Antisense inhibition of Phosphorylase kinase beta expression

Details Antisense inhibition of Phosphorylase kinase beta expression Antisense inhibition of Phosphorylase kinase beta expression

2000-09-14

2002-04-09

McGarry, Sean (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, C536S023200, C536S024300, C536S024310, C536S024330, C536S024500

Reexamination Certificate

active

06368856

ABSTRACT:

FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of Phosphorylase kinase beta. In particular, this invention relates to antisense compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding Phosphorylase kinase beta. Such oligonucleotides have been shown to modulate the expression of Phosphorylase kinase beta.
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 beta (also known as PHKB) is one of the three regulatory subunits of the phosphorylase kinase enzyme and is localized to chromosome 16q12 (Francke et al.,
Am. J. Hum. Genet
., 1989, 45, 276-282; Kilimann,
J. Inherit. Metab. Dis
., 1990, 13, 435-441). This subunit is expressed in all tissues and mutations in the gene have been reported that result in one form of autosomal recessive glycogen storage disease (Burwinkel et al.,
Hum. Mol. Genet
., 1997, 6, 1109-1115; Burwinkel et al.,
Hum. Genet
., 1997, 101, 170-174; van den Berg et al.,
Am. J. Hum. Genet
., 1997, 61, 539-546; Wullrich-Schmoll and Kilimann,
Eur. J. Biochem
., 1996, 238, 374-380).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of phosphorylase kinase beta and to date, investigative strategies aimed at studying phosphorylase kinase beta function have involved the use of antibodies. Consequently, there remains a long felt need for agents capable of effectively modulating phosphorylase kinase 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 phosphorylase kinase beta expression.
The present invention provides compositions and methods for modulating phosphorylase kinase beta expression.
SUMMARY OF THE INVENTION
The present invention is directed to antisense compounds, particularly oligonucleotides, which are targeted to a nucleic acid encoding Phosphorylase kinase beta, and which modulate the expression of Phosphorylase kinase beta. Pharmaceutical and other compositions comprising the antisense compounds of the invention are also provided. Further provided are methods of modulating the expression of Phosphorylase kinase 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 Phosphorylase kinase 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 Phosphorylase kinase beta, ultimately modulating the amount of Phosphorylase kinase beta produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding Phosphorylase kinase beta. As used herein, the terms “target nucleic acid” and “nucleic acid encoding Phosphorylase kinase beta” encompass DNA encoding Phosphorylase kinase 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 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 beta. 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 beta. 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 intragenic site is the region encompassing the translation initiation or termination codon of the open reading frame (ORF) of the gene. Since, as is known in the art, the translation initiation codon is typically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the “AUG codon,” the “start codon” or the “AUG start codon”. A minority of genes have a translation initiation codon having the

Affiliated with

Also associated with

Rating

Antisense inhibition of Phosphorylase kinase beta expression does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Antisense inhibition of Phosphorylase kinase beta expression, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Antisense inhibition of Phosphorylase kinase beta expression will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2914961