Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of regulating cell metabolism or physiology
Reexamination Certificate
2000-09-11
2002-12-10
Wang, Andrew (Department: 1635)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of regulating cell metabolism or physiology
C435S091100, C536S024500
Reexamination Certificate
active
06492170
ABSTRACT:
FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of caspase 9. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding caspase 9. Such compounds have been shown to modulate the expression of caspase 9.
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.
The most well-characterized apoptotic signaling cascade to date is that orchestrated by a family of cysteine proteases known as caspases. These enzymes activate apoptosis through proteolytic events triggered by one of several described mechanisms; including ligand binding to the cell surface death receptors of either the TNF or NGF receptor families, changes in mitochondrial integrity or chemical induction (Thornberry,
Br. Med. Bull.,
1997, 53, 478-490).
Caspases have been classified into two groups, initiator caspases and effector caspases, based upon their position in the apoptotic signaling pathway.
Initiator caspases include caspase 1, 2, 4, 5, 8, 9, 10 and 14 and these enzymes have the largest prodomains of all the caspase zymogens. These prodomains allow the initiator caspases to interact with other downstream substrates including other caspases. Initiator caspases are further divided into two groups based on their protein binding domains. Caspases 8 and 10 contain the DED (death effector domain) while caspases 1, 2, 4 and 9 contain the CARD (caspase recruitment domain) (Bratton et al.,
Exp. Cell. Res.,
2000, 256, 27-33; Garcia-Calvo et al.,
Cell. Death Differ.,
1999, 6, 362-369).
Effector caspases are activated by initiator caspases and include caspase 3, 6, 7, 11 and 13 and these contain a shorter prodomain. Once activated, effector caspases then cleave a number of structural and regulatory proteins within the cell (Bratton et al.,
Exp. Cell. Res.,
2000, 256, 27-33; Garcia-Calvo et al.,
Cell. Death Differ.,
1999, 6, 362-369).
Caspase 9 (also known as CASP9, MCH6, APAF3, ICE-LAP6 or ICE9) is a ubiquitously expressed initiator caspase which has been shown to be the most upstream caspase in the apoptotic cascade and to induce apoptosis in breast carcinoma cells when overexpressed (Duan et al.,
J. Biol. Chem.,
1996, 271, 16720-16724; Kuida,
Int. J. Biochem. Cell Biol.,
2000, 32, 121-124; Li et al.,
Cell,
1997, 91, 479-489). Caspase 9 exists as two isoforms in both humans and mice and the shorter of the isoforms has been shown to act as a dominant-negative of the longer form in vitro by blocking protein-protein interactions with the caspase 9 adaptor molecule, Apaf-1 (Fujita et al.,
Biochem. Biophys. Res. Commun.,
1999, 264, 550-555; Seol and Billiar,
J. Biol. Chem.,
1999, 274, 2072-2076; Srinivasula et al.,
Cancer Res.,
1999, 59, 999-1002).
Activation of the caspase 9 zymogen occurs upon mitochondrial release of cytochrome c subsequent to triggers of cell death followed by complex formation with Apaf-1 forming the apoptosome. Proteolytic cleavage of the caspase 9 zymogen then results in release of the mature caspase 9 from the apoptosome and initiates the apoptotic cascade (Saleh et al.,
J. Biol. Chem.,
1999, 274, 17941-17945).
Disclosed in U.S. Pat. No. 6,010,878 are the polypeptide and polynucleotide sequences of human caspase 9 as well as an expression vector and host cells for the expression of said vector (Dixit et al., 2000).
Caspase 9 is required for normal brain development and mediates apoptosis induced by chemotherapeutic agents (Hakem et al.,
Cell,
1998, 94, 339-352; Kuida,
Int. J. Biochem. Cell Biol.,
2000, 32, 121-124) and oncogenic transformation (Fearnhead et al.,
Proc. Natl. Acad. Sci. U. S. A.,
1998, 95, 13664-13669; Kuwahara et al.,
Cancer Lett.,
2000, 148, 65-71; Soengas et al.,
Science,
1999, 284, 156-159; Wang et al.,
Eur. J. Cancer,
1999, 35, 1517-1525; Zhuang and Cohen,
Toxicol. Lett.,
1998, 102-103, 121-129).
Caspase 9 also plays a role in ischemic recovery (Krajewski et al.,
Proc. Natl. Acad. Sci. U. S. A.,
1999, 96, 5752-5757) and thymocyte apoptosis induced by sepsis (Tinsley et al.,
Shock,
2000, 13, 1-7). These data suggest that modulation of caspase 9 would render opportunity to treat patients with disorders such as cancer, stroke, brain injury or neurodegenerative diseases.
It is currently believed that modulation of caspase expression represents a potential therapeutic target in a variety of deregulated apoptotic pathologic conditions. Several types of broad-spectrum caspase inhibitors have been identified for the treatment of deregulated bone metabolism (Harada et al., 2000; Reszka, 1999), as immunomodulatory agents (Gunasekera et al., 2000) and as combination therapies for the regulation of blood cholesterol (Reszka, 1999).
These non-specific caspase inhibitors fall into three main classes; peptide-based molecules that mimic caspase substrates, small molecules and naturally-occurring caspase inhibitors or decoys (Deveraux et al.,
Embo J.,
1998, 17, 2215-2223; Dong et al.,
Biochem. J.,
2000, 347 Pt 3, 669-677; Gunasekera et al., 2000; Harada et al., 2000; Reszka, 1999; Reszka, 1999; Robidoux et al., 2000; Spruce et al., 1999).
Currently, however, there are no known therapeutic agents which effectively inhibit the synthesis of caspase 9 and to date, strategies aimed at modulating caspase 9 function have involved the use of antibodies and molecules that block upstream entities such as the death receptors, broad-spectrum caspase inhibitors or targeted gene knockouts in mice. Mice lacking caspase 9 die perinatally with a markedly enlarged and malformed cerebrum caused by reduced apoptosis in the brain and embryonic stem cells lacking caspase 9 show resistance to several apoptotic stimuli (Hakem et al.,
Cell,
1998, 94, 339-352; Kuida et al.,
Cell,
1998, 94, 325-337).
There exists, therefore, a need to identify methods of modulating apoptosis for the therapeutic treatment of human diseases.
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 gene expression and cellular processes.
The present invention satisfies this need and provides compositions and methods for modulating caspase 9 expression, including modulation of aberrant forms of caspase 9, including mutated and alternatively spliced forms.
SUMMARY OF THE INVENTION
The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding caspase 9, and which modulate the expression of caspase 9. Pharmace
ISIS Pharmaceuticals Inc.
Licata & Tyrrell P.C.
Wang Andrew
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