Medicament for treatment of Duchenne muscular dystrophy

Details Medicament for treatment of Duchenne muscular dystrophy Medicament for treatment of Duchenne muscular dystrophy

2000-09-22

2003-11-25

Lacourciere, Karen (Department: 1635)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S024300, C536S024310, C536S024330, C435S006120

Reexamination Certificate

active

06653467

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to medicaments for treatment of Duchenne muscular dystrophy, which medicaments are designed to correct an existing shift of the amino acid reading frame in dystrophin pre-mRNA by inducing in a predetermined manner an exon skipping in the pre-mRNA having the reading frame shift resulting from abnormalities in dystrophin gene. More specifically, the present invention relates to splicing enhancer sequences (SES's) in dystrophin gene which can be used for the preparation of medicaments for treatment of certain types of Duchenne muscular dystrophy, as well as to antisense oligonucleotides against the splicing enhancer sequences, and medicaments comprising thereof.
BACKGROUND OF THE INVENTION
Today, it has become possible to diagnose some hereditary diseases caused by abnormal splicing of corresponding pre-mRNA molecules. An intractable disease, muscular dystrophy, has come to draw particular attention. Muscular dystrophy is divided into Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). DMD is a hereditary muscular disease of highest incidence, occurring in one in 3,500 live male births. Patients of DMD at first exhibit lowered muscular power in their infancy, suffer from progressive muscular atrophy since then on, and eventually die in their age of around 20. No effective medicament is so far available for DMD, and development of a medicament for it has been longed for by the patients around the world. In 1987, dystrophin gene, which is the causative gene of DMD, was found using retrospective genetics, and BMD also was found to result from abnormality of the same dystrophin gene [
Koenig
, M. et al., Cell, 50:509-517(1987)]. As for BMD, its onset is relatively late, observed in the adulthood, and nearly normal survival is allowed, although a mild loss of muscular power is observed after the onset of the disease.
Dystrophin gene is located in the subregion 21 of the short arm of the X-chromosome. The size of dystrophin gene is 3,0 Mb, the largest known human gene. Despite that large size, it is regions of only 14 kb in total of the dystrophin gene that encodes dystrophin protein, and the encoding regions are divided into no less than 79 exons which are distributed within the gene [Roberts, R G., et al.,
Genomics
, 16:536-538(1993)]. Its pre-mRNA, the transcript of dystrophin gene, undergoes splicing into the mature mRNA of 14 kb. The gene includes eight distinct promoter regions, which are also distributed within the gene and responsible for production of respective distinct mRNAs [Nishio, H., et al.,
J. Clin. Invest
., 94:1073-1042(1994), Ann, A H. and Kunkel, L M.,
Nature Genet
., 3:283-291(1993), D'Souza, V N. et al.,
Hum. Mol. Genet
., 4:837-842(1995)]. Thus, dystrophin gene and its transcript are very complex in structure.
Genetic diagnosis of DMD and BMD was performed in early days using fragments of dystrophin gene, and then by Southern blot analysis using cDNAs as probes. Thus, it was revealed that approximately six tenth of DMD/BMD patients have abnormalities such as large loss or multiplication in dystrophin gene [Hoffman, E P. and Kunkel, L M.,
Neuron
, 2:1019-1029(1989)]. Most of the abnormalities found in the gene in DMD/BMD patients was a loss in the gene, with sizes of as big as several kb. As abnormalities in dystrophin gene detected by Southern blotting were concentrated on two “hot-spots” in the gene, multiplex PCR was designed for genetic diagnosis which, by focusing on 19 exons in those hot-spots, can easily identify a deletion using two PCR (polymerase chain reaction) systems [Chamberlain J S., et al.,
Nucleic Acids Res
., 16:11141-11156(1988), Beggs A H., et al.,
Hum. Genet
., 86:45-48(1990)]. Today, the multiplex PCR has become the most popular diagnosing method, for it quickly gives results and can detect 98% of gene abnormalities which are detectable by Southern blotting.
There is known an animal model for DMD, a mdx (X chromosome-linked muscular dystrophy) mouse [Bulfield, G. et al.,
Proc. Natl. Acad. Sci. U.S.A
., 81:1189-1192(1984)].
Due to a nonsense mutation within exon 23 of the mouse dystrophin, this gene in the mdx mouse is inactivated, i.e., translation is terminated within exon 23. No functional dystrophin is expressed in the mdx mouse, although a trace of dystrophin-positive muscle fiber is detected histochemically.
No explanation had been given to the cause of the great difference in pathological conditions clinically observed between the two diseases, DMD and BMD, both resulting from apparently similar abnormalities in the same dystrophin gene, until so-called frameshift hypothesis was proposed [Monaco, A P., et al.,
Genomics
, 2:90-95(1988)1: In DMD, a partial deletion present in the gene results in a (out-of-frame) shift of amino acids reading frame along the dystrophin mRNA and an eventually emerging stop codon puts an end to the dystrophin synthesis halfway. In contrast, in BMD, the reading frame is kept intact (in-frame) in spite of a partial deletion present in the gene and dystrophin protein therefore is synthesized, though it differs size from wild dystrophin. In fact, analyses of dystrophin in patients' muscle demonstrated that dystrophin was lost in DMD, whereas it occurred in BMD, though with an altered staining property. In addition, according to comparisons made of the phenotypes DMD/BMD with the types of reading frames deduced from the abnormalities in dystrophin gene, the frameshift hypothesis has been proved proper in more than 90% of the patients.
Though not established as a method for treatment of muscular dystrophy, introduction of functional dystrophin gene has been attempted by means of myoblasts implantation or utilizing plasmids or viral vectors [Morgan, J.,
Hum. Gene. Ther
. 5:165-173(1994)].
Dystrophin-positive muscle fibers are also found in many DMD patients (Nicholson, L. et al.,
J. Neurol. Sci
., 94:137-146(1989)]. The dystrophin positive fibers found in DMD patients have been said to be produced through exon skipping [Klein, C. et al.,
Am. J. Hum. Genet
., 50:950-959(1992)]. In fact, an in-frame dystrophin transcript was identified which had underwent skipping of an exon containing a major nonsense mutation [Wilton, S. et al.,
Muscle Nerve
, 20:728-734(1997)].
Genetic information transcribed from the gene undergoes splicing to remove introns and thus mature mRNA is produced, which exclusively consists of exons. The mature mRNA is then translated along its reading frame to synthesize a protein strictly in consistent with the genetic information encoded in the gene. In the splicing step of pre-mRNA, there is a mechanism for precisely distinguishing introns from exons in the nucleotide sequence of the pre-mRNA. For this purpose, sequences in intron-exon boundaries are conserved in every gene in certain rules, and thus known as consensus sequences.
Consensus sequences are known at three sites: a splice donor site at the 5′ end of an intron (the site providing an exon-intron junction), a splice acceptor site at the 3′ end of the intron, and a branch site.
It has been reported concerning a number of diseases that substitution of just a single nucleotide in one of these consensus sequences results in abnormal splicing. This indicates that the consensus sequences are the keys to splicing [Sakuraba, H. et al.,
Genomics
, 12: 643-650 (1992)].
The present inventors for the first time in Japan performed a PCR diagnosis of dystrophin gene abnormalities in DMD/BMD patients, and thereby showed that there is no significant difference between Westerners and Japanese in the type of abnormalities in the gene, i.e., no significant racial difference exists. Though the gene abnormalities thus found by the genetic diagnosis were, without exception, gigantic ones involving several kb to several hundred kb nucleotides, further analyses for the first time led to successful identification of the nucleotide sequence of th

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