Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-07-31
2002-04-02
Wang, Andrew (Department: 1635)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C536S024500, C435S325000, C435S375000
Reexamination Certificate
active
06365354
ABSTRACT:
FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of Lysophospholipase I. In particular, this invention relates to antisense compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding Lysophospholipase I. Such oligonucleotides have been shown to modulate the expression of Lysophospholipase I.
BACKGROUND OF THE INVENTION
Phospholipids, considered the building blocks of biological membranes, function not only as structural barriers for the cell but are vital constituents, acting as second messengers, of signal transduction pathways. It has been demonstrated that oxidized phospholipids play a key role in the development of certain diseases, and therefore the study-of phospholipid metabolism has become a field of intense study.
Lysophospholipids (LysoPLs) are intermediates of phospholipid metabolism. These lipids consist of one long hydrophobic acyl chain and one large hydrophilic polar head group, making them amphipathic (having both positive and negatively charged characteristics). This quality gives the lysophospholipids surfactant and detergent properties and thus their levels must be strictly regulated for proper cell function and survival. Increased levels of lysophospholipids could result in disruption of membrane structure and possibly cell lysis. Consequently, increased levels of lysophospholipids have been associated with a variety of disease processes (Wang and Dennis,
Biochim. Biophys. Acta
., 1999, 1439, 1-16).
Regulation of lysophospholipid levels is controlled by a family of enzymes known as lysophospholipases (LysoPLAs). These enzymes control the levels of lysophospholipids through hydrolysis and are the major pathway by which lysophospholipids are degraded (Wang and Dennis,
Biochim. Biophys. Acta
., 1999, 1439, 1-16).
Lysophospholipases are divided into low and high molecular weight isoforms with varying substrate specificity and pH requirements. The high molecular weight isoforms act as hydrolases and transacylases while the low molecular weight isoforms act as hydrolases. Many species have two low molecular weight forms, lysophospholipase A I (LysoPLA I) and lysophospholipase A II (LysoPLA II).
Lysophospholipase I (also known as LPL1, LYPLA1 and LysoPLA I) was first sequenced and cloned from a rat liver cDNA library using antibody techniques. It was subsequently cloned from the mouse and human (Wang et al.,
J. Biol. Chem
., 1997, 272, 12723-12729; Wang et al.,
Biochim. Biophys. Acta
., 1999, 1437, 157-169).
Disclosed in U.S. Pat. Nos. 5,858,756, 5,965,423 and the PCT publication WO 98/49319 are the polynucleotide and polypeptide sequence of the human lysophospholipase A I (NHLP) as well as vectors, host cells and methods for expressing the enzyme. Isolated polynucleotides completely complementary to a polynucleotide encoding NHLP are also disclosed. Further disclosed are antibodies to the enzyme, agonists and antagonists of the polypeptide as well as a purified polynucleotide which hybridizes under stringent conditions to the polynucleotide which encodes lysophospholipase I (Hillman et al., 1998; Hillman et al., 1999).
Tissue distribution studies by Northern and Western blots indicate that the lysophospholipase A I mRNA and protein are widely distributed in all tissues examined (Wang et al.,
Biochim. Biophys. Acta
., 1999, 1437, 157-169). In the human adult tissues, the highest expression is seen in the heart, placenta and skeletal muscle while fetal tissues demonstrated a more uniform pattern of expression (Wang et al.,
Biochim. Biophys. Acta
., 1999, 1437, 157-169).
Lysophospholipase I has also been shown to mediate other functions within the cell including G-protein signal transduction. It has been shown to remove the palmitate group from G-protein alpha subunits (Duncan and Gilman,
J. Biol. Chem
., 1998, 273, 15830-15837).
The pharmacological modulation of lysophospholipase I activity and/or expression may therefore be an appropriate point of therapeutic intervention in pathological conditions involving deregulated phospholipid metabolism.
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of lysophospholipase I. The most potent inhibitor of lysophospholipase I activity is MAFP (methyl arachidonyl fluorophosphonate) which irreversibly inhibits enzyme activity (Wang et al.,
Biochim. Biophys. Acta
., 1999, 1437, 157-169). Consequently, there remains a long felt need for additional agents capable of effectively inhibiting lysophospholipase I 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 lysophospholipase I expression.
The present invention provides compositions and methods for modulating lysophospholipase I expression.
SUMMARY OF THE INVENTION
The present invention is directed to antisense compounds, particularly oligonucleotides, which are targeted to a nucleic acid encoding Lysophospholipase I, and which modulate the expression of Lysophospholipase I. Pharmaceutical and other compositions comprising the antisense compounds of the invention are also provided. Further provided are methods of modulating the expression of Lysophospholipase I 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 Lysophospholipase I 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 Lysophospholipase I, ultimately modulating the amount of Lysophospholipase I produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding Lysophospholipase I. As used herein, the terms “target nucleic acid” and “nucleic acid encoding Lysophospholipase I” encompass DNA encoding Lysophospholipase I, 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 Lysophospholipase I. 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
Bennett C. Frank
Wyatt Jacqueline
Epps Janet
ISIS Pharmaceuticals Inc.
Licata & Tyrrell P.C.
Wang Andrew
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