Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of regulating cell metabolism or physiology
Reexamination Certificate
2000-06-14
2001-08-07
Yucel, Remy (Department: 1635)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of regulating cell metabolism or physiology
C435S006120, C435S091100, C435S325000, C536S023100, C536S024500, C536S024300, C536S024310, C536S024330
Reexamination Certificate
active
06271030
ABSTRACT:
FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of C/EBP beta. In particular, this invention relates to antisense compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding C/EBP beta. Such oligonucleotides have been shown to modulate the expression of C/EBP beta.
BACKGROUND OF THE INVENTION
Transcription factors represent a group of molecules within the cell that function to connect the pathways from extracellular signals to intracellular responses. Immediately after an environmental stimulus, these proteins which reside predominantly in the cytosol are translocated to the nucleus where they bind to specific DNA sequences in the promoter elements of target genes and activate the transcription of these target genes. One family of transcription factors, CCAAT/Enhancer-binding proteins (C/EBPs), regulates the expression of an extensive panel of genes that control normal tissue development and cellular function, cellular proliferation and functional differentiation. Six members of this family have been identified to date all of which form both homo- and heterodimers with other C/EBP family members as well as with members of the NFkB and Fos/Jun families of transcription factors (Lekstrom-Himes and Xanthopoulos,
J. Biol. Chem.,
1998, 273, 28545-28548). While all of the members of the C/EBP family have a similar modular protein structure, expression levels and tissue distributions vary widely leading to a diversity of roles (Lekstrom-Himes and Xanthopoulos,
J. Biol. Chem.,
1998, 273, 28545-28548).
C/EBP beta (also known as C/EBP2, LAP, TCF5, CRP2, NFIL6, IL6DBP, NF-M, AGP/EBP and Apc/EPB) was originally identified as a mediator of IL-6 signaling, binding to IL-6 responsive agents in acute phase response genes such as TNF, IL-8 and G-CSF (Akira et al.,
Embo J.,
1990, 9, 1897-1906; Descombes et al.,
Genes Dev.,
1990, 4, 1541-1551) isolated from the liver and primarily regulates hormone responsiveness and oxidative stress responses (Descombes et al.,
Genes Dev.,
1990, 4, 1541-1551). Studies of tissue distribution and developmental expression patterns showed that C/EBP beta is found the liver, lung, spleen, kidney, brain and testis with the highest expression found in the lung (Descombes et al.,
Genes Dev.,
1990, 4, 1541-1551). Disclosed in U.S. Pats. 5,215,892 and 5,360,894 are the nucleic acid sequence of C/EBP beta as well as plasmids and host cells for the expression of the recombinant protein (Kishimoto et al., 1994; Kishimoto et al., 1993).
C/EBP-deficient mice have been generated for five of the six members of the C/EBP family and these have been characterized for system-specific phenotypic abnormalities. Mice lacking C/EBP beta demonstrate defective carbohydrate metabolism, immunodeficiency, defective Th1 response and female sterility. They also have a low level of expression of phosphoenolpyruvate carboxykinase (Park et al.,
J. Biol. Chem.,
1999, 274, 211-217; Yamada et al.,
J. Biol. Chem.,
1999, 274, 5880-5887) and demonstrate perinatal lethality suggesting involvement of C/EBP beta in gluconeogenic pathways (Arizmendi et al.,
J. Biol. Chem.,
1999, 274, 13033-13040; Greenbaum et al.,
J. Clin. Invest.,
1998, 102, 996-1007). These mice are highly susceptible to infection by a wide range of pathogens indicating a critical role for C/EBP beta in bactericidal responses (Tanaka et al.,
Cell,
1995, 80, 353-361).
In addition to stage-specific expression level variations, the C/EBP members also undergo multiple isoform expression arising from alternative start positions, for the alpha and beta isoforms, in 5′ upstream open reading frames (Geballe and Morris,
Trends Biochem. Sci.,
1994, 19, 159-164; Lincoln et al.,
J. Biol. Chem.,
1998, 273, 9552-9560). The steady-state level of the various pools of transcripts also changes as a function of age and stress challenges (Hsieh et al.,
Mol. Biol. Cell,
1998, 9, 1479-1494). In mice the expression of certain transcripts of one isoform has also been shown to regulate the expression of other C/EBP isoforms (Burgess-Beusse et al.,
Hepatology,
1999, 29, 597-601).
C/EBP beta occurs as two isoforms in the cell, a full-length 32-kDa form, known as LAP and a shorter form known as LIP. The dimerization of these two forms attenuates transcription of the C/EBP beta gene and therefore represents an autoregulatory mechanism of expression (Descombes et al.,
Genes Dev.,
1990, 4, 1541-1551).
In disease states, C/EBP beta has been implicated in the development of diabetes and cancer. Studies comparing normal and tumorigenic tissue from human ovaries demonstrated a higher level of expression of C/EBP alpha and beta in the tumor tissues, irrespective of stage or grade of tumor (Sundfeldt et al.,
Br. J. Cancer,
1999, 79, 1240-1248). In the rat pancreas, it was shown that C/EBP beta expression is upregulated upon chronically elevated levels of glucose, possibly contributing to impaired insulin secretion in severe type II diabetes (Lu et al.,
J. Biol. Chem.,
1997, 272, 28349-28359; Seufert et al.,
J. Clin. Invest.,
1998, 101, 2528-2539).
The pharmacological modulation of C/EBP beta activity and/or expression may therefore be an appropriate point of therapeutic intervention in pathological conditions.
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of C/EBP beta and to date, investigative strategies aimed at modulating C/EBP beta function have involved the use of antibodies and gene knock-outs in mice. However, these strategies are untested as therapeutic protocols and consequently there remains a long felt need for agents capable of effectively inhibiting C/EBP beta 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 C/EBP beta expression.
The present invention provides compositions and methods for modulating C/EBP beta expression, including modulation of both the long and short isoforms of C/EBP beta.
SUMMARY OF THE INVENTION
The present invention is directed to antisense compounds, particularly oligonucleotides, which are targeted to a nucleic acid encoding C/EBP beta, and which modulate the expression of C/EBP beta. Pharmaceutical and other compositions comprising the antisense compounds of the invention are also provided. Further provided are methods of modulating the expression of C/EBP beta 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 C/EBP beta 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 C/EBP beta, ultimately modulating the amount of C/EBP beta produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding C/EBP beta. As used herein, the terms “target nucleic acid” and “nucleic acid encoding C/EBP beta” encompass DNA encoding C/EBP beta, 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 func
Butler Madeline M.
Monia Brett P.
Wyatt Jacqueline
ISIS Pharmaceuticals Inc.
Lacourciere Karen A.
Licata & Tyrrell P.C.
Yucel Remy
LandOfFree
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