Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-08-16
2004-04-27
Lacourciere, Karen (Department: 1635)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S024300, C536S024310, C536S024330, C435S006120, C435S091100
Reexamination Certificate
active
06727355
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to pharmaceutical compositions for treatment of Duchenne muscular dystrophy, which pharmaceutical compositions 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 shifted reading frame as a result of abnormalities in dystrophin gene. More specifically, the present invention relates to a splicing enhancer sequence (SES) in dystrophin gene which can be utilized for the preparation of pharmaceutical compositions for treatment of a specific type of Duchenne muscular dystrophy, as well as to antisense oligonucleotides against the splicing enhancer sequence, and therapeutic pharmaceutical compositions comprising such oligonucleotides.
BACKGROUND OF THE INVENTION
Diagnosis has become available today for hereditary diseases caused by abnormal splicing of pre-mRNA molecules. A so far intractable disease, muscular dystrophy, has thus come to draw particular attention. Muscular dystrophy is divided into two groups: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). DMD is a hereditary muscular disease of the highest incidence, occurring in one in 3,500 live male births. Patients of DMD at first exhibit a lowered muscular power in their infancy, then suffer a constant progression of muscular atrophy thereafter, and eventually die at around the age of 20. It is in contrast to BMD, in which the onset of the disease is relatively late, somewhere in the adulthood, and though a mild loss of muscular power is observed after the onset of the disease, patients can live nearly a normal life. No drug is so far available for effective treatment of DMD, and therefore development of a drug for its treatment has been longed for by the patients across the world. In 1987, dystrophin gene, the causative gene of DMD, was found by means of retrospective genetics, and BMD also was found to result from abnormality in the same dystrophin gene [Koenig, M. et al., Cell, 50:509-517(1987)].
Dystrophin gene is located in the subregion 21 of the short arm of the X-chromosome. The size of the gene is 3.0 Mb, the largest known human gene. Despite that large size, only 14 kb regions in total of the dystrophin gene do encode the whole dystrophin protein, and those encoding regions are divided into no less than 79 exons which are distributed throughout the gene [Roberts, R G., et al., Genomics, 16:536-538(1993)]. The transcript of dystrophin gene, i.e. pre-mRNA, is spliced into the mature 14 kb mRNA. The gene has eight distinct promoter regions also distributed within the gene and they are responsible for production of distinct mRNAs, respectively [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 structurally very complex.
Genetic diagnosis of DMD and BMD was performed by Southern blotting using fragments of dystrophin gene in early years, and then 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 can conveniently identify a deletion using two PCR (polymerase chain reaction) systems by focusing on 19 exons in those hot-spots [Chamberlain J S., et al., Nucleic Acids Res., 16:11141-11156(1988), Beggs A H., et al., Hum. Genet., 86:45-48(1990)]. The multiplex PCR has become the most popular diagnosing method today, for it gives results in a short time and can detect 98% of genetic abnormalities which are detectable by Southern blotting.
There is known an animal model for DMD, mdx (X chromosome-linked muscular dystrophy) mice [Bulfield, G. et al., Proc. Natl. Acad. Sci. U.S.A., 81:1189-1192(1984)].
Due to a nonsense mutation within exon 23 of mouse dystrophin, the gene is inactivated in the mdx mice, i.e., translation is terminated within exon 23. No functional dystrophin molecule is expressed in mdx mice, while a trace of dystrophin-positive muscle fiber is detectable histochemically.
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, no explanation had been given until so-called frameshift hypothesis was proposed [Monaco, A P., et al., Genomics, 2:90-95(1988)]: In DMD, a partial deletion in the gene causes a shift of amino acids reading frame along the dystrophin mRNA (i.e., out-of-frame shift) and an eventually emerging stop codon puts an end to the dystrophin synthesis before completion. In contrast, in BMD, the reading frame is kept intact (i.e., in-frame) in spite of a partial deletion present in the gene and a dystrophin protein therefore is synthesized, though it differs in size from wild dystrophin. Analyses of dystrophin in patients' muscle demonstrated that dystrophin was lost in DMD while it occurred in BMD with an altered staining property. In addition, based on a comparison made of the phenotypes DMD/BMD with the types of reading frames deduced from the respective abnormalities in dystrophin gene, the frameshift hypothesis has been proved proper in more than 90% of the patients.
For a method for treatment of muscular dystrophy, introduction of functional dystrophin gene has been attempted through 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 mdx mice, an in-frame dystrophin transcript was identified, in which an exon containing a major nonsense mutation had been skipped [Wilton, S. et al., Muscle Nerve, 20:728-734(1997)].
Introns-including genetic information which was transcribed from the gene receives splicing to remove the introns and thus mature mRNA is produced which exclusively consists of exon sequences. 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 step of splicing in pre-mRNA, there exists a mechanism for precisely distinguishing introns from exons in the pre-mRNA nucleotide sequence. For this purpose, sequences in intron-exon boundaries are conserved in every gene according to 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.
For a number of diseases, it has been reported that substitution of just a single nucleotide in one of these consensus sequences would lead to 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 performed a PCR diagnosis of dystrophin gene abnormalities in DMD/BMD patients for the first time in Japan, and thereby demonstrated that there was no significant difference between Westerners and Japanese in the type of abnormalities in the gene, i.e., no significant racial difference existed. Though the gene abnormalities thus found by genetic diagnosis were, without exception,
Kamei Shoichiro
Matsuo Masafumi
Greenblum & Bernstein P.L.C.
JCR Pharmaceuticals Co. Ltd.
Lacourciere Karen
LandOfFree
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