US 5,705,388 AGrant

CETP Ribozymes

Issue Date:1998-01-06

•14 Claims

•6 Drawing Sheets

Abstract

A nucleic acid molecule which blocks synthesis and/or expression of mRNAs associated with initial development, progression or regression of vascular disease.

Metadata

Assignees

Ribozyme Pharmaceuticals, Inc.
Warner-Lambert Company

Inventors

Larry Couture
Dan Stinchcomb
James McSwiggen
Charles Bisgaier
Michael Pape

Application Information

Application Number:US 3632407

Filing Date:1994-12-23

Priority Date:1994-12-23

Art Unit:189

Classifications

IPC:

C12N 1585C12Q 168

Field of Search:

4355365146;91.31;172.3;325;366;320.123.1;23.2;24.544

Patent Drawings (6 sheets)

Description

BACKGROUND OF THE INVENTION

This invention relates to the methods for the prevention, inhibition of progression and regression of vascular diseases, in particular, inhibition of cholesterol ester transfer protein (CETP) inhibition.

The following is a discussion of relevant art, none of which is admitted to be prior art to the present invention.

Vascular diseases, which includes etiologies such as peripheral vascular disease, coronary heart disease (CHD), stroke and restenosis, remain the leading cause of death and disability in the United States and throughout the world. In 1990 alone, approximately 500,000 people died in the United States from CHD. Although, diet and life style can accelerate the rate of onset of vascular diseases, genetic predisposition leading to "dyslipidemia" is a major and significant factor attributing to vascular related deaths and disabilities(Anderson et al., 1987 JAMA 257, 2176). By "dyslipidemia" is meant abnormal levels of lipoproteins in plasma.

A variety of risk factors have been identified that are associated with increased risk of vascular disease (Barr et al., 1951 Am. J. Med. 11,480; Kannel et al., 1971 Ann. Intern. Med. 74, 1; Miller et al., 1975 Lancet 1, 16; Levy et al., 1984 Circulation 69, 325.; Lipid Research Clinics Program, 1984 JAMA 251, 351; Lipid Research Clinics Program, 1984 JAMA 251, 365; Anderson et al., 1987 JAMA 257, 2176; Blankenhorn et al., 1987 J. Am. Med. Assoc. 257, 3233.; Frick et al., 1987 N. Engl. J. Med. 317, 1237; Expert Panel, 1988 Arch. Intern. Med. 148, 36.; Grundy et al., 1989 Arch. Intern. Med. 149, 505.; La Rosa, 1990 Am. J. Cardiol. 65, 7F-10F). Among these are the dyslipidemias of high levels of low density lipoproteins (LDL) and low levels of high density lipoproteins (HDL), singly or in combination. Often the ratio of HDL cholesterol to that of LDL cholesterol is used to assess risk of vascular disease. Thus, a high ratio of HDL/LDL cholesterol is desirable, and intervention to increase the ratio by lowering LDL and elevating HDL, singly or in combination is desirable.

Familial hypercholesterolemia (FH), a genetic disorder caused by defective or deficient LDL receptors presents as a marked elevation in LDL and risk in vascular disease (Goldstein et al., 1989 "Familial hypercholesterolemia" In: The Metabolic Basis of Inherited Diseases, 6th Ed., Schiver, C. R., Beaudet, A. L., Sly, W. S., and Valle, D. editors, 1215). Homozygous FH is a relatively rare disorder (1 in 1,000,000). Homozygous FH patients have extremely high levels of LDL with very short life expectancies. Therapy for these individuals include liver transplantation and LDL plasmaphoresis. Heterozygous FH is relatively common disorder (1 in 500). Heterozygous FH patients present with LDL levels approximately twice normal and are at risk for developing premature atherosclerosis followed by the common sequelae associated with vascular diseases, including myocardial infarction and stroke. Conventional therapy for heterozygous FH patients generally includes HMG CoA reductase inhibitors alone or in combination with bile acid sequestrants. Human prospective trials have demonstrated reduction in CHD related endpoints in hypercholesterolemic subjects treated with HMGCoA reductase inhibitors, bile acid sequestrants, Nicotinic acid, and gemfibrozil (La Rosa, 1990 Am. J. Cardiol. 65, 7F; Pedersen et al., 1994 Lancet 344, 1383).

Other conditions, such as apoE 3/4 and apoE4/4 genotype are also associated with increased LDL elevation and risk of CHD. However the direct causal relation between apoE4 polymorphism, elevated LDL and increased risk is unknown (Davignon et al., 1988 Arteriosclerosis 8, 1; Dallongeville et al., 1992 J. Lipid Res. 33, 447; Walden et al., 1994 Ann. Intern. Med. 120, 1026).

Low levels of HDL or hypoalphalipoproteinemia is a relatively common condition. The genetic basis for hypoalphalipoproteinemia is poorly understood, but likely results from multiple factors related to genetic predisposition and life style. Numerous prospective and retrospective studies have shown that HDL is inversely and strongly correlated with vascular disease. Therefore treatment to elevate HDL levels is warranted (Grundy et al., 1989 Arch. Intern. Med. 149, 505). It is also well recognized, that plasma triglyceride elevation is generally associated with low HDL levels, and insulin resistance (Reaven, 1988 Diabetes 37, 1595). Conventional therapies for elevated triglycerides and low HDL generally include treatment with fibrates. Gemfibrozil, a compound of this class, effectively lowers plasma triglycerides, and moderately elevates HDL (Frick, 1987 N. Engl. J. Med. 317, 1237). In a large human prospective trial, gemfibrozil has been shown to cause a significant reduction in vascular endpoints (Frick, 1987 supra).

The process termed reverse cholesterol transport (RCT; Bailey, 1965 Exp. Cell. Res. 37, 175; Glomset, 1968 J. Lipid Res. 9, 155) is a mechanism resulting in a net efflux of cholesterol present in peripheral tissues for disposal in bile. This multistep "hypothesized" pathway invokes removal of cholesteryl from peripheral tissues to HDL. Lecithin: cholesterol acyltransferase (LCAT), a circulating plasma enzyme, primarily mediates the esterification of HDL cholesterol to cholesteryl esters. CETP, also present in plasma, mediates HDL cholesteryl ester net transfer to apolipoprotein B (apoB)-containing lipoproteins including very low density lipoproteins (VLDL), intermediate density lipoproteins or remnants (IDL) and LDL. CETP-mediated cholesteryl ester enrichment of the LDL precursors, VLDL and IDL, ultimately contributes to the cholesterol content of LDL. Hepatic receptor-mediated LDL uptake, and a net flux of these delivered cholesteryl esters to the bile acid pool completes RCT. However, species that lack CETP (Oschry et al., 1982 J. Lipid Res. 23, 1099) or humans deficient in CETP (Koizumi et al., 1985 Atherosclerosis 58, 175) are unable to effectively transfer cholesteryl esters formed in HDL to the LDL precursor pool. Although the break in this link of the RCT pathway might predictably result in a marked deficiency in peripheral tissue cholesterol egress, surprisingly, this is not the case. In CETP deficient species, the HDL cholesteryl ester pool accumulates at the expense of the LDL pool. The HDL particles become enlarged and apoA-I, apoA-IV, and apoE-enriched (Brown et al., 1989 Nature 342, 448-451; Yamashita et al., 1990 J. Clin. Invest. 86, 688; Bisgaier et al., 1991 J. Lipid Res. 32, 21). Particles containing apoE can effectively be delivered to the liver as whole particles by facilitated mechanisms, including those utilizing the LDL receptor and the LDL receptor related protein (LRP) (Goldstein et al., 1985 Ann. Rev. Cell Biol. 1, 1; Mahley, 1988 Science 240, 622.; Beisiegel et al., 1989 Nature 341, 162; Bisgaier et al., 1989 J. Biol. Chem. 264, 862.). Thus, alternative mechanisms exist that facilitate tissue cholesterol egress and delivery of non-LDL cholesterol to liver in the absence of CETP.

Thus, CETP inhibition may inhibit or eliminate the RCT pathway thereby preventing the reduction in size and density of HDL, prolonging HDL half-life, and resulting in increased HDL levels. Additionally, the lack of transport of cholesteryl esters from HDL to apoB-containing lipoproteins may reduce LDL concentrations. Both these effects would result in an elevation of the HDL to LDL ratio. As high HDL/LDL ratios and HDL levels have been associated with anti-atherogenicity, diminishing CETP activity may prevent or inhibit progression and regression of vascular disease.

CETP is a 74 kDa glycoprotein that facilitates neutral lipid (cholesteryl esters and triglycerides) transfer between plasma lipoproteins (Zilversmit et al., 1975 Biochim. Biophys. Acta 409, 393; Ha et al., 1982 Comp. Biochem. Physiol. 71B, 265; Drayna et al., 1987 Nature 327, 632; Hesler et al., 1987 J. Biol. Chem. 262, 2275; Swenson et al., 1987 J. Biol. Chem. 262, 16271; Hesler et al., 1988 J. Biol. Chem. 263, 5020; Nagashima et al., 1988 J. Lipid Res. 29, 1643; Pape et al., 1991 Arterioscler. Thromb. 11, 1759). In non-human primates and rabbits, hepatic non-parenchymal cells are likely the major synthetic source of CETP (Pape et al., 1991 Arterioscler. Thromb. 11, 1759; Pape et al., 1991 J. Biol. Chem. 266, 12829; Rea et al., 1993 J. Lipid Res. 34, 1901), in that these cells have the highest cellular content of CETP mRNA relative to total RNA. Abundant amounts of CETP mRNA has also been shown in hepatic parenchymal cells, adipose, and spleen and to a lesser extent in the intestine and heart.

The level of CETP activity between species is highly variable (Ha et al., 1982 Comp. Biochem. Physiol. 71B, 265; Bisgaier et al., 1993 J. Lipid Res. 34, 1625). In general, species with high CETP activity (e.g., humans and rabbits) are susceptible to dietary induced atherosclerosis, while species with little or no CETP activity (e.g., mice, rats and dogs) are resistant (Koizumi et al., 1985 Atherosclerosis 58, 175; Inazu et al., 1990 N. Engl. J. Med. 323, 1234; Agellon et al., 1991 J. Biol. Chem. 260, 10796; Bisgaier et al., 1991 J. Lipid Res. 32, 21; Marotti et al., 1992 Arterioscler. Thrombosis 12, 736). Likewise, those species with little or no CETP activity have anti-atherosclerotic lipoprotein profiles: plasma HDL levels are elevated and LDL are reduced (Ha et al., 1982 Comp. Biochem. Physiol. 71B, 265). Infusions of inhibitory CETP monoclonal or polyclonal antibodies into rabbits or infusion of CETP into rats will invert the lipoprotein profiles (Ha et al., 1985 Biochim. Biophys. Acta. 833, 203; Abbey et al., 1989 Biochim. Biophys. Acta 1003, 20; Groener et al., 1989 Biochim. Biophys. Acta 1002, 93; Whitlock et al., 1989 J. Clin. Invest. 84, 129.). Unlike control mice of similar genetic background, CETP transgenic mice develop atherosclerotic lipoproteins and atherosclerosis (Marotti et al., 1992 Arterioscler. Thromb.12, 736).

Recent studies of a Japanese family have shown that a deficiency in plasma CETP associated with marked elevation of HDL, its associated apolipoproteins (apoA-I, apoE, apoA-IV) and a rarity of coronary artery disease (Koizumi et al., 1985 Atherosclerosis 58, 175; Brown et al., 1989 Nature 342, 448; Inazu et al., 1990 N. Engl. J. Med. 323, 1234; Bisgaier et al., 1991 J. Lipid Res. 32, 21.; Ikewaki et al., 1991 Arterioscler. Thromb. 11, 1400a; Koizumi et al., 1991 Atherosclerosis 90, 189). These individuals were identified through routine cholesterol screening and have no other hyperlipidemia related disease. The defect has been identified as a G to A substitution in the fourteenth intron of CETP pre-messenger ribonucleic acid (RNA) (Brown et al., 1989 Nature 342, 448). This splice donor defect is also the cause of the deficiency in additional Japanese families (Inazu et al., 1990 N. Engl. J. Med. 323, 1234; Koizumi et al., 1991 Atherosclerosis 90, 189; Hirano et al., 1993 Atherosclerosis 100, 85). In a more recent study, the deficiency (both homozygous and heterozygous) has been shown to be associated with a large proportion of Japanese with hyperalphalipoproteinemia (Inazu et al., 1992 Horm. Metab. Res. 24, 284; Hirano et al., 1993 Atherosclerosis 100, 85). A missense mutation at nucleotide 1506 (G for A) also has been identified in exon 15 of the CETP gene, resulting in a substation of a glycine for aspartic acid at amino acid 442 (Takahashi et al., 1993 J. Clin. Invest. 92, 2060). The two subjects heterozygous for the missense mutation had three times the normal HDL levels. Overall these studies suggest that even partial reduction in CETP levels, as found in heterozygous individuals, is associated with elevated HDL. This apparently benign condition (CETP deficiency) has been coined the "longevity syndrome" (Koizumi et al., 1985 Atherosclerosis 58, 175).

Although CETP facilitates an equimolar exchange of neutral lipids, net transfer of cholesteryl ester to LDL occurs due to (1) concentration and core lipid composition of exchange partners and (2) residence time of lipoproteins (Nichols et al., 1965 J. Lipid. Res. 206; Pattnaik et al., 1978 Biochim. Biophys. Acta 530, 428; Barter et al., 1979 Metabolism 28, 230). Under basal conditions (i.e., overnight fast), CETP facilitates transfer below maximal velocity, while postprandially CETP appears to facilitate transfer at or near maximal velocity (Tall et al., 1986 J. Clin. Invest. 77, 1163; Mann et al., 1991 J. Clin. Invest. 88, 2059; Bisgaier et al., 1993 J. Lipid Res. 34, 1625). It is also likely, but has not been systematically shown, that individuals with elevated triglycerides would have elevated CETP activity (but not necessarily increased CETP mass). In general, these subjects have reduced levels of HDL and elevated LDL. These consequences, in part may be the result of events facilitated by CETP.

The complete amino acid sequence of human, rabbit, cynomolgus monkey and hamster CETP are known (Drayna et al., 1987 Nature 327, 632; Nagashima et al., 1988 J. Lipid Res. 29, 1643; Jiang et al., 1991 J. Biol. Chem. 266, 4631; Pape et al., 1991 Arterioscler. Thromb. 11, 1759). Human plasma levels are approximately 1-2 .mu.g/ml, while rabbit levels are approximately 4 .mu.g/ml. Cholesterol feeding in rabbits elevates tissue CETP mRNA, plasma CETP, and maximal plasma activity approximately 4 fold (Quinet et al., 1990 J. Clin. Invest. 85, 357; McPherson et al., 1991 Arterioscler. Thromb. 11, 797). The protein is stable to heat, limited proteolysis, but not oxidation. CETP has been mapped with neutral and inhibitory monoclonal antibodies and by site-directed mutagenesis (Hesler et al., 1987 J. Biol. Chem. 262, 2275; Hesler et al., 1988 J. Biol. Chem. 263, 5020; Wang et al., 1991 Biochemistry 30, 3484). Stable transfection of the human gene in CHO cells has been accomplished (Wang et al., 1991 Biochemistry 30, 3484; Wang et al., 1992 J. Biol. Chem. 267, 17487). However, the protein has not been crystallized nor have the lipid binding domains been identified. Furthermore, the mechanisms by which CETP facilitates transfer are poorly understood.

Direct pharmacological inhibition of the existing protein in plasma or targeting CETP gene expression might lead to reduced plasma activity and result in a beneficial lipoprotein profile (i.e., HDL elevation and LDL diminution) and a reduced risk of coronary heart disease. However a synthetic compound approach for the direct inhibition of the plasma CETP has not yet been promising (Bisgaier et al., 1994 Lipids 29).

The gene encoding CETP is composed of 16 exons of various sizes (32-250 bp) and spans approximately 25 kb on the long arm (q12-21) of chromosome 16 (Lusis et al., 1987 Genomics 1,232; Agellon et al., 1990 Biochemistry 29, 1372). Cloning and sequencing of the human CETP cDNA has been reported and shown to contain an open reading frame and 3' untranslated region of 1656 nucleotides in length (Drayna et al., 1987 Nature 327, 632). Analysis of amino-acid and nucleic acid sequence has indicated a protein that is unique among eukaryotic species. A pentanucleotide amino acid stretch in the precursor protein signal peptide of CETP is conserved among the lipid metabolism associated proteins, for example apoA-IV, apoA-I, and lipoprotein lipase (Agellon et al., 1990 Biochemistry 29, 1372). This conservation occurs at both the nucleotide and the amino-acid level. This small but highly conserved region is found only in the precursor protein species and is removed before secretion of the mature protein into the blood stream. Other less conserved homologies have been noted with two lipopolysaccaride binding proteins, bacterial permeability increasing protein found in leukocyte granules and plasma lipopolysaccharide binding protein (Tall, 1993 J. Lipid Res. 34, 1255).

A single predominant splicing variant of the CETP message has been identified and characterized. This variant CETP mRNA lacks exon 9 and accounts for between 14-46% of total CETP mRNA with the highest percentage of this variant seen in the spleen. While the function of this abundant splice variant is not clearly understood, when coordinately expressed with full-length CETP in Chinese Hamster Ovary (CHO) cells, it was shown not to be secreted and capable of inhibiting secretion of the full length CETP protein (Quinet et al., 1993 J. Biol. Chem. 268, 16891).

A consequence of inhibiting CETP, besides that of favorably increasing the HDL/LDL cholesterol ratio, is a change in the distribution and level of apoE. In species lacking CETP (e.g., rats), during monoclonal antibody induced inhibition of CETP in hamsters, and in human CETP deficiency, plasma apoE levels are elevated (Yamashita et al., 1990 J. Clin. Invest. 86, 688; Eto et al., 1990 Artery 17, 202; Hirano et al., 1993 Atherosclerosis 100, 85; Takahashi et al., 1993 J. Clin. Invest. 92, 2060; Bisgaier et al., 1991 J. Lipid Res. 32, 21; Evans et al., 1994 J. Lipid Res. 35, 1634). Furthermore, HDL apoE-enrichment was observed (Evans et al., 1994 J. Lipid Res. 35, 1634). Recent in vitro studies have revealed mechanisms by which apoE-enriched HDL are protective (Yamada et al., 1992 J. Clin. Invest. 706; Saxena et al., 1993 J. Biol. Chem. 268, 14812). Additional studies have also demonstrated that apoE deficiency causes profound and accelerated rates of atherosclerosis in mice, a species not normally susceptible to atherosclerosis (Plump et al., 1992 Cell 71, 343; Zhang et al., 1992 Science 258, 468). Thus an expected and desirable consequence of CETP inhibition includes elevation of apoE-rich HDL. In apoE deficiency, overexpression of apoA-I can also afford protection against atherosclerosis (Plump et al., 1994 Proc. Natl. Acad. Sci. USA 91, 9607), and elevation of this protein is also an expected consequence of CETP inhibition (Koizumi et al., 1985 Atherosclerosis 58, 175; Eto et al., 1990 Artery 17, 202; Hirano et al., 1993 Atherosclerosis 100, 85; Takahashi et al., 1993 J. Clin. Invest. 92, 2060).

There currently exists no practical therapeutic treatment for interfering with or blocking CETP activity in humans. Although not practical, repetitive anti-CETP combined with anti-LDL plasmaphoreseis resulted in favorable changes in LDL and HDL levels and HDL/LDL ratios in a limited number of human studies (Davidson, U.S. Pat. No. 5,279,540). Although plasma CETP levels markedly decreased with duration of plasmaphoresis treatments, neither anti-CETP plasmaphoresis alone nor control non-immune plasmaphoresis data were reported. Several potential inhibitors are being explored in various laboratories. These inhibitors include monoclonal antibodies and an inhibitor protein recently found in baboon plasma tentatively identified as the N-terminal fragment of apolipoprotein C-I (Kushwaha et al., 1993 J. Lipid Res. 1993, 1285; Kushwaha et al., WO 93/11782). In the current application, a ribozyme, antisense or 2-5A-antisense or triplex DNA approach is described. The advantage of these approaches is their ability to selectively target specific regions of the CETP mRNA.

SUMMARY OF THE INVENTION

The invention features novel nucleic acid-based techniques e.g., enzymatic RNA molecules (ribozymes), antisense nucleic acids, 2-5A antisense chimeras, triplex DNA! and methods for their use for: (1) treatment of dyslipidemias by decreasing lipoproprotein risk factors, in particular, decreasing high levels of LDL or increasing low levels of HDL, or a combination of both and (2) for the prevention, inhibition of progression, and regression of vascular diseases, particularly, those diseases associated with (but not limited to) peripheral vascular disease, coronary heart disease, stroke, vascular complications of diabetes, transplant, atherectomy, and angloplastic restenosis.

The uniqueness of the CETP coding region and protein afford an increased safety margin when contemplating sequence-specific therapeutics targeting of the mRNA, such as ribozymes or antisense nucleic acids or 2-5A antisense chimeras, since there would be a reduced likelihood of non-specific activity from these therapeutics.

In a preferred embodiment, the invention features use of nucleic acid-based techniques to treat lipoprotein risk factors and/or prevent vascular diseases by inhibiting the synthesis of cholestryl ester transfer protein (CETP).

Those in the art will recognize the other potential targets, for e.g., apolipoprotein B, are also suitable for treatment with nucleic acid-based techniques described in the present invention.

By "inhibit" is meant that the activity of CETP or level of mRNAs encoded by CETP is reduced below that observed in the absence of the nucleic acid, particularly, inhibition with ribozymes and preferably is below that level observed in the presence of an inactive RNA molecule able to bind to the same site on the mRNA, but unable to cleave that RNA.

By "enzymatic nucleic acid (NA) molecule" it is meant a nucleic acid molecule which has complementarity in a substrate binding region to a specified gene target, and also has an enzymatic activity which is active to specifically cleave RNA in that target. That is, the enzymatic nucleic acid molecule is able to intermolecularly cleave RNA and thereby inactivate a target RNA molecule. This complementarity functions to allow sufficient hybridization of the enzymatic nucleic acid molecule to the target RNA to allow the cleavage to occur. One hundred percent complementarity is preferred, but complementarity as low as 50-75% may also be useful in this invention.

By "equivalent" RNA to CETP is meant to include those naturally occuring RNA molecules RNA molecules associated with cardiovascular diseases in various animals, including human, rabbit and monkey. Such a molecule will generally contain some ribonucleotides, but the other nucleotides may be substituted at the 2'-hydroxyl position and in other locations with other moieties as discussed below.

By "antisense nucleic acid" is meant a non-enzymatic nucleic acid molecule that binds to another RNA (target RNA) by means of RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al., 1993 Nature 365, 566) interactions and alters the activity of the target RNA (for a review see Stein and Cheng, 1993 Science 261, 1004). By "2-5A antisense chimera" is meant, an antisense oligonucleotide containing a 5' phosphorylated 2'-5'-linked adenylate residues. These chimeras bind to target RNA in a sequence-specific manner and activate a cellular 2-5A-dependent ribonuclease which in turn cleaves the target RNA (Torrence et al., 1993 Proc. Natl. Acad. Sci. USA 90, 1300).

By "triplex DNA" is meant an oligonucleotide that can bind to a double-stranded DNA in a sequence-specific manner to form a triple-strand helix. Triple-helix formation has been shown to inhibit transcription of the targeted gene (Duval-Valentin et al., 1992 Proc. Natl. Acad. Sci. USA 89, 504).

By "gene" is meant a nucleic acid that encodes an RNA.

By "complementarity" is meant a nucleic acid that can form hydrogen bond(s) with other RNA sequence by either traditional Watson-Crick or other non-traditional types (for example, Hoogsteen type) of base-paired interactions.

Six basic varieties of naturally-occurring enzymatic RNAs are known presently. Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions. Table I summarizes some of the characteristics of these ribozymes. In general, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.

The enzymatic nature of a ribozyme is advantageous over other technologies, since the concentration of ribozyme necessary to affect a therapeutic treatment is lower. This advantage reflects the ability of the ribozyme to act enzymatically. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA. In addition, the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base-pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage. Single mismatches, or base-substitutions, near the site of cleavage can completely eliminate catalytic activity of a ribozyme.

Ribozymes that cleave the specified sites in CETP mRNAs represent a novel therapeutic approach to vascular disease. Applicant indicates that ribozymes are able to inhibit the activity of CETP and that the catalytic activity of the ribozymes is required for their inhibitory effect. Those of ordinary skill in the art, will find that it is clear from the examples described that other ribozymes that cleave these sites in CETP mRNAs may be readily designed and are within the invention.

In preferred embodiments of this invention, the enzymatic nucleic acid molecule is formed in a hammerhead or hairpin motif, but may also be formed in the motif of a hepatitis delta virus, group I intron or RNaseP RNA (in association with an RNA guide sequence) or Neurospora VS RNA. Examples of such hammerhead motifs are described by Rossi et al., 1992, Aids Research and Human Retroviruses 8, 183, of hairpin motifs by Hampel et al., EP0360257, Hampel and Tritz, 1989 Biochemistry 28, 4929, and Hampel et al., 1990 Nucleic Acids Res. 18, 299, and an example of the hepatitis delta virus motif is described by Perrotta and Been, 1992 Biochemistry 31, 16; of the RNaseP motif by Guerrier-Takada et al., 1983 Cell 35, 849, Neurospora VS RNA ribozyme motif is described by Collins (Saville and Collins, 1990 Cell 61, 685-696; Saville and Collins, 1991 Proc. Natl. Acad. Sci. USA 88, 8826-8830; Collins and Olive, 1993 Biochemistry 32, 2795-2799) and of the Group I intron by Cech et al., U.S. Pat. No. 4,987,071. These specific motifs are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule.

In a preferred embodiment the invention provides a method for producing a class of enzymatic cleaving agents which exhibit a high degree of specificity for the RNA of a desired target. The enzymatic nucleic acid molecule is preferably targeted to a highly conserved sequence region of a target mRNAs encoding CETP proteins such that specific treatment of a disease or condition can be provided with either one or several enzymatic nucleic acids. Such enzymatic nucleic acid molecules can be delivered exogenously to specific cells as required. Alternatively, the ribozymes can be expressed from DNA/RNA vectors that are delivered to specific cells.

Synthesis of nucleic acids greater than 100 nucleotides in length is difficult using automated methods, and the therapeutic cost of such molecules is prohibitive. In this invention, small enzymatic nucleic acid motifs (e.g., of the hammerhead or the hairpin structure) are used for exogenous delivery. The simple structure of these molecules increases the ability of the enzymatic nucleic acid to invade targeted regions of the mRNA structure. However, these catalytic RNA molecules can also be expressed within cells from eukaryotic promoters (e.g., Scanlon et al., 1991, Proc. Natl. Acad. Sci. USA, 88, 10591-5; Kashani-Sabet et al., 1992 Antisense Res. Dev., 2, 3-15; Dropulic et al., 1992 J. Virol, 66, 1432-41; Weerasinghe et al., 1991 J. Virol, 65, 5531-4; Ojwang et al., 1992 Proc. Natl. Acad. Sci. USA 89, 10802-6; Chen et al., 1992 Nucleic Acids Res., 20, 4581-9; Sarver et al., 1990 Science 247, 1222-1225). Those skilled in the art realize that any ribozyme can be expressed in eukaryotic cells from the appropriate DNA/RNA vector. The activity of such ribozymes can be augmented by their release from the primary transcript by a second ribozyme (Draper et al., PCT WO 93/23569, and Sullivan et al., PCT WO 94/02595, both hereby incorporated in their totality by reference herein; Ohkawa et al., 1992 Nucleic Acids Symp. Ser., 27, 15-6; Taira et al., 1991, Nucleic Acids Res., 19, 5125-30; Ventura et al., 1993 Nucleic Acids Res., 21, 3249-55; Chowrira et al., 1994 J. Biol. Chem. 269, 25856).

Such ribozymes are useful for the prevention of the diseases and conditions discussed above, and any other diseases or conditions that are related to the level of CETP activity in a cell or tissue. By "related" is meant that the inhibition of CETP mRNAs and thus reduction in the level of protein activity will relieve to some extent the symptoms of the disease or condition.

Ribozymes are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells. The nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex vivo, or in vivo through injection, infusion pump or stent, with or without their incorporation in biopolymers. In preferred embodiments, the ribozymes have binding arms which are complementary to the sequences in Tables II, IV, VI and VII. Examples of such ribozymes are shown in Tables III, V, VI and VII. Examples of such ribozymes consist essentially of sequences defined in these Tables. By "consists essentially of" is meant that the active ribozyme contains an enzymatic center equivalent to those in the examples, and binding arms able to bind mRNA such that cleavage at the target site occurs. Other sequences may be present which do not interfere with such cleavage.

In another aspect of the invention, ribozymes that cleave target molecules and inhibit CETP activity are expressed from transcription units inserted into DNA or RNA vectors. The recombinant vectors are preferably DNA plasmids or viral vectors. Ribozyme expressing viral vectors could be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Preferably, the recombinant vectors capable of expressing the ribozymes are delivered as described above, and persist in target cells. Alternatively, viral vectors may be used that provide for transient expression of ribozymes. Such vectors might be repeatedly administered as necessary. Once expressed, the ribozymes cleave the target mRNA. Delivery of ribozyme expressing vectors could be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that would allow for introduction into the desired target cell.

By "vectors" is meant any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.

In a preferred embodiment nucleic acids targeted to Exon 9 of CETP gene is described. A single predominant alternate-splicing variant of the CETP message that lacks exon 9 has been identified and characterized (Inazu et al., 1991 Biochemistry 31, 2352; Quinet et al., 1993 J. Biol. Chem. 268, 16891). While the function of this abundant splice variant is not clearly understood, it is known to be not secreted and capable of inhibiting secretion of the full-length CETP protein. (Quinet et. al. 1993 J. Biol. Chem. 16891). Inhibition of full-length CETP secretion is believed to occur due to a heterodimeric complex formation between the full-length and the spliced variant of CETP. This suggests that the spliced variant of CETP might be beneficial in regulating the plasma level of CETP. Nucleic acid-based therapeutics of this invention, therefore, may be selectively targeted to block the expression of exon 9-containing CETP.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The drawings will first briefly be described.

DRAWINGS

FIG. 1 is a diagrammatic representation of the hammerhead ribozyme domain known in the art. Stem II can be .gtoreq.2 base-pair long.

FIG. 2a is a diagrammatic representation of the hammerhead ribozyme domain known in the art; FIG. 2b is a diagrammatic representation of the hammerhead ribozyme as divided by Uhlenbeck (1987, Nature, 327, 596-600) into a substrate and enzyme portion; FIG. 2c is a similar diagram showing the hammerhead divided by Haseloff and Gerlach (1988, Nature, 334, 585-591) into two portions; and FIG. 2d is a similar diagram showing the hammerhead divided by Jeffries and Symons (1989, Nucl. Acids. Res., 17, 1371-1371) into two portions.

FIG. 3 is a diagrammatic representation of the general structure of a hairpin ribozyme. Helix 2 (H2) is provided with a least 4 base pairs (i.e., n is 1, 2, 3 or 4) and helix 5 can be optionally provided of length 2 or more bases (preferably 3-20 bases, i.e., m is from 1-20 or more). Helix 2 and helix 5 may be covalently linked by one or more bases (i.e., r is .gtoreq.1 base). Helix 1, 4 or 5 may also be extended by 2 or more base pairs (e.g., 4-20 base pairs) to stabilize the ribozyme structure, and preferably is a protein binding site. In each instance, each N and N' independently is any normal or modified base and each dash represents a potential base-pairing interaction. These nucleotides may be modified at the sugar, base or phosphate. Complete base-pairing is not required in the helices, but is preferred. Helix 1 and 4 can be of any size (i.e., o and p is each independently from 0 to any number, e.g., 20) as long as some base-pairing is maintained. Essential bases are shown as specific bases in the structure, but those in the art will recognize that one or more may be modified chemically (abasic, base, sugar and/or phosphate modifications) or replaced with another base without significant effect. Helix 4 can be formed from two separate molecules, i.e., without a connecting loop. The connecting loop when present may be a ribonucleotide with or without modifications to its base, sugar or phosphate. "q" is .gtoreq.2 bases. The connecting loop can also be replaced with a non-nucleotide linker molecule. H, refers to bases A, U or C. Y refers to pyrimidine bases.

FIG. 4 is a representation of the general structure of the hepatitis delta virus ribozyme domain known in the art.

FIG. 5 is a representation of the general structure of the self-cleaving VS RNA ribozyme domain.

FIG. 6 is a schematic representation of an RNAseH accessibility assay. Specifically, the left side of FIG. 6 is a diagram of complementary DNA oligonucleotides bound to accessible sites on the target RNA. Complementary DNA oligonucleotides are represented by broad lines labeled A, B, and C. Target RNA is represented by the thin, twisted line. The right side of FIG. 6 is a schematic of a gel separation of uncut target RNA from a cleaved target RNA. Detection of target RNA is by autoradiography of body-labeled, T7 transcript. The bands common to each lane represent uncleaved target RNA; the bands unique to each lane represent the cleaved products.

RIBOZYMES

Ribozymes of this invention block to some extent CETP production and can be used to treat disease or diagnose such disease. Ribozymes will be delivered to cells in culture and to cells or tissues in animal models of cardiovascular disorders. Ribozyme cleavage of CETP encoded mRNAs in these systems may alleviate disease symptoms.

Target sites

Targets for useful ribozymes can be determined as disclosed in Draper et al., "Method and reagent for treatment of arthritic conditions U.S. Ser. No. 08/152,487, filed Nov. 12, 1993, and hereby incorporated by reference herein in totality. Rather than repeat the guidance provided in those documents here, below are provided specific examples of such methods, not limiting to those in the art. Ribozymes to such targets are designed as described in those applications and synthesized to be tested in vitro and in vivo, as also described.

The sequence of human and rabbit CETP mRNAs were screened for optimal ribozyme target sites using a computer folding algorithm. Hammerhead or hairpin ribozyme cleavage sites were identified. These sites are shown in Tables II, IV, VI and VII (All sequences are 5' to 3' in the tables) The nucleotide base position is noted in the Tables as that site to be cleaved by the designated type of ribozyme. While rabbit and human sequences can be screened and ribozymes thereafter designed, the human targeted sequences are of most utility. However, as discussed in Stinchcomb et al., "Method and Composition for Treatment of Restenosis and Cancer Using Ribozymes," filed May 18, 1994, U.S. Ser. No. 08/245,466, rabbit targeted ribozymes may be useful to test efficacy of action of the ribozyme prior to testing in humans. The nucleotide base position is noted in the Tables as that site to be cleaved by the designated type of ribozyme.

Hammerhead or hairpin ribozymes are designed that could bind and were individually analyzed by computer folding (Jaeger et al., 1989 Proc. Natl. Acad. Sci. USA, 86, 7706) to assess whether the ribozyme sequences fold into the appropriate secondary structure. Those ribozymes with unfavorable intramolecular interactions between the binding arms and the catalytic core are eliminated from consideration. Varying binding arm lengths can be chosen to optimize activity. Generally, at least 5 bases on each arm are able to bind to, or otherwise interact with, the target RNA.

Referring to FIG. 6, mRNA were screened for accessible cleavage sites by the method described generally in McSwiggen, U.S. patent application Ser. No. 07/883,849 filed on May 1, 1992, entitled "Assay for ribozyme target site", hereby incorporated by reference herein. Briefly, DNA oligonucleotides representing potential hammerhead or hairpin ribozyme cleavage sites were synthesized. A polymerase chain reaction is used to generate substrates for T7 RNA polymerase transcription from human and rabbit CETP cDNA clones. Labeled RNA transcripts are synthesized in vitro from the templates. The oligonucleotides and the labeled transcripts are annealed, RNAseH is added and the mixtures are incubated for the designated times at 37.degree. C. Reactions are stopped and RNA separated on sequencing polyacrylamide gels. The percentage of the substrate cleaved is determined by autoradiographic quantitation using a PhosphorImaging system. From these data, hammerhead or hairpin ribozyme sites are chosen as the most accessible.

Ribozymes of the hammerhead or hairpin motif are designed to anneal to various sites in the mRNA message. The binding arms are complementary to the target site sequences described above. The ribozymes are chemically synthesized. The method of synthesis used follows the procedure for normal RNA synthesis as described in Usman et al., 1987 J. Am. Chem. Soc., 109, 7845 and in Scaringe et al., 1990 Nucleic Acids Res., 18, 5433 and made use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end. The average stepwise coupling yields are >98%. Inactive ribozymes are synthesized by substituting a U for G.sub.5 and a U for A.sub.14 (numbering from Hertel et al., 1992 Nucleic Acids Res., 20, 3252). Hairpin ribozymes are synthesized in two parts and annealed to reconstruct the active ribozyme (Chowrira and Burke, 1992 Nucleic Acids Res., 20, 2835-2840). Ribozymes are also synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol. 180, 51). All ribozymes are modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-O-methyl, 2'-H (for a review see Usman and Cedergren, 1992 TIBS 17, 34). Ribozymes are purified by gel electrophoresis using general methods or are purified by high pressure liquid chromatography (HPLC; See Usman et al., Synthesis, deprotection, analysis and purification of RNA and ribozymes, filed May, 18, 1994, U.S. Ser. No. 08/245,736 the totality of which is hereby incorporated herein by reference) and are resuspended in water.

The sequences of the ribozymes that are chemically synthesized, useful in this study, are shown in Tables III, V, VI and VII. Those in the art will recognize that these sequences are representative only of many more such sequences where the enzymatic portion of the ribozyme (all but the binding arms) is altered to affect activity. For example, stem-loop II sequence of hammerhead ribozymes listed in Tables III and V (5'-GGCCGAAAGGCC-3') can be altered (substitution, deletion, and/or insertion) to contain any sequences provided a minimum of two base-paired stem structure can form. Similarly, stem-loop IV sequence of hairpin ribozymes listed in Tables VI and VII (5'-CACGUUGUG-3') can be altered (substitution, deletion, and/or insertion) to contain any sequence, provided a minimum of two base-paired stem structure can form. The sequences listed in Tables III, V, VI and VII may be formed of ribonucleotides or other nucleotides or non-nucleotides. Such ribozymes are equivalent to the ribozymes described specifically in the Tables.

Optimizing Ribozyme Activity

Ribozyme activity can be optimized as described by Stinchcomb et al., supra. The details will not be repeated here, but include altering the length of the ribozyme binding arms (stems I and III, see FIG. 2c), or chemically synthesizing ribozymes with modifications that prevent their degradation by serum ribonucleases (see e.g., Eckstein et al., International Publication No. WO 92/07065; Perrault et al., 1990 Nature 344, 565; Pieken et al., 1991 Science 253, 314; Usman and Cedergren, 1992 Trends in Biochem. Sci. 17, 334; Usman et al., International Publication No. WO 93/15187; and Rossi et al., International Publication No. WO 91/03162, as well as Usman, N. et al. U.S. patent application Ser. No. 07/829,729, and Sproat, European Patent Application 92110298.4 which describe various chemical modifications that can be made to the sugar moieties of enzymatic RNA molecules, modifications which enhance their efficacy in cells, and removal of stem II bases to shorten RNA synthesis times and reduce chemical requirements. (All these publications are hereby incorporated by reference herein.),

Sullivan, et al., supra, describes the general methods for delivery of enzymatic RNA molecules. Ribozymes may be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres. For some indications, ribozymes may be directly delivered ex vivo to cells or tissues with or without the aforementioned vehicles. Alternatively, the RNA/vehicle combination is locally delivered by direct injection or by use of a catheter, infusion pump or stent. Other routes of delivery include, but are not limited to, intravascular, intramuscular, subcutaneous or joint injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. More detailed descriptions of ribozyme delivery and administration are provided in Sullivan et al; supra and Draper et al., supra which have been incorporated by reference herein.

Another means of accumulating high concentrations of a ribozyme(s) within cells is to incorporate the ribozyme-encoding sequences into a DNA or RNA expression vector. Transcription of the ribozyme sequences are driven from a promoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III). Transcripts from pol II or pol III promoters will be expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type will depend on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby. Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990 Proc. Natl. Acad. Sci. USA, 87, 6743-7; Gao and Huang 1993 Nucleic Acids Res., 21, 2867-72; Lieber et al., 1993 Methods Enzymol., 217, 47-66; Zhou et al., 1990 Mol. Cell. Biol., 10, 4529-37). Several investigators have demonstrated that ribozymes expressed from such promoters can function in mammalian cells (e.g. Kashani-Sabet et al., 1992 Antisense Res. Dev., 2, 3-15; Ojwang et al., 1992 Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al., 1992 Nucleic Acids Res., 20, 4581-9; Yu et al., 1993 Proc. Natl. Acad. Sci. USA, 90, 6340-4; L'Huillier et al., 1992 EMBO J. 11, 4411-8; Lisziewicz et al., 1993 Proc. Natl. Acad. Sci. U.S.A., 90, 8000-4). The above ribozyme transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated virus vectors), or viral RNA vectors (such as retroviral or alphavirus vectors).

In a preferred embodiment of the invention, a transcription unit expressing a ribozyme that cleaves mRNAs encoded by CETP is inserted into a plasmid DNA vector or an adenovirus or adeno-associated virus DNA viral vector or a retroviral RNA vector. Viral vectors have been used to transfer genes and lead to either transient or long term gene expression (Zabner et al., 1993 Cell 75, 207; Carter, 1992 Curr. Opi. Biotech. 3, 533). The adenovirus vector is delivered as recombinant adenoviral particles. The DNA may be delivered alone or complexed with vehicles (as described for RNA above). The recombinant adenovirus or AAV particles are locally administered to the site of treatment, e.g., through incubation or inhalation in vivo or by direct application to cells or tissues ex vivo.

In another preferred embodiments, the ribozyme is administered to the site of CETP expression (e.g., liver cells) in an appropriate liposomal vesicle.

EXAMPLE 1

CETP Hammerhead ribozymes

By engineering ribozyme motifs we have designed several ribozymes directed against CETP encoded mRNA sequences. These ribozymes are synthesized with modifications that improve their nuclease resistance. The ability of ribozymes to cleave target sequences in vitro was evaluated.

Several common human cell lines, such as HepG2, are available that can be induced to express endogenous CETP for experimental purposes. Alternatively, non-human cell lines have been developed which constitutively express a cDNA encoding for human CETP (Wang et al., 1991 Biochemistry 30, 3484; Wang et al., 1992 J. Biol. Chem. 267, 17487). Additional lines expressing human or rabbit full length or exon 9 deleted cDNA under the control of inducible or constitutive promoters could readily be developed by those skilled in the art. Several rabbit animal models of experimental hypercholesterolemia are available. New Zealand white rabbits fed with high cholesterol diets have been shown to develop atherosclerotic disease (Clarkson et al., 1988 in Use of Animal Models For Research in Human Nutrition, Comparative Animal Nutrition vol. 6, Bexnen and West, eds.) and Watanabe rabbits are a model of homozygous FH (defective LDL receptor) and present with increased cholesterol levels and spontaneous development of atherosclerosis and tendinous xanthomas (Watanbe, 1980 Atherosclerosis 36, 261). CETP protein levels can be measured clinically or experimentally by ELISA, or radioimmuno assay. CETP enzyme activity can be measured in vitro or ex vivo by the use of a fluorescently labeled substrate (Bisgaier et al., 1993 J. Lipid Res. 34, 1625; Bisgaier et al., 1994 Lipids 29, in press). CETP encoded mRNA levels can be assessed by Northern analysis, RNAse protection, primer extension analysis or quantitative RT-PCR. Ribozymes that block the induction of CETP activity and/or CETP protein encoding mRNAs by more than 20% in vitro can be identified.

RNA ribozymes and/or genes encoding them will be delivered by either free delivery, liposome delivery, cationic lipid delivery, adeno-associated virus vector delivery, adenovirus vector delivery, retrovirus vector delivery or plasmid vector delivery in these animal model (e.g., transgenic mouse) experiments. One dose of a ribozyme vector that constitutively expresses the ribozyme or one or more doses of a stable anti-CETP ribozyme or a transiently expressing ribozyme vector may reduce the incidence or severity of atherosclerotic lesions or heart disease.

Diagnostic uses

Ribozymes of this invention may be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of CETP RNA in a cell. The close relationship between ribozyme activity and the structure of the target RNA allows the detection of mutations in any region of the molecule which alters the base-pairing and three-dimensional structure of the target RNA. By using multiple ribozymes described in this invention, one may map nucleotide changes which are important to RNA structure and function in vitro, as well as in cells and tissues. Cleavage of target RNAs with ribozymes may be used to inhibit gene expression and define the role (essentially) of specified gene products in the progression of disease. In this manner, other genetic targets may be defined as important mediators of the disease. These experiments will lead to better treatment of the disease progression by affording the possibility of combinational therapies (e.g., multiple ribozymes targeted to different genes, ribozymes coupled with known small molecule inhibitors, or intermittent treatment with combinations of ribozymes and/or other chemical or biological molecules). Other in vitro uses of ribozymes of this invention are well known in the art, and include detection of the presence of mRNAs associated with CETP related condition. Such RNA is detected by determining the presence of a cleavage product after treatment with a ribozyme using standard methodology.

In a specific example, ribozymes which can cleave only wild-type or mutant forms of the target RNA are used for the assay. The first ribozyme is used to identify wild-type RNA present in the sample and the second ribozyme will be used to identify mutant RNA in the sample. As reaction controls, synthetic substrates of both wild-type and mutant RNA will be cleaved by both ribozymes to demonstrate the relative ribozyme efficiencies in the reactions and the absence of cleavage of the "non-targeted" RNA species. The cleavage products from the synthetic substrates will also serve to generate size markers for the analysis of wild-type and mutant RNAs in the sample population. Thus each analysis will require two ribozymes, two substrates and one unknown sample which will be combined into six reactions. The presence of cleavage products will be determined using an RNAse protection assay so that full-length and cleavage fragments of each RNA can be analyzed in one lane of a polyacrylamide gel. It is not absolutely required to quantify the results to gain insight into the expression of mutant RNAs and putative risk of the desired phenotypic changes in target cells. The expression of mRNA whose protein product is implicated in the development of the phenotype (i.e., CETP) is adequate to establish risk. If probes of comparable specific activity are used for both transcripts, then a qualitative comparison of RNA levels will be adequate and will decrease the cost of the initial diagnosis. Higher mutant form to wild-type ratios will be correlated with higher risk whether RNA levels are compared qualitatively or quantitatively.

Other embodiments are within the following claims.

Claims

We claim:

1. An enzymatic RNA molecule which specifically cleaves RNA produced from the gene encoding cholesterol ester transfer protein (CETP).

2. The enzymatic RNA molecule of claim 1, wherein said RNA molecule is in a hammerhead motif.

3. The enzymatic RNA molecule of claim 5, wherein the binding arms of said enzymatic RNA molecule comprises sequences complementary to any of sequences defined as Seq ID Nos 2-258.

4. The enzymatic RNA molecule of claim 1, wherein said RNA molecule is in a hairpin, hepatitis Delta virus, group I intron, VS nucleic acid or RNaseP RNA motif.

5. The hairpin enzymatic RNA molecule of claim 4, wherein said hairpin RNA molecule comprises sequences complementary any of sequences defined as Seq ID Nos 1078-1125.

6. The enzymatic RNA molecule of claim 1, wherein said enzymatic RNA molecule comprises at least one sugar modification.

7. The enzymatic RNA molecule of claim 1, wherein said RNA comprises between 12 and 100 bases complementary to the RNA of said region.

8. The enzymatic RNA molecule of claim 1, wherein said RNA molecule comprises between 14 and 24 bases complementary to the RNA of said region.

9. The Enzymatic RNA molecule of claim 1, wherein said enzymatic RNA molecule comprises any of sequences shown as Seq ID Nos 259-515 and 1030-1077.

10. A mammalian cell including an enzymatic RNA molecule of claim 1 in vitro.

11. The mammalian cell of claim 10, wherein said cell is a human cell.

12. An expression vector comprising nucleic acid encoding the enzymatic RNA molecule of claim 1, in a manner which allows expression and/or delivery of that enzymatic RNA molecule within a mammalian cell in vitro.

13. A mammalian cell including the expression vector of claim 12 in vitro.

14. The mammalian cell of claim 13, wherein said cell is a human cell.

Patent Citations (10)

Patent	Date	Inventor
US4987071	1991-01-01	Cech et al.
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