Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of regulating cell metabolism or physiology
Reexamination Certificate
2000-11-01
2002-08-27
LeGuyader, John L. (Department: 1655)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of regulating cell metabolism or physiology
C435S006120, C435S091100, C435S325000, C435S366000, C536S023100, C536S024310, C536S024330, C536S024500
Reexamination Certificate
active
06440737
ABSTRACT:
FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of cellular apoptosis susceptibility gene. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding cellular apoptosis susceptibility gene. Such compounds have been shown to modulate the expression of cellular apoptosis susceptibility gene.
BACKGROUND OF THE INVENTION
Apoptosis, or programmed cell death, is a naturally occurring process that has been strongly conserved during evolution to prevent uncontrolled cell proliferation. This form of cell suicide plays a crucial role in ensuring the development and maintenance of multicellular organisms by eliminating superfluous or unwanted cells. However, if this process goes awry becoming overstimulated, cell loss and degenerative disorders including neurological disorders such as Alzheimers, Parkinsons, ALS, retinitis pigmentosa and blood cell disorders can result. Stimuli which can trigger apoptosis include growth factors such as tumor necrosis factor (TNF), Fas and transforming growth factor beta (TGF&bgr;), neurotransmitters, growth factor withdrawal, loss of extracellular matrix attachment and extreme fluctuations in intracellular calcium levels (Afford and Randhawa,
Mol. Pathol.,
2000, 53, 55-63).
Alternatively, insufficient apoptosis, triggered by growth factors, extracellular matrix changes, CD40 ligand, viral gene products neutral amino acids, zinc, estrogen and androgens, can contribute to the development of cancer, autoimmune disorders and viral infections (Afford and Randhawa,
Mol. Pathol.,
2000, 53, 55-63). Consequently, apoptosis is regulated under normal circumstances by the interaction of gene products that either induce or inhibit cell death and several gene products which modulate the apoptotic process have now been identified.
Cellular apoptosis susceptibility gene (also known as CAS, CSE1 and CSP) is the human homolog of the yeast chromosome segregation gene, CSE1, and has been simultaneously implicated in the regulation of mitosis, apoptosis and cellular proliferation (Brinkmann et al.,
Biochemistry,
1996, 35, 6891-6899; Scherf et al.,
Proc. Natl. Acad. Sci. U. S. A.,
1996, 93, 2670-2674).
CAS was first identified in a screen for genes that affect the sensitivity of breast cancer cells toward toxins used in experimental cancer therapy. In these screens cellular apoptosis susceptibility gene was isolated as antisense cDNA fragments that rendered MCF-7 breast cancer cells resistent to cell death induced by exotoxins, exotoxin-derived immunotoxins, diptheria toxin and tumor necrosis factor (Brinkmann et al.,
Proc. Natl. Acad. Sci. U. S. A.,
1995, 92, 10427-10431; Ogryzko et al.,
Biochemistry,
1997, 36, 9493-9500). Characterization of the protein has since revealed that the cellular apoptosis susceptibility gene is highly expressed in other cancer cells including lymphoid neoplasms (Wellmann et al.,
American Journal of Pathology,
1997, 150, 25-30), benign and malignant cutaneous melanocytic lesions (Böni et al.,
The American Journal of Dermatopathology,
1999, 21, 125-128), and colon cancer cell lines (Brinkmann,
Am. J. Hum. Genet.,
1998, 62, 509-513; Brinkmann et al.,
Genome Research,
1996, 6, 187-194).
It has been determined that cellular apoptosis susceptibility gene undergoes alternative splicing in a tissue—and development-specific manner (Brinkmann et al.,
Genomics,
1999, 58, 41-49). Northern blot analyses have shown that the predominant transcript in proliferating tissues is a 3147 nucleotide transcript, while a larger transcripts can be detected in fetal and adult brain and smaller transcripts can be detected in trachea, liver and some cancers (Brinkmann et al.,
Genomics,
1999, 58, 41-49). The protein and nucleic acid sequences of the human cellular apoptosis susceptibility gene are disclosed in U.S. Pat. No. 5,759,782 and its corresponding PCT publication WO 96/40713 and U.S. Pat. No. 6,072,031, respectively (Pastan and Brinkmann, 2000; Pastan and Brinkmann, 1996; Pastan and Brinkmann, 1998). Also disclosed are antibodies to the protein, protein fragments and an isolated single-stranded antisense DNA sequence consisting of nucleotides 2100-2536 of the human cellular apoptosis susceptibility gene (Pastan and Brinkmann, 1996; Pastan and Brinkmann, 1998).
The human cellular apoptosis susceptibility gene is located on chromosome 20q13 and is amplified in BT474 breast cancer cells (Brinkmann et al.,
Genome Research,
1996, 6, 187-194). This chromosomal location harbors a remarkable degree of instability in various tumors and amplification in this region is observed frequently in aggressive types of breast cancers (Brinkmann et al.,
Genome Research,
1996, 6, 187-194).
Cellular apoptosis susceptibility gene has also been shown to mediate export of importin-&agr; from the nucleus (Kutay et al.,
Cell,
1997, 90, 1061-1071). Importin-&agr; is the nuclear import receptor for nuclear localization signal-containing proteins. This interaction, which is regulated by phosphorylation events, requires the presence of RanGTP to form a ternary complex; and it has been suggested that deregulation of importin transport can cause cell cycle defects (Kutay et al.,
Cell,
1997, 90, 1061-1071; Scherf et al.,
Biochemical and Biophysical Research Communications,
1998, 250, 623-628).
Collectively, these data suggest that modulation of cellular apoptosis susceptibility gene would render opportunity to treat patients with various cancers and deregulated apoptotic pathologic conditions.
Strategies aimed at modulating cellular apoptosis susceptibility gene function have involved the use of antibodies and antisense expression vectors but currently, there are no known therapeutic agents which effectively inhibit the synthesis of cellular apoptosis susceptibility gene. Consequently, there remains a long felt need for agents capable of effectively inhibiting cellular apoptosis susceptibility gene function.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of cellular apoptosis susceptibility gene expression.
Currently there exists a need to identify methods of modulating apoptosis for the therapeutic treatment of human diseases and it is believed that cellular apoptosis susceptibility gene modulators will be integral to these methods. The present invention, therefore, provides compositions and methods for modulating cellular apoptosis susceptibility gene expression, including modulation of alternatively spliced forms of cellular apoptosis susceptibility gene.
SUMMARY OF THE INVENTION
The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding cellular apoptosis susceptibility gene, and which modulate the expression of cellular apoptosis susceptibility gene. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of modulating the expression of cellular apoptosis susceptibility gene in cells or tissues comprising contacting said cells or tissues with one or more of the antisense compounds or compositions of the invention. Further provided are methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of cellular apoptosis susceptibility gene by administering a therapeutically or prophylactically effective amount of one or more of the antisense compounds or compositions of the invention.
DETAILED DESCRIPTION OF THE INVENTION
The present invention employs oligomeric compounds, particularly antisense oligonucleotides, for use in modulating the function of nucleic acid molecules encoding cellular apoptosis susceptibility gene, ultimately modulating the amount of cellular apoptosis susceptibility gene produced. This is acc
ISIS Pharmaceuticals Inc.
LeGuyader John L.
Licata & Tyrrell P.C.
Schmidt Mary
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