 106 to 210.
In one embodiment, the second unit oligomer comprises or consists of any one base sequence selected from the group consisting of SEQ ID NOS: 114, 124, 151, 201, 203, and 205.
In one embodiment, the first unit oligomer comprises or consists of any one base sequence selected from the group consisting of SEQ ID NOS: 907 to 1602, the second unit oligomer comprises or consists of any one base sequence selected from the group consisting of SEQ ID NOs: 106 to 210, and the first antisense oligomer comprises the first unit oligomer and the second unit oligomer from the 5′ ends in this order.
In one embodiment, the first antisense oligomer comprises the first unit oligomer and the second unit oligomer from the 5′ ends in this order, and
Table 6 below shows examples of the target sequence of the second antisense oligomer of the present invention, and a complementary sequence (antisense sequence) thereof.
In one embodiment, the second antisense oligomer of the present invention comprises a base sequence complementary to:
Herein, the base sequence (c) is a mutant type of the base sequence (a), and examples of such a mutant type also include:
In one embodiment, the second antisense oligomer of the present invention comprises or consists of:
Herein, the base sequence (b) is a mutant type of the base sequence (a), and examples of such a mutant type also include:
In one embodiment, the second antisense oligomer of the present invention comprises or consists of any one base sequence selected from the group consisting of SEQ ID Nos: 4299 to 5090.
In one embodiment, the second antisense oligomer comprises or consists of any one base sequence selected from the group consisting of SEQ ID NOs: 4698, 4702, 4752, 4923, 4926, 4936, 4950, and 4977.
Table 7 below shows examples of the target sequence of the third antisense oligomer of the present invention, and a complementary sequence (antisense sequence) thereof.
In one embodiment, the third antisense oligomer of the present invention comprises a base sequence complementary to:
In one embodiment, the third antisense oligomer comprises or consists of a base sequence complementary to:
In one embodiment, the third antisense oligomer comprises or consists of a base sequence complementary to:
In one embodiment, the third antisense oligomer comprises or consists of a base sequence complementary to:
Herein, the base sequence (c) is a mutant type of the base sequence (a), and examples of such a mutant type also include:
In one embodiment, the third antisense oligomer comprises or consists of:
Herein, the base sequence (b) is a mutant type of the base sequence (a), and examples of such a mutant type also include:
In one embodiment, the third antisense oligomer of the present invention comprises or consists of any one base sequence selected from the group consisting of SEQ ID NOs: 2555 to 3506.
In one embodiment, the third antisense oligomer comprises or consists of a base sequence selected from the group consisting of SEQ ID NOS: 3060, 3065, 3077, 3082, 3087, 3090, 3096, 3108, 3119, and 3320. In one embodiment, the third antisense oligomer comprises or consists of a base sequence selected from the group consisting of SEQ ID NOs: 3077, 3082, 3087, 3090, 3096, 3108, and 3119. In one embodiment, the third antisense oligomer comprises or consists of a base sequence selected from the group consisting of SEQ ID NOs: 3082, 3087, 3090, 3096, 3108, and 3119.
A combination of the first unit oligomer and the second unit oligomer comprised in the first antisense oligomer of the present invention, and the second antisense oligomer of the present invention (optionally the third antisense oligomer of the present invention) is not limited, and any combination can be used.
In one embodiment, the first antisense oligomer comprises the first unit oligomer and the second unit oligomer from the 5′ ends in this order, and
In one embodiment, the first antisense oligomer comprises the first unit oligomer and the second unit oligomer from the 5′ ends in this order, the first unit oligomer comprises any one base sequence selected from SEQ ID NOs: 907 to 1602, the second unit oligomer comprises any one base sequence selected from SEQ ID NOS: 106 to 210, and the second antisense oligomer comprises any one base sequence selected from SEQ ID NOs: 4299 to 5090. In one embodiment, the first antisense oligomer comprises the first unit oligomer and the second unit oligomer from the 5′ ends in this order, the first unit oligomer comprises a base sequence of SEQ ID No: 1201, the second unit oligomer comprises a base sequence of SEQ ID NO: 151, and the second antisense oligomer comprises a base sequence of SEQ ID NO: 4950 or 4880 (preferably 4950).
In one embodiment, the first antisense oligomer comprises the first unit oligomer and the second unit oligomer from the 5′ ends in this order, and
In one embodiment, the first antisense oligomer comprises the first unit oligomer and the second unit oligomer from the 5′ ends in this order, the first unit oligomer comprises any one base sequence selected from SEQ ID NOs: 907 to 1602, the second unit oligomer comprises any one base sequence selected from the SEQ ID NOs: 106 to 210, the second antisense oligomer comprises any one base sequence selected from SEQ ID NOs: 4299 to 5090, and the third antisense oligomer comprises any one base sequence selected from SEQ ID NOs: 2555 to 3506. In one embodiment, the first antisense oligomer comprises the first unit oligomer and the second unit oligomer from the 5′ ends in this order, the first unit oligomer comprises a base sequence of SEQ ID NO: 1201, the second unit oligomer comprises a base sequence of SEQ ID NO: 151, the second antisense oligomer comprises a base sequence of SEQ ID NO: 4950 or 4880 (preferably 4950), and the third antisense oligomer comprises a base sequence of SEQ ID NO: 3082, 3090, or 3096.
The antisense oligomer of the present invention (including the linked-type antisense oligomer of the present invention) may be an oligonucleotide, morpholino oligomer or peptide nucleic acid (PNA) oligomer (hereinafter, also referred to as the “antisense oligonucleotide of the present invention”, the “antisense morpholino oligomer of the present invention”, or the “antisense peptide nucleic acid oligomer of the present invention”).
The antisense oligonucleotide of the present invention is an antisense oligomer composed of nucleotides as constituent units. Such nucleotides may be any of ribonucleotides, deoxyribonucleotides and modified nucleotides.
The modified nucleotide refers to one having fully or partly modified nucleobases, sugar moieties and/or phosphate-binding regions, which constitute the ribonucleotide or deoxyribonucleotide.
The nucleobase includes, for example, adenine, guanine, hypoxanthine, cytosine, thymine, uracil, and modified bases thereof. Examples of such modified bases include, but not limited to, pseudouracil, 3-methyluracil, dihydrouracil, 5-alkylcytosines (e.g., 5-methylcytosine), 5-alkyluracils (e.g., 5-ethyluracil), 5-halouracils (e.g., 5-bromouracil), 6-azapyrimidine, 6-alkylpyrimidines (e.g., 6-methyluracil), 2-thiouracil, 4-thiouracil, 4-acetylcytosine, 5-(carboxyhydroxymethyl) uracil, 5′-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, 1-methyladenine, 1-methylhypoxanthine, 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-isopentenyladenine, uracil-5-oxyacetic acid, 2-thiocytosine, purine, 2, 6-diaminopurine, 2-aminopurine, isoguanine, indole, imidazole, xanthine, etc.
Modification of the sugar moiety may include, for example, modifications at the 2′-position of ribose and modifications of the other positions of the sugar. The modification at the 2′-position of ribose includes a modification of replacing the 2′-OH of ribose with —OR, —R, —R′OR, —SH, —SR, —NH2, —NHR, —NR2, —N3, —CN, —F, —Cl, —Br or —I, wherein R represents an alkyl or an aryl and R′ represents an alkylene.
The modification for the other positions of the sugar includes, for example, replacement of O at the 4′ position of ribose or deoxyribose with S, bridging between 2′ and 4′ positions of the sugar, e.g., LNA (locked nucleic acid) or ENA (2′-O, 4′-C-ethylene-bridged nucleic acids), but is not limited thereto.
A modification of the phosphate-binding region includes, for example, a modification of replacing phosphodiester bond with phosphorothioate bond, phosphorodithioate bond, alkyl phosphonate bond, phosphoramidate bond or boranophosphate bond (cf., e.g., Enya et al: Bioorganic & Medicinal Chemistry, 2008, 18, 9154-9160) (cf., e.g., Japan Domestic Re-Publications of PCT Application Nos. 2006/129594 and 2006/038608).
As used herein, the alkyl is preferably a straight or branched alkyl having 1 to 6 carbon atoms. Specific examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, n-hexyl and isohexyl. The alkyl may optionally be substituted. Examples of such substituents are a halogen, an alkoxy, cyano and nitro. The alkyl may be substituted with 1 to 3 substituents.
As used herein, the cycloalkyl is preferably a cycloalkyl having 3 to 12 carbon atoms. Specific examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl and cyclododecyl.
As used herein, the halogen includes fluorine, chlorine, bromine and iodine.
As used herein, the alkoxy is a straight or branched alkoxy having 1 to 6 carbon atoms such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentyloxy, isopentyloxy, n-hexyloxy, isohexyloxy, etc. Among others, an alkoxy having 1 to 3 carbon atoms is preferred.
As used herein, the aryl is preferably an aryl having 6 to 10 carbon atoms. Specific examples include phenyl, α-naphthyl and β-naphthyl. Among others, phenyl is preferred. The aryl may optionally be substituted. Examples of such substituents are an alkyl, a halogen, an alkoxy, cyano and nitro. The aryl may be substituted with one to three of such substituents.
As used herein, the alkylene is preferably a straight or branched alkylene having 1 to 6 carbon atoms. Specific examples include methylene, ethylene, trimethylene, tetramethylene, pentamethylene, hexamethylene, 2-(ethyl) trimethylene and 1-(methyl) tetramethylene.
As used herein, the acyl includes a straight or branched alkanoyl or aroyl. Examples of the alkanoyl include formyl, acetyl, 2-methylacetyl, 2,2-dimethylacetyl, propionyl, butyryl, isobutyryl, pentanoyl, 2, 2-dimethylpropionyl, hexanoyl, etc. Examples of the aroyl include benzoyl, toluoyl and naphthoyl. The aroyl may optionally be substituted at substitutable positions and may be substituted with an alkyl(s).
Preferably, the antisense oligonucleotide of the present invention is the antisense oligomer of the present invention having a group represented by general formula below as a constituent unit wherein the —OH group at position 2′ of ribose is substituted with methoxy and the phosphate-binding region is a phosphorothioate bond:
wherein Base represents a nucleobase.
The antisense oligonucleotide of the present invention may be easily synthesized using various automated synthesizer (e.g., AKTA oligopilot plus 10/100 (GE Healthcare)). Alternatively, the synthesis may also be entrusted to a third-party organization (e.g., Promega Corp. or Takara Co.), etc.
The antisense morpholino oligomer of the present invention is an antisense oligomer comprising the constituent unit represented by general formula below:
wherein Base has the same significance as defined above, and,
wherein X represents —CH2R1, —O—CH2R1, —S—CH2R1, —NR2R3, or F;
Examples of morpholino monomer compounds that are used in synthesis of the antisense morpholino oligomer of the present invention include, but not limited to, the following morpholino monomer compound (A), morpholino monomer compound (C), morpholino monomer compound (T), and morpholino monomer compound (G) shown in Table 8.
In the present invention, preferably, the morpholino oligomer is an oligomer having a group represented by general formula below as a constituent unit (phosphorodiamidate morpholino oligomer (hereinafter referred to as “PMO”)).
wherein Base, R2 and R3 have the same significance as defined above.
The morpholino oligomer may be produced by the procedure described in, e.g., WO 1991/009033 or WO 2009/064471. In particular, PMO can be produced by the procedure described in WO 2009/064471 or WO2013/100190.
The antisense peptide nucleic acid oligomer of the present invention is an antisense oligomer having a group represented by general formula below as a constituent unit:
wherein Base has the same significance as defined above.
The peptide nucleic acid oligomer can be produced in accordance with, e.g., the following literatures:
The antisense oligomer of the present invention (including the linked-type antisense oligomer of the present invention) may be in the form of a pharmaceutically acceptable salt thereof, in the form of a hydrate thereof, or in the form of a hydrate of the pharmaceutically acceptable salt.
Examples of the pharmaceutically acceptable salt of the antisense oligomer of the present invention are alkali metal salts such as salts of sodium, potassium and lithium; alkaline earth metal salts such as salts of calcium and magnesium; metal salts such as salts of aluminum, iron, zinc, copper, nickel, cobalt, etc.; ammonium salts; organic amine salts such as salts of t-octylamine, dibenzylamine, morpholine, glucosamine, phenylglycine alkyl ester, ethylenediamine, N-methylglucamine, guanidine, diethylamine, triethylamine, dicyclohexylamine, N,N′-dibenzylethylenediamine, chloroprocaine, procaine, diethanolamine, N-benzylphenethylamine, piperazine, tetramethylammonium, tris (hydroxymethyl) aminomethane; hydrohalide salts such as salts of hydrofluorates, hydrochlorides, hydrobromides and hydroiodides; inorganic acid salts such as nitrates, perchlorates, sulfates, phosphates, etc.; lower alkane sulfonates such as methanesulfonates, trifluoromethanesulfonates and ethanesulfonates; arylsulfonates such as benzenesulfonates and p-toluenesulfonates; organic acid salts such as acetates, malates, fumarates, succinates, citrates, tartarates, oxalates, maleates, etc.; and, amino acid salts such as salts of glycine, lysine, arginine, ornithine, glutamic acid and aspartic acid. These salts may be produced by known methods. Alternatively, the antisense oligomer of the present invention may be in the form of a hydrate thereof.
The third antisense oligomer of the present invention may have a function as a suppressor antisense oligomer. In the present invention, a suppressor antisense oligomer means an antisense oligomer which suppresses single exon skipping (hereinafter, referred to as “single skipping”). The suppressor antisense oligomer can suppress single skipping and thereby enhance an effect of multi-exon skipping by an antisense oligomer. Accordingly, a combination of the present invention comprising the third antisense oligomer may have a higher effect of multi-exon skipping as compared with one not comprising the third antisense oligomer.
Specifically, the third antisense oligomer of the present invention can suppress single skipping of any one exon selected from the group consisting of the 45th exon to the 55th exon in human dystrophin pre-mRNA. More specifically, the third antisense oligomer of the present invention can suppress single skipping of the 45th exon in human dystrophin pre-mRNA.
The third antisense oligomer of the present invention can suppress single skipping by, for example, targeting the site of a splicing silencer sequence, a branch site sequence, or a splice site sequence in human dystrophin pre-mRNA and inhibiting splicing. The third antisense oligomer of the present invention reduces the efficiency of single skipping of an intended exon as compared with a control.
In one embodiment, the third antisense oligomer of the present invention targets a recognition sequence of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) that is a splicing silencer sequence. A splicing silencer sequence refers to a base sequence element that functions to suppress recognition of an exon in pre-mRNA. A target sequence of the third antisense oligomer has been herein described.
Whether the suppressor antisense oligomer enhances a multi-exon skipping effect or not can be confirmed by providing (i) an experimental system for multi-exon skipping using only the antisense oligomer of the present invention alone and (ii) an experimental system for multi-exon skipping using the antisense oligomer and the suppressor antisense oligomer of the present invention such that the other conditions are the same therebetween, and observing the difference between a multi-exon skipping effect obtained in the experimental system (ii) and a multi-exon skipping effect obtained in the experimental system (i).
The antisense oligomer of the present invention may be PMO. An aspect of PMO is, for example, the compound represented by general formula (I) below (hereinafter, referred to as PMO (I)).
wherein Base, R2 and R3 have the same significance as defined above; and,
PMO (I) can be produced in accordance with a known method (cf., e.g., WO2009/064471 or WO2013/100190).
In the antisense oligomer of the present invention, the 5′ end may be a group represented by any of chemical structures (1) to (3) below, and preferably is (3)-OH.
Hereinafter, the groups shown by (1), (2) and (3) above are referred to as “Group (1),” “Group (2)” and “Group (3),” respectively.
The antisense oligomer of the present invention may be in the form of a complex formed together with a functional peptide for purpose of improving effectiveness (for example, a cell-penetrating peptide for purpose of improving transport efficiency to a target cell) or an antibody fragment (for example, a Fab of an antibody to a muscle cell specific receptor such as a transferrin receptor) (International Publications WO2008/036127, WO2009/005793, WO2012/150960, WO2016/187425, WO2018/118662, WO2011/013700, WO2018/118599, and WO2018/118627, Japanese Patent Laid-Open No. 2022-47613, J. D. Ramsey, N. H. Flynn, Pharmacology & Therapeutics 154, 78-86 (2015), M. K. Tsoumpra et al., EBioMedicine, 45, 630-645 (2019), International Publications WO2020/028832, WO2021/142307, WO2021/142313, WO2022/020107, and WO2022/020108). A binding site is not especially limited, and it is preferable that the 5′ end or the 3′ end of the antisense oligomer is bonded to the amino terminal or carboxyl terminal of a functional peptide or an antibody fragment.
In another aspect, the antisense oligomer of the present invention and a functional peptide or an antibody fragment may form a complex via a linker. The linker is not especially limited, and it is preferable that the 5′ end or the 3′ end of the antisense oligomer is bonded to one end of the linker, and that the amino terminal or the carboxyl terminal of the functional peptide or the antibody fragment is bounded to the other end of the linker. An additional amino acid may be present between the functional peptide or the antibody fragment and the linker.
In one embodiment, the present invention provides a pharmaceutical composition comprising the first antisense oligomer and the second antisense oligomer of the present invention (also including a pharmaceutically acceptable salt thereof, or a hydrate thereof) (hereinafter, also referred to as the “pharmaceutical composition of the present invention”). The pharmaceutical composition of the present invention may further comprise the third antisense oligomer of the present invention (also including a pharmaceutically acceptable salt thereof, or a hydrate thereof) and/or a pharmaceutically acceptable carrier.
In one embodiment, the present invention provides a pharmaceutical combination of a pharmaceutical composition comprising the first antisense oligomer of the present invention and a pharmaceutical composition comprising the second antisense oligomer of the present invention (hereinafter, also referred to as the “pharmaceutical combination of the present invention”). The pharmaceutical combination of the present invention may further comprise the third antisense oligomer and/or a pharmaceutically acceptable carrier.
The pharmaceutical composition of the present invention comprises any combination of the antisense oligomers of the present invention. The pharmaceutical combination of the present invention also comprises any combination of the antisense oligomers of the present invention. Details of the combinations of the antisense oligomers are as described herein.
In one embodiment, the antisense oligomers in the combination of the present invention are comprised in one pharmaceutical composition to be simultaneously administered. In another embodiment, the antisense oligomers in the combination of the present invention are comprised in a plurality of pharmaceutical compositions (pharmaceutical combination of the present invention) to be separately (simultaneously or sequentially) administered. As used herein, the term “simultaneously” administering a plurality of pharmaceutical compositions means that a plurality of pharmaceutical compositions are administered at the same time. As used herein, the term “sequentially” administering a plurality of pharmaceutical compositions means that these are administered at different times. Specifically, one pharmaceutical composition may be administered before or after another pharmaceutical composition, and an administration interval in this case is not limited, but may be, for example, a few minutes, a few hours, or a few days.
The pharmaceutical composition of the present invention and the pharmaceutical combination of the present invention can each be used for the treatment of, for example, Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy (LGMD), congenital muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, oculopharyngeal muscular dystrophy, cerebral autosomal dominant arteriopathy with subcortical infarct and leukoencephalopathy (CADASIL), and Alport's syndrome. The pharmaceutical combination of the present invention and the pharmaceutical composition of the present invention can each be administered to a human patient and in particular, a human patient with muscular dystrophy. The patient to receive the pharmaceutical combination of the present invention or the pharmaceutical composition of the present invention may be a human patient having a mutation that is the target of skipping of two or more exons selected from the group consisting of exons 45 to 55 in the dystrophin gene. Herein, the mutation that is the target of exon skipping is not limited, and an example includes a patient having deletion of exon (for example, having deletion of exon 46, exon 46 to 47, exon 46 to 48, exon 46 to 50, exon 46 to 51, exon 46 to 52, exon 46 to 53, exon 46 to 55, exon 47 to 50, exon 47 to 52, exon 48 to 50, exon 48 to 52, exon 48 to 54, exon 49 to 50, exon 49 to 52, exon 49 to 54, exon 50, exon 50 to 52, exon 51, exon 51 to 53, exon 52, exon 53, or exon 53 to 54) in the dystrophin gene.
One aspect of the present invention provides a method for treatment of muscular dystrophy, which comprises administering to a patient with muscular dystrophy a combination of the antisense oligomer of the present invention. Another aspect of the present invention provides a method for treatment of muscular dystrophy, which comprises administering to a patient with muscular dystrophy the pharmaceutical composition of the present invention or the pharmaceutical combination of the present invention.
The method for treatment may involve performing skipping of any two or more numerically consecutive exons selected from the group consisting of the 45th exon to the 55th exon in human dystrophin pre-mRNA. In the method for treatment, the patient with muscular dystrophy may be a patient having a mutation that is the target of exon 45 to 55 skipping in the dystrophin gene. The patient may be a human and may be a human patient having a mutation that is the target of exon 45 to 55 skipping in the dystrophin gene.
The present invention further provides use of a combination of the antisense oligomer of the present invention, or the pharmaceutical composition of the present invention or the pharmaceutical combination of the present invention in manufacturing of a medicament for the treatment of muscular dystrophy.
The present invention further provides a combination of the antisense oligomer of the present invention, or the pharmaceutical composition of the present invention or the pharmaceutical combination of the present invention for use in the treatment of muscular dystrophy. The treatment may involve performing skipping of any two or more numerically consecutive exons selected from the group consisting of the 45th exon to the 55th exon in human dystrophin pre-mRNA. In the treatment, the patient with muscular dystrophy may be a patient having a mutation that is the target of exon 45 to 55 skipping in the dystrophin gene. The patient may be a human and may be a human patient having a mutation that is the target of exon 45 to 55 skipping in the dystrophin gene.
Administration route for the combination of the antisense oligomer of the present invention, or the pharmaceutical composition of the present invention or the pharmaceutical combination of the present invention is not particularly limited so long as it is pharmaceutically acceptable route for administration, and can be chosen depending upon method of treatment. In view of easiness in delivery to muscle tissues, preferred are intravenous administration, intraarterial administration, intramuscular administration, subcutaneous administration, oral administration, tissue administration, transdermal administration, etc. Also, dosage forms which are available for the composition of the present invention are not particularly limited, and include, for example, various injections, oral agents, drips, inhalations, ointments, lotions, etc.
In administration of the antisense oligomer of the present invention to patients with muscular dystrophy, preferably, the composition of the present invention contains a carrier to promote delivery of the oligomer to muscle tissues. Such a carrier is not particularly limited as far as it is pharmaceutically acceptable, and examples include cationic carriers such as cationic liposomes, cationic polymers, etc., or carriers using viral envelope. The cationic liposomes are, for example, liposomes composed of 2-O-(2-diethylaminoethyl) carabamoyl-1, 3-O-dioleoylglycerol and phospholipids as the essential constituents (hereinafter referred to as “liposome A”), Oligofectamine (registered trademark) (manufactured by Invitrogen Corp.), Lipofectin (registered trademark) (manufactured by Invitrogen Corp.), Lipofectamine (registered trademark) (manufactured by Invitrogen Corp.), Lipofectamine 2000 (registered trademark) (manufactured by Invitrogen Corp.), DMRIE-C (registered trademark) (manufactured by Invitrogen Corp.), GeneSilencer (registered trademark) (manufactured by Gene Therapy Systems), TransMessenger (registered trademark) (manufactured by QIAGEN, Inc.), TransIT TKO (registered trademark) (manufactured by Mirus) and Nucleofector II (Lonza). Among others, liposome A is preferred. Examples of cationic polymers are JetSI (registered trademark) (manufactured by Qbiogene, Inc.) and Jet-PEI (registered trademark) (polyethylenimine, manufactured by Qbiogene, Inc.). An example of carriers using viral envelop is GenomeOne (registered trademark) (HVJ-E liposome, manufactured by Ishihara Sangyo). Alternatively, the medical devices described in Japanese Patent Nos. 2924179 and the cationic carriers described in Japanese Domestic Re-Publication PCT Nos. 2006/129594 and 2008/096690 may be used as well.
A concentration of the antisense oligomer of the present invention contained in the pharmaceutical composition of the present invention and/or the pharmaceutical combination of the present invention may vary depending on kind of the carrier, etc., and is appropriately in a range of 0.1 nM to 100 μM, preferably in a range of 1 nM to 10 μM, and more preferably in a range of 10 nM to 1 μM. A weight ratio of the antisense oligomer of the present invention contained in the composition of the present invention and the carrier (carrier/antisense oligomer of the present invention) may vary depending on property of the oligomer, type of the carrier, etc., and is appropriately in a range of 0.1 to 100, preferably in a range of 1 to 50, and more preferably in a range of 10 to 20.
In one embodiment, the antisense oligomers in the combination of the present invention are comprised in one pharmaceutical composition to be simultaneously administered. In another embodiment, the antisense oligomers in the combination of the present invention are comprised in a plurality of pharmaceutical compositions (pharmaceutical combination of the present invention) to be separately (simultaneously or sequentially) administered. When the antisense oligomers in the combination of the present invention are comprised in one or a plurality of pharmaceutical compositions, concentrations of the antisense oligomers are as follows.
The pharmaceutical composition of the present invention and/or the pharmaceutical combination of the present invention may be in the form of an aqueous solution. In this case, the pharmaceutical composition of the present invention and/or the pharmaceutical combination of the present invention may comprise the antisense oligomer of the present invention in a concentration 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, 20 to 250 mg/mL, 20 to 200 mg/mL, 20 to 150 mg/mL, 20 to 100 mg/mL, 20 to 50 mg/mL, 20 to 40 mg/mL, 20 to 30 mg/mL, 23 to 27 mg/mL, 24 to 26 mg/mL, or 25 mg/mL. The pharmaceutical composition of the present invention and/or the pharmaceutical combination of the present invention may comprise the antisense oligomer of the present invention in a concentration 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/mL, 40 to 60 mg/mL, 45 to 55 mg/mL, 47 to 53 mg/mL, 48 to 52 mg/mL, 49 to 51 mg/mL, or 50 mg/mL.
The pharmaceutical composition of the present invention and/or the pharmaceutical combination of the present invention may be in a dry form. In this case, in order to prepare the pharmaceutical composition of the present invention and/or the pharmaceutical combination of the present invention in an aqueous solution form, for example, 125 mg or 250 mg of the antisense oligomer of the present invention in a dry form may be mixed with 0.5 mL to 100 ml of water (which corresponds to a concentration of 1.25 mg/mL to 250 mg/mL or 2.5 mg/mL to 500 mg/mL of the antisense oligomer of the present invention), preferably with 1 mL to 50 mL of water (which corresponds to a concentration of 2.5 mg/mL to 125 mg/mL or 5 mg/mL to 250 mg/mL of the antisense oligomer of the present invention), more preferably with 5 mL to 10 mL of water (which corresponds to a concentration of 12.5 mg/mL to 25 mg/mL or 25 mg/mL to 50 mg/mL of the antisense oligomer of the present invention) for use.
When the antisense oligomers in the combination of the present invention are comprised in one or a plurality of pharmaceutical compositions, a total concentration of the antisense oligomers is as follows.
When the pharmaceutical composition of the present invention and/or the pharmaceutical combination of the present invention is in an aqueous solution form, the pharmaceutical composition of the present invention and/or the pharmaceutical combination of the present invention may comprise the antisense oligomers of the present invention in a total concentration 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, 20 to 250 mg/mL, 20 to 200 mg/mL, 20 to 150 mg/mL, 20 to 100 mg/mL, 20 to 50 mg/mL, 20 to 40 mg/mL, 20 to 30 mg/mL, 23 to 27 mg/mL, 24 to 26 mg/mL, or 25 mg/mL, or 5 to 1000 mg/mL, 10 to 900 mg/mL, 20 to 800 mg/mL, 30 to 700 mg/mL, 40 to 600 mg/mL, 40 to 500 mg/mL, 40 to 400 mg/mL, 40 to 300 mg/mL, 40 to 200 mg/mL, 40 to 100 mg/mL, 40 to 80 mg/mL, 40 to 60 mg/mL, 46 to 54 mg/mL, 48 to 52 mg/mL, or 50 mg/mL, or 7.5 to 1500 mg/mL, 15 to 1350 mg/mL, 30 to 1200 mg/mL, 45 to 1150 mg/mL, 60 to 900 mg/mL, 60 to 750 mg/mL, 60 to 600 mg/mL, 60 to 450 mg/mL, 60 to 300 mg/mL, 60 to 150 mg/mL, 60 to 120 mg/mL, 60 to 90 mg/mL, 69 to 81 mg/mL, 72 to 78 mg/mL, or 75 mg/mL. Alternatively, the pharmaceutical composition of the present invention and/or the pharmaceutical combination of the present invention may comprise the antisense oligomers of the present invention in a total concentration 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/mL, 40 to 60 mg/mL, 45 to 55 mg/mL, 47 to 53 mg/mL, 48 to 52 mg/mL, 49 to 51 mg/mL, or 50 mg/mL, or 20 to 200 mg/mL, 30 to 190 mg/mL, 40 to 160 mg/mL, 50 to 150 mg/mL, 60 to 140 mg/mL, 70 to 130 mg/mL, 80 to 120 mg/mL, 90 to 110 mg/mL, 94 to 106 mg/mL, 96 to 104 mg/mL, 98 to 102 mg/mL, or 100 mg/mL, or 30 to 300 mg/mL, 45 to 285 mg/mL, 60 to 240 mg/mL, 75 to 225 mg/mL, 90 to 210 mg/mL, 105 to 195 mg/mL, 120 to 180 mg/mL, 130 to 165 mg/mL, 141 to 159 mg/mL, 144 to 156 mg/mL, 147 to 153 mg/mL, or 150 mg/mL.
When the pharmaceutical composition of the present invention and/or the pharmaceutical combination of the present invention is in a dry form, in order to prepare the pharmaceutical composition of the present invention and/or the pharmaceutical combination of the present invention in an aqueous solution form, for example, 125 mg or 250 mg of the antisense oligomer of the present invention in a dry form may be mixed with 0.5 mL to 100 mL of water (which corresponds to a total concentration of 1.25 mg/mL to 250 mg/mL or 2.5 mg/mL to 500 mg/mL of the antisense oligomers of the present invention), preferably with 1 mL to 50 ml of water (which corresponds to a total concentration of 2.5 mg/mL to 125 mg/mL or 5 mg/mL to 250 mg/mL of the antisense oligomers of the present invention), more preferably with 5 mL to 10 mL of water (which correspond to a total concentration of 12.5 mg/mL to 25 mg/mL or 25 mg/mL to 50 mg/mL of the antisense oligomers of the present invention), or for example, 250 mg or 500 mg in total of the antisense oligomers of the present invention in a dry form may be mixed with 0.5 mL to 100 ml of water (which corresponds to a total concentration of 2.5 mg/mL to 500 mg/mL or 5 mg/mL to 1000 mg/mL of the antisense oligomers of the present invention), preferably with 1 mL to 50 mL of water (which corresponds to a total concentration of 5 mg/mL to 250 mg/mL or 10 mg/mL to 500 mg/mL of the antisense oligomers of the present invention), more preferably with 5 mL to 10 mL of water (which correspond to a total concentration of 25 mg/mL to 50 mg/mL or 50 mg/mL to 100 mg/mL of the antisense oligomers of the present invention), or for example, 375 mg or 750 mg in total of the antisense oligomers of the present invention in a dry form may be mixed with 0.5 mL to 100 ml of water (which corresponds to a total concentration of 3.75 mg/mL to 750 mg/mL or 7.5 mg/mL to 150 mg/mL of the antisense oligomers of the present invention), preferably with 1 mL to 50 mL of water (which corresponds to a total concentration of 7.5 mg/mL to 375 mg/mL or 15 mg/mL to 750 mg/mL of the antisense oligomers of the present invention), more preferably with 5 mL to 10 ml of water (which corresponds to a total concentration of 37.5 mg/mL to 75 mg/mL or 75 mg/mL to 150 mg/mL of the antisense oligomers of the present invention) for use.
In addition to the antisense oligomer of the present invention and the carrier described above, pharmaceutically acceptable additives may also be optionally formulated in the pharmaceutical composition of the present invention and/or the pharmaceutical combination of the present invention. Examples of such additives are emulsification aids (e.g., fatty acids having 6 to 22 carbon atoms and their pharmaceutically acceptable salts, albumin and dextran), stabilizers (e.g., cholesterol, phosphatidic acid, mannitol, and sorbitol), isotonizing agents (e.g., sodium chloride, glucose, maltose, lactose, sucrose, and trehalose), and pH controlling agents (e.g., hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, sodium hydroxide, potassium hydroxide and triethanolamine). One or more of these additives can be used. The content of the additive in the composition of the present invention is appropriately 90 wt % or less, preferably 70 wt % or less and more preferably, 50 wt % or less.
The pharmaceutical composition of the present invention and/or the pharmaceutical combination of the present invention can be prepared by adding the antisense oligomer of the present invention to a carrier dispersion and adequately stirring the mixture. Additives may be added at an appropriate step either before or after addition of the antisense oligomer of the present invention. An aqueous solvent that can be used in adding the antisense oligomer of the present invention is not particularly limited as far as it is pharmaceutically acceptable, and examples are injectable water or injectable distilled water, electrolyte fluid such as physiological saline, etc., and sugar fluid such as glucose fluid, maltose fluid, etc. A person skilled in the art can appropriately choose conditions for pH and temperature for such matter.
The pharmaceutical composition of the present invention and/or the pharmaceutical combination of the present invention may be prepared into, e.g., a liquid form and its lyophilized preparation. The lyophilized preparation can be prepared by lyophilizing the composition of the present invention in a liquid form in a conventional manner. The lyophilization can be performed, for example, by appropriately sterilizing the composition of the present invention in a liquid form, dispensing an aliquot into a vial container, performing preliminary freezing for 2 hours at conditions in a range of about −40° C. to −20° C., performing a primary drying in a range of about 0° C. to 10° C. under reduced pressure, and then performing a secondary drying in a range of about 15° C. to 25° C. under reduced pressure. In general, the lyophilized preparation of the composition of the present invention can be obtained by replacing the content of the vial with nitrogen gas and capping.
The lyophilized preparation of the pharmaceutical composition of the present invention and/or the pharmaceutical combination of the present invention can be used in general upon reconstitution by adding an optional suitable solution (reconstitution liquid) and redissolving the preparation. Such a reconstitution liquid includes injectable water, physiological saline and other infusion fluids. A volume of the reconstitution liquid may vary depending on the intended use, etc., is not particularly limited, and is suitably 0.5-fold to 2-fold greater than the volume prior to lyophilization or no more than 500 mL.
It is desired to control a dose of the pharmaceutical composition of the present invention and/or the pharmaceutical combination of the present invention to be administered, by taking the following factors into account: the type and dosage form of the antisense oligomer of the present invention contained; patients' conditions including age, body weight, etc.; administration route; and the characteristics and extent of the disease. A single dose calculated as the amount of the antisense oligomer of the present invention can be 0.1 mg to 1 g per kg body weight, preferably 1 mg to 100 mg per kg body weight, more preferably 1 mg to 90 mg per kg body weight, and further preferably 1 mg to 80 mg per kg body weight. The frequency of administration may be once per 1 to 3 days, once per week, or once per 2 to 3 weeks. This numerical range may vary occasionally depending on type of the target disease, administration route and target molecule. Therefore, a dose or frequency of administration lower than the range may be sufficient in some occasion and conversely, a dose or frequency of administration higher than the range may be required occasionally.
In still another aspect of the pharmaceutical composition of the present invention and/or the pharmaceutical combination of the present invention, there is provided a pharmaceutical composition comprising a vector capable of expressing the antisense oligomer of the present invention and the carrier described above. Such an expression vector may be a vector capable of expressing a plurality of the antisense oligomers of the present invention of the present invention. The composition may be formulated with pharmaceutically acceptable additives as in the case with the composition of the present invention containing the antisense oligomer of the present invention. A concentration of the expression vector contained in the composition may vary depending upon type of the career, etc., and is appropriately in a range of 0.1 nM to 100 μM, preferably in a range of 1 nM to 10 μM, and more preferably in a range of 10 nM to 1 μM. A weight ratio of the expression vector contained in the composition and the carrier (carrier/expression vector) may vary depending on property of the expression vector, type of the carrier, etc., and is appropriately in a range of 0.1 to 100, preferably in a range of 1 to 50, and more preferably in a range of 10 to 20. The content of the carrier contained in the composition is the same as in the case with the composition of the present invention containing the antisense oligomer of the present invention, and a method for producing the same is also the same as in the case with the composition of the present invention.
Hereinafter, the present invention will be described in more detail with reference to Examples and Test Examples below, but is not limited thereto.
In accordance with the method described in Example 1 of International Publication WO2013/100190, antisense oligomers shown in Table 9 (PMO Nos. 1 to 5 (SEQ ID NOS: 5098 to 5102)) were synthesized. Theoretical values and actual values measured by ESI-TOF-MS of the molecular weights of the antisense oligomers are also shown. The 5′ end of each PMO is Group (1) below. The synthesized PMO was dissolved in water for injection (manufactured by Otsuka Pharmaceutical Factory, Inc.).
The target base sequence of the antisense oligomer of the present invention was described as “Ma1_b1-C1”, “Ma2_b2-C2_Ma3_b3-C3”.
“Ma1 ” represents the ath exon of the mouse dystrophin gene, “b1” represents the 5′-terminal base of the target base sequence, and “C1” represents the 3′-terminal base of the target base sequence.
When “b1” and “C1” are positive integers, “b1” and “C1” each represent a base number in the downstream direction when the 5′-terminal base of the ath exon is counted as the 1st base. On the other hand, when “b1” and “C1” are negative numbers, “b1” and “C1” each represent a base number in the upstream direction when the 3′-terminal base of the (a-1) th intron is counted as the 1st base.
For example, “M55_(-4)-24” means a base sequence in which the 5′ end of the target base sequence is the 4th base in the upstream direction from the 3′ end of the 54th intron and the 3′ end of the target base sequence is the 24th base in the downstream direction from the 5′ end of the 55th exon.
“Ma2_b2-C2” which is the first part of “Ma2_b2-C2_Ma3 b3-C3” means the target base sequence of a 3′ unit oligomer constituting the antisense oligomer, and the second part “Ma3_b3-C3” means the target base sequence of a 5′ unit oligomer constituting the antisense oligomer.
When “Ma2” and “Ma3” are the same, the “_Ma3” part may be omitted.
For example, “M45_(-66)-(-61)_19-40” or “M45_(-66)-(-61)_M45_19-40” means a base sequence in which the target base sequence of the 3′ unit oligomer constituting the antisense oligomer is “M45_(-66)-(-61)” and the target base sequence of the 5′ unit oligomer constituting the antisense oligomer is “M45_19-40”.
H2K-mdx52 cells (immortalized myoblasts established from a crossbred individual of a mdx52 mouse, that is, Duchenne muscular dystrophy model, and a H-2 kb-tsA58 transgenic mouse) were seeded in a 0.4% Gelatine-coated 48-well plate (manufactured by AGC Techno Glass Co., Ltd.) at 1×104/well, and were cultured for 3 days under conditions of 37° C. and 5% CO2 in 0.5 mL of a growth medium (High glucose Dulbecco's Modified Eagle Medium (DMEM) (containing GlutaMax) (manufactured by Thermo Fisher Scientific) supplemented with 20% FBS (manufactured by Sigma Aldrich), 2% chick embryo extract (manufactured by US Biological, hereinafter the same), 2% L-glutamine (manufactured by Sigma Aldrich, hereinafter the same), 1% penicillin/streptomycin (manufactured by Sigma Aldrich, hereinafter the same), and 20 U/mL Recombinant Murine IFN-γ (manufactured by PeproTech)). After 48 hours, the growth medium was changed to a differentiation medium (DMEM supplemented with 5% horse serum (manufactured by Thermo Fisher Scientific), 2% L-glutamine, and 1% penicillin/streptomycin). After culturing for 3 days, transfection was performed with 30 UM PMO using 6 μM Endo-Porter (manufactured by Gene Tools, hereinafter the same). PMOs used here are shown in Table 10.
After culturing for another 3 days, the resultant cells were washed once with PBS (manufactured by Takara Bio Inc.), and then, the total RNA was extracted with RNeasy Mini Kit (manufactured by Qiagen K. K.). 350 μL of Buffer RLT (manufactured by Qiagen K. K.) containing 1%2-mercaptoethanol (manufactured by Nacalai Tesque, Inc.) was added to the cells, and after the cells were allowed to stand at room temperature for a few minutes to lyse the cells, the lysate was collected into a QIAshredder homogenizer (manufactured by Qiagen K. K.). A homogenate was produced by centrifugation at 15,000 rpm for 2 minutes. The total RNA was extracted according to the protocol attached to RNeasy Mini Kit (manufactured by Qiagen K. K.). The concentration of the total RNA extracted was determined using a NanoDrop One C (manufactured by Thermo Fisher Scientific). One-Step RT-PCR was performed with 400 ng of the extracted total RNA using a QIAGEN One Step RT-PCR Kit (manufactured by Qiagen K. K.). A reaction solution was prepared in accordance with the protocol attached to the kit. Veriti 96 Well Thermal Cycler (manufactured by Thermo Fisher Scientific) was used as the thermal cycler. The RT-PCR program used was as follows.
The base sequences of the forward primer and reverse primer used for RT-PCR are given below.
Transcripts (429 bp) having no skipping and transcripts (253 bp) having single exon skipping of exon 45 can be detected by a combination of the forward primer and the reverse primer 1, and transcripts (218 bp) having multi-exon skipping of exons 45 to 55 can be detected by a combination of the forward primer and the reverse primer 2.
The reaction product of the PCR above was analyzed using MultiNA (manufactured by Shimadzu Corp.).
The polynucleotide level “A” of the band with skipping of exons 45 to 55, the polynucleotide level “B” of the band with skipping of exon 45, and the polynucleotide level “C” of the band having no skipping were measured. Based on these measurement values of “A”, “B”, and “C”, the skipping efficiencies of exon 45 to 55 skipping and exon 45 skipping were determined by the following equations:
The results are shown in
As compared with the mixture of PMO No. 1 and PMO No. 2 (15 μM each, Mixture 2) used singly, the mixture additionally containing PMO No. 3 targeting hnRNP A1 (10 UM each, Mixture 2+PMO No. 3) increased the skipping efficiency of exon 45 to 55 skipping (
H2K-mdx52 cells were seeded in a 0.4% Gelatine-coated 24-well plate at 5×104/well and cultured for 48 hours under conditions of 37° C. and 5% CO2 in 1 mL of a growth medium, and then the growth medium was changed to a differentiation medium. After culturing for 3 days, transfection was performed with 15 μM PMO using 6 μM Endo-Porter.
A PMO shown in Table 11 was used in addition to those used in Test Example 1.
After culturing for another 3 days, the resultant cells were collected in the same manner as in Test Example 1, the total RNA was extracted, and subjected to One-Step RT-PCR, and the reaction product of the PCR thus obtained was analyzed to obtain the skipping efficiencies of exon 45 to 55 skipping and exon 45 skipping.
The results are shown in
As compared with the mixture of PMO No. 1 and PMO No. 2 (Mixture 2) used singly, the mixture additionally containing PMO No. 3 targeting hnRNP A1 (Mixture 2+hnRNP A1) increased the skipping efficiency of exon 45 to 55 skipping (
The mixture of Mixture 2 and PMO No. 4 targeting hnRNP A1 (Mixture 2+PMO No. 4) caused exon 45 to 55 skipping (
As a result of studying the case where the ratio between Mixture 2 and PMO No. 3 targeting hnRNP A1 was changed, when these were formulated at 3:1, the skipping efficiency of exon 45 to 55 skipping was the highest (
H2K-mdx52 cells were seeded in a 0.4% Gelatine-coated 24-well plate at 6.7×104/well and cultured for 1 day under conditions of 37° C. and 5% CO2 in 2 mL of a growth medium. After culturing for 2 days, the growth medium was changed to a differentiation medium. After culturing for 3 days, transfection was performed with 50 UM PMO using 6 μM Endo-Porter. After culturing for another 3 days, the resultant cells were collected in the same manner as in Test Example 1, the total RNA was extracted, and subjected to One-Step RT-PCR, and the reaction product of the PCR thus obtained was analyzed to obtain skipping efficiencies of exon 45 to 55 skipping and exon 45 skipping.
The results are shown in
As compared with the mixture of PMO No. 1 and PMO No. 2 (Mix 2) used singly, the mixture additionally containing PMO No. 3 targeting hnRNP A1 (Mix 2+hnRNP A1) increased the skipping efficiency of exon 45 to 55 skipping (
H2K-mdx52 cells were seeded in a 0.4% Gelatine-coated 24-well plate at 6.7×104/well and cultured for 1 day under conditions of 37° C. and 5% CO2 in 2 mL of a growth medium. After culturing for 2 days, the growth medium was changed to a differentiation medium. After culturing for 3 days, transfection was performed with 50 UM PMO using 6 μM Endo-Porter. After culturing for another 3 days, the medium was changed to a differentiation medium, and after culturing for another 1 day, the resultant cells were collected with a cell lysis buffer, Pierce RIPA Buffer (Thermo Fisher Scientific) containing protease inhibitor cocktail, complete, Mini (manufactured by Roche Diagnostics) added thereto. The cells were crushed with a sonicator, Bioruptor UCD-250 (manufactured by Sonicbio Co., Ltd.) (output: H, three times each for 30 seconds), and centrifuged (15,000 rpm, 4° C., 15 minutes) with a cooling centrifuge (TOMY MX-305, rotor: AR015-24, manufactured by Tomy Seiko Co., Ltd.) to obtain a supernatant as a cell lysate. Pierce BCA Protein Assay Kit (manufactured by Thermo Fisher Scientific) was used to measure an absorbance at 562 nm with a plate reader, Synergy HTX Multi-Mode Microplate Reader (manufactured by BioTek Instruments), and a protein concentration in the cell lysate was obtained with data analysis software, Gen5 version 2.09.2 (manufactured by BioTek Instruments). The cell lysate (in an amount corresponding to 30 μg of protein) was subjected to electrophoresis (150 V, 75 minutes) with polyacrylamide gel NuPAGE 3 to 8%, Tris-Acetate, 1.5 mm, Mini Protein Gel, 15-well (manufactured by Thermo Fisher Scientific). As a molecular weight marker, HiMark Pre-Stained Protein Standard (manufactured by Thermo Fisher Scientific) was used.
After the electrophoresis, transcription (4 mA/cm2, 30 minutes) was conducted into Immobilon-P Transfer membrane (manufactured by Merck Millipore) by semi-dry blotting. Western blotting was conducted by using, as a primary antibody, a 100-fold diluted anti-dystrophin antibody (NCL-Dys1, manufactured by Leica Biosystems Newcastle Ltd.), and as a secondary antibody, a 2,500-fold diluted goat anti-mouse IgG (H+L)—Horseradish Peroxidase complex (manufactured by Bio-Rad Laboratories). After completing the antibody reaction, light emission was caused with ECL Prime Western Blotting Detection System (manufactured by Cytiva), and the light emission was detected with a chemiluminescence gel imaging apparatus, ChemiDoc Touch MP Imaging System (manufactured by Bio-Rad Laboratories) to take an image.
The results are shown in
In a negative control, or the mixture of PMO No. 1 and PMO No. 2 (Mix 2), the dystrophin protein was not expressed, but in the mixture additionally containing PMO No. 3 targeting hnRNP A1 (Mix 2+hnRNP A1), the expression of the dystrophin protein corresponding to exon 45 to 55 skipping was confirmed (
In the same manner as in Example 1, antisense oligomers shown in Table 12 (PMO Nos. 6 to 33) were synthesized. Theoretical values and actual values measured by ESI-TOR-MS of the molecular weights of the antisense oligomers are also shown. The 5′ end of each PMO is Group (1) as in Example 1. The synthesized PMO was dissolved in water for injection (manufactured by Otsuka Pharmaceutical Factory, Inc.).
A target base sequence of the antisense oligomer of the present invention was described as “Ha1_b1-C1” or “Ha2_b2-C2_Ha3_b3-C3”.
“Ha1” represents the ath exon of the human dystrophin gene, “b1” represents the 5′-terminal base of the target base sequence, and “C1” represents the 3′-terminal base of the target base sequence.
When “b1” and “C1” are positive integers, “b1” and “C1” each represent a base number in the downstream direction when the 5′-terminal base of the ath exon is counted as the 1st base. On the other hand, when “b1” and “C1” are negative numbers, “b1” and “C1” each represent a base number in the upstream direction when the 3′-terminal base of the (a-1) th intron is counted as the 1st base.
For example, “H55_(-18)-10” means a base sequence in which the 5′ end of the target base sequence is the 18th base in the upstream direction from the 3′ end of the 54th intron and the 3′ end of the target base sequence is the 10th base in the downstream direction from the 5′ end of the 55th intron.
“Ha2_b2-C2” which is the first part of “Ha2_b2-C2_Ha3_b3-C3” means the target base sequence of a 3′ unit oligomer constituting the antisense oligomer, and the second part “Ha3_b3-C3” means the target base sequence of a 5′ unit oligomer constituting the antisense oligomer.
When “Ha2” and “Has” are the same, the “Ha3” part may be omitted.
For example, “H45_(-66)-(-61) 19-40” or “H45 (-66)-(-61)_H45_19-40” means a base sequence in which the target base sequence of the 3′ unit oligomer constituting the antisense oligomer is “H45_(-66)-(-61)” and the target base sequence of the 5′ unit oligomer is “H45_19-40”.
Normal human-derived myoblasts (manufactured by LONZA) were subjected to direct immunofluorescence staining with PE anti-human CD82 antibody (manufactured by BioLegend, hereinafter the same), and the resultant was sorted with Cell Sorter SH800S (manufactured by Sony, hereinafter the same) to obtain CD82-positive normal human-derived myoblasts. The CD82-positive normal human-derived myoblasts were seeded in a collagen I coat microplate 96-well (manufactured by AGC Techno Glass Co., Ltd.) coated with Corning (R) Matrigel Basement Membrane Matrix (manufactured by Corning, hereinafter the same) at 5×104/well, and cultured for 1 day under conditions of 37° C. and 5% CO2 in 0.1 mL of a growth medium for normal human myoblasts (DMEM, high glucose, GlutaMAX (TM) Supplement, Pyruvate (manufactured by Thermo Fisher Scientific, hereinafter the same) supplemented with 20% fetal bovine serum (FBS) (manufactured by Corning, hereinafter the same), 0.1% hBFGF (manufactured by Sigma Aldrich), and 1% penicillin/streptomycin (P/S) (manufactured by Sigma Aldrich, hereinafter the same)). On the next day of the seeding, the medium was changed from the growth medium to 0.2 mL of a differentiation medium for normal human myoblasts (DMEM, High Glucose, GlutaMAX (TM) Supplement, Pyruvate supplemented with 2% horse serum (manufactured by Thermo Fisher Scientific), 1% ITS liquid medium supplement (100×) (manufactured by Sigma Aldrich), and P/S). After culturing for 7 days, transfection was performed with PMO using 6 μM Endo-Porter (manufactured by Gene Tools). PMOs used here are shown in Table 13 below. PMOs targeting the same position in the human dystrophin gene as PMO Nos. 1 to 3 targeting the mouse dystrophin gene were used.
The used PMOs and concentrations thereof in the medium are shown in Table 14 below.
After culturing for another 3 days, the medium was changed to 0.25 mL of a differentiation medium for normal human myoblasts. Seven days after the addition of PMO, the cells were washed once with PBS (manufactured by Takara Bio Inc.), and the total RNA was extracted with RNeasy Micro Kit (manufactured by Qiagen K. K.). 75 μL of Buffer RLT (manufactured by Qiagen K. K.) containing 1%2-mercaptoethanol (manufactured by Nacalai Tesque, Inc.) was added to the cells, and after the cells were allowed to stand at room temperature for a few minutes to lyse the cells, the total RNA was extracted according to the protocol attached to RNeasy Mini Kit (manufactured by Qiagen K. K.). The concentration of the total RNA extracted was determined using a NanoDrop One C (manufactured by Thermo Fisher Scientific). One-Step RT-PCR was performed with 100 ng of the extracted total RNA using QIAGEN One Step RT-PCR Kit (manufactured by Qiagen K. K.). A reaction solution was prepared in accordance with the protocol attached to the kit. Veriti 96 Well Thermal Cycler (manufactured by Thermo Fisher Scientific) was used as the thermal cycler. The RT-PCR program used here was as follows.
The base sequences of forward primers and reverse primers used in the RT-PCR are shown in Table 15 below.
Transcripts (301 bp) having multi-exon skipping of exons 45 to 55 can be detected by a combination of the forward primer 1 and the reverse primer 1. Transcripts (245 bp) of a region of exons 37 to 38 not affected by skipping can be detected by a combination of the forward primer 2 and the reverse primer 2.
The reaction product of the PCR was analyzed with MultiNA (manufactured by Shimadzu Corporation). The polynucleotide level “A” of the band with skipping of exons 45 to 55, and the polynucleotide level “B” of the band having no skipping were measured. Based on these measurement values of “A” and “B”, the skipping efficiency of exon 45 to 55 skipping was determined by the following equation:
Skipping efficiency of exon 45 to 55 skipping (%)=A/B×100
One-Step RT-PCR was performed for exon 45 skipping in the same manner as in the detection of exon 45 to 55 skipping by using primers shown in Table 16 below.
Transcripts (268 bp) having skipping of exon 45 and transcripts (444 bp) having no skipping can be detected by a combination of the forward primer and the reverse primer.
The reaction product of the PCR was analyzed with MultiNA (manufactured by Shimadzu Corporation). The polynucleotide level “A” of the band with skipping of exon 45, and the polynucleotide level “B” of the band having no skipping were measured. Based on these measurement values of “A” and “B”, the skipping efficiency of exon 45 skipping was determined by the following equation:
The results are shown in
DMD patient-derived myoblasts with exon 48 to 50 deletion obtained from NCNP BioBank were subjected to direct immunofluorescence staining with PE anti-human CD82 antibody and APC anti-human CD56 antibody (manufactured by Milternyi Biotec, hereinafter the same), and the resultant was sorted with Cell Sorter SH800S to obtain CD56- and CD82-positive DMD patient-derived myoblasts with exon 48 to 50 deletion. The DMD patient-derived myoblasts with exon 48 to 50 deletion (CD56-positive and CD82-positive) were seeded in a Corning BioCoat collagen I 48-well transparent microplate coated with Corning (R) Matrigel Basement Membrane Matrix at 5×104/well, and cultured for 1 day under conditions of 37° C. and 5% CO2 in 0.25 mL of a growth medium for DMD patient-derived myoblasts (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMD/F12) (manufactured by Thermo Fisher Scientific, hereinafter the same) supplemented with 20% fetal bovine serum (FBS) and 1% P/S). On the next day of the seeding, the medium was changed from the growth medium to 0.5 mL of a differentiation medium for DMD patient-derived myoblasts (DMEM/F12 supplemented with 2% horse serum, 1% ITS liquid medium supplement (100×), and 1% P/S). After culturing for 6 days in the differentiation medium, transfection was performed with PMO using 6 μM Endo-Porter. The same PMOs as those used in Text Example 1 were used, and concentrations thereof in the medium are shown in Table 17 below.
After culturing for another 3 days, the medium was changed to 0.5 mL of a differentiation medium. Seven days after the addition of PMO, the total RNA was extracted from the cells in the same manner as in Test Example 1 of Example 2, and One-Step RT-PCR was performed with 200 ng of the extracted total RNA in the same manner as in Test Example 1, and the reaction product of the PCR thus obtained was analyzed to obtain skipping efficiencies of exon 45 to 55 skipping and exon 45 skipping.
The results are shown in
In the DMD patient-derived myoblasts with exon 48 to 50 deletion, exon 45 to 55 skipping was confirmed to be caused by the mixture of PMO No. 6 and PMO No. 8 (condition 4). Exon 45 to 55 skipping was confirmed to be induced also by the mixtures further containing PMO NO. 7 targeting hnRNP A1 (conditions 2 and 3) (FIG. 14), but the skipping efficiency of exon 45 skipping was reduced, and single skipping was thus suppressed (
DMD patient-derived myoblasts with exon 48 to 50 deletion (CD56-positive and CD82-positive) prepared in the same manner as in Test Example 2 were seeded in a Corning BioCoat collagen I 48-well transparent microplate coated with Corning (R) Matrigel Basement Membrane Matrix at 2×104/well, and cultured for 3 days under conditions of 37° C. and 5% CO2 in 0.25 mL of a growth medium for DMD patient-derived myoblasts. 3 days after the seeding, the medium was changed from the growth medium to 0.5 mL of a differentiation medium for DMD patient-derived myoblasts. After culturing for 8 days in the differentiation medium, transfection was performed with PMO using 6 μM Endo-Porter. The same PMOs as those used in Text Examples 1 and 2 were used, and concentrations thereof in the medium are shown in Table 18 below.
After culturing for another 3 days, the medium was changed to 0.5 mL of a differentiation medium. Six days after the addition of PMO, the total RNA was extracted in the same manner as in Test Example 2, and One-Step RT-PCR was performed with 200 ng of the extracted total RNA in the same manner as in Test Examples 1 and 2, and the reaction product of the PCR thus obtained was analyzed to obtain skipping efficiencies of exon 45 to 55 skipping and exon 45 skipping.
The results are shown in
As compared with PMO No. 6 used singly (condition 4), the mixtures additionally containing PMO No. 8 or PMO No. 7 and PMO No. 8 in addition to PMO No. 6 (conditions 2 and 3) increased the skipping efficiency of exon 45 to 55 skipping (
DMD patient-derived myoblasts with exon 48 to 50 deletion (CD56-positive and CD82-positive) were seeded in a collagen I coat microplate 24-well (manufactured by AGC Techno Glass Co., Ltd., hereinafter the same) coated with Corning (R) Matrigel Basement Membrane Matrix at 1.0×105/well, and cultured for 1 day under conditions of 37° C. and 5% CO2 in 1 mL of a growth medium for DMD patient-derived myoblasts. On the next day of the seeding, the medium was changed from the growth medium for DMD patient derived myoblasts to a differentiation medium for DMD patient-derived myoblasts. After culturing for 6 days, transfection was performed with PMO using 6 μM Endo-Porter. After culturing for another 3 days, the medium was changed to a differentiation medium, and after culturing for 7 days after the addition of PMO, a cell lysate was prepared in the same manner as in Test Example 4 of Example 2, and a protein concentration in the cell lysate was obtained. Western blotting was performed in the same manner as in Test Example 4 of Example 2 except that the cell lysate was used in an amount corresponding to 30 μg of protein, that the electrophoresis time was 120 minutes, and that a 250-fold diluted anti-dystrophin antibody (NCL-DYS1) was used, and thus the dystrophin protein was detected. Samples subjected to the electrophoresis are shown in Table 19. As a positive control of dystrophin expression, a lysate of mouse C2C12 cells having been muscle differentiation cultured for 12 days (normal dystrophin control), and a lysate of skeletal muscle of exon 45 to 55 deletion transgenic mouse (exon 45 to 55 deletion dystrophin expression control) were used.
The results are shown in
In the negative control (condition 3), the dystrophin protein was not expressed, but expression of the dystrophin protein corresponding to exon 45 to 55 skipping caused by the mixture of PMO No. 6 and PMO No. 8 (condition 5) and the mixtures of PMO Nos. 6 to 8 (conditions 4 and 6) was confirmed (
DMD patient-derived myoblasts with exon 46 to 51 deletion (CD56-positive and CD82-positive) obtained by sorting DMD patient-derived myoblasts with exon 46 to 51 deletion obtained from NCNP BioBank in the same manner as in Test Example 2 were seeded in a Corning BioCoat collagen I 48-well transparent microplate coated with Corning (R) Matrigel Basement Membrane Matrix at 5×104/well, and cultured for 1 day under conditions of 37° C. and 5% CO2 in 0.25 mL of a growth medium for DMD patient-derived myoblasts. On the next day of the seeding, the medium was changed from the growth medium to 0.3 mL of a differentiation medium for DMD patient-derived myoblasts. After culturing for 6 days in the differentiation medium, transfection was performed with PMO using 6 μM Endo-Porter. The same PMOs as those used in Text Examples 1 to 4 were used, and concentrations thereof in the medium are shown in Table 20 below.
After culturing for another 3 days, the medium was changed to 0.3 mL of a differentiation medium. Five days after the addition of PMO, the total RNA was extracted in the same manner as in Test Examples 2 to 3, One-Step RT-PCR was performed with 100 ng of the extracted total RNA in the same manner as in Test Examples 1 to 3, and the reaction product of the PCR thus obtained was analyzed to obtain skipping efficiency of exon 45 to 55 skipping. Besides, One-Step RT-PCR was performed in the same manner as in Test Examples 1 to 3 except that primers shown in Table 21 below were used, and the reaction product of the PCR thus obtained was analyzed to obtain skipping efficiency of exon 45 skipping. Transcripts (162 bp) having exon 45 skipping and transcripts (338 bp) having no skipping can be detected by a combination of the forward primer and the reverse primer.
The results are shown in
In the DMD patient-derived myoblasts with exon 46 to 51 deletion, exon 45 to 55 skipping was confirmed to be induced by PMO No. 6 used singly (condition 3) and the mixture of PMO Nos. 6 to 8 (condition 2) (
DMD patient-derived myoblasts with exon 46 to 51 deletion (CD56-positive and CD82-positive) prepared in the same manner as in Test Example 5 were seeded in a Corning BioCoat collagen I 48-well transparent microplate coated with Corning (R) Matrigel Basement Membrane Matrix at 6.3×103/well, and cultured for 4 days under conditions of 37° C. and 5% CO2 in 0.25 mL of a growth medium for DMD patient-derived myoblasts. Four days after the seeding, the medium was changed from the growth medium to 0.3 mL of a differentiation medium for DMD patient-derived myoblasts. After culturing for 7 days in the differentiation medium, transfection was performed with PMO using 6 μM Endo-Porter. The same PMOs as those used in Text Examples 1 to 5 were used, and concentrations thereof in the medium are shown in Table 22 below.
After culturing for another 3 days, the medium was changed to 0.3 mL of a differentiation medium. Seven days after the addition of PMO, the total RNA was extracted in the same manner as in Test Examples 2 to 3 and 5, and One-Step RT-PCR was performed in the same manner as in Test Example 5 except that 100 ng of the extracted total RNA was used, and the reaction product of the PCR thus obtained was analyzed to obtain skipping efficiencies of exon 45 to 55 skipping and exon 45 skipping.
The results are shown in
DMD patient-derived myoblasts with exon 46 to 51 deletion (CD56-positive and CD82-positive) were seeded in a collagen I coat microplate 24-well coated with Corning (R) Matrigel Basement Membrane Matrix at 8.0×104/well, and cultured for 1 day under conditions of 37° C. and 5% CO2 in 1 mL of a growth medium for DMD patient-derived myoblasts. On the next day of the seeding, the medium was changed from the growth medium for DMD patient derived myoblasts to 1 mL of a differentiation medium for DMD patient-derived myoblasts. After culturing 4 days, the medium was changed, and after culturing for 7 days, transfection was performed with PMO using 6 μM Endo-Porter. After culturing for another 3 days, the medium was changed to a differentiation medium, and after culturing for 7 days after the addition of PMO, a cell lysate was prepared in the same manner as in Test Example 4 of Example 2 and Test Example 4 of the present example, and Western blotting was performed in the same manner as in Test Example 4 of Example 2 and Test Example 4 of the present example except that the cell lysate was used in an amount corresponding to 24 μg of protein to detect the dystrophin protein. Samples subjected to the electrophoresis are shown in Table 23 below.
The results are shown in
In the negative control (condition 3), the dystrophin protein was not expressed, but expression of the dystrophin protein corresponding to exon 45 to 55 skipping caused by the mixture of PMO No. 6 and PMO No. 8 (condition 5) and the mixture of PMO Nos. 6 to 8 (condition 4) was confirmed (
DMD patient-derived myoblasts with exon 51 deletion (CD-56 positive, CD-82 positive) obtained by sorting DMD patient-derived myoblasts with exon 51 deletion obtained from NCNP BioBank in the same manner as in Example 3 and 6 were seeded in a collagen I coat microplate 24-well (manufactured by AGC Techno Glass Co., Ltd.) coated with Corning (R) Matrigel Basement Membrane Matrix at 5×104/well, and cultured for 3 days under conditions of 37° C. and 5% CO2 in 0.5 mL of a growth medium for DMD patient-derived myoblasts. Three days after the seeding, the medium was changed from the growth medium to 0.5 mL of a differentiation medium for DMD patient-derived myoblasts. After culturing for 4 days in the differentiation medium for DMD patient-derived myoblasts, transfection was performed with PMO using 6 μM Endo-Porter. In addition to the PMOs used in Text Examples 1 to 7, PMOs shown in Table 24 below were also used.
The PMOs were added in concentrations in the medium shown in Table 25 below.
After culturing for another 3 days, the medium was changed to 0.5 mL of a differentiation medium for DMD patient-derived myoblasts. Seven days after the addition of PMO, the total RNA was extracted in the same manner as in Test Examples 2, 3, 5, and 6, One-Step RT-PCR was performed with 200 ng of the extracted total RNA in the same manner as in Test Examples 1 to 3, and the reaction product of the PCR thus obtained was analyzed to obtain skipping efficiencies of exon 45 to 55 skipping and exon 45 skipping.
The results are shown in
DMD patient-derived myoblasts with exon 51 deletion (CD-56 positive, CD-82 positive) prepared in the same manner as in Test Example 8 were seeded in a Corning BioCoat collagen I 48-well transparent microplate coated with Corning (R) Matrigel Basement Membrane Matrix at 5×104/well, and cultured for 1 day under conditions of 37° C. and 5% CO2 in 0.25 mL of a growth medium for DMD patient-derived myoblasts. On the next day of the seeding, the medium was changed from the growth medium to 0.25 mL of a differentiation medium for DMD patient-derived myoblasts. After culturing for 7 days in the differentiation medium for DMD patient-derived myoblasts, transfection was performed with PMO using 6 μM Endo-Porter. In addition to the PMOs used in Text Examples 1 to 7, PMOs shown in Table 26 below were used.
The PMOs were added in concentrations in the medium shown in Table 27 below.
After culturing for another 3 days, the medium was changed to 0.3 mL of a differentiation medium for DMD patient-derived myoblasts. Seven days after the addition of PMO, the total RNA was extracted in the same manner as in Test Examples 2, 3, 5, 6, and 8, One-Step RT-PCR was performed with 200 ng of the extracted total RNA in the same manner as in Test Examples 1 to 3 and 8, and the reaction product of the PCR thus obtained was analyzed to obtain skipping efficiencies of exon 45 to 55 skipping and exon 45 skipping.
The results are shown in
DMD patient-derived myoblasts with exon 51 deletion (CD-56 positive, CD-82 positive) prepared in the same manner as in Test Examples 8 and 9 were seeded in a Corning BioCoat collagen I 48-well transparent microplate coated with Corning (R) Matrigel Basement Membrane Matrix at 5×104/well, and cultured for 1 day under conditions of 37° C. and 5% CO2 in 0.25 mL of a growth medium for DMD patient-derived myoblasts. On the next day of the seeding, the medium was changed from the growth medium to 0.25 mL of a differentiation medium for DMD patient-derived myoblasts. After culturing for 7 days in the differentiation medium for DMD patient-derived myoblasts, transfection was performed with PMO using 6 μM Endo-Porter. In addition to the PMOs used in Text Examples 1 to 7, PMOs shown in Table 28 below were used.
The PMOs were added in concentrations in the medium shown in Table 29 below.
After culturing for another 3 days, the medium was changed to 0.3 mL of a differentiation medium for DMD patient-derived myoblasts. Seven days after the addition of PMO, the total RNA was extracted in the same manner as in Test Examples 2, 3, 5, 6, 8, and 9, One-Step RT-PCR was performed with 200 ng of the extracted total RNA in the same manner as in Test Examples 1 to 3, 8, and 9, and the reaction product of the PCR thus obtained was analyzed to obtain skipping efficiencies of exon 45 to 55 skipping and exon 45 skipping.
The results are shown in
DMD patient-derived myoblasts with exon 51 deletion (CD-56 positive, CD-82 positive) prepared in the same manner as in Test Examples 8 to 10 were seeded in a Corning BioCoat collagen I 48-well transparent microplate coated with Corning (R) Matrigel Basement Membrane Matrix at 5×104/well, and cultured for 1 day under conditions of 37° C. and 5% CO2 in 0.25 mL of a growth medium for DMD patient-derived myoblasts. On the next day of the seeding, the medium was changed from the growth medium to 0.25 mL of a differentiation medium for DMD patient-derived myoblasts. After culturing for 3 days in the differentiation medium for DMD patient-derived myoblasts, transfection was performed with PMO using 6 μM Endo-Porter. In addition to the PMOs used in Text Examples 1 to 7, PMOs shown in Table 30 below were used.
The PMOs were added in concentrations in the medium shown in Table 31 below.
After culturing for another 3 days, the medium was changed to 0.3 mL of a differentiation medium for DMD patient-derived myoblasts. Seven days after the addition of PMO, the total RNA was extracted in the same manner as in Test Examples 2, 3, 5, 6, and 8 to 10, One-Step RT-PCR was performed with 200 ng of the extracted total RNA in the same manner as in Test Examples 1 to 3 and 8 to 10, and the reaction product of the PCR thus obtained was analyzed to obtain skipping efficiencies of exon 45 to 55 skipping and exon 45 skipping.
The results are shown in
DMD patient-derived myoblasts with exon 51 deletion (CD-56 positive, CD-82 positive) prepared in the same manner as in Test Examples 8 to 11 were seeded in a Corning BioCoat collagen I 48-well transparent microplate coated with Corning (R) Matrigel Basement Membrane Matrix at 5×104/well, and cultured for 1 day under conditions of 37° C. and 5% CO2 in 0.25 mL of a growth medium for DMD patient-derived myoblasts. On the next day of the seeding, the medium was changed from the growth medium to 0.25 mL of a differentiation medium for DMD patient-derived myoblasts. After culturing for 7 days in the differentiation medium for DMD patient-derived myoblasts, transfection was performed with PMO using 6 μM Endo-Porter.
In addition to the PMOs used in Text Examples 1 to 7, PMOs shown in Table 32 below were used.
The PMOs were added in concentrations in the medium shown in Table 33 below.
After culturing for another 3 days, the medium was changed to 0.3 mL of a differentiation medium for DMD patient-derived myoblasts. Seven days after the addition of PMO, the total RNA was extracted in the same manner as in Test Examples 2, 3, 5, 6, and 8 to 11, One-Step RT-PCR was performed with 200 ng of the extracted total RNA in the same manner as in Test Examples 1 to 3 and 8 to 11, and the reaction product of the PCR thus obtained was analyzed to obtain skipping efficiencies of exon 45 to 55 skipping and exon 45 skipping.
The results are shown in
As a result of adding PMO Nos. 27 to 33 as the second antisense oligomer together with PMO No. 6 (conditions 2 to 7, and 9), exon 45 to 55 skipping was confirmed to be induced to the same extent as in a case where PMO No. 8 was added (condition 8) (
DMD patient-derived myoblasts with exon 51 deletion (CD-56 positive, CD-82 positive) prepared in the same manner as in Test Examples 8 to 12 were seeded in a Corning BioCoat collagen I 48-well transparent microplate coated with Corning (R) Matrigel Basement Membrane Matrix at 5×104/well, and cultured for 1 day under conditions of 37° C. and 5% CO2 in 0.25 mL of a growth medium for DMD patient-derived myoblasts. On the next day of the seeding, the medium was changed from the growth medium to 0.25 mL of a differentiation medium for DMD patient-derived myoblasts. After culturing for 3 days in the differentiation medium for DMD patient-derived myoblasts, transfection was performed with PMO using 6 μM Endo-Porter. The PMOs used in Text Example 12 were used in concentrations in the medium shown in Table 34 below.
After culturing for another 3 days, the medium was changed to 0.3 mL of a differentiation medium for DMD patient-derived myoblasts. Seven days after the addition of PMO, the total RNA was extracted in the same manner as in Test Examples 2, 3, 5, 6, and 8 to 12, One-Step RT-PCR was performed with 200 ng of the extracted total RNA in the same manner as in Test Examples 1 to 3 and 8 to 12, and the reaction product of the PCR thus obtained was analyzed to obtain skipping efficiencies of exon 45 to 55 skipping and exon 45 skipping.
The results are shown in
DMD patient-derived myoblasts with exon 51 deletion (CD-56 positive, CD-82 positive) prepared in the same manner as in Test Examples 8 to 13 were seeded in a Corning BioCoat collagen I 48-well transparent microplate coated with Corning (R) Matrigel Basement Membrane Matrix at 5×104/well, and cultured for 1 day under conditions of 37° C. and 5% CO2 in 0.25 mL of a growth medium for DMD patient-derived myoblasts. On the next day of the seeding, the medium was changed from the growth medium to 0.25 mL of a differentiation medium for DMD patient-derived myoblasts. After culturing for 7 days in the differentiation medium for DMD patient-derived myoblasts, transfection was performed with PMO using 6 μM Endo-Porter. The PMOs used in Text Example were added in concentrations in the medium shown in Table 35 below.
After culturing for another 3 days, the medium was changed to 0.3 mL of a differentiation medium for DMD patient-derived myoblasts. Seven days after the addition of PMO, the total RNA was extracted in the same manner as in Test Examples 2, 3, 5, 6, and 8 to 13, One-Step RT-PCR was performed with 200 ng of the extracted total RNA in the same manner as in Test Examples 1 to 3 and 8 to 13, and the reaction product of the PCR thus obtained was analyzed to obtain skipping efficiencies of exon 45 to 55 skipping and exon 45 skipping.
The results are shown in
As a result of respectively adding PMO No. 30, 31, or 33 as the second antisense oligomer together with PMO No. 6 and PMO No. 7 (conditions 3, 4, and 6), exon 45 to 55 skipping was confirmed to the same extent as in a case where PMO No. 8 was added (condition 5) (
DMD patient-derived myoblasts with exon 51 deletion (CD56-positive and CD82-positive) were seeded in a collagen I coat microplate 24-well (manufactured by AGC Techno Glass Co., Ltd.) coated with Corning (R) Matrigel Basement Membrane Matrix at 2.0×105/well, and cultured for 1 day under conditions of 37° C. and 5% CO2 in 1 mL of a growth medium for DMD patient-derived myoblasts. On the next day of the seeding, the medium was changed from the growth medium for DMD patient derived myoblasts to a differentiation medium for DMD patient-derived myoblasts. After culturing for 3 days in the differentiation medium, transfection was performed with PMO using 6 μM Endo-Porter. After culturing for 3 days, the medium was changed to a differentiation medium. After culturing for 7 days or 11 days after the addition of PMO, Western blotting was performed in the same manner as in Test Examples 4 and 7 of the present example to detect the dystrophin protein. Samples subjected to electrophoresis are shown in Table 36. As a positive control of dystrophin expression, a lysate of mouse C2C12 cells having been muscle differentiation cultured for 12 days (normal dystrophin control), and a lysate of skeletal muscle of exon 45 to 55 deletion transgenic mouse (exon 45 to 55 deletion dystrophin expression control) were used.
The results are shown in
DMD patient-derived myoblasts with exon 51 deletion (CD56-positive and CD82-positive) prepared in the same manner as in Test Examples 8 to 15 were seeded in a collagen I coat microplate 24-well (manufactured by AGC Techno Glass Co., Ltd.) coated with Corning (R) Matrigel Basement Membrane Matrix at 2.0×105/well, and cultured for 1 day under conditions of 37° C. and 5% CO2 in 1 mL of a growth medium for DMD patient-derived myoblasts. On the next day of the seeding, the medium was changed from the growth medium for DMD patient-derived myoblasts to a differentiation medium for DMD patient-derived myoblasts. After culturing for 7 days in the differentiation medium, transfection was performed with PMO using 6 μM Endo-Porter. The PMOs used here are shown in Table 37 below.
After culturing for another 3 days, the medium was changed to a differentiation medium. After culturing for 7 days after the addition of PMO, Western blotting was performed in the same manner as in Test Examples 4, 7, and 15 of the present example to detect the dystrophin protein. Samples subjected to the electrophoresis are shown in Table 38 below.
The results are shown in
In the negative control (condition 3), the band was not confirmed in the same position as the band of exon 45 to 55 deletion dystrophin-positive control (condition 2), but in the samples transfected with a cocktail of PMOs (conditions 4, 5, and 6), expression of the dystrophin protein corresponding to exon 45 to 55 skipping was confirmed (
| Number | Date | Country | Kind |
|---|---|---|---|
| 2021-104145 | Jun 2021 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP22/25120 | 6/23/2022 | WO |
