Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2000-10-04
2003-05-20
McGarry, Sean (Department: 1635)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S006120, C435S091100, C536S023100, C536S024500
Reexamination Certificate
active
06566135
ABSTRACT:
FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of caspase 6. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding caspase 6. Such compounds have been shown to modulate the expression of caspase 6.
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 6 (also known as CASP6, ced-3 homology 2 and MCH2) is an effector caspase first identified in Jurkat T lymphocytes. Characterization of the gene revealed that caspase 6 exists as two different isoforms, the longer of which induced apoptosis in Sf9 insect cells (Fernandes-Alnemri et al.,
Cancer Res
., 1995, 55, 2737-2742). These isoforms have been shown to have differential expression patterns suggesting different or alternate roles in apoptosis (Faleiro et al.,
Embo J
., 1997, 16, 2271-2281). In fact, the longer form of caspase 6, MCH2&agr;, preferentially cleaves lamin A, an event required for nuclear reorganization during apoptosis (Orth et al.,
J. Biol. Chem
., 1996, 271, 16443-16446; Takahashi et al.,
Proc. Natl. Acad. Sci. U.S.A
., 1996, 93, 8395-8400).
Disclosed in U.S. Pat. No. 5,985,640 are the nucleotide and polypeptide sequence of the caspase 6 gene as well as the sequence of the splice variants thereof. Also generally disclosed are antisense oligonucleotides, at least 10 nucleotides in length, which hybridize with the caspase 6 mRNA. The antisense oligonucleotides include a single-stranded DNA sequence and an antisense RNA oligonucleotide produced from an expression vector within the cell (Litwack et al., 1999).
Caspase 6 has been localized to human chromosome 4q25, a region frequently affected in disorders such as Reiger syndrome, a dominantly inherited disorder characterized by hypodontia and malformation of the eye (Tiso et al.,
Biochem. Biophys. Res. Commun
., 1996, 225, 983-989). It has also been proposed that the protein product of a gene at this locus interacts with the ataxia telangiectasia gene product accounting for the genetic instability seen in this disorder. Caspase 6 has also been implicated in the inhibition of replicative DNA synthesis after certain insults such as ionizing radiation (Nasir et al.,
Mamm. Genome
, 1997, 8, 56-59).
Increased expression of caspase 6 has been noted in several neoplastic disorders including gallbladder carcinomas and dysplasias (Turunen et al.,
Histol. Histopathol
., 2000, 15, 53-60), malignant non-Hodgkin's lymphoma (Soini and Paakko,
Apmis
, 1999, 107, 1043-1050), breast cancer (Vakkala et al.,
Br. J. Cancer
, 1999, 81, 592-599) and osteosarcomas (Seki et al.,
Cancer Chemother. Pharmacol
., 2000, 45, 199-206).
In addition, caspase 6 has been shown to play a role in the processing of the amyloid precursor protein in Alzheimer's disease (LeBlanc et al.,
J. Biol. Chem
., 1999, 274, 23426-23436; Pellegrini et al.,
J. Biol. Chem
., 1999, 274, 21011-21016).
Collectively, these data suggest that modulation of caspase 6 would render opportunity to treat patients with various types of cancers or neurological conditions such as Alzheimer's disease as well as a variety of deregulated apoptotic pathologic conditions.
Several types of broad-spectrum caspase inhbitors 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).
Strategies aimed at modulating caspase 6 function have involved the use of antibodies and molecules that block upstream entities such as the death receptors and broad-spectrum caspase inhibitors.
Currently there exists a need to identify methods of modulating apoptosis for the therapeutic treatment of human diseases and it is believed that caspases modulators will be integral to these methods.
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.
There are no known therapeutic agents which effectively inhibit the synthesis of caspase 6 and the present invention provides compositions and methods for modulating caspase 6 expression, including modulation of aberrant forms of caspase 6, including 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 6, and which modulate the expression of caspase 6
Isis Pharmaceuticals , Inc.
Licata & Tyrrell P.C.
McGarry Sean
Zara Jane
LandOfFree
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