Antisense inhibition of PKA regulatory subunit RII alpha...

Details Antisense inhibition of PKA regulatory subunit RII alpha... Antisense inhibition of PKA regulatory subunit RII alpha...

2001-09-11

2003-02-25

Wang, Andrew (Department: 1635)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Method of regulating cell metabolism or physiology

C435S006120, C435S091100, C435S091300, C435S325000, C536S023100, C536S023200, C536S024500, C536S024300, C536S024310, C536S024330

Reexamination Certificate

active

06524854

ABSTRACT:

FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of PKA regulatory subunit RII alpha. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding PKA regulatory subunit RII alpha. Such compounds have been shown to modulate the expression of PKA regulatory subunit RII alpha.
BACKGROUND OF THE INVENTION
One of the principal mechanisms by which cellular responses are mediated by biologically active molecules including growth factors, cytokines, neurotransmitters and hormones, occurs through the transduction of extracellular signals across the membrane via specific cell-surface receptors and the concomitant generation of second messengers. One such second messenger system is that of cyclic AMP (cAMP). As a second messenger, cAMP acts to amplify the signal from several ligand effectors and is generated through the action of G proteins coupled to adenyl cyclase.
The cAMP signaling pathway is a signal transduction system comprising a sequence of molecules whose function is to govern the generation, degradation and biological response of cAMP within the cell (Perez et al.,
Bipolar Disord
., 2000, 2, 27-36). To accomplish this, cells have a network of proteins including G proteins, adenylate cyclase, cyclic nucleotide phosphodiesterases, protein phosphatases and cAMP-dependent protein kinase (Perez et al.,
Bipolar Disord
., 2000, 2, 27-36).
cAMP-dependent protein kinase (also known as protein kinase A; PKA) is the primary target of elevated levels of cAMP. PKA is a serine-threonine kinase that catalyzes the phosphorylation of several substrate proteins which, as a result of phosphorylation, undergo conformational changes leading to changes in their functional properties. In some cases, the phosphorylated proteins act as immediate effectors of a biological response and in other cases, act in intermediate steps of signal cascade networks (Perez et al.,
Bipolar Disord
., 2000, 2, 27-36). A noteworthy feature of cAMP signal routing is that, unlike other second messengers, almost all of the cellular effects of cAMP are mediated by phosphorylation reactions catalyzed by PKA (Perez et al.,
Bipolar Disord
., 2000, 2, 27-36). The cAMP/PKA signaling pathway is involved in metabolism, gene regulation, cell growth and division, cell differentiation, sperm motility and ion channel conductivity (Skalhegg and Tasken,
Front. Biosci
., 2000, 5, D678-693).
At the molecular level, the inactive PKA species is a tetramer consisting of two C (catalytic) subunits and one R (regulatory) subunit dimer. Activation of PKA occurs when cAMP binds to the R subunits and the PKA complex dissociates into an R dimer and two free C subunits. Once released from the holoenzyme state, C subunits are able to phosphorylate many different classes of substrate proteins (Perez et al.,
Bipolar Disord
., 2000, 2, 27-36).
There are two isozymes of PKA, known as PKAI and PKAII which are differentiated by two different regulatory subunits known as RI and RII (Skalhegg and Tasken,
Front. Biosci
., 2000, 5, D678-693). Cloning of the cDNAs for the regulatory subunits has resulted in the further discovery of additional forms of RI and RII subunits which are now known as RI alpha, RI beta, RII alpha and RII beta (Skalhegg and Tasken,
Front. Biosci
., 2000, 5, D678-693).
The PKA regulatory subunit RII alpha was cloned (Oeyen et al.,
FEBS Lett
., 1989, 246, 57-64) and mapped to chromosome 3p21.3-p21.2 (Tasken et al.,
Genomics
, 1998, 50, 378-381). This gene product is ubiquitously expressed as a 6.0 kb mRNA transcript (Oyen et al.,
Biol. Reprod
., 1987, 37, 947-956). However, a truncated form (2.2 kb mRNA) of PKA regulatory subunit RII alpha has been identified in rat germ cells, which may be responsible for anchoring kinase activity to sperm flagella where cAMP-dependent phosphorylation of certain substrates is believed to be essential for sperm motility (Oeyen et al.,
Biol. Reprod
., 1990, 43, 46-54).
The involvement of the cAMP/PKA signaling pathway in metabolism, gene regulation, cell growth and division, cell differentiation, sperm motility and ion channel conductivity, make the modulation of its activity a potential strategy with which to treat metabolic or developmental disorders. Modulation of the expression of the PKA regulatory RII subunit alpha may provide such a means of regulating the expression of PKA.
To date, investigative strategies aimed at modulating PKA regulatory RII subunit alpha expression include the use of cycloheximide (Austlid Tasken et al.,
Mol. Endocrinol
., 1991, 5, 21-28) and adrenal glucocorticoids (Dwivedi and Pandey,
J. Pharmacol. Exp. Ther
., 2000, 294, 103-116). However, these inhibitors are not specific for the PKA regulatory RII subunit alpha.
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of PKA regulatory RII subunit beta.
Consequently, there remains a long felt need for additional agents capable of effectively inhibiting PKA regulatory RII subunit alpha function.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of PKA RII regulatory subunit alpha expression.
The present invention provides compositions and methods for modulating PKA regulatory RII subunit alpha expression, including modulation of truncated forms of PKA regulatory RII subunit alpha.
SUMMARY OF THE INVENTION
The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding PKA regulatory subunit RII alpha, and which modulate the expression of PKA regulatory subunit RII alpha. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of modulating the expression of PKA regulatory subunit RII alpha 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 PKA regulatory subunit RII alpha 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 PKA regulatory subunit RII alpha, ultimately modulating the amount of PKA regulatory subunit RII alpha produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding PKA regulatory subunit RII alpha. As used herein, the terms “target nucleic acid” and “nucleic acid encoding PKA regulatory subunit RII alpha” encompass DNA encoding PKA regulatory subunit RII alpha, 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 PKA regulatory subunit RII alpha. In the context of the pres

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