Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-07-31
2003-11-25
Lacourciere, Karen (Department: 1635)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S024310, C536S024330, C536S024500, C435S006120, C435S091100
Reexamination Certificate
active
06653466
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the use of an antisense oligonucleotide for the manufacture of a therapeutic pharmaceutical composition for a certain hereditary disease, and more specifically to a therapeutic pharmaceutical composition for Duchenne muscular dystrophy intended to induce an exon skipping in the pre-mRNA of a certain abnormal dystrophin gene.
BACKGROUND OF THE INVENTION
Antisense oligonucleotide strategy has been widely studied for the purpose of inhibiting expression of oncogenes or viral genes. Antisense oligonucleotides have been known to efficiently inhibit de novo synthesis of their respective targeted proteins. For example, it is known that an antisense oligonucleotide against the mRNA encoding IGF-I (Insulin-like Growth Factor-I) inhibits proliferation of rat glioblastoma cells [Askari, F. K., and McDonnell, W. M., N. Engl. J. Med, 334: 316-318 (1996); Trojan, et al., Science, 259: 94-97 (1993), Trojan, et al., Proc. Natl Acad. Sci. U.S.A., 89: 4874-4878 (1992)].
In addition, a method has been reported to inhibit an abnormal splicing of a pre-mRNA by means of its antisense oligonucleotide [Japanese Laid-Open Patent Publication No. H08-510130].
Today, diagnosis has become available for some hereditary diseases caused by abnormal splicing of the corresponding pre-mRNA, and an intractable disease, muscular dystrophy, has come to draw particular attention. Muscular dystrophy is grossly classified into Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). DMD is a hereditary muscular disease of highest incidence, occurring in one out of 3,500 newborn boys. Patients of DMD exhibit lowered muscular power in their infancy at first, and, after suffering from consistent muscular atrophy from then on, eventually die at the age of about 20. At present, no effective therapeutic drug is available for DMD, and therefore development of such a therapeutic has been longed for by patients all over the world. In 1987, dystrophin gene as the causative gene of DMD was found with the aid of retrospective genetics, and BMD also was found to occur from abnormality of the same dystrophin gene [Koenig, M. et al., Cell, 50:509-517(1987)]. As for BMD, its onset is relatively late, which is noted in the adulthood, and, though a mild loss of muscular power is observed after the onset of the disease, nearly normal life is allowed.
Dystrophin gene is located in the subregion 21 of the short arm or the X-chromosome. Nishio et al. revealed the size of dystrophin gene to be 3,000 kb, which is the largest known human gene [Nishio, H. et al., J. Clin. Invest., 94:1037-1042(1994)]. Despite that large size, regions of only 14 kb in total of the dystrophin gene encode dystrophin protein, and the encoding regions are divided into 79 exons distributed within the gene [Roberts, R G., et al., Genomics, 16:536-538(1993)]. Furthermore, the gene includes seven 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, there is high structural complexity resides in dystrophin gene and its transcript.
Genetic diagnosis of DMD and BMD was performed in early days using fragments of dystrophin gene, and then by Southern blot analysis with cDNA probes. It thereby was revealed that about six tenth of DMD/BMD patients have abnormalities of 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 is a loss occurring in the gene, with sizes of as big as several kb. For genetic diagnosis, as the abnormalities are concentrated on two hot-spots in dystrophin gene, multiplex PCR was designed, which can conveniently identify a deletion using two PCR (polymerase chain reaction) systems 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)]. Today, the multiplex PCR has become the most popular diagnosing method, for it quickly gives results and can detect 98% of gene abnormalities detectable by Southern blotting.
No explanation was given to the cause of the big difference in pathological conditions clinically observed between the two diseases, DMD and BMD, 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)]: 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 different in size from wild dystrophin. In fact, analyses of dystrophin in patients' muscle demonstrated that dystrophin is lost in DMD, whereas it occurs in BMD, with an altered staining property, though. In addition, the according to comparisons made of the phenotypes, DMD and BMD, with the types of reading frames deduced from the abnormalities in dystrophin gene, frameshift hypothesis has been found proper in more than 90% of the patients.
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 in accordance with 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, indicating that the consensus sequences are the keys to splicing [Sakuraba, H. et al., Genomics, 12: 643-650 (1992)].
Upon this background, the present inventors investigated for the purpose of providing a pharmaceutical agent to correct the expression of abnormal genes through artificial regulation of pre-mRNA splicing.
The present inventors for the first time in Japan performed a PCR diagnosis of dystrophin gene abnormalities for DMD/BMD patients, and thereby revealed that there is no significant difference between westerners and Japanese in the type of abnormalities in the gene, i.e., no significant difference exists among these races. 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 the deleted part of a dystrophin gene, and the result was reported along with the corresponding case named “dystrophin Kobe” [Matsuo, M, et al., Biochem. Biophys. Res. Commun., 170:963-967(1990)].
The one with the gene abnormality named “dystrophin Kobe” is a DMD case. The results of its multiplex PCR analyses revealed that no band of amplified genomic DNA corresponding to exon 19 was found at its expected position, apparently indicating loss of exon 19. However, after a reaction attempted to amplify the exon 19 region of the genomic DNA, exon 19, though smaller than its normal siz
Greenblum & Bernstein P.L.C.
JCR Pharmaceuticals Co. Ltd.
Lacourciere Karen
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