JP2002325582A - Therapeutic agent for duchenne muscular dystrophy - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a measure for therapy of Duchenne muscular dystrophy by inducing skipping of dystrophine exon 45. SOLUTION: Disclosed are an anti-sense oligonucleotide comprising a base sequence complementary to a sequence represented by a specific base sequence derived from a human dystrophine and a therapeutic agent for Duchenne muscular dystrophy comprising the same.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a precursor mRNA having an amino acid reading frame (reading frame) shifted due to an abnormality in the dystrophin gene, which induces a predetermined exon skipping to shift the reading frame. To eliminate Duchenne (Du
chenne) muscular dystrophy. More specifically, the present invention relates to a splicing promoting sequence (SES) of the dystrophin gene, which can be used for the manufacture of a therapeutic agent for a specific type of Duchenne muscular dystrophy, and an antisense oligo against the splicing promoting sequence. The present invention relates to nucleotides and therapeutic agents containing the same.

[0002]

2. Description of the Related Art At present, it is possible to diagnose a genetic disease caused by aberrant splicing of a precursor mRNA molecule. In particular, muscular dystrophy, which is an intractable disease, has attracted attention. Muscular dystrophy is Duchenne muscular dystrophy (DMD:
Duchenne Muscular Dystrophy) and Becker Muscular Dystrophy (BMD). DMD is the most frequent genetic muscular disease, affecting one out of every 3,500 male births. Patients with this disease develop muscle weakness in early childhood, followed by muscular atrophy that consistently progresses to death around the age of 20. Currently, D
There is no effective treatment for MD, and there is a strong demand for the development of treatments from patients all over the world. In 1987, the dystrophin gene, the causative gene of DMD, was discovered by retrograde genetics, and it was revealed that BMB also develops from the same dystrophin gene abnormality [Koen]
ig, M. et al., Cell, 50: 509-517 (1987)]. BMD
In, the age of onset is relatively late in adulthood, and although there is mild muscle weakness after onset, almost normal survival is possible.

[0003] The dystrophin gene is present in the short arm 21 region of the X chromosome, its gene size is 3.0 megabases,
It is the largest known gene in humans. Despite its large size, the dystrophin protein encodes only 14 kb of the dystrophin protein in the dystrophin gene, and its coding region is divided into 79 exons and distributed throughout the gene [ Roberts, RG., Et a
l., Genomics, 16: 536-538 (1993)]. Pre-mRNA, a transcript of the dystrophin gene, is spliced into a 14 kb mature mRNA. In addition, this gene also has eight different promoter regions dispersed within the gene, each producing a different mRNA [Nishio, H., et al., J. Clin. Invest.,
94: 1073-1042 (1994), Ann, AH. And Kunkel, LM., Na
ture Genet., 3: 283-291 (1993), D'Souza, VN. et a
l., Hum. Mol. Genet., 4: 837-842 (1995)]. From these, the dystrophin gene and its transcript are:
It has a very complicated configuration.

[0004] In the genetic diagnosis of DMD and BMD, a gene fragment of dystrophin is used initially, and then cDN
Southern blotting using A as a probe revealed that approximately 60% of DMD / BMD patients had a large deletion or duplication abnormality in the dystrophin gene [Hoffman, EP. And Kunkel, LM]. ., Neu
ron, 2: 1019-1029 (1989)]. Most of the genetic abnormalities found in DMD / BMD were gene deletions, and their size was as large as several kb. Regarding genetic diagnosis, since the abnormalities of the dystrophin gene found by Southern blotting are concentrated in two hot spots of the gene, the target was targeted at the 19 exons of these hot spots and two PCRs ( Polymerase Cha
in Reaction) using a reaction system to easily detect deletions,
An overlapping PCR method has been devised [Chamberlain JS., Et al.,
Nucleic Acids Res., 16: 11141-11156 (1988), Beggs
AH., Et al., Hum. Genet., 86: 45-48 (1990)]. This overlapping PCR method is the most popular genetic diagnosis method today because it can obtain a result in a short time and 98% of the gene abnormalities that can be detected by the Southern blot method can be detected by this method.

As an animal model of DMD, mdx (X chr
omosome-linked muscular dystrophy) mice [Bu
lfield, G. et al., Proc. Natl. Acad. Sci. USA,
81: 1189-1192 (1984)].

[0006] A nonsense mutation in exon 23 of the mouse dystrophin gene inactivates this gene in the mdx mouse, resulting in termination of translation in exon 23. The mdx mice do not express any functional dystrophin, but immunochemically detect trace amounts of dystrophin-positive muscle fibers.

[0007] The major difference in the clinical pathology between DMD and BMD, two diseases caused by the same gene, namely the dystrophin gene, and also caused by similar genetic abnormalities, has been enigmatic, but the so-called frameshift theory [Mo
naco, AP., et al., Genomics, 2: 90-95 (1988)]. That is, "In DMD,
Dystrophin mRNA due to partial deletion in gene
Occurs in the reading frame (reading frame) encoded by the amino acid (out-frame: out-of-fram)
e) As a result, a stop codon appears and the synthesis of dystrophin stops halfway. On the other hand, in BMD, even if there is a partial deletion in the gene, the reading frame is maintained (in-frame), and although the size is different from the original dystrophin, a dystrophin protein can be produced. " Things. In fact,
Examination of dystrophin in the muscle of the patient reveals that dystrophin disappears in DMD, whereas dystrophin whose staining property is different from normal exists in BMD. When comparing the type of the reading frame calculated from the dystrophin gene abnormality with the phenotype of the patient in DMD / BMD patients, this frameshift theory is applicable to 90% or more of the patients.

As trial treatments for muscular dystrophy, transplantation of myoblasts, introduction of a functional dystrophin gene by using a plasmid or viral vector, and the like have been attempted [Morgan, J., Hum. Gene.
her. 5: 195-173 (1994)].

[0009] Dystrophin-positive myofibers are found in many DM
D patients [Nicholson, L. et al., J.
Neurol. Sci., 94: 137-146 (1989)]. Dystrophin-positive muscle fibers found in DMD patients have been proposed to be generated by an exon skipping mechanism [Klein, C. et al.
et al., Am. J. Hum. Genet., 50: 950-959 (1992)] Indeed, in the example of the mdx mouse, the reading frame was skipped exon containing the major nonsense mutation. Dystrophin gene transcript identified
[Wilton, S. et al., Muscle Nerve, 20: 728-734 (199
7)].

[0010] Genetic information transcribed from a gene containing an intron undergoes modification such as removal of the intron sequence by splicing, and becomes a mature mRNA consisting of only exon sequences. Further, the mature mRNA is translated according to its reading frame, and a protein can be synthesized according to the genetic information encoded by the gene. During splicing of the pre-mRNA, the mRNA
There is a mechanism to accurately determine the intron sequence and exon sequence in the precursor base sequence. Therefore, the sequence of the intron / exon boundary is conserved in all genes according to a certain rule, and is known as a consensus sequence.

As the consensus sequence, a site extending from the exon called the splice donor site at the 5 'end of the intron to the intron, a site called the splice acceptor site at the 3' end of the intron, and a site called the branching point are Three sequences are known.

In these consensus sequences, it has been reported that various splices can cause splicing abnormalities when a single base substitution occurs [Saku
raba, H. et al., Genomics, 12: 643-650 (1992)],
Consensus sequences are the key to driving splicing.

First, the present inventor, for the first time in Japan, diagnosed a dystrophin gene abnormality in a DMD / BMD patient using the PCR method. He also revealed that the abnormal form of the dystrophin gene did not differ greatly between Westerners and Japanese, that is, that there was no significant racial difference. The genetic abnormalities found by such genetic diagnosis were only huge, ranging from several kb to several hundred kb in the number of bases. However, through further detailed analysis, the nucleotide sequence of the deletion site of a certain dystrophin gene was determined. Was the first in the world to succeed in the determination of dystrophin, and was reportedly named "Dystrophin Kobe" [Matsuo, M, et al., Biochem. Biophy.
s. Res. Commun., 170: 963-967 (1990)].

The case having a genetic abnormality named “dystrophin Kobe” is DMD, and according to the result of the overlap PCR analysis, the band of the amplification product of genomic DNA corresponding to exon 19 is located at the position where it should originally exist. It was not recognized and seemed to be a deletion of exon 19. However, when an attempt was made to amplify the exon 19 region alone in genomic DNA, exon 19 was detected as an amplification product having a size smaller than the normal size, and it was found that the exon 19 was not a simple exon deletion often found in the dystrophin gene. By amplifying the exon 19 region of dystrophin of a member of the patient's family by PCR, an amplification product of the same size as that of the patient was obtained from the DNA of the mother and sister together with a normal amplification product. Turned out to be a carrier.

Next, when the nucleotide sequence of the abnormal amplification product obtained from the patient was determined, exon 19 consisting of 88 bases was determined.
It was found that 52 bases were deleted in the sequence. The deletion of this 52 bases in the exon results in a shift in the reading frame of the patient's dystrophin mRNA (out-frame) and the appearance of a stop codon in exon 20. The results of this genetic diagnosis were consistent with the clinical diagnosis of DMD for this patient.

To examine the effect of exon 19 deletions found in dystrophin Kobe on splicing, patient dystrophin mRNA was analyzed [Ma
tsuo, M., et al., J. Clin. Invest., 87: 2127-2131
(1991)].

First, the mRNA of the patient's leukocytes is converted into cDNA by reverse transcriptase, and this is nested PCR (nested PCR).
(ed-PCR) method. Amplification of the region spanning exons 18 to 20 resulted in an amplified fragment that was even shorter than the size calculated based on abnormalities found in the genome. This suggested that mRNA may have a different level of abnormality than that of genomic DNA, or that mRNA differs between leukocytes and muscle. Therefore, in order to confirm that this mRNA abnormality is also present in muscle mRNA associated with the disease, the region from exon 18 to exon 20 was amplified by PCR using cDNA synthesized from muscle mRNA as a template. The size of the resulting amplification product was exactly the same as the amplification product of exons 18-20 of leukocytes.

Next, by determining the nucleotide sequence of the obtained small abnormal amplification product, the exon 18 sequence was directly linked to the exon 20 sequence in the dystrophin cDNA of the dystrophin Kobe patient, and the exon 19 sequence was completely lost. Turned out to be. This result contradicts that in the genome, only 52 bases in exon 19 were deleted and 36 bases remained as exons. From these facts, dystrophin Kobe has a
In the maturation process, exon 19 of 36 bases was spliced out, indicating that exon skipping occurred.

There have been many reports of exon skipping resulting from genetic abnormalities. The present inventors have already discovered, for the first time in the world, that exon skipping occurs in point mutations in the dystrophin gene [Hagiwara, Y., Am. J. Hum. Genet., 54: 53-61 ( 1
994)]. All of the gene mutations that cause these exon skippings are due to the above-mentioned abnormalities in the consensus sequence that determines the splicing site.

On the other hand, in dystrophin Kobe, only a deletion of 52 bases was found in the “exon”, but there was no abnormality in the consensus sequence, and the cause of exon skipping in this example was unknown.

Since the cause of the exon skipping observed in dystrophin Kobe is not required to be due to an abnormality in the primary structure of DNA and the pre-mRNA, it is considered that the cause of the exon skipping exists in the secondary structure of the pre-mRNA. It was speculated that its secondary structure was analyzed. Analysis calculates the most energetically stable base bond in secondary structure
[Matsuo, M. et al., Biochem. Biophys. Res. C]
ommun., 182: 495-500 (1992)]. Analysis of 617 bases including the exon 19 of wild-type dystrophin and the base sequences of introns on both sides of the exon 19 revealed that mRNA was
The precursor exhibited a relatively simple stem-loop structure. As a characteristic structure, an intraexon hairpin structure in which the exon 19 sequence itself forms a base pair was confirmed. On the other hand, when the secondary structure of the mRNA precursor was deduced from the base sequence of the exon of dystrophin Kobe having an exon deletion of 52 bases and the intron in the vicinity thereof, it was greatly different from the wild type, and the largest in dystrophin Kobe. The feature was that the exon sequence formed a simple stem structure that base-paired only with the intron sequence.
This result suggests that the intraexon hairpin structure found in the wild strain may be a factor that characterizes the exon structure of the dystrophin gene.

Therefore, 22 exons whose nucleotide sequences of neighboring introns were determined were selected from 79 exons of the dystrophin gene, and the secondary structure of the pre-mRNA was analyzed. All exons analyzed resulted in the formation of an intra-exon hairpin structure, which was considered an essential element of exon function. These strongly suggested that the exon skipping found in dystrophin Kobe was caused by the disappearance of the intra-exon hairpin structure of the pre-mRNA. This also suggested that the sequence of the exon itself played an important role in exon recognition during splicing.

Recently, it has been reported that exon skipping can also occur due to abnormalities in sequences within exons in addition to consensus sequences [Dietz, HC., Et al., Science,
259: 680-683 (1993)], and attention has been paid to the fact that exon sequences in addition to consensus sequences also function as splicing site determinants. These facts break the conventional concept of splicing in molecular biology.

Since it was suggested that the sequence in exon 19 is important for determining the splice site, an in vitro splicing reaction system was constructed and an attempt was made to clarify this experimentally [Takeshima, Y. ., et al., J. Cli
n. Invest., 95: 515-520 (1995)]. First, exons 18 and 19 of the dystrophin gene and intron 18
Was prepared, and a radioisotope-labeled mRNA precursor was synthesized from the minigene. The obtained mRNA precursor was mixed with a nuclear extract of HeLa cells, a splicing reaction was allowed to proceed in a test tube, and the produced mature mRNA was separated by electrophoresis. When an mRNA precursor having a normal exon 19 base sequence was used in this reaction system, splicing proceeded normally, and a mature mRNA in which exon 18 was linked to 19 could be obtained. However, when the nucleotide sequence of exon 19 was replaced with that of dystrophin, mature mRNA was not produced. This indicated that 52 bases deleted from exon 19 in dystrophin Kobe play an important role in splicing.

However, since this splicing abnormality may be the result of the exon 19 "size" being shortened to 36 bases, the deleted gene was inverted to complement the deletion of exon 19 in dystrophin Kobe. A similar experiment was performed. In this pre-mRNA,
Splicing proceeded, but its efficiency was low. This result indicates that even if the exon has a normal length, the splicing efficiency is reduced if the base sequence in the exon is different, and the exon (not the size)
This indicates that the nucleotide sequence itself in the exon is important.

Therefore, in order to further analyze the effect of the base sequence itself in the exon on splicing, mRNA having two different sequences inserted in place of the 52 base deletion sequence was used.
Precursors were made and their splicing efficiency was investigated. β
Splicing progressed in both of the two types of mRNA precursors into which a part of the globin gene or a part of the ampicillin resistance gene was inserted, but both had extremely low efficiency. However, the insertion of the β-globin gene showed relatively higher splicing efficiency than the insertion of the ampicillin resistance gene. The former nucleotide sequence contains many purine groups, and the purine sequence in the exon is thought to be involved in exon recognition [Watanabe, A., et al., Genes Dev., 7: 407
-418 (1993)].

These experimental results show experimentally that not only the consensus sequence but also the sequence of exons downstream thereof are involved in splicing, and introduce a new concept into the genetic information processing process. Was something.

<Splicing Control by Antisense Oligonucleotide> From the above finding that the sequence in exon 19 of the dystrophin gene is extremely important for the progress of splicing, splicing abnormalities can be artificially induced by disrupting this sequence. Paying attention to the possibility that this may be the case, the present inventors continued their investigation. That is, of the 52 bases deleted in dystrophin Kobe, 2′-O complementary to 31 bases shown in SEQ ID NO: 2 in the sequence listing including the base sequence shown in SEQ ID NO: 1 in the sequence listing was added. -Methyl oligo RNA is synthesized, and its effect on the splicing of the pre-mRNA consisting of exon 18-intron 18-exon 19
The test was performed using the in vitro splicing reaction system described above. As a result, the splicing reaction was inhibited depending on the amount of the antisense oligonucleotide added and the reaction time. This was the first experimental demonstration that antisense oligonucleotides could block dystrophin intron splicing. This indicated that splicing in the nucleus could be controlled by artificial means [Takeshim
a, Y. etal., J. Clin. Invest., 95: 515-520 (199
Five)].

<Splicing control in cell nucleus> In order to demonstrate that antisense oligonucleotides can control splicing of dystrophin pre-mRNA even in the nucleus of living cells, the present inventors prepared normal human lymphoblast cells. Was used to introduce an antisense oligo DNA having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 2 including the nucleotide sequence shown in SEQ ID NO: 1 in the Sequence Listing. Dystrophin mature mRNA produced in the presence thereof was analyzed [Zacharias A. DP. Et al., BBRC, 226: 4
45-449 (1996)]. That is, the antisense oligo DN
A was introduced into the nucleus by mixing with lipofectamine and then adding it to a lymphoblast culture. As a result, unlike the results previously obtained in the in vitro splicing reaction system, antisense oligo DNA against the base sequence of dystrophin exon 19 induces exon 19 skipping in human lymphoblastoid cells,
It was confirmed that RNA in which exon 18 was directly linked to exon 20 was obtained. Further, by extending the culture time, the effect of inducing exon skipping was completed, and all mRNAs were exon 1
9 as a deletion. Furthermore, it was confirmed that the antisense oligo DNA used did not cause a problem in splicing of other exons.

Today, antisense oligonucleotides
(AOs: Antisense oligonucleotides) have been applied to suppress gene expression to inhibit protein translation. AOs have also been used to target specific regions of DNA to inhibit transcription by RNA polymerase II. Further, as another approach, a method of inhibiting abnormal splicing of a precursor mRNA molecule using an antisense oligonucleotide has been reported (Japanese Patent Application Laid-Open No. 8-510130). In addition, phosphorothioate-
Since 2'-O-methyl oligonucleotides do not induce ribonuclease-H activity, they have been used in thalassemia symptomatic patients with the aim of restoring correct splicing by inhibiting shifted splicing sites in precursor mRNA. .

<Application of Artificial Exon Skipping to Treatment> As described above, in DMD, dystrophin mR
It has a genetic abnormality such that the reading frame of NA shifts and becomes out-of-frame. If the abnormalities in the reading frame could be converted to in-frame, the DMD would become BMD and the improvement of the symptoms would be expected. For example, assuming a patient in which only exon 20 is deleted alone, exon 20
It consists of 242 bases. Naturally, the single deletion causes a frame shift, a stop codon appears during translation, and dystrophin synthesis stops in the middle.
Phenotype. However, for this case, if the antisense oligonucleotide to exon 19 as used in the above experiment was administered to artificially induce the skipping of exon 19,
Precursor mRNA contains 88 bases of exon 19 in addition to 242 bases
, Resulting in a total deletion of 330 bases, which could reverse the reading frame to become in-frame, that is, convert the DMD phenotype to BMD by using an antisense oligonucleotide. But there is in theory.

However, the dystrophin gene has a very complex structure as described above, and its pre-mRNA also has a complex secondary structure containing a large number of long introns to be spliced out. Controls the normal progress of splicing. Therefore, whether the antisense oligonucleotide against exon 19 can cause skipping of exon 19 as expected in not only normal human lymphoblast cells but also myoblasts of a patient having a single deletion of exon 20 Alternatively, even if exon 19 is successfully skipped, the mRNA having no abnormality that originally causes exon 20 splicing out has no effect on splicing out of exon 20 or other sites. Whether the reading frame can be converted from out-frame to in-frame, and furthermore, even if the mRNA can be converted into in-frame, whether its mRNA can effectively produce a protein similar to dystrophin, That real Potential was unknown.

Against this background, one of the present inventors has identified exon 20 in dystrophin mature mRNA.
Cells of a DMD patient with a complete deletion of exon 19
It was demonstrated that the antisense oligonucleotides could be used to induce its splice-out and thereby correct the reading frame shift of dystrophin mature mRNA and convert dystrophin negative cells to positive cells, and based on the results DMD
Therapeutic agents for the drug were disclosed earlier (Japanese Patent Application No. 11-140930)
issue).

That is, dystrophin exon 19
Is administered into the culture of myoblasts from a DMD patient with a single deletion of exon 20, which is taken up into the myoblasts and then into the nucleus, resulting in the exon Deletion of exon 19 and exon 20 completely, but the amino acid reading frame is restored from out-frame to in-frame, and it becomes possible to produce full-length dystrophin except for the portion encoded by exon 19 and exon 20 Was confirmed. This suggests that administration of an antisense oligonucleotide against exon 19 to a DMD patient based on a single deletion of exon 20 can reduce DMD, a very serious disease, to BMD, a relatively mild disease.
We strongly support the possibility of converting to MD.

Thus, mRN transcribed from the genome
When the precursor A is spliced into mature mRNA, the splicing promoting sequence present in the exon (in addition to the conventionally known consensus sequence existing at the exon / intron boundary) is used for determining the splicing site. Splicing Enhancer Sequence (SES) plays an important role. As described above, one of the present inventors has shown that SES is present in exon 19 of the dystrophin gene, and further revealed that exon 19 skipping can be introduced by an antisense oligonucleotide against the SES.

Further studies have shown that the inventor has
Using the splicing system described above, a new SES represented by SEQ ID NO: 3 and SEQ ID NO: 4 in the sequence listing was successfully identified in exons 43 and 53 of dystrophin, respectively. Based on these SES sequences, a therapeutic agent for Duchenne muscular dystrophy was created, comprising a complementary nucleotide sequence to them, specifically, an antisense oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 5 and SEQ ID NO: 6 in the sequence listing, respectively [ Japanese Patent Application 2000-12544
8]. These therapeutics include human dystrophin mRNA
Is a Duchenne muscular dystrophy caused by a change in the number of bases due to deletion of a base from a normal base sequence in a base sequence constituting an exon adjacent to exon 43 or 53, wherein the net change in the base number is (3 × N
Each is used to treat Duchenne muscular dystrophy, which is expressed as a decrease of +1) bases (N is zero or a natural number).

[0037]

When the reading frame is repaired against DMD by inducing exon skipping during splicing of dystrophin precursor mRNA, a partially restored dystrophin protein is produced. , Whereby it is possible to change the DMD to a BMD. However, there may be many types of mutation sites in the dystrophin gene in DMD. To treat DMD that develops in response to each mutation, it is necessary to identify SES for additional dystrophin exons and provide antisense to it. An object of the present invention is to provide a further therapeutic agent for DMD by identifying SES in a dystrophin exon other than the above-mentioned exon and inducing the skipping of exon 45 based thereon.

[0038]

Under the above background, the present inventors have succeeded in identifying a new SES within exon 45 of the dystrophin gene using an in vitro splicing system, and based on this, A new treatment for Duchenne muscular dystrophy has been created.

That is, the present invention is selected from the group consisting of DNA having the base sequence shown in SEQ ID NO: 15 in the sequence listing and RNA having a base sequence complementary to the base sequence complementary to the base sequence. An oligonucleotide is provided.

RN of these oligonucleotides
A is the SES portion within exon 45 of the pre-mRNA of human dystrophin. These DNA and RNA
Can be used as a template for the production of antisense as a therapeutic agent for Duchenne muscular dystrophy of the type described below.

The present invention also relates to the present invention.
An antisense oligonucleotide comprising a nucleotide sequence complementary to the nucleotide sequence shown in 5 is also provided.

Since the antisense oligonucleotides are complementary to SES in human dystrophin mRNA exon 45, their administration can induce exon 45 skipping during human dystrophin mRNA splicing. Therefore, the antisense oligonucleotide can be used as a therapeutic agent based on repairing the deviation of the reading frame in a specific Duchenne muscular dystrophy.

The antisense oligonucleotide may be a DNA or a phosphorothioate DNA having the base sequence shown in SEQ ID NO: 19 in the sequence listing. These sequences are complementary to the sequence including the SES in exon 45 and the adjacent base sequences at both ends thereof. Therefore, DNAs (or phosphorothioate DNAs) having these sequences are each mRNA. It can more strongly hybridize to the SES of the precursor exon 45 and block its function.

Further, the present invention provides a human dystrophin mRN
A Duchenne muscular dystrophy caused by a change in the number of bases due to deletion of a base from a normal base sequence in a base sequence constituting an exon adjacent to exon 45 of A, wherein the net change in the base number is (3 × N + 1) (N
Is zero or a natural number). For the production of a therapeutic agent for Duchenne muscular dystrophy, which is expressed as a decrease in bases, a base sequence complementary to the base sequence represented by SEQ ID NO: 15 in the sequence listing is used. Also provided is the use of an antisense oligonucleotide having. Here, the antisense oligonucleotide may be a DNA or a phosphorothioate DNA having a base sequence represented by SEQ ID NO: 19 in the sequence listing.

The present invention further provides a therapeutic agent comprising any of the above antisense oligonucleotides against SES of exon 45 in a pharmaceutically acceptable injectable medium. . The therapeutic agent is a Duchenne muscular dystrophy caused by a change in the number of bases due to deletion of a base from a normal base sequence in a base sequence constituting an exon adjacent to exon 45 in human dystrophin mRNA, Used for Duchenne muscular dystrophy, where the net change is expressed as a reduction of (3 × N + 1) (N is zero or a natural number) bases.

In the present invention, "oligonucleotide"
Includes not only oligo DNA and oligo RNA, but also phosphorothioate analogs such as phosphorothioate oligo DNA. Phosphorothioate DNA is a nucleotide in which the oxygen atom of the phosphate group has been replaced with a sulfur atom, and is resistant to various nucleases. Is a widely used nucleotide analog,
Its preparation and properties, as well as its various uses, are well known to those skilled in the art. Phosphorothioate DNA forms base pairs similarly to ordinary DNA, but has high resistance to various degrading enzymes and is particularly advantageous for use in the present invention. Here, the “phosphorothioate analog” has a structure in which one or more phosphorodiester groups between nucleotides of ordinary DNA are replaced with phosphorothioate groups.

The therapeutic agent of the present invention is preferably used in an amount of 0.05 to 5
μmoles / ml of the antisense oligonucleotide,
0.02-10% w / v carbohydrate or polyhydric alcohol and
A more preferred range of the antisense oligonucleotide containing from 0.1 to 0.4% w / v of a pharmaceutically acceptable surfactant is from 0.1 to 1 μmoles / ml.

The carbohydrates include monosaccharides and / or 2
Sugars are particularly preferred. Examples of these carbohydrates and polyhydric alcohols include glucose, galactose, mannose, lactose, maltose, mannitol and sorbitol. These may be used alone or in combination.

Preferred examples of the surfactant include:
Polyoxyethylene sorbitan mono-tri-ester,
Alkylphenyl polyoxyethylene, sodium taurocholate, sodium cholate, and polyhydric alcohol esters are included. Of these, particularly preferred are polyoxyethylene sorbitan mono- to tri-esters, wherein particularly preferred esters are oleate, laurate, stearate and palmitate. These may be used alone or in combination.

The therapeutic agent of the present invention is more preferably
0.03-0.09M pharmaceutically acceptable neutral salts, for example,
Contains sodium chloride, potassium chloride and / or calcium chloride.

Further, the therapeutic agent of the present invention is more preferably
It may contain 0.002-0.05M pharmaceutically acceptable buffer. Examples of preferred buffers include sodium citrate, sodium glycinate, sodium phosphate, tris (hydroxymethyl) aminomethane. These buffers may be used alone or in combination.

Further, the above-mentioned therapeutic agent may be supplied in a solution state. However, it is usually preferable to freeze-dry it in order to stabilize the antisense oligonucleotide and prevent a decrease in the therapeutic effect, for example, when it is necessary to store the solution for a certain period of time. It may be reconstituted with distilled water for injection, etc., that is, used in the liquid state to be administered. Accordingly, the therapeutic agents of the present invention also include those that contain the antisense oligonucleotide in a lyophilized state so that each component can be reconstituted prior to use in a patient so as to be in a predetermined concentration range. Amino acids such as albumin and glycine may be further contained for the purpose of enhancing the solubility of the lyophilized product. When designing the lyophilized preparation, the lysing solution for reconstitution may be distilled water for injection, or may contain a part of the therapeutic agent other than the antisense oligonucleotide.

[0053]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail based on various tests.

1. Induction of Exon Skipping in Patient-Derived Lymphoblast Cells As described above, in the splicing reaction of a pre-mRNA transcribed from a gene having a normal dystrophin gene structure, the inventors designed the nucleotide sequence in exon 19 for the splicing reaction. It was confirmed that the antisense oligonucleotide effectively induced exon 19 skipping. On the other hand, in a DMD patient in which exon 20 has been deleted, the secondary or tertiary structure of the dystrophin pre-mRNA is expected to be different from normal because the gene structure is different from normal. It was examined whether or not the 31-base antisense oligonucleotide was effective even in DMD patients in which such exon 20 had been deleted. That is, as described in detail below, a lymphoblast cell line transformed with EB virus was established from two DMD patients lacking dystrophin exon 20, and exon skipping was performed using an antisense oligonucleotide using this. I confirmed that I could guide.

(A) Establishment of DMD patient-derived lymphoblast cell line DMD patient 2 lacking dystrophin exon 20
From the name, a lymphoblastoid cell line transformed with the EB virus was established as follows. That is, 2 ml of whole blood collected from a patient is mixed with 2 ml of RPMI1640 medium (supplemented with 10% FBS), and 3 ml of Ficoll Paque (Pharmacia)
Layered on top and subjected to concentration gradient centrifugation. Thereafter, only the lymphocyte layer was collected, washed twice with RPMI1640 medium (supplemented with 10% FBS), and finally suspended in 0.5 ml of RPMI1640 medium (supplemented with 10% FBS) to obtain a lymph suspension. This solution was mixed with 0.5 ml of an EB virus solution prepared in advance, and the mixed solution was cultured at 37 ° C. for one week. One week later, the cells were washed with RPMI1640 medium (supplemented with 10% FBS) to remove the EB virus, and then the culture was continued using the same culture solution. Thus, cells which became morphologically large and became lymphoblasts were obtained by infecting the patient's lymphocytes with the EB virus.

(B) Introduction of Antisense Oligo DNA The cell culture solution of the lymphoblastoid cell line obtained as described above was centrifuged to separate only cell components. And it consists of a sequence complementary to the base sequence shown by SEQ ID NO: 2 in the sequence listing
A 31-base antisense oligo DNA was prepared at about 200 nM (200 pm).
oles / ml) and 2% fetal calf serum (FBS) in a maintenance medium at 36 ° C. for 5 hours. Next, after the medium was replaced with a serum medium, culture was continued for 12 hours. After completion of the culture, cells were collected by a conventional method to extract total RNA.

(C) Analysis of dystrophin cDNA Using the obtained total RNA as a substrate and a random oligonucleotide consisting of hexa-oligonucleotide as a primer, cDNA was synthesized by the action of reverse transcriptase according to a conventional method. A region extending from exons 18 to 21 of dystrophin was amplified from the synthesized cDNA by using nested PCR. First, amplification was performed once with primers designed for exons 18 and 21. Using this amplification product as a template, a primer matching a region located inside the primer used for the first time was designed, and a second PCR amplification was performed. This amplification was performed with the annealing temperature set at 60 ° C.

(D) Confirmation of Exon 19 Skipping Exon 18 to 2 of dystrophin cDNA
When the region of No. 1 was amplified, no antisense was added.
A clean band of 384 base pairs was obtained. When the base sequence of this amplification product was determined by a conventional method, it was shown that it consisted of exons 18, 19, and 21. This was in good agreement with the patient's genetic analysis.

On the other hand, with the cDNA of the cells to which the antisense oligo DNA had been added, not only an amplification product of the same size as that obtained from the cells into which the antisense oligo DNA had not been introduced, but also a small size product from the fourth day of culture was obtained. A product with the reading frame maintained was obtained. Also,
In the lymphoblast cells established from Case 2, two types of bands were obtained by the same treatment. When the base sequence of the small-sized amplification product was determined, it was found that the sequence of exon 18 was directly linked to the sequence of exon 21, and that exons 19 and 20 were deleted. This indicates that exon 19
Indicates that skipping has occurred. On the other hand, in the lymphoblast cells established from a normal person, only transcripts having a small size, that is, those having exon 19 skipping were obtained under these conditions. In addition, dystrophin cDN
When the whole region of A was divided into 10 antibodies and amplified and examined, no fragment showing abnormal splicing was obtained.

(E) Discussion The cause of the difference in the exon skipping-inducing effect of this antisense oligonucleotide between normal and DMD patients is due to the difference in the secondary structure or tertiary structure of the mRNA precursor around exon 19. Was considered one. By changing the concentration of the antisense oligonucleotide,
Furthermore, the exon skipping induction efficiency in DMD patients was examined. However, conditions were not obtained such that all transcripts exhibited exon skipping, as seen in cells from normal patients. In addition, this antisense induction was not observed with the sense oligonucleotide or the antisense oligonucleotide with respect to other regions.

These results indicate that dystrophin mRNA
Controlling precursor splicing induces exon skipping, indicating that the reading frame can be modified. However, it was still unclear whether the mRNA whose amino acid reading frame was able to be maintained by such an mRNA modification in a muscle cell could effectively synthesize a protein.

[0062] 2. Expression of dystrophin-like protein in DMD patient-derived muscle cells Then, it was examined whether or not a dystrophin-like protein is expressed using myoblasts of a DMD patient lacking exon 20.

(A) Establishment of a DMD patient-derived muscle cell line From a patient lacking exon 20 of the dystrophin gene, muscle tissue was aseptically removed, cut into small pieces, and free cells were obtained with trypsin. After washing, the cells were cultured in a growth medium (Growth medium: Ham-F10 supplemented with 20% FCS and 0.5% chicken embryo extract). At the time of passage, a cover glass was placed in a dish for cell culture, and the myocytes were cultured thereon. When the myoblasts proliferated until the ratio reached about 80%, the culture medium was replaced with Fusion medium (DMEM supplemented with 2% HS) to induce differentiation, and the cells were used as myocytes.

(B) Introduction of antisense oligo DNA On day 4 after the induction of differentiation, antisense oligo DNA (20
0 pmol) was introduced into the cells by lipofectamine (6 μl), and the cells were further cultured for 3, 7, and 10 days.

(C) Dystrophin Immunostaining Each cell after the above culture was subjected to dystrophin immunostaining using an antibody against the C-terminal of dystrophin. As a result, dystrophin was stained in cells that had no dystrophin staining at first. In each case, dystrophin-positive cells were confirmed. In addition, even when an antibody recognizing the N-terminal region of dystrophin was used, the same staining result as that of staining of the C-terminal region was obtained, and it was confirmed that the dystrophin produced extends from the N-terminal to the C-terminal. .

As described above, dystrophin staining was observed in the myoblasts to which the antisense oligo DNA had been added, whereas the antisense oligo DNA was treated in the same manner.
When dystrophin staining was not observed when myoblasts without additives were examined.

(D) Analysis of dystrophin cDNA Subsequently, RNA was extracted from the cultured myoblasts to which the antisense oligo DNA had been added by an ordinary method. Obtained RNA
More cDNA was synthesized and the region of exons 18-21 of dystrophin was amplified in the same manner as described above for RNA from lymphoblast cells.

The nucleotide sequence of the obtained amplification product was determined by a conventional method. As a result, from day 4 of culture, exon 1
The base sequence of No. 8 was directly linked to the base sequence of exon 21 to obtain an amplification product in which the amino acid reading frame was in frame.

Next, the cDNA prepared from the cultured myoblasts to which the antisense oligo DNA had been added was amplified by the PCR method by dividing the entire region into 10 portions, and the size of each fragment obtained as an amplification product was determined by a conventional method. And electrophoresis. As a result, except for the skipping of exons 19 and 20, no fragment suggesting abnormal splicing was found. These results indicate that the resulting dystrophin mature mRNA was exon 19 and exon 20
This indicates that the mRNA is a full-length mRNA in which the reading frame is maintained, except for the fact that is completely deleted.

3. Transfer of the antisense oligo DNA into the nucleus Then, in order to confirm that the antisense oligo DNA is actually transferred into the nucleus and acting, the fluorescently labeled antisense oligo DNA is used to transfer the antisense oligo DNA The state of migration into the inside was observed.

The antisense oligo DNA used above was labeled with FITC (fluorescein isothiocyanate) by a conventional method, and the transfer of the antisense oligo DNA into the nucleus was examined. That is, a muscle cell collected from a DMD patient was used as a growth medium (Growth medium: 20% FCS, 0.5%
The cells were cultured in Ham-F10) supplemented with a chicken embryo extract. Culture is performed on a cover glass placed in a cell culture dish, and when cells become semi-confluent.
Differentiation was induced by replacing the cells with Fusion medium (DMEM supplemented with 2% HS) to obtain muscle cells. On day 4 after differentiation induction, FIT
C-labeled antisense oligo DNA (200 pmol) was introduced into cells by lipofectan (6 μl),
After 2, 3, 7 and 10 days, the localization of FITC was confirmed with a fluorescence microscope.

As a result, a fluorescent signal was detected in accordance with the nucleus. This confirms that the antisense oligo DNA actually translocated into the nucleus and skipped exon 19 splicing.

As shown in the above test results, a protein corresponding to dystrophin can be synthesized by converting the amino acid reading frame into in-frame in myoblasts of a DMD patient. This indicates the possibility of converting a patient having a single deletion of exon 20 into a relatively mild BMD in DMD, which was conventionally a very serious incurable disease.

4. Induction of Exon Skipping by Intraperitoneal Administration of Antisense Oligonucleotides to mdx Mice To examine the effects of administration to living organisms, male mdx mice 6-8 weeks old were challenged with exon 19 of dystrophin mRNA.
20 m of the antisense oligonucleotide to
g / kg was administered intraperitoneally. Intraperitoneal administration of 2, 4, 7, and
Fourteen days later, mouse heart muscle and skeletal muscle tissue samples were collected, and the mRNA contained therein was extracted by a conventional method. Using this as a template, exon 18 of dystrophin mRNA
After amplifying the region of ２０20 by RT-PCR, the amplification products were separated by gel electrophoresis. As a result, both the myocardial and skeletal muscle samples showed exon 1 two days after administration.
A skipped nucleotide fragment of exon 19, consisting only of 8 and exon 20, was clearly identified. This fragment was slightly observed 4 days after the administration.
These results indicate that not only in in vitro tests, but also by injection administration to animals, antisense oligonucleotides to exons in dystrophin pre-mRNA prevent skipping of the exons in cardiac and skeletal muscle cells of the animals. It indicates that it is induced. In addition, in order to examine the presence or absence of localization of the administered antisense oligonucleotide in skeletal muscle and its change with time, FITC-labeled 20 mg / kg of the antisense oligonucleotide was intraperitoneally administered to mdx mice. 4, 7, and 14 days later, mouse skeletal muscle tissue samples were collected and observed with a fluorescence microscope. as a result,
Two days after intraperitoneal administration of the FITC-labeled antisense oligonucleotide, it was observed that the skeletal muscle cell membrane became fluorescently positive. Four days after administration, fluorescence was also observed in myocyte nuclei. This result indicates that the injected antisense oligonucleotide was transferred to the muscle cell nucleus. Furthermore, to see the dose-effect relationship of the antisense oligonucleotide, the mdx mice were challenged with the antisense oligonucleotide for 0.2, 2, 20 or 200 m respectively.
g / kg was administered intraperitoneally. Two days after the administration, cardiac and skeletal muscle tissues were collected, and mRNA contained therein was extracted by a conventional method. RT- region covering exons 18-20
The product was amplified by PCR, and the amplified products were separated by gel electrophoresis. As a result, in the tissue of the mouse to which the 20 mg / kg or 200 mg / kg antisense oligonucleotide was administered, a skipped nucleotide fragment of exon 19 consisting of only exon 18 and exon 20 was clearly confirmed. Production of the fragment was highest in samples from mice that received 20 mg / kg of the antisense oligonucleotide.

5. Search for SES sequences in other exons-
1 Based on the above results, the present invention further provides an exon consisting of a fraction of bases relative to the reading frame around exons 45 to 55, which are deletion-prone sites in the dystrophin gene (ie, when the deletion occurs, (Out of frame for amino acid readings) was examined for the presence of sequences that provide SES as a transcript. As described above, SES is rich in purine residues (particularly, aag repeats) by analysis in an in vitro system. Based on this, the present inventors have proposed that a base sequence consisting of 26 bases in exon 43 (sequence table) can serve as a template for providing a transcript relatively rich in purine residues in exons of the human dystrophin gene. (2) a base sequence consisting of 28 bases in exon 46; and (3) a base sequence consisting of 28 bases in exon 53 (shown as SEQ ID No. 4 in the sequence listing). Base sequence complementary to the base sequence)
Were selected as candidates, and it was next examined whether or not those sequences give transcripts having SES activity.

For the preparation and evaluation of the pre-mRNA for the evaluation of SES activity, the doublesex (ds
x) exon 3, intron 3, and exon 4 of the gene
Watakabe, A., et al., Genes
& Development, 7: 407-418 (1993) was used as the basic plasmid. This plasmid contains a female-specific acce site from exon 3 of the Drosophila doublesex (dsx) gene.
pspdsxE34f, a plasmid obtained by subcloning a dsx genomic fragment containing up to 1128 bases downstream of ptor site) into pSP73 (Promega) [Inoue et al., Proc. Natl.
Acad. Sci. USA, 89: 8092-8096 (1992)] BglII-HincII
It was obtained by inserting the fragment into the BglII-SmaI site of plasmid pSP72. In this transcript, splicing between exons sandwiching intron 3 is not observed in the transcript unless SES is added immediately downstream of the 5′-terminal portion of exon 4 which is a female-specific exon. To provide a system in which splicing can be seen when SES is added to the system. On the other hand, for the base sequence to be analyzed, single-stranded DNAs in both forward and reverse directions were synthesized. At this time, 5 'of the DNA in the forward direction
A restriction enzyme BamHI cleavage site was added to the end, and the D
A restriction enzyme XhoI cleavage site was added to the 5 'end of NA. The synthesized forward and reverse single-stranded DNAs are mixed and heat-treated (at 94 ° C., 2
After that, the temperature was returned to room temperature and both strands were annealed to form double strands. This double-stranded DNA was ligated to the Bam immediately downstream of the 5 'end of dsx exon 4 in the basic plasmid for the test.
Inserted into HI-Xhol site. Thus, a plasmid containing a minigene consisting of the base sequence from exon 3 of dsx to the 5 'end of exon 4 and the base sequence to be analyzed inserted downstream thereof was obtained. Using these plasmids as templates, pre-mRNA labeled with radioisotope by RNA polymerase was synthesized by a conventional method. This mRNA precursor was used for Hela in the same manner as described above.
After reacting with the cell nucleus extract for 1 hour to allow the splicing reaction to proceed, analysis was performed by gel electrophoresis according to a conventional method.

As a result, the SES of exons 43 and 53
In the splicing reactions using the pre-mRNAs incorporating the candidates, the production of spliced mRNAs between the exons sandwiching intron 3 was clearly observed. This indicates that these two candidate sequences have SES activity. In contrast, the SE
Exon 43 had higher S activity. On the other hand, in the splicing reaction using the pre-mRNA incorporating the SES candidate for exon 46, although the mRNA subjected to the splicing reaction was observed, the activity was very weak.

Thus, the present inventors have found that SES exists in exons 43 and 53 of human dystrophin mRNA. their SES in mRNA
Are the ribonucleotide sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 in the sequence listing, respectively.

As described above, the present inventors have found that SES is present in exon 19 of the precursor mRNA which is a transcript of the dystrophin gene, and that skipping of exon 19 can be induced by an antisense oligonucleotide against the SES. It has been found that this enables the recovery of the reading frame in patients with exon 20 deletion. Similarly, for SES present in exon 43 and exon 53 further identified above, respectively, exons 43 (173 bases, that is, 3 × 57 + 2 bases) and 53 (212 bases) can be obtained by using antisense oligonucleotides against them. Base, ie 3
(× 70 + 2 bases) can be induced.

Therefore, the precursor mRNA of dystrophin
In DMD cases in which the number of bases is reduced by (3 × N + 1) (N is zero or a natural number) due to deletion of a base sequence in an exon adjacent to exon 43, exon 43 has an antiserum against SES. Exon 4 during splicing by administration of sense oligonucleotide
3 skipping is induced. As a result, 173 bases constituting exon 43 are further deleted during splicing.
Mutations that were out-of-frame can be returned to in-frame by making the number of deleted bases in NA a multiple of three.
For this reason, although the amino acid corresponding to the skipped nucleotide sequence is deleted, the amino acid sequence on the downstream side thereof is not affected by the genetic abnormality, and the dystrophin protein is synthesized.
Is converted to Examples of such DMDs include deletions of exon 44 (148 bases, ie, 3 × 49 + 1 bases), and exons 44 to 46 (148 + 176 + 148 = 472 bases, ie, 3
X157 + 1 bases), exons 44 to 47
Up to (148 + 176 + 148 + 150 = 622 bases, ie 3 × 207 + 1 bases)
Deleted, exons 44 to 48 (148 + 176 +
148 + 150 + 186 = 808 bases, ie, 3 × 269 + 1 bases, and exons 44 to 49 (148 + 176 + 148 + 15 bases)
0 + 186 + 102 = 910 bases, that is, 3 × 303 + 1 bases).

Similarly, for example, the number of bases is reduced by (3 × N + 1) (N is zero or a natural number) due to deletion of a base sequence in an exon adjacent to exon 53 of dystrophin precursor mRNA of a DMD patient. For DMD cases of the type, administration of an antisense oligonucleotide to the SES of exon 53 induces exon 53 skipping during splicing. Applicable examples include deletion of exon 52 (118 bases, ie, 3 × 39 + 1 bases), or deletion of exon 50, exon 51, exon 52 (109 + 233 + 118 = 460 bases,
That is, there is a DMD case in which 3 × 153 + 1 bases have been deleted. In contrast, by introducing an antisense oligonucleotide against SES of exon 53 to induce skipping of exon 53 during splicing, the length of the deletion in the mRNA after splicing is set to 330 bases and 672 bases, respectively. It is possible. By doing so, the mRNA after splicing
Since the number of the deleted bases is a multiple of 3, the displacement of the reading frame caused by the original deletion is restored to normal.

6. Search for SES Sequence in Additional Exon-2 The present invention and the like further identify SES in exon 45 (this nucleotide sequence is shown in SEQ ID NO: 7 in the sequence listing) at the site of deletion-prone site in the dystrophin gene. The study was conducted for the purpose. That is, a base sequence consisting of 163 bases obtained by removing a splicing site (7 bases on the 5 ′ side and 6 bases on the 3 ′ side) from the base sequence of exon 45 (176 bases) is converted into a fragment consisting of about 30 bases ( Fragments 1-5)
Into 5 parts. Thus, from the 5 'side to the 3' side, 31 bases (fragment 1: shown by SEQ ID NO: 8 in the sequence listing), 32 bases (fragment 2: shown by SEQ ID NO: 9 in the sequence listing), and 33 bases (fragment 3: represented by SEQ ID NO: 10 in the sequence listing), 32 bases (fragment 4: SEQ ID NO: 11 in the sequence listing)
) And 35 bases (fragment 5: shown as SEQ ID NO: 12 in the sequence listing). For each fragment, as described above, the Drosophila double sex
(dsx) gene exon 3, intron 3, exon 4
A DNA having a sequence serving as a template for these SES candidates was inserted into the 3′-side of the plasmid into which the DNA was inserted to prepare a minigene. As a positive control, the exon 19 SE
A minigene in which a DNA having the S sequence (shown by SEQ ID NO: 1 in the sequence listing) was similarly incorporated was also prepared. This SES
The SES activity of each fragment was measured using an activity measurement system. That is, using those plasmids as templates, mRNA precursors labeled with radioisotope by RNA polymerase were synthesized in a conventional manner. This mRNA precursor was reacted with a Hela cell nucleus extract for 1 hour in the same manner as described above to allow the splicing reaction to proceed, and then analyzed by gel electrophoresis according to a conventional method. As a result, fragment 4
It was found that the fragment had a slightly stronger SES activity than other fragments.

Next, the sequences before and after the fragment 4 were subjected to SE
The S activity was examined. That is, 1) a fragment consisting of a base sequence of a region of the same length located 16 bases upstream from the region corresponding to fragment 4 in the base sequence of exon 45 (fragment 4a: shown by SEQ ID NO: 13 in the sequence listing); 2) Fragment consisting of the base sequence of the same length region located 13 bases upstream (fragment 4b: shown by SEQ ID NO: 14 in the sequence listing), from the base sequence of the same length region located 16 bases downstream Fragment (fragment 4c: SEQ ID NO: 1 in the sequence listing)
5) were prepared, and the SES activity was examined for each in the same manner as described above. As a result, it was found that the fragment 4c had a stronger activity than the original fragment 4. The activity of fragment 4c was comparable to that of exon 19 SES.

For further identification of the active region, fragment 4c
The SES activity was also examined for sequences with shortened. That is, a fragment obtained by deleting 10 bases on the 5 'side of fragment 4c (fragment 4
ca: represented by SEQ ID NO: 16 in the sequence listing), 3 ′
A fragment in which bases have been deleted (fragment 4cb: represented by SEQ ID NO: 17 in the sequence listing) and a fragment in which 5 bases have been removed from both the 5 ′ and 3 ′ sides (fragment 4cc: represented by SEQ ID NO: 18 in the sequence listing) They were prepared, and the SES activity of each of them was examined by the above procedure. As a result, each of the sequences had weaker SES activity than the original fragment 4c. This indicates that fragment 4c (shown as SEQ ID NO: 15 in the sequence listing)
Indicates that the base sequence of exon 45 constitutes the region with the highest SES activity in exon 45. This means that skipping of exon 45 can be induced by the antisense oligonucleotide to the nucleotide sequence of fragment 4c. An example of the antisense oligonucleotide is a DNA or a phosphorothioate DNA having the base sequence shown in SEQ ID NO: 19 in the sequence listing.

Exon 45 has 176 bases (3 × 58 + 2 bases)
Consisting of Therefore, in a Duchenne muscular dystrophy caused by a change in the number of bases due to deletion of a base from a normal base sequence in a base sequence constituting an exon adjacent to exon 45 in human dystrophin mRNA, the net change in the number of bases Can be expressed as a reduction of (3 × N + 1) bases (N is zero or a natural number), and it is possible to recover the reading frame by inducing a further reduction of 176 bases by skipping exon 45. Become. Examples of such DMDs include exon 44 (148 bases, ie, 3 × 49 + 1 bases), exon 46 (148 bases, ie, 3 × 49 + 1 bases).
Exons 46 and 47 (148 + 150 = 298
Bases, ie 3 × 99 + 1 bases), exon 4
6 to 48 (148 + 150 + 186 = 484 bases, ie 3 × 161 + 1
Bases), exons 46, 47 and 49
(148 + 150 + 102 = 400 bases, ie 3 × 133 + 1 bases), exons 46, 47, 49, 50 and 51 (14
8 + 150 + 102 + 109 + 233 = 742 bases, ie, 3 × 247 + 1 bases), exons 46, 47, 49, 50, 51,
52, 53, 54 and 55 (148 + 150 + 102 + 109 + 233 + 118 +
212 + 155 + 190 = 1417 bases, ie, 3 × 472 + 1 bases).

7. Preparation of Antisense Oligo DNA and Phosphorothioate DNA The production of DNA and a phosphorothioate DNA having a nucleotide sequence complementary to the nucleotide sequence shown by SEQ ID NO: 15 in the sequence listing is performed by Applied Biosystems M
Using a commercially available DNA synthesizer such as odel 1380B, etc.
Zon et al., "Oligonucleotides and Analogue
s ": A Practical Approach, F. Eckstein, Ed., p. 87
-108, Oxford University Press, Oxford, England, U.S. Patent No. 5,151,510.

7. Clinical Use of Antisense Nucleotide for Exon 45 SES Administration of the antisense oligonucleotide to a compatible DMD patient can be performed, for example, as follows. That is, an antisense DNA or antisense phosphorothioate DNA comprising a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 15 in the sequence listing, for example, consisting of the nucleotide sequence shown in SEQ ID NO: 19 in the sequence listing Is manufactured by a method well known to those skilled in the art, and this is sterilized by a conventional method to prepare a solution for injection at, for example, 1200 μg / ml. This solution is injected intravenously into a patient at a dose of the antisense oligonucleotide of, for example, 20 m / kg body weight.
g, for example, in the form of an infusion. The administration is repeated, for example, four times at intervals of two weeks, and thereafter, the treatment is repeated as appropriate, while confirming the therapeutic effect based on the expression of dystrophin protein in the muscle biopsy tissue, the serum creatine kinase value, and the clinical symptoms. . As long as there is a therapeutic effect and no obvious side effects are observed, the treatment is continued and, in principle, administered for life.

Hereinafter, the present invention will be described more specifically with reference to typical examples. However, it is not intended that the present invention be limited to the examples.

[0089]

EXAMPLES <Formulation Example 1> A required amount of a base component was mixed and dissolved in accordance with the following formulation, and an antisense oligonucleotide was dissolved therein to make a predetermined liquid volume.
The solution is filtered through a 0.22 μm membrane filter to give a preparation for intravenous administration. Antisense oligonucleotide (Note 1): 500 mg Sodium chloride: 8.6 g Potassium chloride: 0.3 g Calcium chloride: 0.33 g Distilled water for injection: 1000 ml Note 1: Shown in SEQ ID NO: 19 in the sequence listing Phosphorothioate DNA consisting of an isolated base sequence

<Formulation Example 2> According to the following formulation, a required amount of a base component was mixed and dissolved, and an antisense oligonucleotide was dissolved therein to make a predetermined volume. ) To give a preparation for intravenous administration. Antisense oligonucleotide (Note 2): 100 mg Sodium chloride: 8.3 g Potassium chloride: 0.3 g Calcium chloride 0.33 g Sodium hydrogen phosphate / 12 hydrate 1.8 g 1N hydrochloric acid ... Appropriate amount (pH 7.4) Distilled water for injection ... Total amount 1000ml Note 2: Phosphorothioate DNA consisting of the base sequence shown in SEQ ID NO: 19 in the sequence listing

<Formulation Example 3> A required amount of a base component is mixed and dissolved according to the following formulation, and an antisense oligonucleotide is dissolved therein to make a predetermined liquid volume. A filter having a pore size of 35 nm (PLANOVE 35: Asahi Kasei Corporation) ) To give a preparation for intravenous administration. Antisense oligonucleotide (Note 3): 100 mg Sodium chloride: 8.3 g Potassium chloride: 0.3 g Calcium chloride: 0.33 g Glucose: 0.4 g Sodium hydrogen phosphate, dodecahydrate …… 1.8g 1N hydrochloric acid …… Appropriate amount (pH 7.4) Distilled water for injection …… Total amount 1000m
l Note 3: Phosphorothioate DNA consisting of the base sequence shown in SEQ ID NO: 19 in the sequence listing

[0092]

[Sequence List] Sequence Listing <110> JCR Pharmaceuticals Co., Ltd .; Matsuo, Masafumi <120> Medicament for Treatment of Duchenne Muscular Dystrophy <130> P128-01 <150> JP2000-256547 <151> 2000-08-25 <160> 19 <210> 1 <211> 16 <212> DNA <213> Homo sapience <400> 1 gcaagatgcc agcaga 16 <210> 2 <211> 31 <212> DNA <213> Homo sapience <400> 2 aactgcaaga tgccagcaga tcagctcagg c 31 <210> 3 <211> 26 <212> RNA <213> Homo sapience <400> 3 agcaagaaga cagcagcauu gcaaag 26 <210> 4 <211> 26 <212> RNA <213> Homo sapience <400> 4 ggaagcuaag gaagaagcug agcagg 26 <210> 5 <211> 31 <212> DNA <213> Homo sapience <400> 5 gcactttgca atgctgctgt cttcttgcta t 31 <210> 6 <211> 31 <212> DNA <213> Homo sapience <400> 6 gacctgctca gcttcttcct tagcttccag c 31 <210> 7 <211> 176 <212> DNA <213> Homo sapience <400> 7 gaactccagg atggcattgg gcagcggcaa actgttgtca gaacattgaa tgcaactggg 60 gaagaaataa ttcagcaatc ctcaaaaaca gatgcaagta ttctacagga aaaattggga 120 agcctgaatc tgcggtggca ggaggtctgc aaacagctgt cagacagaaa aaagag 176 < 210> 8 <211> 31 <212> DNA <213> Homo sapience <400> 8 aggatggcat tgggcagcgg caaactgttg t 31 <210> 9 <211> 32 <212> DNA <213> Homo sapience <400> 9 cagaacattg aatgcaactg gggaagaaat aa 32 <210> 10 <211> 33 <212> DNA <213> Homo sapience <400> 10 ttcagcaatc ctcaaaaaca gatgccagta ttc 33 <210> 11 <211> 32 <212> DNA <213> Homo sapience <400> 11 tacaggaaaa attgggaagc ctgaatctgc gg 32 <210> 12 <211> 35 <212> DNA <213> Homo sapience <400> 12 tggcaggagg tctgcaaaca gctgtcagac agaaa 35 <210> 13 <211> 32 <212> DNA <213> Homo sapience <400> 13 acagatgcca gtattctaca ggaaaaattg gg 32 <210> 14 <211> 32 <212> DNA <213> Homo sapience <400> 14 gatgccagta ttctacagga aaaattggga ag 32 <210> 15 <211> 32 <212> DNA <213> Homo sapience <400 > 15 aagcctgaat ctgcggtggc aggaggtctg ca 32 <210> 16 <211> 22 <212> DNA <213> Homo sapience <400> 16 ctgcggtggc aggaggtctg ca 22 <210> 17 <211> 22 <212> DNA <213> Homo sapience < 400> 17 aagcctgaat ctgcggtggc ag 22 <210> 18 <211> 22 <212> DNA <213> Homo sapience <400> 18 tgaatct gcg gtggcaggag gt 22 <210> 19 <211> 32 <212> DNA <213> Homo sapience <400> 19 tgcagacctc ctgccaccgc agattcaggc tt 32

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 47/10 A61K 47/12 47/12 47/14 47/14 47/18 47/18 47/26 47 / 26 47/28 47/28 47/34 47/34 48/00 48/00 A61P 21/00 A61P 21/00 25/14 25/14 C12N 15/00 ZNAA F-term (reference) 4B024 AA01 CA01 CA11 4C076 BB11 CC01 CC09 DD01 DD09 DD22 DD23 DD26Z DD38 DD43Z DD46 DD50Z DD51Z DD66 DD67 DD70 FF16 FF61 4C084 AA13 MA05 MA66 NA14 ZA012 ZA942 4C086 AA01 AA02 AA03 EA16 MA01 MA02 MA04 MA05 MA66 NA14 ZA01 ZA94 ZA94

Claims (16)

[Claims] 1. An oligonucleotide selected from the group consisting of DNA having the base sequence shown in SEQ ID NO: 15 in the sequence listing and RNA having a base sequence complementary to a base sequence complementary to the base sequence. 2. An antisense oligonucleotide comprising a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 15 in the sequence listing. 3. The antisense oligonucleotide according to claim 2, which is a DNA or a phosphorothioate DNA having a base sequence represented by SEQ ID NO: 19 in the sequence listing. 4. Exon 4 of human dystrophin mRNA
5. A Duchenne muscular dystrophy caused by a change in the number of bases due to deletion of a base from a normal base sequence in a base sequence constituting an exon adjacent to 5, wherein the net change in the number of bases is (3 × N + 1) Use of the antisense oligonucleotide of claim 2 or 3 for the manufacture of a therapeutic agent for Duchenne muscular dystrophy, which can be expressed as (N is zero or a natural number) base reduction. 5. A therapeutic agent for Duchenne muscular dystrophy, comprising the antisense oligonucleotide according to claim 2 or 3 in a pharmaceutically acceptable injectable medium. 6. The therapeutic agent according to claim 5, comprising 0.05 to 5 μmoles / ml of the antisense oligonucleotide. 7. The therapeutic agent according to claim 5, comprising 0.02 to 10% w / v of carbohydrate or polyhydric alcohol and 0.01 to 0.4% w / v of a pharmaceutically acceptable surfactant. 8. The therapeutic agent according to claim 5, which comprises 0.03 to 0.09 M of a pharmaceutically acceptable neutral salt. 9. The therapeutic agent according to claim 8, wherein said neutral salt is selected from the group consisting of sodium chloride, potassium chloride and calcium chloride. 10. The therapeutic agent according to claim 5, further comprising 0.002 to 0.05M of a pharmaceutically acceptable buffer. 11. The therapeutic agent according to claim 10, wherein said buffer is selected from the group consisting of sodium citrate, sodium glycinate, sodium phosphate, and tris (hydroxymethyl) aminomethane. 12. The therapeutic agent according to claim 7, wherein said carbohydrate is selected from the group consisting of monosaccharides and disaccharides. 13. The therapeutic agent according to claim 7, wherein said carbohydrate or polyhydric alcohol is selected from the group consisting of glucose, galactose, mannose, lactose, maltose, mannitol and sorbitol. 14. The surfactant according to claim 1, wherein the surfactant is selected from the group consisting of polyoxyethylene sorbitan mono- to tri-esters, alkylphenyl polyoxyethylene, sodium taurocholate, sodium cholate and polyhydric alcohol esters. 7 or 12 therapeutic agents. 15. The therapeutic agent according to claim 14, wherein said polyoxyethylene sorbitan ester is an ester selected from the group consisting of oleate, laurate, stearate and palmitate. 16. The therapeutic agent according to claim 5, comprising the lyophilized antisense oligonucleotide for reconstitution prior to use in a patient.