Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-09-07
2002-07-30
Wang, Andrew (Department: 1635)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S375000, C536S023100, C536S024500
Reexamination Certificate
active
06426188
ABSTRACT:
FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of Phosphorylase kinase alpha 1. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding Phosphorylase kinase alpha 1. Such compounds have been shown to modulate the expression of Phosphorylase kinase alpha 1.
BACKGROUND OF THE INVENTION
Balanced energy metabolism is critical to the regulation of all biological processes. In higher organisms, energy stores are in the form of glycogen. Upon energy deficit, these stores are mobilized through enzymatic digestion to glucose-1-phosphate by a diverse set of signals and are used to maintain blood-glucose levels, as a source of energy during muscle contraction and as source of fuel for a broad range of cellular activities.
The protein kinase, phosphorylase kinase (PHK) plays a central role in the regulation of glycogen degradation or glycogenolysis by phosphorylating glycogen phosphorylase b, a unique reaction catalyzed only by phosphorylase kinase. It also lies at the interface between signaling and metabolic pathways and translates the pleiotropic actions of extracellular signals, including hormonal and neuronal, into specific and directional intracellular responses. In addition, phosphorylase kinase can express varying degrees of activity depending on pH, metal ion concentration, allosteric effectors and covalent modifications (Brushia and Walsh,
Front. Biosci
., 1999, 4, D618-641).
Structurally, phosphorylase kinase is one of the most complex enzymes isolated to date, a hexadecamer, having three distinct regulatory subunits, alpha, beta and delta (also known as calmodulin), and one catalytic subunit, gamma. Each holoenzyme is composed of four heterotetramers of the component subunits and the subunit stoichiometry has been shown to vary depending on the tissue source. The phosphorylase kinase subunits also exist as multiple isoforms adding an additional layer of complexity. The alpha, beta, and gamma isoforms are found expressed in the liver and muscle with minor amounts in the gut, while the delta (calmodulin) isoforms are expressed in all tissues examined (Brushia and Walsh,
Front. Biosci
., 1999, 4, D618-641).
Due to the direct relationship between phosphorylase kinase enzyme activity and maintenance of blood-glucose homeostasis, modifications to the regulatory properties of this enzyme could provide great therapeutic benefit in the arena of metabolic disorders, especially diabetes.
Phosphorylase kinase alpha 1 (also known as PHKA, PHKA1 and &agr;M) is one of the three regulatory alpha subunit isoforms identified to date and is localized solely in muscle tissue (Wullrich et al.,
J. Biol. Chem
., 1993, 268, 23208-23214).
The gene is located on chromosome Xq13 and several types of mutations in this gene have been reported which result in differential mRNA processing and certain forms of glycogen storage diseases. These mutations include a splice junction mutation in a patient with myopathy (Bruno et al.,
Biochem. Biophys. Res. Commun
., 1998, 249, 648-651) and nonsense mutations that resemble the X-linked phosphorylase kinase deficiency seen in I-strain mice (Wehner et al.,
Hum. Mol. Genet
., 1994, 3, 1983-1987). In mice this nonsense mutation results in a frameshift of the coding region and therefore disrupts the expression of both the liver and muscle isoforms of the alpha subunit (Bender, Biochem.
Biophys. Res. Commun
., 1991, 179, 707-712; Bender and Lalley,
Proc. Natl. Acad. Sci. U.S.A
., 1989, 86, 9996-10000; Schneider et al.,
Nat. Genet
., 1993, 5, 381-385).
Currently however, there are no known therapeutic agents which effectively inhibit the synthesis of phosphorylase kinase alpha 1 and to date, investigative strategies aimed at studying phosphorylase kinase alpha 1 function have involved the use of antibodies and crosslinking agents.
Consequently, there remains a long felt need for agents capable of effectively modulating phosphorylase kinase alpha 1 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 phosphorylase kinase alpha 1 expression.
The present invention provides compositions and methods for modulating phosphorylase kinase alpha 1 expression.
SUMMARY OF THE INVENTION
The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding Phosphorylase kinase alpha 1, and which modulate the expression of Phosphorylase kinase alpha 1. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of modulating the expression of Phosphorylase kinase alpha 1 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 Phosphorylase kinase alpha 1 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 Phosphorylase kinase alpha 1, ultimately modulating the amount of Phosphorylase kinase alpha 1 produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding Phosphorylase kinase alpha 1. As used herein, the terms “target nucleic acid” and “nucleic acid encoding Phosphorylase kinase alpha 1” encompass DNA encoding Phosphorylase kinase alpha 1, 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 Phosphorylase kinase alpha 1. 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 Phosphorylase kinase alpha 1. The targeting process also includes determination of a site or sites within this gene for the antisense interaction to occur such that the desired effect, e.g., detection or modulation of expression of the protein, will result. Within the context of the present invention, a preferred
ISIS Pharmaceuticals Inc.
Licata & Tyrrell P.C.
Wang Andrew
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