Antisense modulation of daxx expression

Details Antisense modulation of daxx expression Antisense modulation of daxx expression

2000-01-24

2001-01-30

Schwartzman, Robert A. (Department: 1635)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091100, C435S325000, C536S024500

Reexamination Certificate

active

06180353

ABSTRACT:

FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of daxx. In particular, this invention relates to antisense compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding daxx. Such oligonucleotides have been shown to modulate the expression of daxx.
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 the development and maintenance of multicellular organisms by eliminating superfluous or unwanted cells. However, if this process goes awry, excessive apoptosis results in cell loss and degenerative disorders including neurological disorders such as Alzheimers, Parkinsons, ALS, retinitis pigmentosa and blood cell disorders, while insufficient apoptosis contributes to the development of cancer, autoimmune disorders and viral infections (Thompson,
Science,
1995, 267, 1456-1462).
Although several stimuli can induce apoptosis, little is known about the intermediate signaling events, including inhibition, that connect the apoptotic signal to a common cell death pathway conserved across many species. Recently, major advances have been made in understanding the signaling pathways mediated by the tumor necrosis factor receptor (TNFR) family which signals apoptosis. Two cell surface cytokine receptors of the TNFR family, TNFR-1 and CD95 (Fas/APO-1), act as death receptors and a number of binding proteins have been identified which mediate apoptosis through these receptors (Baker and Reddy,
Oncogene,
1998, 17, 3261-3270).
Daxx (also known as Fas binding protein, CENP-C binding protein, dap6 for death associated protein 6 and EAP for Ets-1 associated protein) is a novel signaling protein which interacts with the cytoplasmic domain of Fas/APO-1 acting as a downstream effector in the process of apoptosis (Yang et al.,
Cell,
1997, 89, 1067-1076). The nucleic acid and protein sequences for human daxx are disclosed in the PCT publication WO 98/34946. Also disclosed are methods for decreasing Jun N-terminal kinase (JNK) signaling pathways by contacting cells with an inhibitor of daxx, wherein the inhibitors are antisense nucleic acids (Yang et al., 1998). Overexpression of daxx enhances Fas-mediated apoptosis and activates the JNK pathway (Chang et al.,
Science,
1998, 281, 1860-1863; Yang et al.,
Cell,
1997, 89, 1067-1076). This signaling pathway was originally identified as an oncogene- and ultraviolet light-stimulated kinase pathway but is now known to be activated by growth factors, cytokines and T-cell costimulation (Moriguchi et al.,
Adv. Pharmacol.,
1996, 36, 121-137).
Daxx is widely expressed in human tissues and cloning of the human gene revealed its localization to chromosome 6p21, a genomic region that includes the major histocompatibility complex (MHC) and which is implicated in the pathway for deletion of autoreactive lymphocytes (Herberg et al.,
J. Mol. Biol.,
1998, 277, 839-857; Kiriakidou et al.,
DNA Cell. Biol.,
1997, 16, 1289-1298). This may suggest a role for daxx in autoimmune diseases.
Daxx also interacts with a centromeric protein known as CENP-C. CENP-C is crucial to proper chromosome segregation and mitotic progression. Pluta et al. have demonstrated that daxx colocalizes with CENP-C at interphase-specific centromeres in human cell nucleii and suggest that daxx may play a role in regulating cellular responses to apoptotic stimuli (Pluta et al.,
J. Cell. Sci.,
1998, 111, 2029-2041).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of daxx, and there remains a long felt need for agents capable of effectively inhibiting daxx function.
To date, investigative strategies aimed at modulating daxx function have involved the use of antibodies, molecules that block upstream entities, and gene knock-outs in mice.
Knockout of the daxx gene in mice resulted in extensive apoptosis and embryonic lethality. This was in contrast to the expected result from the loss of a pro-apoptotic gene, that being a hyperproliferative disorder. These findings argue against a role for Daxx in promoting Fas-induced cell death and suggest that Daxx either directly or indirectly suppresses apoptosis in the early embryo (Michaelson et al.,
Genes Dev.,
1999, 13, 1918-1923).
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 daxx expression.
The pharmacological modulation of daxx activity and/or function may therefore be an appropriate point of therapeutic intervention in pathological conditions and the present invention provides compositions and methods for modulating daxx expression.
SUMMARY OF THE INVENTION
The present invention is directed to antisense compounds, particularly oligonucleotides, which are targeted to a nucleic acid encoding daxx, and which modulate the expression of daxx. Pharmaceutical and other compositions comprising the antisense compounds of the invention are also provided. Further provided are methods of modulating the expression of daxx 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 daxx 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 antisense compounds, particularly oligonucleotides, for use in modulating the function of nucleic acid molecules encoding daxx, ultimately modulating the amount of daxx produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding daxx. As used herein, the terms “target nucleic acid” and “nucleic acid encoding daxx” encompass DNA encoding daxx, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA. The specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid. This modulation of function of a target nucleic acid by compounds which specifically hybridize to it is generally referred to as “antisense”. The functions of DNA to be interfered with include replication and transcription. The functions of RNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA. The overall effect of such interference with target nucleic acid function is modulation of the expression of daxx. In the context of the present invention, “modulation” means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene. In the context of the present invention, inhibition is the preferred form of modulation of gene expression and mRNA is a preferred target.
It is preferred to target specific nucleic acids for antisense. “Targeting” an antisense compound to a particular nucleic acid, in the context of this invention, is a multistep process. The process usually begins with the identification of a nucleic acid sequence whose function is to be modulated. This may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In the present invention, the target is a nucleic acid molecule encoding daxx. The targeting process also includes determination

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