Ribozyme treatment of diseases or conditions related to levels of NF-kappaB

Abstract

Enzymatic RNA molecules which cleave rel A mRNA.

Description

RELATED APPLICATIONS

[0001] This application is a continuation-in-part of 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 which is a continuation-in-part of Draper, “Method and Reagent for Treatment of a Stenotic Condition”, filed Dec. 7, 1992, U.S. Ser. No. 07/987,132, both hereby incorporated by reference herein.

FIELD OF THE INVENTION

[0002] The present invention relates to therapeutic compositions and methods for the treatment or diagnosis of diseases or conditions related to NF-&kgr;B levels, such as restenosis, rheumatoid arthritis, asthma, inflammatory or autoimmune disorders and transplant rejection.

BACKGROUND OF THE INVENTION

[0003] The following is a brief description of the physiological role of NF-&kgr;B. 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 invention.

[0004] The nuclear DNA-binding activity, NF-&kgr;B, was first identified as a factor that binds and activates the immunoglobulin K light chain enhancer in B cells. NF-&kgr;B now is known to activate transcription of a variety of other cellular genes (e.g., cytokines, adhesion proteins, oncogenes and viral proteins) in response to a variety of stimuli (e.g., phorbol esters, mitogens, cytokines and oxidative stress). In addition, molecular and biochemical characterization of NF-&kgr;B has shown that the activity is due to a homodimer or heterodimer of a family of DNA binding subunits. Each subunit bears a stretch of 300 amino acids that is homologous to the oncogene, v-rel. The activity first described as NF-&kgr;B is a heterodimer of p49 or p50 with p65. The p49 and p50 subunits of NF-&kgr;B (encoded by the nf-&kgr;B2 or nf-&kgr;B1 genes, respectively) are generated from the precursors NF-&kgr;B1 (p105) or NF-&kgr;B2 (p100). The p65 subunit of NF-&kgr;B (now termed Rel A) is encoded by the rel A locus.

[0005] The roles of each specific transcription-activating complex now are being elucidated in cells (N. D. Perkins, et al., 1992 Proc. Natl. Acad. Sci USA 89, 1529-1533). For instance, the heterodimer of NF-&kgr;B1 and Rel A (p50/p65) activates transcription of the promoter for the adhesion molecule, VCAM-1, while NF-&kgr;B2/RelA heterodimers (p49/p65) actually inhibit transcription (H. B. Shu, et al., Mol. Cell. Biol. 13, 6283-6289 (1993)). Conversely, heterodimers of NF-&kgr;B2/RelA (p49/p65) act with Tat-I to activate transcription of the HIV genome, while NF-&kgr;B1/RelA (p50/p65) heterodimers have little effect (J. Liu, N. D. Perkins, R. M. Schmid, G. J. Nabel, J. Virol. 1992 66, 3883-3887). Similarly, blocking rel A gene expression with antisense oligonucleotides specifically blocks embryonic stem cell adhesion; blocking NF-&kgr;B1 gene expression with antisense oligonucleotides had no effect on cellular adhesion (Narayanan et al., 1993 Mol. Cell. Biol. 13, 3802-3810). Thus, the promiscuous role initially assigned to NF-&kgr;B in transcriptional activation (M. J. Lenardo, D. Baltimore, 1989 Cell 58, 227-229) represents the sum of the activities of the rel family of DNA-binding proteins. This conclusion is supported by recent transgenic “knock-out” mice of individual members of the rel family. Such “knock-outs” show few developmental defects, suggesting that essential transcriptional activation functions can be performed by more than one member of the rel family.

[0006] A number of specific inhibitors of NF-&kgr;B function in cells exist, including treatment with phosphorothioate antisense oliogonucleotide, treatment with double-stranded NF-&kgr;B binding sites, and over expression of the natural inhibitor MAD-3 (an I&kgr;B family member). These agents have been used to show that NF-&kgr;B is required for induction of a number of molecules involved in inflammation, as described below.

[0007] NF-&kgr;B is required for phorbol ester-mediated induction of IL-6 (I. Kitajima, et al., Science 258, 1792-5 (1992)) and IL-8 (Kunsch and Rosen, 1993 Mol. Cell. Biol. 13, 6137-46).

[0008] NF-&kgr;B is required for induction of the adhesion molecules ICAM-1 (Eck, et al., 1993 Mol. Cell. Biol. 13, 6530-6536), VCAM-1 (Shu et al., supra), and E-selectin (Read, et al., 1994 J. Exp. Med. 179, 503-512) on endothelial cells.

[0009] NF-&kgr;B is involved in the induction of the integrin subunit, CD18, and other adhesive properties of leukocytes (Eck et al., 1993 supra).

[0010] The above studies suggest that NF-&kgr;B is integrally involved in the induction of cytokines and adhesion molecules by inflammatory mediators. Two recent papers point to another connection between NF-&kgr;B and inflammation: glucocorticoids may exert their anti-inflammatory effects by inhibiting NF-&kgr;B. The glucocorticoid receptor and p65 both act at NF-&kgr;B binding sites in the ICAM-1 promoter (van de Stolpe, et al., 1994 J. Biol. Chem. 269, 6185-6192). Glucocorticoid receptor inhibits NF-&kgr;B-mediated induction of IL-6 (Ray and Prefontaine, 1994 Proc. Natl Acad. Sci USA 91, 752-756). Conversely, overexpression of p65 inhibits glucocorticoid induction of the mouse mammary tumor virus promoter. Finally, protein cross-linking and co-immunoprecipitation experiments demonstrated direct physical interaction between p65 and the glucocorticoid receptor (Id.).

SUMMARY OF THE INVENTION

[0011] This invention relates to ribozymes, or enzymatic RNA molecules, directed to cleave mRNA species encoding Rel A protein (p65). In particular, applicant describes the selection and function of ribozymes capable of cleaving this RNA and their use to reduce activity of NF-&kgr;B in various tissues to treat the diseases discussed herein. Such ribozymes are also useful for diagnostic applications.

[0012] Ribozymes that cleave rel A mRNA represent a novel therapeutic approach to inflammatory or autoimmune disorders. Antisense DNA molecules have been described that block NF-&kgr;B activity. See Narayanan et al., supra. However, 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.

[0013] Applicant indicates that these ribozymes are able to inhibit the activity of NF-&kgr;B 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 rel A encoding mRNAs may be readily designed and are within the invention.

[0014] 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.

[0015] 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 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 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. 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.

[0016] 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 Sep. 20, 1989, which is a continuation-in-part of U.S. Ser. No. 07/247,100 filed Sep. 20, 1988, Hampel and Tritz, 1989, Biochemistry, 28, 4929, and Hampel et al., 1990, Nucleic Acids Res.earch, 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.

[0017] 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 Rel 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.

[0018] 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 al., PCT WO93/23569, and Sullivan et al., PCT WO94/02595, both hereby incorporated in their totality by reference herein; Ohkawa, J., et al., 1992, Nucleic Acids Symp. 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).

[0019] Inflammatory mediators such as lipopolysaccharide (LPS), interleukin-1 (IL-1) or tumor necrosis factor-a (TNF-&agr;) act on cells by inducing transcription of a number of secondary mediators, including other cytokines and adhesion molecules. In many cases, this gene activation is known to be mediated by the transcriptional regulator, NF-&kgr;B. One subunit of NF-&kgr;B, the relA gene product (termed RelA or p65) is implicated specifically in the induction of inflammatory responses. Ribozyme therapy, due to its exquisite specificity, is particularly well-suited to target intracellular factors that contribute to disease pathology. Thus, ribozymes that cleave mRNA encoded by rel A may represent novel therapeutics for the treatment of inflammatory and autoimmune disorders.

[0020] Thus, in a first aspect, the invention features ribozymes that inhibit RelA 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 RelA encoding mRNAs, preventing translation and p65 protein accumulation. In the absence of the expression of the target gene, a therapeutic effect may be observed.

[0021] By “inhibit” is meant that the activity or level of RelA 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.

[0022] 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 NF-&kgr;B activity in a cell or tissue. By “related” is meant that the inhibition of relA mRNA and thus reduction in the level of NF-&kgr;B activity will relieve to some extent the symptoms of the disease or condition.

[0023] 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 ex vivo, or in vivo through injection or 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, III, VI-VII. Examples of such ribozymes are shown in Tables IV-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.

[0024] In another aspect of the invention, ribozymes that cleave target molecules and inhibit NF-&kgr;B 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.

[0025] 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

[0026] The drawings will first briefly be described.

DRAWINGS

[0027] FIG. 1 is a diagrammatic representation of the hammerhead ribozyme domain known in the art.

[0028] FIG. 2a is a diagrammatic representation of the hammerhead ribozyme domain known in the art;

[0029] 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;

[0030] FIG. 2c is a similar diagram showing the hammerhead divided by Haseloff and Gerlach (1988, Nature, 334, 585-591) into two portions; and

[0031] FIG. 2d is a similar diagram showing the hammerhead divided by Jeffries and Symons (1989, Nucl. Acids. Res., 17, 1371-1371) into two portions.

[0032] FIG. 3 is a representation of the general structure of the hairpin ribozyme domain known in the art.

[0033] FIG. 4 is a representation of the general structure of the hepatitis delta virus ribozyme domain known in the art.

[0034] FIG. 5 is a representation of the general structure of the VS RNA ribozyme domain known in the art.

[0035] 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.

[0036] Ribozymes

[0037] Ribozymes of this invention block to some extent NF-&kgr;B expression 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 restenosis, transplant rejection and rheumatoid arthritis. Ribozyme cleavage of relA mRNA in these systems may prevent inflammatory cell function and alleviate disease symptoms.

[0038] Target Sites

[0039] Targets for useful ribozymes can be determined as disclosed in Draper et al supra, Sullivan et al., supra, as well as by 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. Such ribozymes can also be optimized and delivered as described therein. While specific examples to mouse 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 targeting human RNA sequences are present in the ribozyme. Such targets may also be selected as described below.

[0040] The sequence of human and mouse relA mRNA can be screened for accessible sites using a computer folding algorithm. Potential hammerhead or hairpin ribozyme cleavage sites were identified. These sites are shown in Tables II, III, and VI-VII. (All sequences are 5′ to 3′ in the tables.) While mouse 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. supra, mouse targetted 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. (In Table II, lower case letters indicate positions that are not conserved between the Human and the Mouse relA sequences.)

[0041] Hammerhead ribozymes are designed that could bind and are individually analyzed by computer folding (Jaeger, J. A., 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.

[0042] Referring to FIG. 6, mRNA is screened for accessible cleavage sites by the method described generally in Draper et al., WO/US93/04020 hereby incorporated by reference herein. Briefly, DNA oligonucleotides representing potential hammerhead ribozyme cleavage sites are synthesized. A polymerase chain reaction is used to generate a substrate for T7 RNA polymerase transcription from human or murine rel 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 ribozyme sites are chosen as the most accessible.

[0043] Ribozymes of the hammerhead 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, N.; Ogilvie, K. K.; Jiang, M. -Y.; Cedergren, R. J. 1987, J. Am. Chem. Soc., 109, 7845-7854 and in Scaringe, S. A.; Franklyn, C.; Usman, N., 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 were synthesized by substituting a U for G5 and a U for A14 (numbering from (Hertel, K. J., et al., 1992, Nucleic Acids Res., 20, 3252)). Hairpin ribozymes are synthesized in two parts and annealed to reconstruct the active ribozyme (Chowrira, B. M. and Burke, J. M., 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′-O-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. Ser. No. 08/245,736 the totality of which is hereby incorporated herein by reference.) and are resuspended in water.

[0044] The sequences of the chemically synthesized ribozymes useful in this study are shown in Tables IV-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 and may be formed of ribonucleotides or other nucleotides or non-nucleotides. Such ribozymes are equivalent to the ribozymes described specifically in the Tables.

[0045] Optimizing Ribozyme Activity

[0046] 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., Nature 1990, 344:565; Pieken et al., Science 1991, 253:314; Usman and Cedergren, Trends in Biochem. Sci. 1992, 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, B. European Patent Application 92110298.4 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.

[0047] 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, intrvascular, 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.

[0048] 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, O. and Moss, B., 1990, Proc. Natl. Acad. Sci. USA, 87, 6743-7; Gao, X. and Huang, L., 1993, Nucleic Acids Res., 21, 2867-72; Lieber, A., et al., 1993, Methods Enzymol., 217, 47-66; Zhou, Y., 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, M., et al., 1992, Antisense Res. Dev., 2, 3-15; 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; Yu, M., et al., 1993, Proc. Natl. Acad. Sci. USA, 96, 6340-4; L'Huillier, P. J., et al., 1992, Embo J., 11, 4411-8; Lisziewicz, J., 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 vectors), or viral RNA vectors (such as retroviral vectors).

[0049] In a preferred embodiment of the invention, a transcription unit expressing a ribozyme that cleaves relA RNA is inserted into a plasmid DNA vector or an adenovirus DNA viral vector. Both vectors have been used to transfer genes to the intact vasculature or to joints of live animals (Willard, J. E., et al., 1992, Circulation, 86, 1-473.; Nabel, E. G., et al., 1990, Science, 249, 1285-1288.) and both vectors lead to transient gene expression. The adenovirus vector is delivered as recombinant adenoviral particles. DNA may be delivered alone or complexed with vehicles (as described for RNA above). The DNA, DNA/vehicle complexes, or the recombinant adenovirus particles are locally administered to the site of treatment, e.g., through the use of an injection catheter, stent or infusion pump or are directly added to cells or tissues ex vivo.

EXAMPLE 1

NF-&kgr;B Hammerhead Ribozymes

[0050] By engineering ribozyme motifs we have designed several ribozymes directed against relA mRNA sequences. These ribozymes are synthesized with modifications that improve their nuclease resistance. The ability of ribozymes to cleave relA target sequences in vitro is evaluated.

[0051] The ribozymes will be tested for function in vivo by analyzing cytokine-induced VCAM-1, ICAM-1, IL-6 and IL-8 expression levels. Ribozymes will be delivered to cells by incorporation into liposomes, by complexing with cationic lipids, by microinjection, or by expression from DNA vectors. Cytokine-induced VCAM-1, ICAM-1, IL-6 and IL-8 expression will be monitored by ELISA, by indirect immunofluoresence, and/or by FACS analysis. Rel A mRNA levels will be assessed by Northern analysis, RNAse protection or primer extension analysis or quantitative RT-PCR. Activity of NF-&kgr;B will be monitored by gel-retardation assays. Ribozymes that block the induction of NF-&kgr;B activity and/or rel A mRNA by more than 50% will be identified.

[0052] RNA ribozymes and/or genes encoding them will be locally delivered to transplant tissue ex vivo in animal models. Expression of the ribozyme will be monitored by its ability to block ex vivo induction of VCAM-1, ICAM-1, IL-6 and IL-8 mRNA and protein. The effect of the anti-rel A ribozymes on graft rejection will then be assessed. Similarly, ribozymes will be introduced into joints of mice with collagen-induced arthritis or rabbits with Streptococcal cell wall-induced arthritis. Liposome delivery, cationic lipid delivery, or adeno-associated virus vector delivery can be used. One dose (or a few infrequent doses) of a stable anti-relA ribozyme or a gene construct that constitutively expresses the ribozyme may abrogate inflammatory and immune responses in these diseases.

[0053] Uses

[0054] A therapeutic agent that inhibits cytokine gene expression, inhibits adhesion molecule expression, and mimics the anti-inflammatory effects of glucocorticoids (without inducing steroid-responsive genes) is ideal for the treatment of inflammatory and autoimmune disorders. Disease targets for such a drug are numerous. Target indications and the delivery options each entails are summarized below. In all cases, because of the potential immunosuppressive properties of a ribozyme that cleaves rel A mRNA, uses are limited to local delivery, acute indications, or ex vivo treatment.

[0055] *Rheumatoid Arthritis (RA).

[0056] Due to the chronic nature of RA, a gene therapy approach is logical. Delivery of a ribozyme to inflamed joints is mediated by adenovirus, retrovirus, or adeno-associated virus vectors. For instance, the appropriate adenovirus vector can be administered by direct injection into the synovium: high efficiency of gene transfer and expression for several months would be expected (B. J. Roessler, E. D. Allen, J. M. Wilson, J. W. Hartman, B. L. Davidson, J. Clin. Invest. 92, 1085-1092 (1993)). It is unlikely that the course of the disease could be reversed by the transient, local administration of an anti-inflammatory agent. Multiple administrations may be necessary. Retrovirus and adeno-associated virus vectors would lead to permanent gene transfer and expression in the joint. However, permanent expression of a potent anti-inflammatory agent may lead to local immune deficiency.

[0057] Restenosis.

[0058] Expression of NF-&kgr;B in the vessel wall of pigs causes a narrowing of the luminal space due to excessive deposition of extracellular matrix components. This phenotype is similar to matrix deposition that occurs subsequent to coronary angioplasty. In addition, NF-&kgr;B is required for the expression of the oncogene c-myb (F. A. La Rosa, J. W. Pierce, G. E. Soneneshein, Mol. Cell. Biol. 14, 1039-44 (1994)). Thus NF-&kgr;B induces smooth muscle proliferation and the expression of excess matrix components: both processes are thought to contribute to reocclusion of vessels after coronary angioplasty.

[0059] *Transplantation.

[0060] NF-&kgr;B is required for the induction of adhesion molecules (Eck et al., supra, K. O'Brien, et al., J. Clin. Invest. 92, 945-951 (1993)) that function in immune recognition and inflammatory responses. At least two potential modes of treatment are possible. In the first, transplanted organs are treated ex vivo with ribozymes or ribozyme expression vectors. Transient inhibition of NF-&kgr;B in the transplanted endothelium may be sufficient to prevent transplant-associated vasculitis and may significantly modulate graft rejection. In the second, donor B cells are treated ex vivo with ribozymes or ribozyme expression vectors. Recipients would receive the treatment prior to transplant. Treatment of a recipient with B cells that do not express T cell co-stimulatory molecules (such as ICAM-1, VCAM-1, and/or B7 an B7-2) can induce antigen-specific anergy. Tolerance to the donor's histocompatibility antigens could result; potentially, any donor could be used for any transplantation procedure.

[0061] *Asthma.

[0062] Granulocyte macrophage colony stimulating factor (GM-CSF) is thought to play a major role in recruitment of eosinophils and other inflammatory cells during the late phase reaction to asthmatic trauma. Again, blocking the local induction of GM-CSF and other inflammatory mediators is likely to reduce the persistent inflammation observed in chronic asthmatics. Aerosol delivery of ribozymes or adenovirus ribozyme expression vectors is a feasible treatment.

[0063] Gene Therapy.

[0064] Immune responses limit the efficacy of many gene transfer techniques. Cells transfected with retrovirus vectors have short lifetimes in immune competent individuals. The length of expression of adenovirus vectors in terminally differentiated cells is longer in neonatal or immune-compromised animals. Insertion of a small ribozyme expression cassette that modulates inflammatory and immune responses into existing adenovirus or retrovirus constructs will greatly enhance their potential.

[0065] Thus, ribozymes of the present invention that cleave rel A mRNA and thereby NF-&kgr;B activity have many potential therapeutic uses, and there are reasonable modes of delivering the ribozymes in a number of the possible indications. Development of an effective ribozyme that inhibits NF-&kgr;B function is described above; available cellular and activity assays are number, reproducible, and accurate. Animal models for NF-&kgr;B function (Kitajima, et al., supra) and for each of the suggested disease targets exist and can be used to optimize activity.

[0066] Diagnostic Uses

[0067] 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 NF-&kgr;B related condition. Such RNA is detected by determining the presence of a cleavage product after treatment with a ribozyme using standard methodology.

[0068] 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., NF-&kgr;B) 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.

[0069] Other embodiments are within the following claims. 1 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 (M1 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 (FIGS. 1 and 2 show examples of various manifestations as used in the art). 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 (FIG. 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 (FIG. 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 (FIG. 5).

[0070] 2 TABLE II Mouse rel A HH Target sequence nt. HH Target Seq. ID nt. HH Target Seq. Pos. Sequence No. Pos. Sequence ID No. 19 AAUGGCU a caCaGgA 7 467 cCAGGCU c cuguUCg 108 22 aGCUCcU a cGUgGUG 8 469 AaGCcAU u AGcCAGC 109 26 CcUCcaU u GcGgACa 9 473 UuUgAGU C AGauCAg 110 93 GAuCUGU U uCCCCUC 10 481 AGCGaAU C CAGACCA 111 94 AuCUGUU u CCCCUCA 11 501 AACCCCU U uCAcGUU 112 100 UuCCCCU C AUCUUuC 12 502 ACCCCUU u CAcGUUC 113 103 CCCUCAU C UuuCCcu 13 508 UuCAcGU U CCUAUAG 114 105 CUCAUCU U uCCcuCA 14 509 uCAcGUU C CUAUAGA 115 106 UCAUCUU u CccuCAG 15 512 cGUUCCU A UAGAgGA 116 129 CAGGCuU C UGGgCCu 16 514 UUCCUAU A GAgGAGC 117 138 GGgCCuU A UGUGGAG 17 534 GGGGACU A uGACuUG 118 148 UGGAGAU C AucGAaC 18 556 UGCGcCU C UGCUUCC 119 151 AGAUCAU c GaaCAGC 19 561 CUCUGCU U CCAGGUG 120 180 AUGCGaU U CCGCUAu 20 562 UCUGCUU C CAGGUGA 121 181 UGCGaUU C CGCUAuA 21 585 aAgCCAU u AGcCAGc 122 186 UUCCGCU A uAAaUGC 22 598 GGCCCCU C CuCCUGa 123 204 GGGCGCU C aGCGGGC 23 613 CcCCUGU C CUcuCaC 124 217 GCAGuAU U CcuGGCG 24 616 CUGUCCU c uCaCAUC 125 239 CACAGAU A CCACCAA 25 617 gucCCUU C CUCAgCC 126 262 CCACCAU C AAGAUCA 26 620 CCUUCCU C AgCCaug 127 268 UCAAGAU C AAUGGCU 27 623 UCCUgcU u CCAUCUc 128 276 AAUGGCU A CACAGGA 28 628 AUCCGAU U UUUGAUA 129 301 UuCGaAU C UCCCUGG 29 630 CCgAUuU U UGAuAAc 130 303 CGaAUCU C CCUGGUC 30 631 CgAUuUU U GAuAAcC 131 310 CCCUGGU C ACCAAGG 31 638 UGgCcAU u GUGuuCC 132 323 GGcCCCU C CUCcuga 32 661 CCGAGCU C AAGAUCU 133 326 uCCaCCU C ACCGGCC 33 667 UCAAGAU C UGCCGAG 134 335 CCGGCCU C AuCCaCA 34 687 CGgAACU C UGGgAGC 135 349 AuGAaCU U GugGGgA 35 700 GCUGCCU C GGUGGGG 136 352 AGaUcaU c GaAcAGc 36 715 AUGAGAU C UUCuUgC 137 375 GAUGGCU a CUAUGAG 37 717 GAGAUCU U CuUgCUG 138 376 AUGGucU C UccGgaG 38 718 AGAUCUU C uUgCUGU 139 378 GGCUaCU A UGAGGCU 39 721 UucUCCU c CauUGcG 140 391 CUGAcCU C UGCCCaG 40 751 AaGACAU U GAGGUGU 141 409 GCaGuAU C CauAGcU 41 759 GAGGUGU A UUUCACG 142 416 CCgCAGU a UCCAuAg 42 761 GGUGUAU U UCACGGG 143 417 CAuAGcU U CCAGAAC 43 762 GUGUAUU U CACGGGA 144 418 AuAGcUU C CAGAACC 44 763 UGUAUUU C ACGGGAC 145 433 UGGGgAU C CAGUGUG 45 792 CGAGGCU C CUUUUCu 146 795 GGCUCCU U UUCuCAA 46 1167 GAUGAGU U UuCCcCC 147 796 GCUCCUU U UcuCAAG 47 1168 AUGAGUU U uCCcCCA 148 797 CUCCUUU U CuCAAGC 48 1169 UGAGUUU u CCcCCAU 149 798 UCCUUUU C uCAAGCU 49 1182 AUGcUGU U aCCaUCa 150 829 UGGCCAU U GUGUUCC 50 1183 UGcUGUU a CCaUCaG 151 834 AUUGUGU U CCGGACu 51 1184 GGccccU C CUcCUGa 152 835 UUGUGUU C CGGACuC 52 1187 GUccCuU c CUcAGCc 153 845 GACuCCU C CgUACGC 53 1188 UUaCCaU C aGGGCAG 154 849 CCUCCgU A CGCcGAC 54 1198 GGgAGuU u AGuCuGa 155 872 cCAGGCU C CUGUuCG 55 1209 CAGcCCU a caCCUUc 156 883 UuCGaGU C UCCAUGC 56 1215 cuGGCCU U aGCaCCG 157 885 CGaGUCU C CAUGCAG 57 1229 GGuCCCU u CCucAGc 158 905 GCGGCCU U CuGAuCG 58 1237 CCCAgcU C CUGCCCC 159 906 CGGCCUU C uGAuCGc 59 1250 CCAGcCU C CAGgCUC 160 919 GcGAGCU C AGUGAGC 60 1268 CCCaGCU C CuGCCcc 161 936 AUGGAgU U CCAGUAC 61 1279 CCAUGGU c cCuuCcu 162 937 UGGAgUU C CAGUACu 62 1281 gUGGgcU C AGCUgcG 163 942 UUCCAGU A CuUGCCA 63 1286 AUgAGuU u UccCCCA 164 953 GCCucAU c CacAuGA 64 1309 CuCCUGU u CgAGUCu 165 962 AGAuGAU C GcCACCG 65 1315 cCCCAGU u CUAaCCC 166 965 CagUacU u gCCaGAc 66 1318 CAGUuCA A aCCCCgG 167 973 ACCGGAU U GaaGAGA 67 1331 gGGuCCU C CcCAGuC 168 986 GAgACcU u cAAGagu 68 1334 CuuUuCU C AaGCUGa 169 996 AGGACcU A UGAGACC 69 1389 ACGCUGU C gGAaGCC 170 1005 GAGACCU U CAAGAGu 70 1413 CUGCAGU U UGAUGcU 171 1006 AGACCUU C AAGAGuA 71 1414 UGCAGUU U GAUGcUG 172 1015 AGAGuAU C AUGAAGA 72 1437 GGGGCCU U GCUUGGC 173 1028 GAAGAGU C CUUUCAa 73 1441 CCUUGCU U GGCAACA 174 1031 GAGUCCU U UCAauGG 74 1467 GgaGUGU U CACAGAC 175 1032 AGUCCUU U CaauGGA 75 1468 gaGUGUU C ACAGACC 176 1033 GUCCUUU C AauGGAC 76 1482 CUGGCAU C uGUgGAC 177 1058 CCGGCCU C CaaCcCG 77 1486 CuUCgGU a GggAACU 178 1064 UaCACCU u GaucCAa 78 1494 GACAACU C aGAGUUU 179 1072 GgCGuAU U GCUGUGC 79 1500 UCaGAGU U UCAGCAG 180 1082 UGUGCCU a CCCGaAa 80 1501 CaGAGUU U CAGCAGC 181 1083 aaGCCUU C CCGaAGu 81 1502 aGAGUUU C AGCAGCU 182 1092 CGaAaCU C AaCUUCU 82 1525 gGuGCAU c CCUGUGu 183 1097 CUCAaCU U CUGUCCC 83 1566 AUGGAGU A CCCUGAa 184 1098 UCAaCUU C UGUCCCC 84 1577 UGAaGCU A UAACUCG 185 1102 CUUCUGU C CCCAAGC 85 1579 AaGCUAU A ACUCGCC 186 1125 CAGCCCU A caCCUUc 86 1583 UAUAACU C GCCUgGU 187 1127 GCCaUAU a gCcUUAC 87 1588 CUCuCCU A GaGAggG 188 1131 cAUCCCU c agCacCA 88 1622 CCCAGCU C CUGCcCC 189 1132 AcaCCUU c cCagCAU 89 1628 UCCUGCU u CggUaGG 190 1133 UCCaUcU c CagCuUC 90 1648 CGGGGCU u CCCAAUG 191 1137 UUUACuU u AgCgCgc 91 1660 cUGaCCU C ugccCAG 192 1140 cCagCAU C CCUcAGC 92 1663 cuCUgCU U cCAGGuG 193 1153 GCACCAU C AACUuUG 93 1664 uCUgCUU c CAGGuGA 194 1158 AUCAACU u UGAUGAG 94 1665 CUCgcUU u cGGAGgU 195 1680 GAAGACU U CUCCUCC 95 1681 AAGACUU C UCCUCCA 96 1683 GACUUCU C CUCCAUU 97 1686 UUCUCCU C CAUUGCG 98 1690 CCUCCAU U GCGGACA 99 1704 AUGGACU U CUCuGCu 100 1705 UGGACUU C UCuGCuC 101 1707 GACUUCU C uGCuCUu 102 1721 uuUGAGU C AGAUCAG 103 1726 GUCAGAU C AGCUCCU 104 1731 AUCAGCU C CUAAGGu 105 1734 AGCUCCU A AGGuGcU 106 1754 CaGugCU C CCaAGAG 107

[0071] 3 TABLE III Human rel A HH Target Sequences nt. HH Target Seq. ID nt. HH Target Seq. ID Pos. Sequence No. Pos. Sequence No. 19 AAUGGCU C GUCUGUA 196 467 GCAGGCU A UCAGUCA 297 22 GGCUCGU C UGUAGUG 197 469 AGGCUAU C AGUCAGC 298 26 CGUCUGU A GUGCACG 198 473 UAUCAGU C AGCGCAU 299 93 GAACUGU U CCCCCUC 199 481 AGCGCAU C CAGACCA 300 94 AACUGUU C CCCCUCA 200 501 AACCCCU U CCAAGUU 301 100 UCCCCCU C AUCUUCC 201 502 ACCCCUU C CAAGUUC 302 103 CCCUCAU C UUCCCGG 202 508 UCCAAGU U CCUAUAG 303 105 CUCAUCU U CCCGGCA 203 509 CCAAGUU C CUAUAGA 304 106 UCAUCUU C CCGGCAG 204 512 AGUUCCU A UAGAAGA 305 129 CAGGCCU C UGGCCCC 205 514 UUCCUAU A GAAGAGC 306 138 GGCCCCU A UGUGGAG 206 534 GGGGACU A CGACCUG 307 148 UGGAGAU C AUUGAGC 207 556 UGCGGCU C UGCUUCC 308 151 AGAUCAU U GAGCAGC 208 561 CUCUGCU U CCAGGUG 309 180 AUGCGCU U CCGCUAC 209 562 UCUGCUU C CAGGUGA 310 181 UGCGCUU C CGCUACA 210 585 GACCCAU C AGGCAGG 311 186 UUCCGCU A CAAGUGC 211 598 GGCCCCU C CGCCUGC 312 204 GGGCGCU C CGCGGGC 212 613 CGCCUGU C CUUCCUC 313 217 GCAGCAU C CCAGGCG 213 616 CUGUCCU U CCUCAUC 314 239 CACAGAU A CCACCAA 214 617 UGUCCUU C CUCAUCC 315 262 CCACCAU C AAGAUCA 215 620 CCUUCCU C AUCCCAU 316 268 UCAAGAU C AAUGGCU 216 623 UCCUCAU C CCAUCUU 317 276 AAUGGCU A CACAGGA 217 628 AUCCCAU C UUUGACA 318 301 UGCGCAU C UCCCUGG 218 630 CCCAUCU U UGACAAU 319 303 CGCAUCU C CCUGGUC 219 631 CCAUCUU U GACAAUC 320 310 CCCUGGU C ACCAAGG 220 638 UGACAAU C GUGCCCC 321 323 GGACCCU C CUCACCG 221 661 CCGAGCU C AAGAUCU 322 326 CCCUCCU C ACCGGCC 222 667 UCAAGAU C UGCCGAG 323 335 CCGGCCU C ACCCCCA 223 687 CGAAACU C UGGCAGC 324 349 ACGAGCU U GUAGGAA 224 700 GCUGCCU C GGUGGGG 325 352 AGCUUGU A GGAAAGG 225 715 AUGAGAU C UUCCUAC 326 375 GAUGGCU U CUAUGAG 226 717 GAGAUCU U CCUACUG 327 376 AUGGCUU C UAUGAGG 227 718 AGAUCUU C CUACUGU 328 378 GGCUUCU A UGAGGCU 228 721 UCUUCCU A CUGUGUG 329 391 CUGAGCU C UGCCCGG 229 751 AGGACAU U GAGGUGU 330 409 GCUGCAU C CACAGUU 230 759 GAGGUGU A UUUCACG 331 416 CCACAGU U UCCAGAA 231 761 GGUGUAU U UCACGGG 332 417 CACAGUU U CCAGAAC 232 762 GUGUAUU U CACGGGA 333 418 ACAGUUU C CAGAACC 233 763 UGUAUUU C ACGGGAC 334 433 UGGGAAU C CAGUGUG 234 792 CGAGGCU C CUUUUCG 335 795 GGCUCCU U UUCGCAA 235 1167 GAUGAGU U UCCCACC 336 796 GCUCCUU U UCGCAAG 236 1168 AUGAGUU U CCCACCA 337 797 CUCCUUU U CGCAAGC 237 1169 UGAGUUU C CCACCAU 338 798 UCCUUUU C GCAAGCU 238 1182 AUGGUGU U UCCUUCU 339 829 UGGCCAU U GUGUUCC 239 1183 UGGUGUU U CCUUCUG 340 834 AUUGUGU U CCGGACC 240 1184 GGUGUUU C CUUCUGG 341 835 UUGUGUU C CGGACCC 241 1187 GUUUCCU U CUGGGCA 342 845 GACCCCU C CCUACGC 242 1188 UUUCCUU C UGGGCAG 343 849 CCUCCCU A CGCAGAC 243 1198 GGCAGAU C AGCCAGG 344 872 GCAGGCU C CUGUGCG 244 1209 CAGGCCU C GGCCUUG 345 883 UGCGUGU C UCCAUGC 245 1215 UCGGCCU U GGCCCCG 346 885 CGUGUCU C CAUGCAG 246 1229 GGCCCCU C CCCAAGU 347 905 GCGGCCU U CCGACCG 247 1237 CCCAAGU C CUGCCCC 348 906 CGGCCUU C CGACCGG 248 1250 CCAGGCU C CAGCCCC 349 919 GGGAGCU C AGUGAGC 249 1268 CCCUGCU C CAGCCAU 350 936 AUGGAAU U CCAGUAC 250 1279 CCAUGGU A UCAGGUC 351 937 UGGAAUU C CAGUACC 251 1281 AUGGUAU C AGCUCUG 352 942 UUCCAGU A CCUGCCA 252 1286 AUCAGCU C UGGCCCA 353 953 GCCAGAU A CAGACGA 253 1309 CCCCUGU C CCAGUCC 354 962 AGACGAU C GUCACCG 254 1315 UCCCAGU C CUAGCCC 355 965 CGAUCGU C ACCGGAU 255 1318 CAGUCCU A GCCCCAG 356 973 ACCGGAU U GAGGAGA 256 1331 AGGCCCU C CUCAGGC 357 986 GAAACGU A AAAGGAC 257 1334 CCCUCCU C AGGCUGU 358 996 AGGACAU A UGAGACC 258 1389 ACGCUGU C AGAGGCC 359 1005 GAGACCU U CAAGAGC 259 1413 CUGCAGU U UGAUGAU 360 1006 AGACCUU C AAGAGCA 260 1414 UGCAGUU U GAUGAUG 361 1015 AGAGCAU C AUGAAGA 261 1437 GGGGCCU U GCUUGGC 362 1028 GAAGAGU C CUUUCAG 262 1441 CCUUGCU U GGCAACA 363 1031 GAGUCCU U UCAGCGG 263 1467 GCUGUGU U CACAGAC 364 1032 AGUCCUU U CAGCGGA 264 1468 CUGUGUU C ACAGACC 365 1033 GUCCUUU C AGCGGAC 265 1482 CUGGCAU C CGUCGAC 366 1058 CCGGCCU C CACCUCG 266 1486 CAUCCGU C GACAACU 367 1064 UCCACCU C GACGCAU 267 1494 GACAACU C CGAGUUU 368 1072 GACGCAU U GCUGUGC 268 1500 UCCGAGU U UCAGCAG 369 1082 UGUGCCU U CCCGCAG 269 1501 CCGAGUU U CAGCAGC 370 1083 GUGCCUU C CCGCAGC 270 1502 CGAGUUU C AGCAGCU 371 1092 CGCAGCU C AGCUUCU 271 1525 AGGGCAU A CCUGUGG 372 1097 CUCAGCU U CUGUCCC 272 1566 AUGGAGU A CCCUGAG 373 1098 UCAGCUU C UGUCCCC 273 1577 UGAGGCU A UAACUCG 374 1102 CUUCUGU C CCCAAGC 274 1579 AGGCUAU A ACUCGCC 375 1125 CAGCCCU A UCCCUUU 275 1583 UAUAACU C GCCUAGU 376 1127 GCCCUAU C CCUUUAC 276 1588 CUCGCCU A GUGACAG 377 1131 UAUCCCU U UACGUCA 277 1622 CCCAGCU C CUGCUCC 378 1132 AUCCCUU U ACGUCAU 278 1628 UCCUGCU C CACUGGG 379 1133 UCCCUUU A CGUCAUC 279 1648 CGGGGCU C CCCAAUG 380 1137 UUUACGU C AUCCCUG 280 1660 AUGGCCU C CUUUCAG 381 1140 ACGUCAU C CCUGAGC 281 1663 GCCUCCU U UCAGGAG 382 1153 GCACCAU C AACUAUG 282 1664 CCUCCUU U CAGGAGA 383 1158 AUCAACU A UGAUGAG 283 1665 CUCCUUU C AGGAGAU 384 1680 GAAGACU U CUCCUCC 284 1681 AAGACUU C UCCUCCA 285 1683 GACUUCU C CUCCAUU 286 1686 UUCUCCU C CAUUGCG 287 1690 CCUCCAU U GCGGACA 288 1704 AUGGACU U CUCAGCC 289 1705 UGGACUU C UCAGCCC 290 1707 GACUUCU C AGCCCUG 291 1721 GCUGAGU C AGAUCAG 292 1726 GUCAGAU C AGCUCCU 293 1731 AUCAGCU C CUAAGGG 294 1734 AGCUCCU A AGGGGGU 295 1754 CUGCCCU C CCCAGAG 296

[0072] 4 TABLE IV Mouse rel A HH Ribozyme Sequences nt. Seq. HH Ribozyme Sequence Seq. ID No. 19 UCCUGUG CUGAUGAGGCCGAAAGGCCGAA AGCCAUU 385 22 CACCACG CUGAUGAGGCCGAAAGGCCGAA AGGAGCU 386 26 UGUCCGC CUGAUGAGGCCGAAAGGCCGAA AUGGAGG 387 93 GAGGGGA CUGAUGAGGCCGAAAGGCCGAA ACAGAUC 388 94 UGAGGGG CUGAUGAGGCCGAAAGGCCGAA AACAGAU 389 100 GAAAGAU CUGAUGAGGCCGAAAGGCCGAA AGGGGAA 390 103 AGGGAAA CUGAUGAGGCCGAAAGGCCGAA AUGAGGG 391 105 UGAGGGA CUGAUGAGGCCGAAAGGCCGAA AGAUGAG 392 106 CUGAGGG CUGAUGAGGCCGAAAGGCCGAA AAGAUGA 393 129 AGGCCCA CUGAUGAGGCCGAAAGGCCGAA AAGCCUG 394 138 CUCCACA CUGAUGAGGCCGAAAGGCCGAA AAGGCCC 395 148 GUUCGAU CUGAUGAGGCCGAAAGGCCGAA AUCUCCA 396 151 GCUGUUC CUGAUGAGGCCGAAAGGCCGAA AUGAUCU 397 180 AUAGCGG CUGAUGAGGCCGAAAGGCCGAA AUCGCAU 398 181 UAUAGCG CUGAUGAGGCCGAAAGGCCGAA AAUCGCA 399 186 GCAUUUA CUGAUGAGGCCGAAAGGCCGAA AGCGGAA 400 204 GCCCGCU CUGAUGAGGCCGAAAGGCCGAA AGCGCCC 401 217 CGCCAGG CUGAUGAGGCCGAAAGGCCGAA AUACUGC 402 239 UUGGUGG CUGAUGAGGCCGAAAGGCCGAA AUCUGUG 403 262 UGAUCUU CUGAUGAGGCCGAAAGGCCGAA AUGGUGG 404 268 AGCCAUU CUGAUGAGGCCGAAAGGCCGAA AUCUUGA 405 276 UCCUGUG CUGAUGAGGCCGAAAGGCCGAA AGCCAUU 406 301 CCAGGGA CUGAUGAGGCCGAAAGGCCGAA AUUCGAA 407 303 GACCAGG CUGAUGAGGCCGAAAGGCCGAA AGAUUCG 408 310 CCUUGGU CUGAUGAGGCCGAAAGGCCGAA ACCAGGG 409 323 UCAGGAG CUGAUGAGGCCGAAAGGCCGAA AGGGGCC 410 326 GGCCGGU CUGAUGAGGCCGAAAGGCCGAA AGGUGGA 411 335 UGUGGAU CUGAUGAGGCCGAAAGGCCGAA AGGCCGG 412 349 UCCCCAC CUGAUGAGGCCGAAAGGCCGAA AGUUCAU 413 352 GCUGUUC CUGAUGAGGCCGAAAGGCCGAA AUGAUCU 414 375 CUCAUAG CUGAUGAGGCCGAAAGGCCGAA AGCCAUC 415 376 CUCCGGA CUGAUGAGGCCGAAAGGCCGAA AGACCAU 416 378 AGCCUCA CUGAUGAGGCCGAAAGGCCGAA AGUAGCC 417 391 CUGGGCA CUGAUGAGGCCGAAAGGCCGAA AGGUCAG 418 391 CUGGGCA CUGAUGAGGCCGAAAGGCCGAA AGGUCAG 428 409 AGCUAUG CUGAUGAGGCCGAAAGGCCGAA AUACUGC 419 416 CUAUGGA CUGAUGAGGCCGAAAGGCCGAA ACUGCGG 420 417 GUUCUGG CUGAUGAGGCCGAAAGGCCGAA AGCUAUG 421 418 GGUUCUG CUGAUGAGGCCGAAAGGCCGAA AAGCUAU 422 433 CACACUG CUGAUGAGGCCGAAAGGCCGAA AUCCCCA 423 467 CGAACAG CUGAUGAGGCCGAAAGGCCGAA AGCCUGG 424 469 GCUGGCU CUGAUGAGGCCGAAAGGCCGAA AUGGCUU 425 473 CUGAUCU CUGAUGAGGCCGAAAGGCCGAA ACUCAAA 426 481 UGGUCUG CUGAUGAGGCCGAAAGGCCGAA AUUCGCU 427 501 AACGUGA CUGAUGAGGCCGAAAGGCCGAA AGGGGUU 428 502 GAACGUG CUGAUGAGGCCGAAAGGCCGAA AAGGGGU 429 508 CUAUAGG CUGAUGAGGCCGAAAGGCCGAA ACGUGAA 430 509 UCUAUAG CUGAUGAGGCCGAAAGGCCGAA AACGUGA 431 512 UCCUCUA CUGAUGAGGCCGAAAGGCCGAA AGGAACG 432 514 GCUCCUC CUGAUGAGGCCGAAAGGCCGAA AUAGGAA 433 534 CAAGUCA CUGAUGAGGCCGAAAGGCCGAA AGUCCCC 434 556 GGAAGCA CUGAUGAGGCCGAAAGGCCGAA AGGCGCA 435 561 CACCUGG CUGAUGAGGCCGAAAGGCCGAA AGGAGAG 436 562 UCACCUG CUGAUGAGGCCGAAAGGCCGAA AAGCAGA 437 585 GCUGGCU CUGAUGAGGCCGAAAGGCCGAA AUGGCUU 438 598 UCAGGAG CUGAUGAGGCCGAAAGGCCGAA AGGGGCC 439 613 GUGAGAG CUGAUGAGGCCGAAAGGCCGAA ACAGGGG 440 616 GAUGUGA CUGAUGAGGCCGAAAGGCCGAA AGGACAG 441 617 GGCUGAG CUGAUGAGGCCGAAAGGCCGAA AAGGGAC 442 620 CAUGGCU CUGAUGAGGCCGAAAGGCCGAA AGGAAGG 443 623 GAGAUGG CUGAUGAGGCCGAAAGGCCGAA AGCAGGA 444 628 UAUCAAA CUGAUGAGGCCGAAAGGCCGAA AUCGGAU 445 630 GUUAUCA CUGAUGAGGCCGAAAGGCCGAA AAAUCGG 446 631 GGUUAUC CUGAUGAGGCCGAAAGGCCGAA AAAAUCG 447 638 GGAACAC CUGAUGAGGCCGAAAGGCCGAA AUGGCCA 448 661 AGAUCUU CUGAUGAGGCCGAAAGGCCGAA AGCUCGG 449 667 CUCGGCA CUGAUGAGGCCGAAAGGCCGAA AUCUUGA 450 687 GCUCCCA CUGAUGAGGCCGAAAGGCCGAA AGUUCCG 451 700 CCCCACC CUGAUGAGGCCGAAAGGCCGAA AGGCAGC 452 715 GCAAGAA CUGAUGAGGCCGAAAGGCCGAA AUCUCAU 453 717 CAGCAAG CUGAUGAGGCCGAAAGGCCGAA AGAUCUC 454 718 ACAGCAA CUGAUGAGGCCGAAAGGCCGAA AAGAUCU 455 721 CGCAAUG CUGAUGAGGCCGAAAGGCCGAA AGGAGAA 456 751 ACACCUC CUGAUGAGGCCGAAAGGCCGAA AUGUCUU 457 759 CGUGAAA CUGAUGAGGCCGAAAGGCCGAA ACACCUC 458 761 CCCGUGA CUGAUGAGGCCGAAAGGCCGAA AUACACC 459 762 UCCCGUG CUGAUGAGGCCGAAAGGCCGAA AAUACAC 460 763 GUCCCGU CUGAUGAGGCCGAAAGGCCGAA AAAUACA 461 792 AGAAAAG CUGAUGAGGCCGAAAGGCCGAA AGCCUCG 462 795 UUGAGAA CUGAUGAGGCCGAAAGGCCGAA AGGAGCC 463 796 CUUGAGA CUGAUGAGGCCGAAAGGCCGAA AAGGAGC 464 797 GCUUGAG CUGAUGAGGCCGAAAGGCCGAA AAAGGAG 465 798 AGCUUGA CUGAUGAGGCCGAAAGGCCGAA AAAAGGA 466 829 GGAACAC CUGAUGAGGCCGAAAGGCCGAA AUGGCCA 467 834 AGUCCGG CUGAUGAGGCCGAAAGGCCGAA ACACAAU 468 835 GAGUCCG CUGAUGAGGCCGAAAGGCCGAA AACACAA 469 845 GCGUACG CUGAUGAGGCCGAAAGGCCGAA AGGAGUC 470 849 GUCGGCG CUGAUGAGGCCGAAAGGCCGAA ACGGAGG 471 872 CGAACAG CUGAUGAGGCCGAAAGGCCGAA AGCCUGG 472 883 GCAUGGA CUGAUGAGGCCGAAAGGCCGAA ACUCGAA 473 885 CUGCAUG CUGAUGAGGCCGAAAGGCCGAA AGACUCG 474 905 CGAUCAG CUGAUGAGGCCGAAAGGCCGAA AGGCCGC 475 906 GCGAUCA CUGAUGAGGCCGAAAGGCCGAA AAGGCCG 476 919 GCUCACU CUGAUGAGGCCGAAAGGCCGAA AGCUCGC 477 936 GUACUGG CUGAUGAGGCCGAAAGGCCGAA ACUCCAU 478 937 AGUACUG CUGAUGAGGCCGAAAGGCCGAA AACUCCA 479 942 UGGCAAG CUGAUGAGGCCGAAAGGCCGAA ACUGGAA 480 953 UCAUGUG CUGAUGAGGCCGAAAGGCCGAA AUGAGGC 481 962 CGGUGGC CUGAUGAGGCCGAAAGGCCGAA AUCAUCU 482 965 GUCUGGC CUGAUGAGGCCGAAAGGCCGAA AGUACUG 483 973 UCUCUUC CUGAUGAGGCCGAAAGGCCGAA AUCCGGU 484 986 ACUCUUG CUGAUGAGGCCGAAAGGCCGAA AGGUCUC 485 1005 ACUCUUG CUGAUGAGGCCGAAAGGCCGAA AGGUCUC 486 1006 UACUCUU CUGAUGAGGCCGAAAGGCCGAA AAGGUCU 487 1015 UCUUCAU CUGAUGAGGCCGAAAGGCCGAA AUACUCU 488 1028 UUGAAAG CUGAUGAGGCCGAAAGGCCGAA ACUCUUC 490 1031 CCAUUGA CUGAUGAGGCCGAAAGGCCGAA AGGACUC 491 1032 UCCAUGG CUGAUGAGGCCGAAAGGCCGAA AAGGACU 492 1033 GUCCAUU CUGAUGAGGCCGAAAGGCCGAA AAAGGAC 493 1058 CGGGUUG CUGAUGAGGCCGAAAGGCCGAA AGGCCGG 494 1064 UUGGAUC CUGAUGAGGCCGAAAGGCCGAA AGGUGUA 495 1072 GCACAGC CUGAUGAGGCCGAAAGGCCGAA AUACGCC 496 1082 UUUCGGG CUGAUGAGGCCGAAAGGCCGAA AGGCACA 497 1083 ACUUCGG CUGAUGAGGCCGAAAGGCCGAA AAGGCUU 498 1092 AGAAGUU CUGAUGAGGCCGAAAGGCCGAA AGUUUCG 499 1097 GGGACAG CUGAUGAGGCCGAAAGGCCGAA AGUUGAG 500 1098 GGGGACA CUGAUGAGGCCGAAAGGCCGAA AAGUUGA 501 1102 GCUUGGG CUGAUGAGGCCGAAAGGCCGAA ACAGAAG 502 1125 GAAGGUG CUGAUGAGGCCGAAAGGCCGAA AGGGCUG 503 1127 GUAAGGC CUGAUGAGGCCGAAAGGCCGAA AUAUGGC 504 1131 UGGUGCU CUGAUGAGGCCGAAAGGCCGAA AGGGAUG 505 1132 AUGCUGG CUGAUGAGGCCGAAAGGCCGAA AAGGUGU 506 1133 GAAGCUG CUGAUGAGGCCGAAAGGCCGAA AGAUGGA 507 1137 GCGCGCU CUGAUGAGGCCGAAAGGCCGAA AAGUAAA 508 1140 GCUGAGG CUGAUGAGGCCGAAAGGCCGAA AUGCUGG 509 1153 CAAAGUU CUGAUGAGGCCGAAAGGCCGAA AUGGUGC 510 1158 CUCAUCA CUGAUGAGGCCGAAAGGCCGAA AGUUGAU 511 1167 GGGGGAA CUGAUGAGGCCGAAAGGCCGAA ACUCAUC 512 1168 UGGGGGA CUGAUGAGGCCGAAAGGCCGAA AACUCAU 513 1169 AUGGGGG CUGAUGAGGCCGAAAGGCCGAA AAACUCA 514 1182 UGAUGGU CUGAUGAGGCCGAAAGGCCGAA ACAGCAU 515 1183 CUGAUGG CUGAUGAGGCCGAAAGGCCGAA AACAGCA 516 1184 UCAGGAG CUGAUGAGGCCGAAAGGCCGAA AGGGGCC 517 1187 GGCUGAG CUGAUGAGGCCGAAAGGCCGAA AAGGGAC 518 1188 CUGCCCU CUGAUGAGGCCGAAAGGCCGAA AUGGUAA 519 1198 UCAGACU CUGAUGAGGCCGAAAGGCCGAA AACUCCC 520 1209 GAAGGUG CUGAUGAGGCCGAAAGGCCGAA AGGGCUG 521 1215 CGGUGCU CUGAUGAGGCCGAAAGGCCGAA AGGCCAG 522 1229 GCUGAGG CUGAUGAGGCCGAAAGGCCGAA AGGGACC 523 1237 GGGGCAG CUGAUGAGGCCGAAAGGCCGAA AGCUGGG 524 1250 GAGCCUG CUGAUGAGGCCGAAAGGCCGAA AGGCUGG 525 1268 GGGGCAG CUGAUGAGGCCGAAAGGCCGAA AGCUGGG 526 1279 AGGAAGG CUGAUGAGGCCGAAAGGCCGAA ACCAUGG 527 1281 CGCAGCU CUGAUGAGGCCGAAAGGCCGAA AGCCCAC 528 1286 UGGGGGA CUGAUGAGGCCGAAAGGCCGAA AACUCAU 529 1309 AGACUCG CUGAUGAGGCCGAAAGGCCGAA ACAGGAG 530 1315 GGGUUAG CUGAUGAGGCCGAAAGGCCGAA ACUGGGG 531 1318 CCGGGGU CUGAUGAGGCCGAAAGGCCGAA AGAACUG 532 1331 GACUGGG CUGAUGAGGCCGAAAGGCCGAA AGGACCC 533 1334 UCAGCUU CUGAUGAGGCCGAAAGGCCGAA AGAAAAG 534 1389 GGCUUCC CUGAUGAGGCCGAAAGGCCGAA ACAGCGU 535 1413 AGCAUCA CUGAUGAGGCCGAAAGGCCGAA ACUGGAG 536 1414 CAGCAUC CUGAUGAGGCCGAAAGGCCGAA AACUGCA 537 1437 GCCAAGC CUGAUGAGGCCGAAAGGCCGAA AGGCCCC 538 1441 UGUUGCC CUGAUGAGGCCGAAAGGCCGAA AGCAAGG 539 1467 GUCUGUG CUGAUGAGGCCGAAAGGCCGAA ACACUCC 540 1468 GGUCUGU CUGAUGAGGCCGAAAGGCCGAA AACACUC 541 1482 GUCCACA CUGAUGAGGCCGAAAGGCCGAA AUGCCAG 542 1486 AGUUCCC CUGAUGAGGCCGAAAGGCCGAA ACCGAAG 543 1494 AAACUCU CUGAUGAGGCCGAAAGGCCGAA AGUUGUC 544 1500 CUGCUGA CUGAUGAGGCCGAAAGGCCGAA ACUCUGA 545 1501 GCUGCUG CUGAUGAGGCCGAAAGGCCGAA AACUCUG 546 1502 AGCUGCU CUGAUGAGGCCGAAAGGCCGAA AAACUCU 547 1525 ACACAGG CUGAUGAGGCCGAAAGGCCGAA AUGCACC 548 1566 UUCAGGG CUGAUGAGGCCGAAAGGCCGAA ACUCCAU 549 1577 CGAGUUA CUGAUGAGGCCGAAAGGCCGAA AGCUUCA 550 1579 GGCGAGU CUGAUGAGGCCGAAAGGCCGAA AUAGCUU 551 1583 ACCAGGC CUGAUGAGGCCGAAAGGCCGAA AGUUAUA 552 1588 CCCUCUC CUGAUGAGGCCGAAAGGCCGAA AGGAGAG 553 1622 GGGGCAG CUGAUGAGGCCGAAAGGCCGAA AGCUGGG 554 1628 CCUACCG CUGAUGAGGCCGAAAGGCCGAA AGCAGGA 555 1648 CAUUGGG CUGAUGAGGCCGAAAGGCCGAA AGCCCCG 556 1660 CUGGGCA CUGAUGAGGCCGAAAGGCCGAA AGGUCAG 557 1663 CACCUGG CUGAUGAGGCCGAAAGGCCGAA AGCAGAG 558 1664 UCACCUG CUGAUGAGGCCGAAAGGCCGAA AAGCAGA 559 1665 ACCUCCG CUGAUGAGGCCGAAAGGCCGAA AAGCGAG 560 1680 GGAGGAG CUGAUGAGGCCGAAAGGCCGAA AGUCUUC 561 1681 UGGAGGA CUGAUGAGGCCGAAAGGCCGAA AAGUCUU 562 1683 AAUGGAG CUGAUGAGGCCGAAAGGCCGAA AGAAGUC 563 1686 CGCAAUG CUGAUGAGGCCGAAAGGCCGAA AGGAGAA 564 1690 UGUCCGC CUGAUGAGGCCGAAAGGCCGAA AUGGAGG 565 1704 AGCAGAG CUGAUGAGGCCGAAAGGCCGAA AGUCCAU 566 1705 GAGCAGA CUGAUGAGGCCGAAAGGCCGAA AAGUCCA 567 1707 AAGAGCA CUGAUGAGGCCGAAAGGCCGAA AGAAGUC 568 1721 CUGAUCU CUGAUGAGGCCGAAAGGCCGAA ACUCAAA 569 1726 AGGAGCU CUGAUGAGGCCGAAAGGCCGAA AUCUGAC 570 1731 ACCUUAG CUGAUGAGGCCGAAAGGCCGAA AGCUGAU 571 1734 AGCACCU CUGAUGAGGCCGAAAGGCCGAA AGGAGCU 572 1754 CUCUUGG CUGAUGAGGCCGAAAGGCCGAA AGCACUG 573

[0073] 5 TABLE V Human rel A HH Ribozyme Sequences nt. Sequence HH Ribozyme Sequence SEQ ID NO. 19 UACAGAC CUGAUGAGGCCGAAAGGCCGAA AGCCAUU 574 22 CACUACA CUGAUGAGGCCGAAAGGCCGAA ACGAGCC 575 26 CGUGCAC CUGAUGAGGCCGAAAGGCCGAA ACAGACG 576 93 GAGGGGG CUGAUGAGGCCGAAAGGCCGAA ACAGUUC 577 94 UGAGGGG CUGAUGAGGCCGAAAGGCCGAA AACAGUU 578 100 GGAAGAU CUGAUGAGGCCGAAAGGCCGAA AGGGGGA 579 103 CCGGGAA CUGAUGAGGCCGAAAGGCCGAA AUGAGGG 580 105 UGCCGGG CUGAUGAGGCCGAAAGGCCGAA AGAUGAG 581 106 CUGCCGG CUGAUGAGGCCGAAAGGCCGAA AAGAUGA 582 129 GGGGCCA CUGAUGAGGCCGAAAGGCCGAA AGGCCUG 583 138 CUCCACA CUGAUGAGGCCGAAAGGCCGAA AGGGGCC 584 148 GCUCAAU CUGAUGAGGCCGAAAGGCCGAA AUCUCCA 585 151 GCUGCUC CUGAUGAGGCCGAAAGGCCGAA AUGAUCU 586 180 GUAGCGG CUGAUGAGGCCGAAAGGCCGAA AGCGCAU 587 181 UGUAGCG CUGAUGAGGCCGAAAGGCCGAA AAGCGCA 588 186 GCACUUG CUGAUGAGGCCGAAAGGCCGAA AGCGGAA 589 204 GCCCGCG CUGAUGAGGCCGAAAGGCCGAA AGCGCCC 590 217 CGCCUGG CUGAUGAGGCCGAAAGGCCGAA AUGCUGC 591 239 UUGGUGG CUGAUGAGGCCGAAAGGCCGAA AUCUGUG 592 262 UGAUCUU CUGAUGAGGCCGAAAGGCCGAA AUGGUGG 593 268 AGCCAUU CUGAUGAGGCCGAAAGGCCGAA AUCUUGA 594 276 UCCUGUG CUGAUGAGGCCGAAAGGCCGAA AGCCAUU 595 301 CCAGGGA CUGAUGAGGCCGAAAGGCCGAA AUGCGCA 596 303 GACCAGG CUGAUGAGGCCGAAAGGCCGAA AGAUGCG 597 310 CCUUGGU CUGAUGAGGCCGAAAGGCCGAA ACCAGGG 598 323 CGGUGAG CUGAUGAGGCCGAAAGGCCGAA AGGGUCC 599 326 GGCCGGU CUGAUGAGGCCGAAAGGCCGAA AGGAGGG 600 335 UGGGGGU CUGAUGAGGCCGAAAGGCCGAA AGGCCGG 601 349 UUCCUAC CUGAUGAGGCCGAAAGGCCGAA AGCUCGU 602 352 CCUUUCC CUGAUGAGGCCGAAAGGCCGAA ACAAGCU 603 375 CUCAUAG CUGAUGAGGCCGAAAGGCCGAA AGCCAUC 604 376 CCUCAUA CUGAUGAGGCCGAAAGGCCGAA AAGCCAU 605 378 AGCCUCA CUGAUGAGGCCGAAAGGCCGAA AGAAGCC 606 391 CCGGGCA CUGAUGAGGCCGAAAGGCCGAA AGCUCAG 607 409 AACUGUG CUGAUGAGGCCGAAAGGCCGAA AUGCAGC 608 416 UUCUGGA CUGAUGAGGCCGAAAGGCCGAA ACUGUGG 609 417 GUUCUGG CUGAUGAGGCCGAAAGGCCGAA AACUGUG 610 418 GGUUCUG CUGAUGAGGCCGAAAGGCCGAA AAACUGU 611 433 CACACUG CUGAUGAGGCCGAAAGGCCGAA AUUCCCA 612 467 UGACUGA CUGAUGAGGCCGAAAGGCCGAA AGCCUGC 613 469 GCUGACU CUGAUGAGGCCGAAAGGCCGAA AUAGCCU 614 473 AUGCGCU CUGAUGAGGCCGAAAGGCCGAA ACUGAUA 615 481 UGGUCUG CUGAUGAGGCCGAAAGGCCGAA AUGCGCU 616 501 AACUUGG CUGAUGAGGCCGAAAGGCCGAA AGGGGUU 617 502 GAACUUG CUGAUGAGGCCGAAAGGCCGAA AAGGGGU 618 508 CUAUAGG CUGAUGAGGCCGAAAGGCCGAA ACUUGAA 619 509 UCUAUAG CUGAUGAGGCCGAAAGGCCGAA AACUUGG 620 512 UCUUCUA CUGAUGAGGCCGAAAGGCCGAA AGGAACU 621 514 GCUCUUC CUGAUGAGGCCGAAAGGCCGAA AUAGGAA 622 534 CAGGUCG CUGAUGAGGCCGAAAGGCCGAA AGUCCCC 623 556 GGAAGCA CUGAUGAGGCCGAAAGGCCGAA AGCCGCA 624 561 CACCUGG CUGAUGAGGCCGAAAGGCCGAA AGCAGAG 625 562 UCACCUG CUGAUGAGGCCGAAAGGCCGAA AAGCAGA 626 585 CCUGCCU CUGAUGAGGCCGAAAGGCCGAA AUGGGUC 627 598 GCAGGCG CUGAUGAGGCCGAAAGGCCGAA AGGGGCC 628 613 GAGGAAG CUGAUGAGGCCGAAAGGCCGAA ACAGGCG 629 616 GAUGAGG CUGAUGAGGCCGAAAGGCCGAA AGGACAG 630 617 GGAUGAG CUGAUGAGGCCGAAAGGCCGAA AAGGACA 631 620 AUGGGAU CUGAUGAGGCCGAAAGGCCGAA AGGAAGG 632 623 AAGAUGG CUGAUGAGGCCGAAAGGCCGAA AUGAGGA 633 628 UGUCAAA CUGAUGAGGCCGAAAGGCCGAA AUCGGAU 634 630 AUUGUCA CUGAUGAGGCCGAAAGGCCGAA AGAUGGG 635 631 GAUUGUC CUGAUGAGGCCGAAAGGCCGAA AAGAUGG 636 638 GGGGCAC CUGAUGAGGCCGAAAGGCCGAA AUUGUCA 637 661 AGAUCUU CUGAUGAGGCCGAAAGGCCGAA AGCUCGG 638 667 CUCGGCA CUGAUGAGGCCGAAAGGCCGAA AUCUUGA 639 687 GCUGCCA CUGAUGAGGCCGAAAGGCCGAA AGUUUCG 640 700 CCCCACC CUGAUGAGGCCGAAAGGCCGAA AGGCAGC 641 715 GUAGGAA CUGAUGAGGCCGAAAGGCCGAA AUCUCAU 642 717 CAGUAAG CUGAUGAGGCCGAAAGGCCGAA AGAUCUC 643 718 ACAGUAG CUGAUGAGGCCGAAAGGCCGAA AAGAUCU 644 721 CACACAG CUGAUGAGGCCGAAAGGCCGAA AGGAAGA 645 751 ACACCUC CUGAUGAGGCCGAAAGGCCGAA AUGUCCU 646 759 CGUGAAA CUGAUGAGGCCGAAAGGCCGAA ACACCUC 647 761 CCCGUGA CUGAUGAGGCCGAAAGGCCGAA AUACACC 648 762 UCCCGUG CUGAUGAGGCCGAAAGGCCGAA AAUACAC 649 763 GUCCCGU CUGAUGAGGCCGAAAGGCCGAA AAAUACA 650 792 CGAAAAG CUGAUGAGGCCGAAAGGCCGAA AGCCUCG 651 795 UUGCGAA CUGAUGAGGCCGAAAGGCCGAA AGGAGCC 652 796 CUUGCGA CUGAUGAGGCCGAAAGGCCGAA AAGGAGC 653 797 GCUUGCG CUGAUGAGGCCGAAAGGCCGAA AAAGGAG 654 798 AGCUUGC CUGAUGAGGCCGAAAGGCCGAA AAAAGGA 655 829 GGAACAC CUGAUGAGGCCGAAAGGCCGAA AUGGCCA 656 834 GGUCCGG CUGAUGAGGCCGAAAGGCCGAA ACACAAU 657 835 GGGUCCG CUGAUGAGGCCGAAAGGCCGAA AACACAA 658 845 GCGUAGG CUGAUGAGGCCGAAAGGCCGAA AGGGGUC 659 849 GUCUGCG CUGAUGAGGCCGAAAGGCCGAA AGGGAGG 660 872 CGCACAG CUGAUGAGGCCGAAAGGCCGAA AGCCUGC 661 883 GCAUGGA CUGAUGAGGCCGAAAGGCCGAA ACACGCA 662 885 CUGCAUG CUGAUGAGGCCGAAAGGCCGAA AGACACG 662 905 CGGUCGG CUGAUGAGGCCGAAAGGCCGAA AGGCCGC 664 906 CCGGUCG CUGAUGAGGCCGAAAGGCCGAA AAGGCCG 665 919 GCUCAGU CUGAUGAGGCCGAAAGGCCGAA AGCUCCC 666 936 GUACUGG CUGAUGAGGCCGAAAGGCCGAA AUUCCAU 667 937 GGUACUG CUGAUGAGGCCGAAAGGCCGAA AAUUCCA 668 942 UGGCAGG CUGAUGAGGCCGAAAGGCCGAA ACUGGAA 669 953 UCGUCUG CUGAUGAGGCCGAAAGGCCGAA AUCUGGC 670 962 CGGUGAC CUGAUGAGGCCGAAAGGCCGAA AUCGUCU 671 965 AUCCGGU CUGAUGAGGCCGAAAGGCCGAA ACGAUCG 672 973 UCUCCUC CUGAUGAGGCCGAAAGGCCGAA AUCCGGU 673 986 GUCCUUU CUGAUGAGGCCGAAAGGCCGAA AGGUUUC 674 996 GGUCUCA CUGAUGAGGCCGAAAGGCCGAA AUGUCCU 675 1005 GCUCUUG CUGAUGAGGCCGAAAGGCCGAA AGGUCUC 676 1006 UGCUCUU CUGAUGAGGCCGAAAGGCCGAA AAGGUCU 677 1015 UCUUCAU CUGAUGAGGCCGAAAGGCCGAA AUGCUCU 678 1028 CUGAAAG CUGAUGAGGCCGAAAGGCCGAA ACUCUUC 679 1031 CCGCUGA CUGAUGAGGCCGAAAGGCCGAA AGGACUC 680 1032 UCCGCUG CUGAUGAGGCCGAAAGGCCGAA AAGGACU 681 1033 GUCCGCU CUGAUGAGGCCGAAAGGCCGAA AAAGGAC 682 1058 CGAGGUG CUGAUGAGGCCGAAAGGCCGAA AGGCCGG 683 1064 AUGCGUC CUGAUGAGGCCGAAAGGCCGAA AGGUGGA 684 1072 GCACAGC CUGAUGAGGCCGAAAGGCCGAA AUGCGUC 685 1082 CUGCGGG CUGAUGAGGCCGAAAGGCCGAA AGGCACA 686 1083 GCUGCGG CUGAUGAGGCCGAAAGGCCGAA AAGGCAC 687 1092 AGAAGCU CUGAUGAGGCCGAAAGGCCGAA AGCUGCG 688 1097 GGGACAG CUGAUGAGGCCGAAAGGCCGAA AGCUGAG 689 1098 GGGGACA CUGAUGAGGCCGAAAGGCCGAA AAGCUGA 690 1102 GCUUGGG CUGAUGAGGCCGAAAGGCCGAA ACAGAAG 691 1125 AAAGGGA CUGAUGAGGCCGAAAGGCCGAA AGGGCUG 692 1127 GUAAAGG CUGAUGAGGCCGAAAGGCCGAA AUAGGGC 693 1131 UGACGUA CUGAUGAGGCCGAAAGGCCGAA AGGGAUA 694 1132 AUGACGU CUGAUGAGGCCGAAAGGCCGAA AAGGGAU 695 1133 GAUGACG CUGAUGAGGCCGAAAGGCCGAA AAAGGGA 696 1137 CAGGGAU CUGAUGAGGCCGAAAGGCCGAA ACGUAAA 697 1140 GCUCAGG CUGAUGAGGCCGAAAGGCCGAA AUGACGU 698 1153 CAUAGUU CUGAUGAGGCCGAAAGGCCGAA AUGGUGC 699 1158 CUCAUCA CUGAUGAGGCCGAAAGGCCGAA AGUUGAU 700 1167 GGUGGGA CUGAUGAGGCCGAAAGGCCGAA ACUCAUC 701 1168 UGGUGGG CUGAUGAGGCCGAAAGGCCGAA AACUCAU 702 1169 AUGGUGG CUGAUGAGGCCGAAAGGCCGAA AAACUCA 703 1182 AGAAGGA CUGAUGAGGCCGAAAGGCCGAA ACACCAU 704 1183 CAGAAGG CUGAUGAGGCCGAAAGGCCGAA AACACCA 705 1184 CCAGAAG CUGAUGAGGCCGAAAGGCCGAA AAACACC 706 1187 UGCCCAG CUGAUGAGGCCGAAAGGCCGAA AAGAAAC 707 1188 CUGCCCA CUGAUGAGGCCGAAAGGCCGAA AAGGAAA 708 1198 CCUGGCU CUGAUGAGGCCGAAAGGCCGAA AUCUGCC 709 1209 GAAGGCC CUGAUGAGGCCGAAAGGCCGAA AGGCCUG 710 1215 CGGGGCC CUGAUGAGGCCGAAAGGCCGAA AGGCCGA 711 1229 ACUUGGG CUGAUGAGGCCGAAAGGCCGAA AGGGGCC 712 1237 GGGGCAG CUGAUGAGGCCGAAAGGCCGAA ACUUGGG 713 1250 GGGGCUG CUGAUGAGGCCGAAAGGCCGAA AGCCUGG 714 1268 AUGGCUG CUGAUGAGGCCGAAAGGCCGAA AGCAGGG 715 1279 GAGCUGA CUGAUGAGGCCGAAAGGCCGAA ACCAUGG 716 1281 CAGAGCU CUGAUGAGGCCGAAAGGCCGAA AUACCAU 717 1286 UGGGCCA CUGAUGAGGCCGAAAGGCCGAA AGCUGAU 718 1309 GGACUGG CUGAUGAGGCCGAAAGGCCGAA ACAGGGG 719 1315 GGGCUAG CUGAUGAGGCCGAAAGGCCGAA ACUGGGA 720 1318 CUGGGGC CUGAUGAGGCCGAAAGGCCGAA AGGACUG 721 1331 GCCUGAG CUGAUGAGGCCGAAAGGCCGAA AGGGCCU 722 1334 ACAGCCU CUGAUGAGGCCGAAAGGCCGAA AGGAGGG 723 1389 GGCCUCU CUGAUGAGGCCGAAAGGCCGAA ACAGCGU 724 1413 AUCAUCA CUGAUGAGGCCGAAAGGCCGAA ACUGCAG 725 1414 CAUCAUC CUGAUGAGGCCGAAAGGCCGAA AACUGCA 726 1437 GCCAAGC CUGAUGAGGCCGAAAGGCCGAA AGGCCCC 727 1441 UGUUGCC CUGAUGAGGCCGAAAGGCCGAA AGCAAGG 728 1467 GUCUGUG CUGAUGAGGCCGAAAGGCCGAA ACACAGC 729 1468 GGUCUGU CUGAUGAGGCCGAAAGGCCGAA AACACAG 730 1482 GUCGACG CUGAUGAGGCCGAAAGGCCGAA AUGCCAG 731 1486 AGUUGUC CUGAUGAGGCCGAAAGGCCGAA ACGGAUG 732 1494 AAACUCG CUGAUGAGGCCGAAAGGCCGAA AGUUGUC 733 1500 CUGCUGA CUGAUGAGGCCGAAAGGCCGAA ACUCGGA 734 1501 GCUGCUG CUGAUGAGGCCGAAAGGCCGAA AACUCGG 735 1502 AGCUGCU CUGAUGAGGCCGAAAGGCCGAA AAACUCG 736 1525 CCACAGG CUGAUGAGGCCGAAAGGCCGAA AUGCCCU 737 1566 CACAGGG CUGAUGAGGCCGAAAGGCCGAA ACUCCAU 738 1577 CGAGUUA CUGAUGAGGCCGAAAGGCCGAA AGCCUCA 739 1579 GGCGAGU CUGAUGAGGCCGAAAGGCCGAA AUAGCCU 740 1583 ACCAGGC CUGAUGAGGCCGAAAGGCCGAA AGUUAUA 741 1588 CUGUCAC CUGAUGAGGCCGAAAGGCCGAA AGGCGAG 742 1622 GGAGCAG CUGAUGAGGCCGAAAGGCCGAA AGCUGGG 743 1628 CCCAGUG CUGAUGAGGCCGAAAGGCCGAA AGCAGGA 744 1648 CAUUGGG CUGAUGAGGCCGAAAGGCCGAA AGCCCCG 745 1660 CUGAAAG CUGAUGAGGCCGAAAGGCCGAA AGGCCAU 746 1663 CUCCUGA CUGAUGAGGCCGAAAGGCCGAA AGGAGGC 747 1664 UCUCCUG CUGAUGAGGCCGAAAGGCCGAA AAGGAGG 748 1665 AUCUCCU CUGAUGAGGCCGAAAGGCCGAA AAAGGAG 749 1680 GGAGGAG CUGAUGAGGCCGAAAGGCCGAA AGUCUUC 750 1681 UGGAGGA CUGAUGAGGCCGAAAGGCCGAA AAGUGUU 751 1683 AAUGGAG CUGAUGAGGCCGAAAGGCCGAA AGAAGUC 752 1686 CGCAAUG CUGAUGAGGCCGAAAGGCCGAA AGGAGAA 753 1690 UGUCCGC CUGAUGAGGCCGAAAGGCCGAA AUGGAGG 754 1704 GGCUGAG CUGAUGAGGCCGAAAGGCCGAA AGUCCAU 755 1705 GGGCUGA CUGAUGAGGCCGAAAGGCCGAA AAGUCCA 756 1707 CAGGGCU CUGAUGAGGCCGAAAGGCCGAA AGAAGUC 757 1721 CUGAUCU CUGAUGAGGCCGAAAGGCCGAA ACUCAGC 758 1726 AGGAGCU CUGAUGAGGCCGAAAGGCCGAA AUCUGAC 759 1731 CCCUUAG CUGAUGAGGCCGAAAGGCCGAA AGCUGAU 760 1734 ACCCCCU CUGAUGAGGCCGAAAGGCCGAA AGGAGCU 761 1754 CUCUGGG CUGAUGAGGCCGAAAGGCCGAA AGGGCAG 762

[0074] 6 TABLE VI Human rel A Hairpin Ribozyme/Target Sequences nt. Seq ID Seq ID Position Hairpin Ribozyme sequence No. Substrate No. 90 UGAGGGGG AGAA GUUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 763 GAACU GUU CCCCCUCA 778 156 GCUGCUUG AGAA GCUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 764 GAGCA GCC CAAGCAGC 779 362 GCCAUCCC AGAA GUCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 765 GGACU GCC GGGAUGGC 780 413 GUUCUGGA AGAA GUGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 766 CCACA GUU UCCAGAAC 781 606 GAAGGACA AGAA GCAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 767 CUGCC GCC UGUCCUUC 782 652 UUGAGCUC AGAA GUGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 768 ACACU GCC GAGCUCAA 783 695 CCCACCGA AGAA GCUG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 769 CAGCU GCC UCGGUGGG 784 853 AGGCUGGG AGAA GCGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 770 ACGCA GAC CCCAGCCU 785 900 GGUCGGAA AGAA GCCG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 771 CGGCG GCC UUCCGACC 786 955 UGACGAUC AGAA GUAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 772 AUACA GAC GAUCGUCA 787 1037 GUCGGUGG AGAA GCUG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 773 CAGCG GAC CCACCGAC 788 1045 GGCCGGGG AGAA GUGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 774 CCACC GAC CCCCGGCC 789 1410 CAUCAUCA AGAA GCAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 775 CUGCA GUU UGAUGAUG 790 1453 ACAGCUGG AGAA GUGC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 776 GCACA GAC CCAGCUGU 791 1471 GAUGCCAG AGAA GUGA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 777 UCACA GAC CUGGCAUC 792

[0075] 7 TABLE VII Mouse rel A Hairpin Ribozyme/Target Sequences nt. Seq. ID Seq. ID Position Hairpin Ribozyme sequence No. Substrate No. 137 GUUGCUUC AGAA GUUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 793 GAACA GCC GAAGCAAC 812 273 GAGAUUCG AGAA GUUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 794 GAACA GUU CGAAUCUC 813 343 GCCAUCCC AGAA GUCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 795 GGACU GCC GGGAUGGC 814 366 GGGCAGAG AGAA GCCU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 796 AGGCU GAC CUCUGCCC 815 633 UUGAGCUC AGAA GUGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 797 ACACU GCC GAGCUCAA 816 676 CCCACCGA AGAA GCUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 798 GAGCU GCC UCGGUGGG 817 834 AGGCUGGG AGAA GCGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 799 ACGCC GAC CCCAGCCU 818 881 GAUCAGAA AGAA GCCG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 800 CGGCG GCC UUCUGAUC 819 1100 AGGUGUAG AGAA GCGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 801 CCGCA GCC CUACACCU 820 1205 GGGCAGAG AGAA GUGC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 802 GCACC GUC CUCUGCCC 821 1361 GGGCUUCC AGAA GCGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 803 ACGCU GUC GGAAGCCC 822 1385 CAGCAUCA AGAA GCAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 804 CUGCA GUU UGAUGCUG 823 1431 ACUCCUGG AGAA GUGC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 805 GCACA GAC CCAGGAGU 824 1449 GAUGCCAG AGAA GUGA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 806 UCACA GAC CUGGCAUC 825 1802 AAGUCGGG AGAA GCUG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 807 CAGCU GCC CCCGACUU 826 2009 UGGCUCCA AGAA GUCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 808 GGACA GAC UGGAGCCA 827 2124 UGGUGUCG AGAA GCAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 809 GUGCU GCC CGACACCA 828 2233 AUUCUGAA AGAA GCCA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 810 UGGCC GCC UUCAGAAU 829 2354 UCAGUAAA AGAA GUCU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 811 AGACA GCC UUUACUGA 830

[0076]

Claims

1. An enzymatic RNA molecule which cleaves rel A mRNA.

2. An enzymatic RNA molecule of claim 1, the binding arms of which contain sequences complementary to the sequences defined 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 III, and IV-VII

4. The enzymatic RNA molecule of claim 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, VS RNA or RNAseP RNA motif.

6. The enzymatic RNA molecule of claim 6, 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 IV, V, VI, and VII.

9. A mammalian cell including an enzymatic RNA molecule of claim 1, 2, or 3.

10. The cell of claim 8, wherein said cell is a human cell.

11. An expression vector including nucleic acid encoding an enzymatic RNA molecule or multiple enzymatic molecules of claim 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 the level of NF-&kgr;B activity by administering to a patient an enzymatic nucleic acid molecule of claim 1, 2, or 3,

15. A method for treatment of a condition related to the level of NF-&kgr;B activity by administering to a patient an expression vector of claim 11.

16. The method of claim 14 or 15, wherein said patient is a human.

17. The method of claim 14 wherein said condition is selected from the group consisting of restenosis, rheumatoid arthritis, asthma, inflammatory or autoimmune disorders, and transplant rejection.

18. The method of claim 15 wherein said condition is selected from the group consisting of restenosis, rheumatoid arthritis, asthma, inflammatory or autoimmune disorders, and transplant rejection.