RIBOZYME TREATMENT OF DISEASES OR CONDITIONS RELATED TO LEVELS OF PLASMA LIPOPROTEIN (a) [Lp(a)] BY INHIBITING APOLIPOPROTEIN (a) [APO(a)]

Abstract: Enzymatic RNA molecules which cleave apo(a) mRNA. Use of these catalytic RNA molecules for the treatment of conditions related to lipoprotein A levels, such as atherosclerosis, myocardial infarction, strokes; testenosis and heart diseases.

Description: DESCRIPTION RIBOZYME TREATMENT OF DISEASES OR CONDITIONS RELATED TO LEVEL9QF PLASMA LIPOPROTEIN (a) fLpfaπ BY INHIBITING APOLIPOPROTEIN fa. APOla)} Field of the Invention The present invention relates to therapeutic compositions and methods for the treatment or diagnosis of diseases or conditions related to Lp(a) levels, such as atherosclerosis, myocardial infarction, stroke, and restenosis. Background Of The Invention The following is a brief description of the physiological role of Lp(a). The discussion is not meant to be complete and is provided only for understanding of the invention that follows. This summary is not an admission that any of the work described below is prior art to the claimed i /ention. Low density lipoproteins (LDLs) are mainly composed of cholesterol, phospholipids and a single hydrophobic protein, apolipoprotein B [apoB]. They are considered as the major carriers of cholesterol in human plasma (for review see Uterman, G. (1989) Science 246, 904-910). ApoB, the only protein subunit of LDL, recognizes and binds to LDL receptors on the surface of cells. This LDL-LDL receptor inter -;tion results in the intemalization of the LDL and the eventual release of cholesterol inside the cell. A modified form of LDL, termed as lipoprotein (a) [Lp(a)], was discovered in 1963 [Berg, K '1963) Acta Pathol. Microbiol. Scand. 59, 369]. Covalent linkage of at. additional glycoprotein, apo(a), to the LDL distinguishes Lp(a) from LDL. Several studies have recently suggested that elevated levels of Lp(a) in * human plasma is linked to heart disease (Gurakar, et al., (1985) Atherosclerosis 57, 293-301 ; Leren, et al., (1988) Atherosclerosis 73, 135-141 ; Utermann, Supra). The Lp(a) levels range over 1000 fold and individuals with top quartile of plasma Lp(a) levels have two-to five-fold increased probability of developing atherosclerosis. Atherosclerosis is a disease associated with hardening and loss of elasticity of arterial walls. High concentrations of cholesterol, in the form of Lp(a), in human blood plasma is one of the most important factors responsible for atherosclerosis. Deposition of cholesterol in the Macrophages and smooth muscle cells associated with arterial walls cause plaques (atheromatous lesions) which cause proliferation of adjoining smooth muscle cells. With time, these plaques grow in size causing hardening of the arterial walls and loss of elasticity, which in turn results in rupturing of the arterial walls* blood clotting and blockage of blood flow in the artery (for datails see Textbook of medical phvsioloov Guyton, A.C., (Saunders Company, Philadelphia, 1991) pp. 761-764). Lp(a) and/or apo(a) levels correlate well with an increased risk of atherosclerosis and its subsequent manifestations such as myocardial infarction, stroke, and restenosis. The apo(a) protein is unique to humans, Old World primates and hedgehogs; its absence in common laboratory animals has made exploration of the physiological role of apo(a) levels difficult. Recently, a transgenic mouse expressing the human gene encoding apo(a) was constructed [Lawn et al., (1992) Nature 360, 670- 672]. The transgenic mice are more susceptible than control liter-mates to the development of lipid-rich regions in the aorta. Moreover, human apo(a) expression colocalizes to the regions of fat deposition. Thus, overexpression of apo(a) directly leads to atherosclerotic-like lesions in experimental animals. This observation lends credence to the hypothesis that elevated levels of apo(a) in humans contribute to atherosclerotic disease. Apolipoprotein(a) is a large glycoprotein which varies in size from 300-800 KDa. Thirty four different isoforms have been characterized from human plasma. The only human cDNA clone currently available encompass 14 kilobase message that encodes apo(a) [McLean et al., (1987) Nature 330, 132-137]. A Rhesus monkey cDNA representing a part of the 3' end of the apo(a) mRNA has also been cloned and sequenced (Tomlinson et al., 1989 J. Biol. Chem. 264, 5957-5965). Sequence analysis of the cloned cDNA revealed two unique facets of the apo(a) structure. First, the apo(a) cDNA is remarkably repetitious. The reconstructed apo(a) cDNA encodes a protein of 4,529 amino acids; 4,210 of the residues are present in 37 repeats of 1 14 amino acids each. The ΛΛ , M 09392 repeated units themselves are especially homologous; 24 are identical in nucleotide sequence, four more share a sequence that differs in only three nucleotides and the remaining repeats differ by only 11 to 71 bases. Secondly, apolipoprotein(a) is highly homologous to the serine protease, plasminogen. Plasminogen consists of five repeated homologous domains termed kringles (which are approximately 50% homologous in their amino sequences) followed by a trypsin-like protease domain. Kringle IV of plasminogen is very homologous to the 37 repeats of apo(a) [75-85% at the protein level). In addition, the 5 ' untranslated region, the signal peptide region, kringle V, the protease domain, and the 3' untranslated region of plasminogen are 98%, 100%, 91 %, 94% and 87% homologous to apo(a) sequences, respectively. Relative to plasminogen, apo(a) is missing kringles I, II, and III and, as mentioned above, has extensively duplicated kringle IV. Despite the high degree of homology apo(a) cannot be converted into a protease by tissue type plasminogen activator (tPA). This is because of a single amino acid substitution in apo(a) at the site of activation of plasminogen by tPA (Utermann, supra). IN vitro studies have indicated that apo(a) and Lp(a) compete with plasminogen for binding to the plasminogen receptor and fibrin which supports the correlation between high Lp(a) levels and myocardial infraction (Gonzalez-Gronow et al., (1989) Biochemistry 28. 2374-2378; Hajjar et al., (1989) Nature 339, 303-305; Miles et al., (1989) Nature 339, 301-303). Recent in vivo studies in human (Moliterno et al., 1993 Circulation 88, 935-940) and monkey (Williams et al., 1993 Atheroscler. Thromb. 13, 548-554) support a role for Lp(a) in preventing clot lysis. The extraordinary homology between apo(a) and plasminogen presents several barriers to drug development. Srr l molecule inhibitors of apo(a) would have to selectively bind apo without negatively impacting plasminogen function. Similarly, antiser approaches will be limited by the overall nucleotide seqmnce homol between the two genes. Current dietary and drug therap.es (Gurakar, al., supra; Leren et al., supra), with the exception of nicot.nic acid, have little or no effect on apo(a) levels. Applicant now shows that these sa. ^ limitations are opportunities for ribozyme therapy. The cleavage site specificity of ribozymes allows one to identify ribozyme target sites present in apo(a) mRNA but completely absent in the mRNA of plasminogen. For instance, there are 13 hammerhead cleavage sites present in the highly conserved kringles of apo(a) that are not present in kringle IV of plasminogen. Likewise, the last kringle repeat, protease domain and 3 ' untranslated region of apo(a) contain 21 hammerhead ribozyme cleavage sites present in apo(a) that are not present in plasminogen. Thus, ribozymes that target apo(a) mRNA represent unique therapeutics and diagnostic tools for the treatment and diagnosis of those at high risk of atherosclerosis. Summary of the Invention This invention relates to ribozymes, or enzymatic RNA molecules, directed to cleave mRNA species encoding apo(a). In particular, applicant describes the selection and function of ribozymes capable of cleaving this RNA and their use to reduce levels of apo(a) in various tissues to treat the diseases discussed herein. Such ribozymes are also useful for diagnostic uses. Ribozymes that cleave apo(a) mRNA represent a novel therapeutic approach to atherosclerosis.' Ribozymes may show greater perdurance or lower effective doses than antisense molecules due to their catalytic properties and their inherent secondary and tertiary structures. Such ribozymes, with their catalytic activity and increased site specificity (as described below), represent more potent and safe therapeutic molecules than antisense oligonucleotides. Applicant indicates that these ribozymes are able to inhibit expression of apo(a) 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 target apo(a) encoding mRNAs may be readily designed and are within the invention. 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 ,. -_ * 6/09392 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, such as antisense technology (where a nucleic acid molecule simply binds to a nucleic acid target to block its processing and translation) since the concentration of ribozyme necessary to affect a therapeutic treatment is lower than that of an antisense oligonucleotide. This advantage reflects the ability of the ribozyme to act enzymatically. Thus, a single ribozyme molecule is able to cle many molecules of target RNA. In addition, the ribozyme is a highly s ific 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. Similar mismatches in antisense molecules do not prevent their action (Woolf, T. M., et al., 1992, Proc. Natl. Acad. Sci. USA. 89, 7305-7309). Thus, the specificity of action of a ribozyme is greater than that of an antisense oligonucleotide binding the same RNA site. 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., "RNA Catalyst for Cleaving Specific RNA Sequences," filed September 20, 1989, which is a continuation-in-part of U.S. Serial No. 07/247,100 filed September 20, 1988, Hampel and Tritz, 1989, Biochemistry , 28, 4929, and Hampel et al., 1990, Nucleic Acids Res. 18,299, and an e .ample 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.Ce// . 35, 849, Neurospora VS RNA ribozyme motif is described by Collins (Saville and Collins, 1990 QM 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. Patent 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. 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 apo(a) encoding mRNA 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 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, K. J., et al., 1991 , Proc. Natl. Acad. Sci. USA. 88, 10591-5; Kashani-Sabet, M., et al., 1992, Antisense Res. Dev.. 2, 3-15; Dropulic, B., et al., 1992, J Virol. 66, 1432-41 ; Weerasinghe, M., et al., 1991 , J Virol. 65, 5531-4; Ojwang, J. O., et al., 1992, Proc. Natl. Acad. Sci. USA , 89, 10802-6; Chen, C. J., et al., 1992, Nucleic Acids Res.. 20, 4581-9; Sarver, H., 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 vector. The activity of such ribozymes can be augmented by their release from the primary transcript by a second ribozyme (Draper et a!., PCT W093/23569, and Sullivan et al., PCT WO94/02595, both hereby incorporated in their totality by reference herein; Ohkawa, J., et al., 1992, Nucleic Acids Svmp. Ser.. 27, 15-6; Taira, K., et al., 1991. Nucleic Acids Res.. 19, 5125-30; Ventura, M., et al., 1993, Nucleic Acids Res.. 21 , 3249-55) . Thus, in a first aspect, the invention features ribozymes that inhibit apo(a) production. These chemically or enzymatically synthesized RNA molecules contain substrate binding domains that bind to accessible regions of their target mRNAs. The RNA molecules also contain domains that catalyze the cleavage of RNA. The RNA molecules are preferably ribozymes of the hammerhead or hairpin motif. Upon binding, the ribozymes cleave the target apo(a) encoding mRNAs, preventing translation and protein accumulation. In the absence of the expression of the target gene, a therapeutic effect may be observed. By "inhibit" is meant that the activity or level of apo(a) encoding mRNA is reduced below that observed in the absense of the ribozyme, 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. 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 apo(a) activity in a cell or tissue. By "related" is meant that the inhibition of apo(a) mRNA translation, and thus reduction in the level of apo(a), 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 RNA or RNA complexes can be locally administered to relevant tissues through the use of a catheter, 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 apo(a) activity are expressed from transcription units inserted into DNA, RNA, or viral vectors. Preferably, the recombinant vectors capable of expressing the ribozymes are locally delivered as described above, and transiently persist in target cells. Once expressed, the ribozymes cleave the target mRNA. The recombinant vectors are preferably DNA plasmids or adenovirus vectors. However, other mammalian cell vectors that direct the expression of RNA may be used for this purpose. 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: Figure 1 is a diagrammatic representation of the hammerhead ribozyme domain known in the art. Stem II can be > 2 base-pair long or may be a loop region without base pairing. Figure 2a is a diagrammatic representation of the hammerhead ribozyme domain known in the art; Figure 2b is a diagrammatic representation of the hammerhead ribozyme as divided by Uhlenbeck i ι9Q7.Nature. 327, 596-600) into a substrate and enzyme portion; Figure 2c is a similar diagram showing the hammerhead divided by Haseloff and Gerlach (1988, Nature , 334, 585-591) into two portions; and Figure 2d is a similar diagram showing the hammerhead divided by Jeffries and Symons (1989, Nucl. Acids. Res.. 17, 1371-1371) into two portions. Figure 3 is a representation of the general structure of the hairpin ribozyme domain known in the art. H, is A, U or C. Y is U or C. N is A, U, G, or C. N' is the complementary sequence of N. Helix 4 can be > 2 base-pair long Figure 4 is a representation of the general structure of the hepatitis delta virus ribozyme domain known in the art. Figure 5 is a representation of the general structure of the Neurospora VS RNA enzyme motif. Figure 6 is a schematic representation of an RNase H accessibility assay. Specifically, the left side of Figure 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 Figure 5 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. Ribozvmes Ribozymes of this invention block to some extent apo(a) expression and can be used to treat disease or diagnose such disease. Ribozymes will be delivered to cells in culture and to tissues in animal models of Lp(a). Ribozyme cleavage of apo(a) mRNA in these systems may prevent or alleviate disease symptoms or conditions. Target sites Targets for useful ribozymes can be determined as disclosed in Draper et al supra. Sullivan et a/., supra, as well as by Draper et al., "Method and reagent for treatment of arthritic conditions U.S.S.N. 08/152,487, filed 1 1/12/93, 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. Such ribozymes can also be optimized and delivered as described therein. While specific examples to monkey and human RNA are provided, those in the art will recognize that the equivalent human RNA targets described can be used as described below. Thus, the same target may be used, but binding arms suitable for targetting human RNA sequences are present in the ribozyme. Such targets may also be selected as described below. The sequence of human and monkey apo(a) mRNA can be screened for accessible sites using a computer folding algorithm. Regions of the mRNA that did not form secondary folding structures and that contain potential hammerhead or hairpin ribozyme cleavage sites can be identified. These sites are shown in Tables II, IV, and VI - VII. (All sequences are 5' to 3' in the tables.) While monkey and human sequences can be screened and ribozymes thereafter designed, the human targetted sequences are of most utility. However, as discussed in Stinchcomb et al. "Method and Composition for Treatment of Restenosis and Cancer Using Ribozymes," U.S.S.N. 08/245,466, filed 5/18/94, and hereby incorporated by reference herein, monkey targeted ribozmes are 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. Ribozyme target sites were chosen such that the cleavage sites are present in apo(a) mRNA but completely absent in the mRNA of plasminogen (Tables II, IV, VI and VII). This is because there exists extraordinary homology between apo(a) and plasminogen (see above). It must be established that the sites predicted by the computer-based RNA folding algorithm correspond to potential cleavage sites. Hammerhead and hairpin ribozymes are designed that could bind and are individually analyzed by computer folding (Jaeger et al., 1989 Proc. Natl. Acad. Sci. USA. 86, 7706-7710) 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 Figure 6, mRNA is screened for accessible cleavage sites by the method described generally in McSwiggen,L S Patent Application 07/883,849 filed 5/1/92, entitled "Assay for ribozyme target site," hereby incorporated by reference herein. Briefly, DNA oligonucleotides representing potential hammerhead or hairpin ribozyme cleavage sites are synthesized. A polymerase chain reaction is used to generate a substrate for T7 RNA polymerase transcription from human or monkey apo(a) cDNA clones. Labeled RNA transcripts are synthesized in vitro from the two templates. The oligonucleotides and the labeled transcripts are annealed, RNaseH is added and the mixtures are incubated for the designated times at 37°C. Reactions are stopped and RNA separated on sequencing polyacrylamide gels. The percentage of the substrate cleaved is determined by autoradiographic quantitation using a phosphor imaging 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, 7845-7854 and in Scaringe et al., 1990 Nucleic Acids Res.. 18, 5433-5441 and makes 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 were >98%. Inactive ribozymes are synthesized by substituting a U for G5 and a U for A14 (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). All ribozymes are modified to enhance stability by modification of five ribonucleotides at both the 5' and 3' ends with 2'-0- methyl groups. 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.S.N. 08/245,736, the totality of which is hereby incorporated herein by reference.) and were resuspended in water. The sequences of the chemically synthesized ribozymes useful in this study are shown in Tables III, V, VI, and VII. Those in the art will recognize Λ , ΛΛ , Λ , 96/09392 12 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 sequence 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 - 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 Ribozvme 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 Figure 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. US Patent Application 07/829,729, and Sproat, European Patent Application 92110298.4 and U.S. Patent 5,334,711 and Jennings et al., WO 94/13688, which describe various chemical modifications that can be made to the sugar moieties of enzymatic RNA molecules. All these publications are hereby incorporated by reference herein.), modifications which enhance their efficacy in cells, and removal of stem II bases to shorten RNA synthesis times and reduce chemical requirements. 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. The ΛΛ , Λ , 09392 13 RNA/vehicle combination is locally delivered by direct injection or by use of a catheter, infusion pump or stent. Alternative routes of delivery include, but are not limited to, intravenous injection, intramuscular injection, subcutaneous injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intrathecai 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 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. U S A. 87, 6743-7; Gao and Huang 1993 Nucleic Acids Res.. 21 , 2867-72; Lieber et al., 1993 Methods EnzvmoL 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. U S A. 89, 10802-6; Chen et al., 1992 Nucleic Acids Res.. 20, 4581-9; Yu et al., 1993 Proc. Natl. Acad. Sci. U S A. 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, Sindbis virus, Semliki forest virus vectors). In a preferred embodiment of the invention, a transcription unit expressing a ribozyme that cleaves apo(a) RNA is inserted into a plasmid DNA vector, a retrovirus DNA viral vector, an adenovirus DNA viral vector or an adeno-associated virus vector. These and other vectors have been used to transfer genes to live animals (for a review see Friedman, 1989 Science 244, 1275-1281 ; Roemer and Friedman, 1992 Eur. J. Biochem. 208, 211-225) and leads to transient or stable gene expression. The vectors are delivered as recombinant viral particles. DNA may be delivered alone or complexed with vehicles (as described for RNA above). The DNA, DNA/vehicle complexes, or the recombinant virus particles are locally administered to the site of treatment, e.g., through the use of a catheter, stent or infusion pump. Example 1 : apo(a) Hammerhead ribozymes By engineering ribozyme motifs we have designed several ribozymes directed against apo(a) mRNA sequences. These have been synthesized with modifications that improve their nuclease resistance. These ribozymes cleave apo(a) target sequences in vitro. The ribozymes will be tested for function in vivo by exogenous delivery to cells expressing apo(a). Ribozymes are delivered by incorporation into liposomes, by complexing with cationic lipids, by microinjection, or by expression from DNA vectors. Expression of apo(a) is monitored by ELISA, by indirect immunofluoresence, and/or by FACS analysis. Levels of apo(a) mRNA are assessed by Northern analysis, RNase protection, by primer extension analysis or by quantitative RT-PCR techniques. Ribozymes that block the induction of apo(a) protein and mRNA by more than 90% are identified. Diagnostic uses Ribozymes of this invention may be used as diagnostic tools to examine genetic drift and mutations within diseased cells. 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 mRNA associated with an apo(a) 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., apo(a)) 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. TABLE I Characteristics of Ribozymes Group I Introns Size: -200 to >1000 nucleotides. Requires a U in the target sequence immediately 5' of the cleavage site. Binds 4-6 nucleotides at 5' side of cleavage site. Over 75 known members of this class. Found in Tetrahymena thermophila rRNA, fungal mitochondria, chloroplasts, phage T4, blue-green algae, and others. RNAseP RNA ( 1 RNA) Size: -290 to 400 nucleotides. RNA portion of a ribonucleoprotein enzyme. Cleaves tRNA precursors to form mature tRNA. Roughly 10 known members of this group all are bacterial in origin. Hammerhead Ribozyme Size: -13 to 40 nucleotides. Requires the target sequence UH immediately 5' of the cleavage site. Binds a variable number nucleotides on both sides of the cleavage site. 14 known members of this class. Found in a number of plant pathogens (virusoids) that use RNA as the infectious agent (Figures 1 and 2) Hairpin Ribozyme Size: -50 nucleotides. Requires the target sequence GUC immediately 3' of the cleavage site. Binds 4-6 nucleotides at 5' side of the cleavage site and a variable number to the 3' side of the cleavage site. Only 3 known member of this class. Found in three plant pathogen (satellite RNAs of the tobacco ringspot virus, arabis mosaic virus and chicory yellow mottle virus) which uses RNA as the infectious agent (Figure 3). Hepatitis Delta Virus (HDV) Ribozyme Size: 50 - 60 nucleotides (at present). Cleavage of target RNAs recently demonstrated. Sequence requirements not fully determined. Binding sites and structural requirements not fully determined, although no sequences 5' of cleavage site are required. Only 1 known member of this class. Found in human HDV (Figure 4). Neurospora VS RNA Ribozyme Size: -144 nucleotides (at present) Cleavage of target RNAs recently demonstrated. Sequence requirements not fully determined. Binding sites and structural requirements not fully determined. Only 1 known member of this class. Found in Neurospora VS RNA (Figure 5). Table I Unique Human apo(a) HH Target sequence nt HH Target nt HH Target Position Sequence Position Sequence 127 CCAGGAU U GCUACCA 11186 ACAGAAU A UUAUCCA 151 ACAGAGU U AUCGAGG 11254 UUGGUGU U AUACCAU 154 GAGUUAU C GAGGCAC 11257 GUGUUAU A CCAUGGA 199 CCAAGCU U GGUCAUC 11266 CAUGGAU C CCAAUGU 362 CAAUGOJ c AGACGCA 11305 ACAAUGU C CAGUGAC 400 GACUGUU A CCCCGGU 11347 GGCUGUU U CUGAACA 408 CCCCGGU U CCAAGCC 11348 GCUGUUU C UGAACAA 409 CCCGGUU C CAAGCCU 11423 CGAGGCU C AUUCUCC 417 CAAGCCU A GAGGCUC 11427 GCUCAUU C UCCACCA 481 CCAUGGU A AUGGACA 11429 UCAUUCU C CACCACU 571 GCAUAGU C GGACCCC 11440 CACUGUU A CAGGAAG 9031 CCACGGU A AUGGACA 11653 CACAACU C CCACGGU 10207 UCCAGAU C CUGUGGC 11670 UCCCAGU U CCAAGCA 10222 AGCCCCU U AUUGUUA 11779 CACCACU A UCACAGG 10223 GCCCCUU A UUGUUAU 11797 AACAUGU C AGUCUUG 10225 CCCUUAU U GUUAUAC 11824 ACCACAU U GGCAUCG 10345 GGCUCCU U CUGAACA 11988 GUGUCCU C ACAACUC 10346 GCUCCUU C UGAACAA 12013 CCCGGUU C CAAGCAC 10532 AAGAACU A CUGCCGA 12159 CUAUGAU A CCACACU 10543 CCGAAAU C CAGAUCC 12235 UCCAGAU U CUGGGAA 10564 AGCCCCU U GGUGUUA 12236 CCAGAUU C UGGGAAA 10570 UUGGUGU U AUACAAC 12320 ACAGAAU c AGGUGUC 10622 CGAUGCU c AGAUGCA 12327 CAGGUGU c CUAGAGA 10677 CAAGCCU A GAGGCUU 12330 GUGUCCU A GAGACUC 10687 GGCUUUU U UUGAACA 12337 AGAGACU C CCACUGU 10736 UGCUACU A CCAUUAU 12374 GCUCAUU C UGAAGCA 10741 CUACCAU U AUGGACA 12453 GCACAUU C UCCACCA 10742 UACCAUU A UGGACAG 12481 GACAUGU C AAUCUUG 10792 AAGAACU U GCCAAGC 12592 AGGCCCU U GGUGUUU 10828 CCAGCAU A GUCGGAC 12650 CGAUGCU C AGACACA 10899 CUGAGAU U CGCCCUU 12974 GCAUCCU C UUCAUUU 10900 UGAGAUU c GCCCUUG 12976 AUCCUCU U CAUUUGA 10906 UCGCCCU U GGUGUUA 13119 GCACCUU A AUAUCCC 10924 CAUGGAU c CCAGUGU 13226 CUCGAAU C UCAUGUU 10976 ACAGAAU c AAGUGUC 13228 CGAAUCU C AUGUUCA 10983 CAAGUGU c CUUGCAA 13839 UGGUAUU U UUGUGUA 10986 GUGUCCU U GCAACUC 13848 UGUGUAU A AGCUUUU 11011 CCCAGAU c CAAGCAC 13930 ACUUAUU U UGAUUUG 11098 GAGUUAU c GAGGCUC 13931 CUUAUUU U GAUUUGA 11170 CUGGCAU c AGAGGAC Table III: Unique Human apo(a) HH Ribozyme Sequence nt. .Human apo (a) HH Ribozyme Sequence Position 127 UGGUAGC OJGAUGAGGCCGAAAGGCCGAA AUCCUGG 151 CCUCGAU CUGAUGAGGCCGAAAGGCCGAA ACUCUGU 154 GUGCCUC CUGAUGAGGCCGAAAGGCCGAA AUAACUC 199 GAUGACC CrUGAUGAGGCCGAAAGGCCGAA AGCUUGG 362 UGCGUCU CUGAUGAGGCCGAAAGGCCGAA AGCAUUG 400 ACCGGGG CUGAUGAGGCCGAAAGGCCGAA AACAGUC 408 GGCUUGG CUGAUGAGGCCGAAAGGCCGAA ACCGGGG 409 AGGCUUG CUGAUGAGGCCGAAAGGCCGAA AACCGGG 417 GAGCCUC CUGAUGAGGCCGAAAGGCCGAA AGGCUUG 481 UGUCCAU CUGAUGAGGCCGAAAGGCCGAA ACCAUGG 571 GGGGUCC CUGAUGAGGCCGAAAGGCCGAA ACUAUGC 9031 UGUCCAU CUGAUGAGGCCGAAAGGCCGAA ACCGUGG 10207 GCCACAG CUGAUGAGGCCGAAAGGCCGAA AUCUGGA 10222 UAACAAU CUGAUGAGGCCGAAAGGCCGAA AGGGGCU 10223 AUAACAA CUGAUGAGGCCGAAAGGCCGAA AAGGGGC 10225 GUAUAAC CUGAUGAGGCCGAAAGGCCGAA AUAAGGG 10345 UGUUCAG CUGAUGAGGCCGAAAGGCCGAA AGGAGCC 10346 uuσuucA CUGAUGAGGCCGAAAGGCCGAA AAGGAGC 10532 UCGGCAG CUGAUGAGGCCGAAAGGCCGAA AGUUCUU 10543 GGAUCUG CUGAUGAGGCCGAAAGGCCGAA AUUUCGG 10564 UAACACC CUGAUGAGGCCGAAAGGCCGAA AGGGGCU 10570 GUUGUAU CUGAUGAGGCCGAAAGGCCGAA ACACCAA 10622 UGCAUCU CUGAUGAGGCCGAAAGGCCGAA AGCAUCG 10677 AAGCCUC CUGAUGAGGCCGAAAGGCCGAA AGGCUUG 11-587 UGUUCAA CUGAUGAGGCCGAAAGGC GAA AAAAGCC 10736 AUAAUGG CUGAUGAGGCCGAAAGGC GAA AGUAGCA 10741 UGUCCAU CUGAUGAGGCCGAAAGGCCGAA AUGGUAG 10- 7 42 CUGUCCA CUGAUGAGGCCGAAAGGCCGAA AAUGGUA IC 92 GCUUGGC CUGAUGAGGCCGAAAGGCCGAA AGUUCUU 10128 GUCCGAC CUGAUGAGGCCGAAAGGCCGAA AUGCUGG 10899 AAGGGCG CUGAUGAGGCCGAAAGGCCGAA AUCUCAG 10900 CAAGGGC CUGAUGAGGCCGAAAGGCCGAA AAUCUCA 10906 UAACACC CUGAUGAGGCCGAAAGGCCGAA AGGGCGA 10924 ACACUGG CUGAUGAGGCCGAAAGGCCGAA AUCCAUG 10976 GACACUU CUGAUGAGGCCGAAAGGCCGAA AUUCUGU 10983 UUGCAAG CUGAUGAGGCC z^AAGGCCGAA ACACUUG 10' ) 86 GAGUUGC TJGAUGAGGCCGAAAGGCCGAA AGGACAC 1-J11 GUGCUU' 'GAUGAGGCCGAAAGGCCGAA AUCUGGG 11098 GAGCCT 3AUGAGGCCGAAAGGCCG AUAACUC 11170 GUCCUC JGAUGAGGCCGAAAGGCCG. . AUGCCAG 11186 UGGAUA.. CUGAUGAGGCCGAAAGGCCGAA AUUCUGU 11254 AUGGUAU CUGAUGAGGCCGAAAGGCCGAA ACACCAA 11257 UCCAUGG CUGAUGAGGCCGAAAGGCCGAA AUAACAC 11266 ACAUUGG CUGAUGAGGCCGAAAGGCCGAA AUCCAUG 11305 GUCACUG σJGAUGAGGCCGAAAGGCCGAA ACAUUGU 11347 UGUUCAG CUGAUGAGGCCGAAAGGCCGAA AACAGCC 11348 UUGUUCA CUGAUGAGGCCGAAAGGCCGAA AAACAGC 11423 GGAGAAU CUGAUGAGGCCGAAAGGCCGAA AGCCUCG 11427 UGGUGGA CUGAUGAGGCCGAAAGGCCGAA AAUGAGC 11429 AGUGGUG CUGAUGAGGCCGAAAGGCCGAA AGAAUGA 11440 CUUCCUG CUGAUGAGGCCGAAAGGCCGAA AACAGUG 11653 ACCGUGG CUGAUGAGGCCGAAAGGCCGAA AGUUGUG 11670 UGCUUGG CUGAUGAGGCCGAAAGGCCGAA ACUGGGA 11779 CCUGUGA CUGAUGAGGCCGAAAGGCCGAA AGUGGUG 11797 CAAGACU CUGAUGAGGCCGAAAGGCCGAA ACAUGUU 11824 CGAUGCC CUGAUGAGGCCGAAAGGCCGAA AUGUGGU 11988 GAGUUGU CUGAUGAGGCCGAAAGGCCGAA AGGACAC 12013 GUGCUUG CUGAUGAGGCCGAAAGGCCGAA AACCGGG 12159 AGUGUGG CUGAUGAGGCCGAAAGGCCGAA AUCAUAG 12235 UUCCCAG CUGAUGAGGCCGAAAGGCCGAA AUCUGGA 12236 UUUCCCA CUGAUGAGGCCGAAAGGCCGAA AAUCUGG 12320 GACACCU CUGAUGAGGCCGAAAGGCCGAA AUUCUGU 12327 UCUCUAG CUGAUGAGGCCGAAAGGCCGAA ACACCUG 12330 GAGUCUC CUGAUGAGGCCGAAAGGCCGAA AGGACAC 12337 ACAGUGG CUGAUGAGGCCGAAAGGCCGAA AGUCUCU 12374 UGCUUCA CUGAUGAGGCCGAAAGGCCGAA AAUGAGC 12453 UGGUGGA CUGAUGAGGCCGAAAGGCCGAA AAUGUGC 12481 CAAGAUU CUGAUGAGGCCGAAAGGCCGAA ACAUGUC 12592 AAACACC CUGAUGAGGCCGAAAGGCCGAA AGGGCCU 12650 UGUGUCU CUGAUGAGGCCGAAAGGCCGAA AGCAUCG 12974 AAAUGAA CUGAUGAGGCCGAAAGGCCGAA AGGAUGC 12976 UCAAAUG CUGAUGAGGCCGAAAGGCCGAA AGAGGAU 13119 GGGAUAU CUGAUGAGGCCGAAAGGCCGAA AAGGUGC 13226 AACAUGA CUGAUGAGGCCGAAAGGCCGAA AUUCGAG 13228 UGAACAU CUGAUGAGGCCGAAAGGCCGAA AGAUUCG 13839 UACACAA CUGAUGAGGCCGAAAGGCCGAA AAUACCA 13848 AAAAGCU CUGAUGAGGCCGAAAGGCCGAA AUACACA 13930 CAAAUCA CUGAUGAGGCCGAAAGGCCGAA AAUAAGU 13931 UCAAAUC CUGAUGAGGCCGAAAGGCCGAA AAAUAAG Table IV: Unique Monkey apo(a) HH Target Sequence nt. HH Target nt. HH Target Position Sequence Position Sequence 127 CUGCCGU C GCaCCUC 11170 ACAaUgU C UGGugAC 151 CUGCCGU C GCaCCUC 11186 ACAGAAU C AAGUGUC 154 CUGCCgU C GcaCCUC 11254 gCUUcUU c UgaAGAA 199 CCCCGGU U CCAAGCC 11257 GACUGCU A CCAUGGU 362 AGAGGCU C CUUCCGA 11266 GAGUUAU C GAGGCUC 400 GGCUCCU U CCGAACA 11305 CGAGGCU C AUUCUCC 408 GGCUCCU U CCGAACA 11347 UCAUUCU C CACCACU 409 GGCUCCU U CCGAACA 11348 GACAUGU C AGUCUUG 417 GGCUCCU U CCGAACA 11423 UCUUGGU C CUCUAUG 481 GCUCCUU C CGAACAA 11427 UGGUCCU C UAUGACA 571 ACAGAGU U AUCGAGG 11429 UGGUCCU C UAUGACA 9031 GAGUUAU C GAGGCAc 11440 GUCCUCU A UGACACC 10207 CCACACU C UCAUAGU 11653 auAGAAU A CUACCCA 10222 CCACACU c UCAUAGU 11670 auAGA * U A CUA-rCCA 10223 AGAGGCU c CUUCUGA 11779 aUGgAaJ c AaGTJGUC 10225 AGAGGCU c CUUCUGA 11797 CAAGUGU C CUUGCaA - " 345 GUGUUAU A CAACgGA 11824 UCCC7 7 U CCAAGCA 346 AACgGAU C CCAGUGU 11988 UcGGO " J C GGAGGAU . 532 AGaGGcU u UUCUuga 12013 UCCCAUU A cgCUAUC 10543 AGAGGCU U UUcUUGA 12159 GCUCCUU C UGAACAA 10564 GAGGCuU u UCuUgaA 12235 CCAGGAU U GCUACCA 10570 AGGCUUU U cUUGAAC 12236 CCAGGAU U GCUACCA 10622 UgCUACU a CcaUUAU 12320 gaACUGU c aGUcUuG 10677 GGCACAU A CUCCACC 12327 UCUUGGU C AUCUAUG 10687 CCACUGU u ACAGGAA 12330 UGGUCAU c UAUGAUA 10736 ccACUGU u ACAGGAA 12337 GUCAUCU A UGAUACC 10741 CCACUGU u ACAGGAA 12374 UGGUGUU A CACgACu 10742 CCACUGU u ACAGGAA 12453 AgagaCU c CCACUGU 10792 CACUGUU A CaGGaAg 12481 CUGUUGU U CCgGUUC 10828 GCAUAGU C GGACCCC 12592 GCUCAUU C UGAAGCA 10899 GCAUAGU C :CCC 12650 UCAAUCU u GGUCAUC 10900 GCAUAGU C * 2CC 12974 CCACAUU c CUGGCCC 10906 AaAaACU a * .-. -aaAu 12976 GGCAAGU c AGUCUuA 10924 CΛGGAAU C CAGAUGC 13119 AgGcc U c CUUCUAC 10976 CAGGAAU C CAGAUGC 13226 AGUGUCU A GGuUGUU 1.1983 CAGGAAU c CAGAUGC 13228 aGuGUCU a GGuUGUu 10986 CAGGAAU c CAGAUGC 13839 UGGUAUU a UUGUGUA 11011 CAGGAAU c CAGA -r 13848 UAAGCUU U UcccGUC 11098 UcGcCCU u GGU _. -A Table V: Unique Monkey apo(a) HH Ribozyme Sequence nt. M Moonnkkeey HH Ribozyme Sequence Position 127 GAGGUGC CUGAUGAGGCCGAAAGGCCGAA ACGGCAG 151 GAGGUGC CUGAUGAGGCCGAAAGGCCGAA ACGGCAG 154 GAGGUGC CUGAUGAGGCCGAAAGGCCGAA ACGGCAG 199 GGCUUGG CUGAUGAGGCCGAAAGGCCGAA ACCGGGG 362 UCGGAAG CUGAUGAGGCCGAAAGGCCGAA AGCCUCU 400 UGUUCGG CUGAUGAGGCCGAAAGGCCGAA AGGAGCC 408 UGUUCGG CUGAUGAGGCCGAAAGGCCGAA AGGAGCC 409 UGUUCGG CUGAUGAGGCCGAAAGGCCGAA AGGAGCC 417 UGUUCGG CUGAUGAGGCCGAAAGGCCGAA AGGAGCC 481 UUGUUCG CUGAUGAGGCCGAAAGGCCGAA AAGGAGC 571 CCUCGAU CUGAUGAGGCCGAAAGGCCGAA ACUCUGU 9031 GUGCCUC CUGAUGAGGCCGAAAGGCCGAA AUAACUC 10207 ACUAUGA CUGAUGAGGCCGAAAGGCCGAA AGUGUGG 10222 ACUAUGA CUGAUGAGGCCGAAAGGCCGAA AGUGUGG 10223 UCAGAAG CUGAUGAGGCCGAAAGGCCGAA AGCCUCU 10225 UCAGAAG CUGAUGAGGCCGAAAGGCCGAA AGCCUCU 10345 UCCGUUG CUGAUGAGGCCGAAAGGCCGAA AUAACAC 10346 ACACUGG CUGAUGAGGCCGAAAGGCCGAA AUCCGUU 10532 UCAAGAA CUGAUGAGGCCGAAAGGCCGAA AGCCUCU 10543 UCAAGAA CUGAUGAGGCCGAAAGGCCGAA AGCCUCU 10564 UUCAAGA CUGAUGAGGCCGAAAGGCCGAA AAGCCUC 10570 GUUCAAG ■ CUGAUGAGGCCGAAAGGCCGAA AAAGCCU 10622 AUAAUGG CUGAUGAGGCCGAAAGGCCGAA AGUAGCA 10677 GGUGGAG CUGAUGAGGCCGAAAGGCCGAA AUGUGCC 10687 UUCCUGU CUGAUGAGC-CCGAAAGGCCGAA ACAGUGG 10736 UUCCUGU CUGAUGAGGCCGAAAGGCCGAA ACAGUGG 10741 UUCCUGU CUGAUGAGGCCGAAAGGCCGAA ACAGUGG 10742 UUCCUGU CUGAUGAGGCCGAAAGGCCGAA ACAGUGG 10792 CUUCCUG CUGAUGAGGCCGAAAGGCCGAA AACAGUG 10828 GGGGUCC CUGAUGAGGCCGAAAGGCCGAA ACUAUGC 10899 GGGGUCC CUGAUGAGGCCGAAAGGCCGAA ACUAUGC 10900 GGGGUCC CUGAUGAGGCCGAAAGGCCGAA ACUAUGC 10906 AUUUGGA CUGAUGAGGCCGAAAGGCCGAA AGUUUUU 10924 GCAUCUG CUGAUGAGC-CCGAAAGGCCGAA AUUCCUG 10976 GCAUCUG CUGAUGAGGCCGAAAGGCCGAA AUUCCUG 10983 GCAUCUG CUGAUGAGGCCGAAAGGCCGAA AUUCCUG 10986 GCAUCUG CUGAUGAGGCCGAAAGGCCGAA AUUCCUG 11011 GCAUCUG CUGAUGAGGCCGAAAGGCCGAA AUUCCUG 11098 UAACACC CUGAUGAGGCCGAAAGGCCGAA AGGGCGA 11170 GUCACCA CUGAUGAGGCCGAAAGGCCGAA ACAUUGU 11186 GACACUU CUGAUGAGGCCGAAAGGCCGAA AUUCUGU 11254 UUCUUCA CUGAUGAGGCCGAAAGGCCGAA AAGAAGC 11257 ACCAUGG CUGAUGAGGCCGAAAGGCCGAA AGCAGUC 11266 GAGCCUC CUGAUGAGGCCGAAAGGCCGAA AUAACUC O 96/09392 23 11305 GGAGAAU CUGAUGAGGCCGAAAGGCCGAA AGCCUCG 11347 AGUGGUG CUGAUGAGGCCGAAAGGCCGAA AGAAUGA 11348 CAAGACU CUGAUGAGGCCGAAAGGCCGAA ACAUGUC 11423 CAUAGAG CUGAUGAGGCCGAAAGGCCGAA ACCAAGA 11427 UGUCAUA CUGAUGAGGCCGAAAGGCCGAA AGGACCA 11429 UGUCAUA CUGAUGAGGCCGAAAGGCCGAA AGGACCA 11440 GGUGU . CUGAUGAGGCCGAAAGGCCGAA AGAGGAC 11653 UGGGUA^ CUGAUGAGGCCGAAAGGCCGAA AUUCUAU 11670 UGGGUAG CUGAUGAGGCCGAAAGGCCGAA AUUCUAU 11779 GACACUU CUGAUGAGGCCGAAAGGCCGAA AUUCCAU 11797 UUGCAAG CUGAUGAGGCCGAAAGGCCGAA ACACUUG 11824 UGCUUGG CUGAUGAGGCCGAAAGGCCGAA ACUGGGA 11988 AUCCUCC CUGAUGAGGCCGAAAGGCCGAA AUGCCGA 12013 GAUAGCG CUGAUGAGGCCGAAAGGCCGAA AAUGGGA 12159 UUGUUCA CUGAUGAGGCCGAAAGGCCGAA AAGGACC 12235 UGGUAGC CUGAUGAGGCCGAAAGGCCGAA AUCCUG3 12236 UGGUAGC CUGAUGAGGCCGAAAGGCCGAA AUCCUGG 12320 CAAGACU CUGAUGAGGCCGAAAGGCCGAA ACAGUUC 12327 CAUAGAU CUGAUGAGGCCGAAAGGCCGAA ACCAAGA 12330 UAUCAUA CUGAUGAGGCCGAAAGGCCGAA AUGACCA 12337 GGUAUCA CUGAUGAGGCCGAAAGGCCGAA AGAUGAC 12374 AGUCGUG CUGAUGAGGCCGAAAGGCCGAA AACACCA 12453 ACAGUGG CUGAUGAGGCCGAAAGGCCGAA AGUCUCU 12481 GAACCGG CUGAUGAGGCCGAAAGGCCGAA ACAACAG 12592 UGCUUCA CUGAUGAGGCCGAAAGGCCGAA AAUGAGC 12650 GAUGACC CUGAUGAGGCCGAAAGGCCGAA AGAUUGA 12974 GGGCCAG CUGAUGAGGCCGAAAGGCCGAA AAUGUGG 12976 UAAGACU CUGAUGAGGCCGAAAGGCCGAA ACUUGCC 13119 GUAGAAG CUGAUGAGGCCGAAAGGCCGAA AAGGCCU 13226 AACAACC CUGAUGAGGCCGAAAGGCCGAA AGACACU 13228 AACAACC CUGAUGAGGCCGAAAGGCCGAA AGACACU 13839 UACACAA CUGAUGAGGCCGAAAGGCCGAA AAUACCA 13848 GACGGGA CUGAUGAGGCCGAAAGGCCGAA AAGCUUA Table VI: Unique Human apo(a) Hairpin Ribozyme Sequence nt. H Haaiirrppijn Ribozyme Sequence Substrate position Sequence 378 GGCGCGAC AGAA GUCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GGACU GCC GUCGCGCC 381 GGAGGCGC AGAA GCAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CUGCC GUC GCGCCUCC 440 UUUGCUCA AGAA GUGC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GCACC GAC UGAGCAAA 7964 UCUGCUCA AGAA GUGC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GCACC GAC UGAGCAGA 10215 CAAUAAGG AGAA GCCA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UGGCA GCC CCUUAUUG 10534 UGGAUUUC AGAA GUAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CUACU GCC GAAAUCCA 10557 CACCAAGG AGAA GCCA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UGGCA GCC CCUUGGUG 10638 GGGACGAA AGAA GUCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GGACU GCC UUCGUCCC 10700 UUUCCUCA AGAA GUGC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GCACU GAC UGAGGAAA 11343 UGUUCAGA AGAA GCCG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CGGCU GUU UCUGAACA 11379 CAGUCCUG AGAA GUGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CCACA GUC CAGGACUG 12342 ACUGGAAC AGAA GUGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CCACU GUU GUUCCAGU 12804 GGCUCCUG AGAA GCCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GGGCU GCC CAGGΛGCC 12877 AGGGUUAC AGAA GUAA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UUACU GCC GUAACCCU 13139 GAGCAGCA AGAA GCAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GUGCU GAC UGCUGCUC 13256 GCUCCAAG AGAA GCCU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AGGCU GUU CUUGGAGC 13522 ACCCUGGC AGAA GUCA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UGACA GUU GCCAGGGU 13794 UAGCUGGG AGAA GUGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA ACACU GUU CCCAGCUA Table VII: Unique Monkey apo(a) Hairpin Ribozyme Sequence nt. H Haaiirrppain Ribozyme Sequence Substrate Position Sequence 57 GGUGCGAC AGAA GUCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GGACU GCC GUCGCACC 60 GGAGGUGC AGAA GCAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CUGCC GUC GCACCUCC 119 UUUGCUCA AGAA GUGC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GCACC GAC UGAGCAAA 318 CAAUAAGG AGAA GCCA ACCAr-^ΛAACACACGUUGUGGUACAUUACCUGGUA UGGCA GCC CCUUAUUG 660 CAAUAAGG AGAA GCCA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UGGCA GCC CCUUAUUG 744 GGAGGUGC AGAA GCAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CUGCA GUC GCACCUCC 803 UUUGCUCA AGAA GUGC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GCACC GAC UGAGCAAA 1002 CAAUAAGG AGAA GCCA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UGGCA GCC CCUUAUUG 1083 GGUGCGAC AGAA GUCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GGACU GCC GUCGCACC 1086 GGAGGUGC AGAA GCAG ACCAGAGAAACACACGUUσUGGUACAUUACCUGGUA CUGCC GUC GCACCUCC 1321 UGGAUUUC AGAA GUAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CUACU GCC GAAAUCCA t 1344 CACCAAGG AGAA GCCA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UGGCA GCC CCUUGGUG U1 2130 UGUUCAGA AGAA GCCA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UGGCU GUU UCUGAACΛ 2500 GACCCCAG AGAA GUUU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA ΛAACΛ GCC CUGGGGUC 3129 ACCGGAAC AGAA GUGG ACCAGAGAAACACACGUUGUGGUACAULTΛ'-TIGGUA CCACU GUU GUUCCGGU 3683 AAGCAGCA AGAA GCAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GUGCU GAC UGCUGCUU 3890 AAUUUGGA AGAA GCAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CUGCC GUC UCCAAAUU 3912 UCAGUCCA AGAA GUGA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UCACC GCC UGGACUGA i ' GΪ "TCUGGG AGAA GUGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA ACACU GUC CCCAGCUA

Claims: Claims 1. An enzymatic RNA molecule which cleaves apo(a) mRNA . 2. An enzymatic RNA molecule of claim 1 , the binding arms of which contain sequences complementary to any one of the sequences defined in any of those in Table II. 3. The enzymatic RNA molecule of claim 1 , the binding arms of which contain sequences complementary to the sequences defined in any one of Tables IV, VI and VII 4. The enzymatic RNA molecule of claims 1 , 2, or 3, wherein said RNA molecule is in a hammerhead motif. 5. The enzymatic RNA molecule of claim 1 , 2, or 3, wherein said RNA molecule is in a hairpin, hepatitis delta virus, group 1 intron, Neurospora VS RNA or RNaseP RNA motif. 6. The enzymatic RNA molecule of claim 5, wherein said ribozyme comprises between 12 and 100 bases complementary to said mRNA. 7. The enzymatic RNA molecule of claim 6, wherein said ribozyme comprises between 14 and 24 bases complementary to said mRNA. 8. Enzymatic RNA molecule consisting essentially of any sequence selected from the group of those shown in Tables III, V, VI, and VII. 9. A mammalian cell including an enzymatic RNA molecule of claims 1 , 2, or 3. 10. The cell of claim 8, wherein said cell is a human cell. 1 1. An expression vector including nucleic acid encoding an enzymatic RNA molecule or multiple enzymatic molecules of claims 1 , 2, or 3 in a manner which allows expression of that enzymatic RNA molecule(s) within a mammalian cell. 12. A mammalian cell including an expression vector of claim 11. 13. The cell of claim 13, wherein said cell is a human cell. 14. A method for treatment of a condition related to elevated plasma Lp(a) levels by administering to a patient an enzymatic nucleic acid molecule of claims 1 , 2, or 3, 15. A method for treatment of a condition related to elevated plasma Lp(a) levels by administering to a patient an expression vector of claim 11. 16. The method of claims 14 or 15, wherein said patient is a human. 17. The method of claim 15 wherein said condition is selected from the group consisting of atherosclerosis, myocardial infraction, stroke, restenosis, and heart diseases. 18. The method of claim 17 wherein said condition is restenosis.

Inventors: MCSWIGGEN JAMES, NEWTON ROGER S, RAMHARACK RANDY, STINCHCOMB DAN T, MCSWIGGEN, JAMES, NEWTON, ROGER, S., RAMHARACK, RANDY, STINCHCOMB, DAN, T.
Applicants: RIBOZYME PHARM INC, RIBOZYME PHARMACEUTICALS, INC.
Application year: 1995
Main classification: C12N15
IPC codes: A61K31, A61K38, A61K48, A61P9, C12N5, C12N9, C12N15