Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of regulating cell metabolism or physiology
Reexamination Certificate
2001-07-30
2004-07-27
McGarry, Sean (Department: 1635)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of regulating cell metabolism or physiology
C435S377000, C536S063000, C536S023100, C536S024100, C536S024500
Reexamination Certificate
active
06767739
ABSTRACT:
FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of microsomal triglyceride transfer protein. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding microsomal triglyceride transfer protein. Such compounds have been shown to modulate the expression of microsomal triglyceride transfer protein.
BACKGROUND OF THE INVENTION
Triglycerides are one of the most efficient storage forms of free energy. Because of their insolubility in biological fluids, their transport between cells and tissues requires that they be assembled into lipoprotein particles. Genetic disruption of the lipoprotein assembly/secretion pathway leads to several human disorders associated with malnutrition and developmental abnormalities. In contrast, patients displaying inappropriately high rates of lipoprotein production display increased risk for the development of atherosclerotic cardiovascular disease (Davis,
Biochim. Biophys. Acta,
1999, 1440, 1-31).
The mammalian lipoprotein assembly/secretion pathway requires 2 components: apolipoprotein B (ApoB, an amphipathic protein) and a lipid transfer protein (microsomal triglyceride transfer protein, MTP). In the endoplasmic reticulum, ApoB has two possible metabolic fates: entrance into the lipoprotein assembly pathway within the lumen of the endoplasmic reticulum (ER), or degradation in the cytoplasm by the ubiquitin-dependent proteasome. The destiny of ApoB is determined by the relative availability of individual lipids and the level of expression of microsomal triglyceride transfer protein (Davis,
Biochim. Biophys. Acta,
1999, 1440, 1-31).
Microsomal triglyceride transfer protein binds and shuttles individual lipid molecules between membranes. In particular, microsomal triglyceride transfer protein accelerates the transport of triglycerides, cholesteryl ester, and phospholipid from the ER membrane, where lipid molecules are synthesized, to developing lipoproteins within the lumen of the ER (Berriot-Varoqueaux et al.,
Annu. Rev. Nutr.,
2000, 20, 663-697). In vitro analyses show that microsomal triglyceride transfer protein has a preference for transferring triglycerides and cholesteryl esters (Gordon and Jamil,
Biochim. Biophys. Acta,
2000, 1486, 72-83).
Microsomal triglyceride transfer protein is a heterodimeric neutral lipid transfer protein found in the lumen of the endoplasmic reticulum of ApoB lipoprotein-secreting cells, predominantly hepatocytes and intestinal enterocytes, and has been recently detected in the human heart (Herrmann et al.,
J. Lipid Res.,
1998, 39, 2432-2435). The smaller 55 kDa subunit of microsomal triglyceride transfer protein has been identified as protein disulfide isomerase (PDI). The isomerase activity is not required for the complex to transfer lipid. The larger 97 kDa subunit is a unique polypeptide responsible for the in vitro binding and transfer of lipids (Gordon and Jamil,
Biochim. Biophys. Acta,
2000, 1486, 72-83).
Sharp et al. isolated and sequenced cDNA encoding human microsomal triglyceride transfer protein. The large subunit of human microsomal triglyceride transfer protein spans about 55 kb and is situated on chromosome 4q22-24 (Sharp et al.,
Nature,
1993, 365, 65-69).
Karpe et al. detected a common functional G/T polymorphism 493 bp upstream from the transcriptional start point of the microsomal triglyceride transfer protein gene. The rare allele confers significantly higher transcriptional activity (Karpe et al.,
Arterioscler. Thromb. Vasc. Biol.,
1998, 18, 756-761). The normal T/T genotype has a 25% lower 10-year risk of developing cardiovascular disease than the G/T genotype (Karpe et al.,
Arterioscler. Thromb. Vasc. Biol.,
1998, 18, 756-761). In contrast, two other polymorphisms were discovered (−400 A/T and −164 T/C), but investigators concluded that the polymorphisms were unrelated to lipid variables or coronary heart disease (Herrmann et al.,
J. Lipid Res.,
1998, 39, 2432-2435).
Disclosed and claimed in U.S. Pat. No. 5,595,872 are the nucleic acid sequences encoding the high molecular weight subunit of microsomal triglyceride transfer protein as well as expression vectors containing said nucleic acids. Additionally claimed are nucleotide sequences fully complementary to the microsomal triglyceride transfer protein sequences. Generally disclosed in the same patent are methods using antisense molecules for the reduction of microsomal triglyceride transfer protein expression (Wetterau et al., 1997).
Small molecule inhibitors of microsomal triglyceride transfer protein are well represented in the art. For example, Wetterau et al. used an isoindolinone piperidine derivative as an microsomal triglyceride transfer protein inhibitor to assay microsomal triglyceride transfer protein-mediated lipid transport. The molecule inhibited the production of lipoprotein in rodent models and normalized plasma lipoprotein levels in Watanabe-heritable hyperlipidemic (WHHL) rabbits, which are a model for human homozygous familial hypercholesterolemia (Wetterau et al.,
Science,
1998, 282, 751-754). Disclosed and claimed in U.S. Pat. No. 6,235,730 is the use of 3-piperidyl-4-oxoquinazoline derivatives, shown to inhibit microsomal triglyceride transfer protein, as therapeutic agents for hyperlipemia or arteriosclerotic diseases (Sato et al., 2001).
Elevated plasma lipid levels cause premature atherosclerosis. Studies of the role of microsomal triglyceride transfer protein in abetalipoproteinemia demonstrate that microsomal triglyceride transfer protein is required for both hepatic and intestinal apoB-containing lipoprotein production. An increase in microsomal triglyceride transfer protein protein in relation to very low density lipopotein (VLDL) production and secretion is thought to cause hyperlipoproteinemia, which is an underlying cause of cardiovascular disease (Kuriyama et al.,
Hepatology,
1998, 27, 557-562). These studies suggest that inhibition of microsomal triglyceride transfer protein function may be an effective strategy to prevent very low density lipoprotein (VLDL) and chylomicron assembly and to lower plasma lipid levels (Jamil et al.,
Proc. Natl. Acad. Sci. U.S.A.,
1996, 93, 11991-11995).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of microsomal triglyceride transfer protein.
To date, investigative strategies aimed at modulating microsomal triglyceride transfer protein function have involved the use of small molecule inhibitors and gene knock-outs in mice.
Consequently, there remains a long felt need for additional agents capable of inhibiting microsomal triglyceride transfer protein 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 microsomal triglyceride transfer protein expression.
The present invention provides compositions and methods for modulating microsomal triglyceride transfer protein expression.
SUMMARY OF THE INVENTION
The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding microsomal triglyceride transfer protein, and which modulate the expression of microsomal triglyceride transfer protein. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of modulating the expression of microsomal triglyceride transfer protein 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 microsomal triglyceride transfer protein by administering a therapeutically or prophylactically effective amount of one or mor
Crooke Rosanne M.
Graham Mark J.
Howson and Howson
ISIS Pharmaceuticals Inc.
McGarry Sean
LandOfFree
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