WO1995031541A2

WO1995031541A2 - Methods and compositions for treatment of restenosis and cancer using ribozymes

Info

Publication number: WO1995031541A2
Application number: PCT/US1995/006368
Authority: WO
Inventors: Dan T. Stinchcomb; Kenneth Draper; James Mcswiggen; Thale Jarvis
Original assignee: Ribozyme Pharmaceuticals, Inc.
Priority date: 1994-05-18
Filing date: 1995-05-18
Publication date: 1995-11-23
Also published as: AU2642295A; MX9605716A; WO1995031541A3; CA2190513A1; US5817796A; EP0763106A1; JPH10500309A; US5646042A

Abstract

An enzymatic nucleic acid molecule which cleaves c-myb RNA, wherein the binding arms of said nucleic acid contain sequences complementary to the sequences defined in Tables II, XII-XXIV.

Description

DESCRIPTION

Methods and Compositions for Treatment of Restenosis and Cancer Using Ribozymes

Background Of The Invention

The present invention concerns therapeutic composi¬ tions and methods for the treatment of restenosis and cancer. The following is a brief description of the physi¬ ology, cellular pathology and treatment of restenosis. 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.

Coronary angioplasty is one of the major surgical treatments for heart disease. Its use has been accelerat¬ ing rapidly; over 450,000 procedures are performed in the U.S. annually. The short term success rate of angioplasty is 80 to 90%. However, in spite of a number of technical improvements in the procedure, post-operative occlusions of the arteries, or restenosis, still occur. Thirty-five to forty-five percent of patients who have undergone a single vessel angioplasty develop clinically significant restenosis within 6 months of the procedure. The rate of restenosis is even higher (50 to 60%) in patients who have undergone multivessel angioplasty (Califf, R. M., et al. , 1990, in Textbook of Interventional Cardiolocry. . E.J. Topol, ed., W. B. Saunders, Philadelphia, pp 363-394.). Histopathological studies have shown that restenosis after angioplasty is characterized by migration of medial smooth muscle cells to the intima and a striking hyper- proliferative response of these neointimal cells (Garratt, K. N., et al., 1991, J^". Am . Coll . Cardio . . 17, 442-428; Austin, G. E., et al. , 1985, J . Am. Coll . Cardiol . . 6, 369-375) . Smooth muscle cell proliferation could be an overly robust response to injury. Alternatively, the intimal smooth muscle cells within atherosclerotic lesions are already in an activated or "synthetic" state (Sjolund, M., et al., 1988, J. Cell . Biol . . 106, 403-413 and thus may be poised to proliferate. One recent study demon- strated a positive correlation between the presence of activated smooth muscle cells in coronary lesions and the extent of subsequent luminal narrowing after atherectomy (Simons, M. , et al. , 1993, New Encrl . J. Med. , 328, 608- 613) . In any case, slowing smooth muscle cell prolifera- tion after angioplasty could prevent intimal thickening and restenosis.

The presently preferred therapeutic treatment for restenosis is the use of streptokinase, urokinase or other thrombolytic compounds, such as fish oil, anticoagulants, ACE (angiotensin converting enzyme) inhibitors, aspirin and cholesterol lowering compounds. Alternative treatment includes the surgical incorporation of endoluminal stents. The occurrence of pharmacologic side-effects (particularly bleeding disorders associated with anti-coagulants and platelet inhibitors) is an issue with current therapies. Popoma, J. J., et al. , report that the current therapies have not significantly impacted the rates of restenosis occurrence. ( Circulation, 84, 1426-1436, 1991) .

Recently, the results of a clinical trial of the efficacy of an anti-platelet therapy have been reported. Patients undergoing coronary angioplasty were given a single bolus injection followed by a 12 hour infusion of an antibody directed against the platelet adhesion mole¬ cule, gpllb/gpllla. After six months, patients with the treatment showed a 23% reduction in the occurrence of restenosis than patients receiving placebo (27 vs. 35%; p=0.001) .

A number of growth factors have been shown to induce smooth muscle cell proliferation. In vi tro, platelet- derived growth factor (PDGF) is a potent smooth muscle cell mitogen (Ross, R. , et al., 1974, Proc . Natl . Acad . Sci . USA, 71, 1207-1210) and a smooth muscle cell chemo- attractant (Grotendorst, G. , et al. , 1982, Proc . Natl . Acad. Sci . USA, 71, 3669-3672.) . In vivo, when PDGF is expressed ectopically in porcine arteries, it induces intimal hyperplasia (Nabel, E. B., et al. , 1993, J. Clin . Invest . . 91, 1822-1829) . Furthermore, antibodies to PDGF have been shown to reduce intimal thickening after arterial injury (Ferns, G. A. A., et al., 1991, Science . 253, 1129-1132) . Analysis of ³H-thymidine incorporation in the lesions indicates that the anti-PDGF antibodies primarily inhibit smooth muscle cell migration.

Basic fibroblast growth factor (bFGF) is another smooth muscle cell mitogen in vi tro (Klagsbrun, M. and Edelman, E. R., 1989, Arteriosclerosis . 9, 269-278) . In a rat model, anti-bFGF antibodies inhibit the prolifera- tion of medial smooth muscle cells 24 to 48 hours after balloon catheter injury (Lidner, V. and Reidy, M. A. ,

1991, Proc . Na tl . Acad . Sci . USA. 88, 3739-3743) . In addition to bFGF, heparin binding epidermal growth factor

(HB-EGF) (Higashiyama, S., et al. , 1991, Science, 251, 936-939.), insulin-like growth factor I (IGF-I) (Banskota, N. K., et al., 1989, Mol ec. Endocrinol . . 3, 1183-1190) and endothelin (Komuro, I., et al. , 1988, FEBS Letters . 238, 249-252) have been shown to induce smooth muscle cell pro¬ liferation. A number of other factors (such as inter- leukin-1 and tumor necrosis factor-α) may indirectly affect smooth muscle cell proliferation by inducing the expression of PDGF (Hajjar, K. A., et al. , 1987, J. Exp . Med.. 166, 235-245; Raines, E. . , et al. , 1989, Science. 243, 393-396) . When whole serum is added to serum-starved smooth muscle cells in vitro, the oncogenes, c-myc, c-fos, and c- myb, are induced (Kindy, M. S. and Sonenshein, G. E., 1986, J. Biol . Chem . , 261, 12865-12868; Brown, K. E., et al., 1992, J. Biol . Chem. , 267, 4625-4630) and cell pro- liferation ensues. Blocking c-myb with an antisense oligonucleotide prevents cells from entering S phase (Brown, K. E., et al. , 1992, J. Biol . Chem. . 267, 4625- 4630.) . Thus, c-myb is required for the G to S transition after stimulation by the multitude of growth factors present in serum. In vivo, a c-iτιyj antisense oligonucleo- tide inhibits restenosis when applied to rat arteries after balloon angioplasty (Simons, M. , et al. , 1992, Nature. 359, 67-70) . Similarly, an antisense oligonucleo- tide directed against mRNA of the oncogene c-myc was shown to inhibit human smooth muscle cell proliferation (Shi, Y., et al., 1993, Circulation. 88, 1190-5) and migration (Biro, S., et al. , 1993, Proc. Natl . Acad. Sci . U S A. 90, 654-8) .

Ohno et al. , 1994 Science 265, 781, have shown that a combination of viral thymidine kinase enzyme expression (gene therapy) and treatment with anti-viral drug ganci- clovir inhibits smooth muscle cell proliferation in pigs, following baloon angioplasty.

Epstein et al. , "Inhibition of non-transformed cell proliferation using antisense oligonucleotides, " NTIS publication 1992 discusses use of antisense oligonucleo- tides to c-myc, PCNA or cyclin B. Fung et al. , PCT WO91/15580, describes gene therapy for cell proliferative disease and mentions administration of a ribozyme con¬ struct against a PGR element. Mention is made of inacti¬ vation of c-myb. Rosenberg et al., WO93/08845, Calabretta et al., WO92/20348 and Gewirtz WO93/09789 concern c-myb antisense oligonucleotides for treatment of melanoma or colorectal cancer, and administration locally. Sytkowski, PCT WO 93/02654, describe the uses of antisense oligo¬ nucleotides to inhibit c-myb gene expression in red blood cells to stimulate hemoglobin synthesis.

Nabel and Nabel, U. S. Patent No. 5, 328, 470, describe a method for the treatment of diseases by delivering therapeutic reagents directly to the sites of disease. They state that- "...Method is based on the delivery of proteins by catheterization to discrete blood vessel seg¬ ments using genetically modified or normal cells or other vector systems... In addition, cata¬ lytic RNAs, called ribozymes, can specifically degrade RNA sequences.... The requirements for a successful RNA cleavage include a hammerhead structure with conserved RNA sequence at the region flanking this structure any GUG sequence within the RNA transcript can serve as a target for degradation by the ribozyme.... gene transfer using vectors expressing such proteins as tPA for the treatment of thrombosis and restenosis, angiogenesis or growth factors for the purpose of revascularization... " Sullivan and Draper, International PCT publication WO 94/02595 describe the use of ribozymes against c-myb RNA to treat stenosis.

Summary Of The Invention

This invention relates to ribozymes, or enzymatic RNA molecules, directed to cleave mRNA species that are required for cellular growth responses. In particular, applicant describes the selection and function of ribo¬ zymes capable of cleaving RNA encoded by the oncogene, c- myb. Such ribozymes may be used to inhibit the hyper- proliferation of smooth muscle cells in restenosis and of tumor cells in numerous cancers. To block restenosis, a target molecule required for the induction of smooth muscle cell proliferation by a number of different growth factors is preferred. To this end c-myc, c-fos, and c-myb are useful targets in this invention. Other transcription factors involved in the response to growth and proliferation signals include NF-KB, oct-1 and SRF. NF-/.B protein activates cellular transcription and induces increases in cellular synthetic pathways. In a resting cell, this protein is found in the cytoplasm, complexed with its inhibitor, 1-κB. Upon phosphorylation of the I-κB molecule, the complex dissociates and NF-KB is released for transport to the nucleus, where it binds DNA and induces transcriptional activity in (NF-KB) -responsive genes. One of the (NF-.B) -responsive genes is the NF-κB gene itself. Thus, release of the NF-κB protein from the inhibitory complex results in a cascade of gene expression which is auto-induced. Early inhibition of NF-κB can reduce expression of a number of genes required for growth and proliferation, such as c-myb.

Two other transcription factors, oct-1 and serum response factor (SRF) have been shown to be expressed selectively in dividing cells. Both oσt-1 and SRF are expressed ubiquitously in cultured cells, including smooth muscle cells. However, R. Majack and his colleagues have recently shown that these transcription factors are not expressed by the smooth muscle cells in intact vessels. Both oct-1 and SRF are rapidly expressed upon dispersal of tissue into single cell suspensions. Thus, these tran¬ scription factors are thought to be regulated by their interactions with the extracellular matrix (Weiser, M. C. M., et al., 1994, J. Cell . Biochem. . S18A, 282; Belknap, J. K-, et al., 1994, J. Cell . Biochem. . S18A, 277) . Upon injury during angioplasty, the expression of oct-1 and SRF may be enhanced, leading to increased smooth muscle cell proliferation. Treatment with ribozymes that block the expression of these transcription factors can alleviate the smooth muscle cell proliferation associated with restenosis.

While some of the above mentioned studies demon¬ strated that antisense oligonucleotides can efficiently reduce the expression of factors required for smooth muscle cell proliferation, enzymatic RNAs, or ribozymes have yet to be demonstrated to inhibit smooth muscle cell proliferation. Such ribozymes, with their catalytic activity and increased site specificity (as described below) , represent more potent and safe therapeutic mole- cules than antisense oligonucleotides. In the present invention, ribozymes that cleave c-myb mRNA are described. Moreover, applicant shows that these ribozymes are able to inhibit smooth muscle cell proliferation and that the catalytic activity of the ribozymes is required for their inhibitory effect. From those of ordinary skill in the art, it is clear from the examples described, that other ribozymes that cleave target mRNAs required for smooth muscle cell proliferation may be readily designed and are within the invention.

By "inhibit" is meant that the activity of c- yb or level of mRNAs encoded by c- yb is reduced below that observed in the absence of the nucleic acid, particularly, inhibition with ribozymes and preferably is below that level observed in the presence of an inactive RNA molecule able to bind to the same site on the mRNA, but unable to cleave that RNA. By "enzymatic nucleic acid molecule" it is meant a nucleic acid molecule which has complementarity in a sub¬ strate binding region to a specified gene target, and also has an enzymatic activity which is active to specifically cleave RNA in that target. That is, the enzymatic nucleic acid molecule is able to intermolecularly cleave RNA and thereby inactivate a target RNA molecule. This complemen¬ tarity functions to allow sufficient hybridization of the enzymatic nucleic acid molecule to the target RNA to allow the cleavage to occur. One hundred percent complemen- tarity is preferred, but complementarity as low as 50-75% may also be useful in this invention. By "equivalent" RNA to c-myb is meant to include those naturally occurring RNA molecules associated with restenosis and cancer in various animals, including human, rat and pig. Such a molecule will generally contain some ribonucleotides, but the other nucleotides may be substituted at the 2' -hydroxyl position and in other locations with other moeities as discussed below.

By "complementarity" is meant a nucleic acid that can form hydrogen bond(s) with other RNA sequence by either traditional Watson-Crick or other non-traditional types (for example, Hoogsteen type) of base-paired interactions. Six basic varieties of naturally-occurring enzymatic RNAs are known presently. Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions. Table I summarizes some of the characteristics of these ribozymes. In general, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base- pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets. The enzymatic nature of a ribozyme is advantageous over other technologies, such as antisense technology (where a nucleic acid molecule simply binds to a nucleic acid target to block its translation) since the concentra¬ tion of ribozyme necessary to affect a therapeutic treat- ment 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 mis¬ matches, 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 . , EP0360257, Hampel and Tritz, 1989 Biochemistry 28, 4929, and Hampel et al . , 1990 Nucleic Acids Res . 18, 299, and an example of the hepatitis delta virus motif is described by Perrotta and Been, 1992 Biochemistry 31, 16; of the RNaseP motif by Guerrier-Takada et al . , 1983 Cell 35, 849, Neurospora VS RNA ribozyme motif is described by Collins (Saville and Collins, 1990 Cell 61, 685-696; Saville and Collins, 1991 Proc . Natl . Acad. Sci . USA 88, 8826-8830; Collins and Olive, 1993 Biochemistry 32, 2795-2799) and of the Group I intron by Cech et al . , U.S. 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.

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

Synthesis of nucleic acids greater than 100 nucleo- tides 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 mole- cules increases the ability of the enzymatic nucleic acid to invade targeted regions of the mRNA structure. However, these catalytic RNA molecules can also be expressed within cells from eukaryotic promoters (e.g., Scanlon et al. , 1991, Proc. Natl . Acad. Sci . USA, 88, 10591-5; Kashani-Sabet et al. , 1992 Antisense Res . Dev. , 2, 3-15; Dropulic et al. , 1992 J. Virol , 66, 1432-41; Weerasinghe et al. , 1991 J. Virol , 65, 5531-4; Ojwang et al . , 1992 Proc . Natl . Acad. Sci . USA 89, 10802-6; Chen et al . , 1992 Nucleic Acids Res . , 20, 4581-9; Sarver et al . , 1990 Science 247, 1222-1225) . Those skilled in the art realize that any ribozyme can be expressed in eukary¬ otic cells from the appropriate DNA/RNA vector. The activity of such ribozymes can be augmented by their release from the primary transcript by a second ribozyme (Draper et al. , PCT W093/23569, and Sullivan et al. , PCT WO94/02595, both hereby incorporated in their totality by reference herein; Ohkawa et al. , 1992 Nucleic Acids Symp. Ser. , 27, 15-6; Taira et al. , 1991, Nucleic Acids Res., 19, 5125-30; Ventura et al., 1993 Nucleic Acids Res . , 21, 3249-55; Chowrira et al. , 1994 J. Biol. Chem. 269, 25856) . Thus, in a first aspect, the invention features ribozymes that inhibit cell proliferation. These chemic¬ ally 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 mole¬ cules are preferably ribozymes of the hammerhead or hairpin motif. Upon binding, the ribozymes cleave the target mRNAs, preventing translation and protein accumula¬ tion. In the absence of the expression of the target gene, cell proliferation is inhibited. In a preferred embodiment, the enzymatic RNA mole¬ cules cleave c-myb mRNA and inhibit smooth muscle cell proliferation. Such ribozymes are useful for the preven¬ tion of restenosis after coronary angioplasty. Ribozymes are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to smooth muscle 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. The ribozymes, simi- larly delivered, also are useful for inhibiting prolifer¬ ation of certain cancers associated with elevated levels of the c-myb oncogene, particularly leukemias, neuro- blastomas, and lung, colon, and breast carcinomas. Using the methods described herein, other enzymatic RNA mole- cules that cleave c-myb, c-myc, oct-1, SRF, NF-.B, PDGF receptor, bFGF receptor, angiotensin II, and endothelium- derived relaxing factor and thereby inhibit smooth muscle cell proliferation and/or tumor cell proliferation may be derived and used as described above. Specific examples are provided below in the Tables.

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 c- myb activity in a cell or tissue. By "related" is meant that the inhibition of c-myb mRNAs and thus reduction in the level of protein activity will relieve to some extent the symptoms of the disease or condition.

Ribozymes are added directly, or can be complexed with cationic lipids, packaged within liposomes, or other- wise delivered to target cells. The nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex vivo, or in vivo through injection, infusion pump or stent, with or without their incorporation in biopolymers.

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

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

In preferred embodiments, the ribozymes have binding arms which are complementary to the sequences in the tables II, XII-XXIV. Examples of such ribozymes are shown as Seq. I.D. Nos. 101-129 (table III) and in tables XII- XXIV. By complementary is thus meant that the binding arms are able to cause cleavage of a human or mouse or rat or porcine mRNA target. Examples of such ribozymes con¬ sist essentially of sequences defined in tables III, XII- XXIV. 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 c-myb 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 cell proliferation 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 smooth muscle cells. Once expressed, the ribozymes cleave their target mRNAs and prevent proliferation of their host cells. The recombinant vectors are preferably DNA plas- mids 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 pre- ferred 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.

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 (1987, 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 diagrammatic representation of the general structure of a hairpin ribozyme. Helix 2 (H2) is provided with a least 4 base pairs ( i . e . , n is 1, 2, 3 or 4) and helix 5 can be optionally provided of length 2 or more bases (preferably 3 - 20 bases, i.e., m is from 1 - 20 or more) . Helix 2 and helix 5 may be covalently linked by one or more bases (i.e., r is ≥ 1 base) . Helix 1, 4 or 5 may also be extended by 2 or more base pairs ( e . g. , 4 - 20 base pairs) to stabilize the ribozyme structure, and preferably is a protein binding site. In each instance, each N and N' independently is any normal or modified base and each dash represents a potential base-pairing inter¬ action. These nucleotides may be modified at the sugar, base or phosphate. Complete base-pairing is not required in the helices, but is preferred. Helix 1 and 4 can be of any size ( i . e . , o and p is each independently from 0 to any number, e . g. , 20) as long as some base-pairing is maintained. Essential bases are shown as specific bases in the structure, but those in the art will recognize that one or more may be modified chemically (abasic, base, sugar and/or phosphate modifications) or replaced with another base without significant effect. Helix 4 can be formed from two separate molecules, i . e . , without a con- neeting loop. The connecting loop when present may be a ribonucleotide with or without modifications to its base, sugar or phosphate. "q" is ≥ 2 bases. The connecting loop can also be replaced with a non-nucleotide linker molecule. H refers to bases A, U, or C. Y refers to pyrimidine bases. " " refers to a covalent bond.

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 self-cleaving VS RNA ribozyme domain.

Figure 6 is a schematic representation of an RNAseH accessibility assay. Specifically, the left side of Figure 6 is a diagram of complementary DNA oligonucleo¬ tides bound to accessible sites on the target RNA. Complementary DNA oligonucleotides are represented by broad lines labeled A, B, and C. Target RNA is repre¬ sented by the thin, twisted line. The right side of Figure 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.

Figure 7 is a graph of the results of an RNAseH accessibility assay of murine c-myb RNA. On the abscissa is the sequence number of the DNA oligonucleotide that is homologous to the ribozyme target site. The ordinate represents the percentage of the intact transcript that was cleaved by RNAse H.

Figure 8 is a graph of the outcome of an RNAseH accessibility assay of human c- yb mRNA. The graphs are labeled as in Figure 7.

Figure 9 shows the effect of chemical modifications on the catalytic activity of hammerhead ribozyme targeted to c-myb site 575. A) diagrammatic representation of 575 hammerhead ribozyme»substrate complex. 2'-0-methyl ribo- zyme represents a hammerhead (HH) ribozyme containing 2'- O-methyl substitutions at five nucleotides in the 5' and 3' termini. 2'-0-methyl P=S ribozyme represents a hammer¬ head (HH) ribozyme containing 2'-0-methyl and phosphoro- thioate substitutions at five nucleotides in the 5' and 3' termini. 2'-C-allyl iT ribozyme represents a hammerhead containing ribose residues at five positions. The remain¬ ing 31 nucleotide positions contain 2'-hydroxyl group substitutions, wherein 30 nucleotides contain 2'-0-methyl substitutions and one nucleotide (U₄) contains 2'-C-allyl substitution. Additionally, 3' end of this ribozyme con¬ tains a 3' -3' linked inverted T. 2'-C-allyl P=S ribozyme is similar to 2'-C-allyl iT ribozyme with the following changes: five nucleotides at the 5' and 3' termini contain phosphorothioate substitutions and the ribozyme lacks the 3' -end inverted T modification. B) shows the ability of ribozymes described in Fig. 9A to inhibit smooth muscle cell proliferation. Figure 10 shows the effect of 2'-C-allyl P=S 575 HH ribozyme concentration on smooth muscle cell prolifera¬ tion. A plot of percent inhibition of smooth muscle cell proliferation (normalized to the effect of a catalytically inactive ribozyme) as a function of ribozyme concentration is shown.

Figure 11 shows a comparison of the effects of 2'-C- allyl P=S 575 HH ribozyme and phosphorothioate antisense DNA on the proliferation of smooth muscle cells. Figure 12 shows the inhibition of smooth muscle cell proliferation catalyzed by 2'-C-allyl P=S HH ribozymes targeted to sites 549, 575, and 1533 within c-myb mRNA.

Figure 13 shows the effect of phosphorthioate substi¬ tutions on the catalytic activity of 2'-C-allyl 575 HH ribozyme. A) diagrammatic representation of 575 hammer¬ head ribozyme»substrate complex. 10 P=S 5' and 3' ribozyme is identical to the 2'-C-allyl P=S ribozyme described in Fig. 9. 5 P=S 3' ribozyme is same as 10 P=S 5' and 3' ribozyme, with the exception that only five nucleotides at the 3' termini contain phosphorothioate substitutions. 5 P=S Loop ribozyme is similar to 2'-C- allyl iT described in Fig. 9, with the exception that five nucleotides within loop II of this ribozyme contain phosphorothioate substitutions. 5 P=S 5' ribozyme is same as 10 P=S 5' and 3' ribozyme, with the exception that only five nucleotides at the 5' termini contain phosphoro¬ thioate substitutions. Additionally, this ribozyme con¬ tains a 3' -3' linked inverted T at its 3' end. B) shows the ability of ribozymes described in Fig. 13A to inhibit smooth muscle cell proliferation.

Figure 14 shows the minimum number of phosphoro¬ thioate substitutions required at the 5' termini of 575 HH ribozyme to achieve efficient inhibition of smooth muscle cell proliferation. Figure 15 shows the effect of varying the length of substrate binding arm of 575 HH ribozyme on the inhibition of smooth muscle cell proliferation. Figure 16 shows the effect of various chemical modi¬ fications, at U₄ and/or U₇ positions within 575 HH ribozyme core, on the ability of the ribozyme to inhibit smooth muscle cell proliferation. Figure 17 shows the inhibition of pig smooth muscle cell proliferation by active c-myb 575 HH ribozyme.

Figure 18 shows the inhibition of human smooth muscle cell proliferation by active c- yb 575 HH ribozyme.

Figure 19 shows ribozyme-mediated inhibition of c-myb expression and cell proliferation.

Figure 20 is digrammatic representation of an optimal c-myb HH ribozyme that can be used to treat diseases like restenosis.

Figure 21 shows the inhibition of Rat smooth muscle cells by 2-5A containing nucleic acids.

Target sites

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 PCT No. PCT/US94/13129, U.S.S.N. 08/152,487, filed 11/12/93, and hereby incor¬ porated 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 vi tro and in vivo, as also described. Such ribozymes can also be optimized and delivered as described therein. While specific examples to mouse RNA are provided, those in the art will recognize that equivalent human RNA targets 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. 18

The sequence of human, pig and murine c-myb mRNAs were screened for optimal ribozyme target sites using a computer folding algorithm. Hammerhead or hairpin ribo¬ zyme cleavage sites were identified. These sites are shown in Tables II and XII-XXIV (All sequences are 5' to 3' in the tables) The nucleotide base position is noted in the Tables as that site to be cleaved by the designated type of ribozyme. While murine, pig and human sequences can be screened and ribozymes thereafter designed, the human targeted sequences are of most utility. However, murine and pig targeted ribozymes may be useful to test efficacy of action of the ribozyme prior to testing in humans. The nucleotide base position is noted in the Tables as that site to be cleaved by the designated type of ribozyme.

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

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

Optimizing Ribozyme Activity

Ribozyme activity can be optimized as described in this application. These include altering the length of the ribozyme binding arms (stems I and III, see Figure 2c) , or chemically synthesizing ribozymes with modifica- tions that prevent their degradation by serum ribo- nucleases (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, US Patent No. 5, 334, 711 which describe various chemical modifications that can be made to the sugar moieties of enzymatic RNA molecules, modifications which enhance their efficacy in cells, and removal of stem II bases to shorten RNA synthesis times and reduce chemical requirements. (All these publications are hereby incor¬ porated by reference herein.) Sullivan, et al . , supra, describes the general methods for delivery of enzymatic RNA molecules. Ribozymes may be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by ionto- phoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres. For some indications, ribozymes may be directly delivered ex vivo to cells or tissues with or without the aforementioned vehicles. Alternatively, the RNA/vehicle combination is locally delivered by direct injection or by use of a catheter, infusion pump or stent. Other routes of delivery include, but are not limited to, intravascular, intramuscular, subcutaneous or joint injec¬ tion, aerosol inhalation, oral (tablet or pill form) , topical, systemic, ocular, intraperitoneal and/or intra- thecal delivery. More detailed descriptions of ribozyme delivery and administration are provided in Sullivan et al . , supra and Draper et al . , supra which have been incorporated by reference herein.

Another means of accumulating high concentrations of a ribozyme(s) within cells is to incorporate the ribozyme- encoding sequences into a DNA or RNA expression vector. Transcription of the ribozyme sequences are driven from a promoter for eukaryotic RNA polymerase I (pol I) , RNA polymerase II (pol II) , or RNA polymerase III (pol III) . Transcripts from pol II or pol III promoters will be expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type will depend on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby. Prokaryotic RNA polymer¬ ase promoters are also used, providing that the prokary- otic 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 Enzymol . , 217, 47-66; Zhou et al. , 1990 Mol . Cell . Biol . , 10, 4529-37) . Several investigators have demonstrated that ribozymes expressed from such promoters can function in mammalian cells (e.g. Kashani-Sabet et al. , 1992 Antisense Res . Dev. , 2, 3-15; Ojwang et al. , 1992 Proc . Natl . Acad. Sci . 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 or alphavirus vectors) .

In a preferred embodiment of the invention, a tran¬ scription unit expressing a ribozyme that cleaves mRNAs encoded by c-myb is inserted into a plasmid DNA vector or an adenovirus or adeno-associated virus DNA viral vector or a retroviral RNA vector. Viral vectors have been used to transfer genes and lead to either transient or long term gene expression (Zabner et al. , 1993 Cell 75, 207; Carter, 1992 Curr. Qpi. Biotech. 3, 533) . The adenovirus vector is delivered as recombinant adenoviral particles.

The DNA may be delivered alone or complexed with vehicles

(as described for RNA above) . The recombinant adenovirus or AAV particles are locally administered to the site of treatment, e.g. , through incubation or inhalation in vivo or by direct application to cells or tissues ex vivo.

In another preferred embodiment, the ribozyme is administered to the site of c- yb expression (e.g., smooth muscle cells) in an appropriate liposomal vesicle.

Examples

Ability Of Exogenouslv-Delivered Ribozymes Directed

Against c-myb To Inhibit Vascular Smooth Muscle Cell

Proliferation The following examples demonstrate the selection of ribozymes that cleave c-myb mRNA. The methods described herein represent a scheme by which ribozymes may be derived that cleave other mRNA targets required for cell division. Also provided is a description of how such ribozymes may be delivered to smooth muscle cells. The examples demonstrate that upon delivery, the ribozymes inhibit cell proliferation in culture. Moreover, no inhibition is observed if mutated ribozymes that are catalytically inactive are applied to the cells. Thus, inhibition requires the catalytic activity of the ribozymes. The cell division assay used represents a model system for smooth muscle cell hyperproliferation in restenotic lesions.

Example 1: Identification of Potential Ribozyme Cleavage Sites in Human c-myb mRNA The sequence of human c-myb mRNA was screened for accessible sites using a computer folding algorithm. Regions of the mRNA that did not form secondary folding structures and contained potential hammerhead ribozyme cleavage sites were identified. These sites are shown in Table II and XII-XXIV Sites are numbered using the sequence numbers from (Westin, E. H. , et al. , 1990, Oncogene, 5, 1117-1124) (GenBank Accession No. X52125) ; the sequence is derived from a longer c-myb cDNA isolate and thus is more representative of the full-length RNA.

Example 2 : Selection of Ribozyme Cleavage Sites in Murine and Human c-mγb mRNA.

To test whether the sites predicted by the computer- based RNA folding algorithm corresponded to accessible sites in c-myb RNA, 41 hammerhead sites were selected for analysis. Ribozyme target sites were chosen by comparing cDNA sequences of mouse and human c-myb (GenBank Accession No. X02774 and GenBank Accession No. X52125, respectively) and prioritizing the sites on the basis of overall nucleo- tide sequence homology. Hammerhead ribozymes were designed that could bind each target (see Figure 2C) and were individually analyzed by computer folding (Jaeger, J. A., et al., 1989, Proc . Na tl . 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 were eliminated from consideration. As noted below, 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.

Example 3 : Screening Ribozyme Cleavage Sites bv RNaseH Protection

Murine and human mRNA was screened for accessible cleavage sites by the method described generally in Draper et al., International PCT publication WO 93/23569, hereby incorporated by reference herein. Briefly, DNA oligo¬ nucleotides representing 41 potential hammerhead ribozyme cleavage sites were synthesized. A polymerase chain reac¬ tion was used to generate a substrate for T7 RNA polymerase transcription from human or murine c-myb cDNA clones. Labeled RNA transcripts were synthesized in vi tro from the two templates. The oligonucleotides and the labeled transcripts were annealed, RNAseH was added and the mixtures were incubated for the designated times at 37° C. Reactions were stopped and RNA separated on sequencing polyacrylamide gels. The percentage of the substrate cleaved was determined by autoradiographic quantitation using a phosphor imaging system. The results are shown in Figures 7 and 8. From these data, 20 hammerhead ribozyme sites were chosen as the most accessible (see Table III) .

Example 4 : Chemical Synthesis and Purification of Ribozymes for Efficient Cleavage of c-mvb RNA Ribozymes of the hammerhead or hairpin motif were designed to anneal to various sites in the mRNA message. The binding arms are complementary to the target site sequences described above. The ribozymes were chemically synthesized. The method of synthesis used followed the procedure for normal RNA synthesis as described in Usman et al . , 1987 J. Am. Chem. Soc. , 109, 7845 and in Scaringe et al., 1990 Nucleic Acids Res . , 18, 5433 and made use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5' -end, and phosphoramidites at the 3 '-end. The average stepwise coupling yields were >98%. Inactive ribozymes were 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 were synthesized in two parts and annealed to reconstruct the active ribozyme (Chowrira and Burke, 1992 Nucleic Acids Res . , 20, 2835-2840) . Ribozymes were also synthe- sized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol . 180, 51) . All ribozymes were modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2' -amino, 2'-C-allyl, 2'-flouro, 2'- O-methyl, 2'-H (for a review see Usman and Cedergren, 1992 TIBS 17, 34) . Ribozymes were purified by gel electro- phoresis using general methods or were 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 used in this study are shown below in Table III.

Example 5: Ribozyme Cleavage of Long Substrate RNA Corresponding to c-mγb mRNA Target

Hammerhead-type ribozymes which were targeted to the murine c-myb mRNA were designed and synthesized to test the cleavage activity at the 20 most accessible sites in in vi tro transcripts of both mouse and human c-myb RNAs. The target sequences and the nucleotide location within the c-myb mRNA are given in Table II. All hammerhead ribozymes were synthesized with binding arm (Stems I and III; see Figure 2C) lengths of seven nucleotides. Two hairpin ribozymes were synthesized to sites 1632 and 2231. The relative abilities of these ribozymes to cleave both murine and human RNAs is summarized in Table II. Ribozymes (1 μM) were incubated with ³²P-labeled substrate RNA (prepared as described in Example 3, approximately 20 nM) for 60 minutes at 37°C using buffers described previ- ously. Intact RNA and cleavage products were separated by electrophoresis through polyacrylamide gels. The percent¬ age of cleavage was determined by Phosphor Imager^® quantitation of bands representing the intact substrate and the cleavage products. Five hammerhead ribozymes (directed against sites

549, 575, 1553, 1597, and 1635) and one hairpin ribozyme

(directed against site 1632) were very active; they cleaved >70% of both murine and human c-myb RNA in 60 minutes. Nine of the hammerhead ribozymes (directed against sites 551, 634, 936, 1082, 1597, 1721, 1724, 1895, and 1943) were intermediate in activity, cleaving > 50% of both murine and human c-myb RNA in 60 minutes. All of the sites cleaved by these active ribozymes were predicted to be accessible to ribozyme cleavage in Table II. Six hammerhead ribozymes and one hairpin ribozyme showed low activity on at least one of the substrates. The observed differences in accessibility between the two species of c- yb RNA demonstrate the sensitivity of ribozyme action to RNA structure and suggest that even when homologous target sequences exist, ribozymes may be excluded from cleaving that RNA by structural constraints. This level of specificity minimizes non-specific toxicity of ribozymes within cells.

Example 6 : Ability of Hammerhead Ribozymes to Inhibit Smooth Muscle Cell Proliferation.

The ribozymes that cleaved c-myb RNA described above were assayed for their effect on smooth muscle cell pro¬ liferation. Rat vascular smooth muscle cells were isolated and cultured as follows. Aortas from adult Sprague-Dawley rats were dissected, connective tissue was removed under a dissecting microscope, and 1 mm² pieces of the vessel were placed, intimal side up, in a Petri dish in Modified Eagle's Medium (MEM) with the following additives: 10% FBS, 2% tryptose phosphate broth, 1% penicillin/streptomycin and 2 mM L-Glutamine. The smooth muscle cells were allowed to migrate and grow to conflu¬ ence over a 3-4 week period. These primary cells were frozen and subsequent passages were grown at 37° C in 5% C0₂ in Dulbecco's modified Eagle's medium (DMEM) , 10% fetal bovine serum (FBS), and the following additives: 2 mM L- Glutamine, 1% penicillin/streptomycin, 1 mM sodium pyruvate, non-essential amino acids (0.1 mM of each amino acid) , and 20 mM Hepes pH 7.4. Cells passed four to six times were used in proliferation assays. For the cell proliferation assays, 24-well tissue culture plates were prepared by coating the wells with 0.2% gelatin and washing once with phosphate-buffered saline (PBS) . RASMC were inoculated at lxlO⁴ cells per well in 1 ml of DMEM plus 10% FBS and additives and incubated for 24 hours. The cells were subconfluent when plated at this density. The cells were serum-starved by removing the medium, washing once with PBS, and incubating 48-72 hours in DMEM containing 0.5% FBS plus additives.

In several other systems, cationic lipids have been shown to enhance the bioavailability of oligonucleotides to cells in culture (Bennet, C. F., et al. , 1992, Mol . Pharmacology . 41, 1023-1033) . In many of the following experiments, ribozymes were complexed with cationic lipids. The cationic lipid, Lipofectamine (a 3:1 (w/w) formulation of DOSPA (2,3-dioleyloxy-N- [2 (sperminecarbox- amido)ethyl] -N,N-dimethyl-l-propanaminium trifluoroace- tate) and dioleoyl phosphatidylethanolamine (DOPE) ) , was purchased from Life Technologies, Inc. DMRIE (N- [l- (2 , 3 - ditetradecyloxy) ropyl] -N, N-dimethyl -N-hydroxyethyl - ammonium bromide) was obtained from VICAL. DMRIE was resuspended in CHC1₃ and mixed at a 1:1 molar ratio with dioleoyl phosphatidylethanolamine (DOPE) . The CHC1₃ was evaporated, the lipid was resuspended in water, vortexed for 1 minute and bath sonicated for 5 minutes. Ribozyme and cationic lipid mixtures were prepared in serum-free DMEM immediately prior to addition to the cells. DMEM plus additives was warmed to room temperature (about 20- 25°C) , cationic lipid was added to the final desired concentration and the solution was vortexed briefly. RNA oligonucleotides were added to the final desired concentration and the solution was again vortexed briefly and incubated for 10 minutes at room temperature. In dose response experiments, the RNA/lipid complex was serially diluted into DMEM following the 10 minute incubation.

Serum-starved smooth muscle cells were washed twice with PBS, and the RNA/lipid complex was added. The plates were incubated for 4 hours at 37°C. The medium was then removed and DMEM containing 10% FBS, additives and 10 μM bromodeoxyuridine (BrdU) was added. In some wells, FBS was omitted to determine the baseline of unstimulated proliferation. The plates were incubated at 37°C for 20- 24 hours, fixed with 0.3% H₂0₂ in 100% methanol, and stained for BrdU incorporation by standard methods. In this procedure, cells that have proliferated and incorporated BrdU stain brown; non-proliferating cells are counter-stained a light purple. Both BrdU positive and BrdU negative cells were counted under the microscope. 300-600 total cells per well were counted. In the following experiments, the percentage of the total cells that have incorporated BrdU (% cell proliferation) is presented. Errors represent the range of duplicate wells. Percent inhibition then is calculated from the % cell proliferation values as follows: % inhibition = 100 - 100 ( (Ribozyme - 0% serum) / (Control - 0% serum)) .

Six hammerhead ribozymes, including the best five ribozymes from the in vi tro RNA cleavage test (directed against sites 549, 575, 1553, 1598, and 1635) and one with intermediate cleavage levels (directed against site 1597) and their catalytically inactive controls were synthesized and purified as described above. The ribozymes were delivered at a concentration of 0.3 μM, complexed with DMRIE/DOPE such that the cationic lipid charges and the anionic RNA charges were at 1:1 molar ratio. The results, shown in Table IV, demonstrate a considerable range in the efficacy of ribozymes directed against different sites. Five of the six hammerhead ribozymes (directed against sites 549, 575, 1553, 1597, and 1598) significantly inhibit smooth muscle cell proliferation. The control, inactive ribozymes that cannot cleave c-myb RNA due to alterations in their catalytic core sequence fail to inhibit rat smooth muscle cell proliferation. Thus, inhibition of cell proliferation by these five hammerhead sequences is due to their ability to cleave c-myb RNA, and not because of any antisense activity. The sixth ribozyme (directed against site 1635) fails to function in smooth muscle cells. This ribozyme cleaved c-myb RNA very efficiently in vitro. In this experiment, 10% FBS (no ribozyme added) induced 64 + 1% proliferation; 0% FBS produced a background of 9 + 1% proliferation.

Example 7 : Ability of exogenouslv delivered hairpin ribozyme against c-mvb to inhibit vascular smooth muscle cell proliferation

In addition to the hammerhead ribozymes tested above, a bipartite hairpin ribozyme (Chowrira, B. M., supra, 1992, Nucleic Acids Res . . 20, 2835-2840)_^ was identified that also cleaves c-myb RNA. The effect of this ribozyme on smooth muscle cell proliferation was tested. Ribozymes were delivered at the indicated doses with Lipofectamine at a 1:1 charge ratio. In this experiment, 10% FBS (no ribozyme) induced 87 ± 1% proliferation; 0% FBS produced 5 + 1% proliferation. The results of a dose-response experiment are shown in Table V. In this example, the control was an irrelevant hammerhead ribozyme. The irrelevant ribozyme control contains the same catalytic core sequences, but has binding arms that are directed to a cellular RNA that is not required for smooth muscle cell proliferation. This control failed to significantly inhibit cell proliferation, demonstrating the sequence specificity of these ribozymes. Another control that could be run is an irrelevant catalytically active ribo- zyme having the same G:C content as the test ribozyme.

Example 8 : Ribozymes inhibit proliferation of rat smooth muscle cells in a dose-dependent fashion.

If the inhibition of proliferation observed in Example 6 is caused by the ribozymes, the level of inhibi¬ tion should be proportional to the dose of RNA added. Rat aortic smooth muscle cells were assayed for proliferation in the presence of differing doses of two hammerhead ribo¬ zymes. The results shown in Table VI indicate that two hammerhead ribozymes that cleave c- yb RNA at sites 575 and 549 inhibit SMC proliferation in a dose-dependent fashion. Ribozymes were delivered with the cationic lipid, Lipofectamine at a 1:1 charge ratio. In this experiment, 10% FBS (no ribozyme) gave 92 + 1% proliferation; 0% FBS gave 6 + 1% proliferation. The con¬ trol is an active ribozyme directed against an irrelevant mRNA target and shows no inhibition over the dose range tested. The control ribozyme contains the same catalytic core sequences as the active ribozymes but differs in its binding arm sequences (stems I and III in Figure 2c) . Thus, ribozyme inhibition of smooth muscle cell prolifera¬ tion requires sequence-specific binding by the hammerhead arms to c-myb mRNA.

Example 9 : Delivery of a c-mvb Ribozyme With Different

Cationic Lipids

The experiment in Table VII shows the response of rat smooth muscle cells to a hammerhead ribozyme that cleaves c-myb RNA at site 575 delivered with two different cationic lipids, DMRIE and Lipofectamine. Similar efficacy is observed with either lipid. 10% FBS (no ribozyme) induced 78 ± 2% proliferation; 0% FBS produced a background of 6 + 1% proliferation.

Example 10: Effect of varying arm-lengths on ribozyme activity.

The exact configuration of each ribozyme can be optimized by altering the length of the binding arms

(stems I and III, see Figure 2C) . The length of the binding arms may have an effect on both the binding and the catalytic cleavage step (Herschlag, D., 1991, Proc . Na tl . Acad . Sci . U S A. 88, 6921-5) . For example, Table VIII shows the ability of arm length variants of c-myb hammerhead 575 to inhibit SMC proliferation. Note that the dose used in this experiment (0.1 μM) is 3-fold lower than in previous experiments. At this concentration, the 7/7 arm variant gives relatively little inhibition. In this case, the degree of inhibition increases with concomitant increases in arm length.

The optimum arm length may be site-specific and should be determined empirically for each ribozyme. Towards this end, hammerhead ribozymes target with 7 nucleotide binding arms (7/7) and ribozymes with 12 nucleotide binding arms (12/12) targeted to three different cleavage sites were compared. Ribozymes were delivered at 0.2 μM with the cationic lipid DMRIE at a 1:1 charge ratio of oligonucleotide to cationic lipid as described in Example 6. The data are shown below in Table IX. As can be seen, all three ribozymes demonstrated enhanced inhibition of smooth muscle cell proliferation with twelve nucleotide binding arms. Each ribozyme showed greater inhibition than its catalytically inactive control, again demonstrating that the ribozymes function via their ability to cleave c-myb RNA. In this experiment, 10% stimulation resulted in 54 ± 2 % cell proliferation; unstimulated cells showed 8 ± 0.5 % cell proliferation. Example 11: Effect of chloroquine on ribozyme activity.

A number of substances that effect the trafficking of macromolecules through the endosome have been shown to enhance the efficacy of DNA delivery to cells. These include, but are not limited to, ammonium chloride, carbonyl cyanide p-trifluoromethoxy phenyl hydrazone (FCCP) , chloroquine, monensin, colchicine, and viral particles (Cotten, M. et al.,1990, Proc . Natl . Acad. Sci

USA , 87, 4033-4037; Cotten, M. et al.,1993, J. Virol . 67, 3777-3785; Cotten, M. et al.,1992, Proc. Natl . Acad

Sci . USA , 89, 6094-6098; Cristiano, R. J. et al.,1993 Proc . Na tl . Acad . Sci . U S A , 90, 2122-6; Curiel, D. T et al.,1991, Proc . Nat . Acad. Sci . USA , 88, 8850-8854 Ege, T. et al.,1984, Exp . Cell Res . , 155, 9-16; Harris C. E. et al.,1993, Am . J. Resvir. Cell Mol . Biol . , 9 441-7; Seth, P. et al.,1994, J. Virol . , 68, 933-40 Zenke, M. et al.,1990, Proc . Natl . Acad. Sci . USA , 87, 3655-3659) . It is thought that DNA is taken up by cells by endocytosis, resulting in DNA accumulation in endosomes (Akhtar, S. and Juliano, R. L.,1992, Trends Cell Biol . , 2, 139-144) . Thus, the above agents may enhance DNA expression by promoting DNA release from endosomes. To determine whether such agents may augment the functional delivery of RNA and ribozymes to smooth muscle cells, the effects of chloroquine on ribozyme inhibition of smooth muscle cell proliferation were assessed. A ribozyme with twelve nucleotide binding arms that cleaves c-mby RNA was delivered to rat smooth muscle cells as described in Example 6 (0.2 μM ribozyme complexed with DMRIE/DOPE at a 1:1 charge ratio) . In some cases, 10 μM chloroquine was added upon stimulation of the cells. The addition of chloroquine had no effect on untreated cells (stimulation with 10% serum in the presence or absence of chloroquine resulted in 80.5 ± 1.5 % and 83 ± 2% cell proliferation, respectively; unstimulated cells with and without chloro¬ quine showed 7 ± 0.5% and 7 ± 1% cell proliferation, respectively) . As shown in Table X below, addition of 32 chloroquine augments ribozyme inhibition of smooth muscle cell proliferation two- to three-fold.

Example 12 : Effect of a hammerhead ribozyme on human smooth muscle cell proliferation.

The hammerhead ribozyme that cleaves human c-myb RNA at site 549 was tested for its ability to inhibit human aortic smooth muscle cell proliferation. The binding site for this ribozyme is completely conserved between the mouse and human cDNA sequences. Human aortic smooth muscle cells (AOSMC) were obtained from Clonetics and were grown in SmGM (Clonetics ) . Cells from passage five or six were used for assays. Conditions for the proliferation assay were the same as for the rat cells (see Example 6) , except that the cells were plated in SmGM and starved in SmBM plus 0.5% FBS. The ribozyme that cleaves site 549 was delivered at varying doses complexed with the cationic lipid DMRIE at a 1:1 charge ratio. In this experiment, 10% FBS (no ribozyme) induced 57 + 7% proliferation; the uninduced background was 6 + 1% proliferation. The results in Table XI show that inhibition is observed over a similar concentration range as was seen with rat smooth muscle cells.

Example 13: Inhibition by direct addition of a modified, stabilized ribozyme.

A hammerhead ribozyme that cleaves site 575 was chemically synthesized with 12 nucleotide binding arms (sequence ID NO. 127, in Table III) . Chemically modified nucleotides were incorporated into this ribozyme that have been shown to enhance ribozyme stability in serum without greatly impacting catalytic activity. (See Eckstein et al . , International Publication No. WO 92/07065, Perrault et al . , 1990, Nature , 344, 565-568, Pieken,W. et al . 1991, Science , 253, 314-317, Usman,Ν. ; Cedergren,R.J. , 1992, Trends in Biochem. Sci . , 17, 334-339, Usman,Ν. et al . US Patent Application 07/829, 729, and Sproat,B. European Patent Application 92110298. 4 describe various chemical modifications that can be made to the sugar moieties of enzymatic RNA molecules. All these publica¬ tions are hereby incorporated by reference herein.) The modifications used were as follows. All the nucleotides of the ribozyme contained 2'-0-methyl groups with the following exceptions: U₄ and U₇ contained 2'-amino substi¬ tutions; G₅, A₆, G₈, G₁₂ , and A_{15 1} were 2 '-OH ribonucleo¬ tides (numbering as in Figure 1) . An inactive ribozyme was chemically synthesized in which G₅ and A₁₄ were substi¬ tuted with 2'-0-methyl U. Ribozymes were added to rat smooth muscle cells at the indicated concentrations as per Example 6 except that cationic lipids were omitted. Proliferation was assessed by BrdU incorporation and staining. Table XII shows that the modified ribozyme is capable of inhibiting rat smooth muscle cell proliferation without addition of cationic lipids. In this experiment, 10% serum induced 45 ± 2 % proliferation while uninduced cells showed a background of 2.3 + 0.1 % proliferation.

Optimizing Ribozyme Activity

As demonstrated in the above examples, ribozymes that cleave c-myb RNA are capable of inhibiting 50% of the smooth muscle cells from proliferating in response to serum. This level of inhibition does not represent the maximal effect obtainable with the ribozymes; in each dose response experiment, the highest dose produced the greatest extent of inhibition. Thus, optimizing activity of the ribozyme within the cells and/or optimizing the delivery of the ribozyme to the cells is expected to increase the extent of inhibition.

Tables VIII and IX demonstrate one means of optimizing ribozyme activity. By altering the length of the ribozyme binding arms (stems I and III, see Figure 2c) , the ability of the ribozyme to inhibit smooth muscle cell proliferation is greatly enhanced. Ribozymes with increasing arm lengths will be synthesized either chemic- ally in one or two parts (see above and see Mamone, U.S. Serial No. 07/882,689, filed May 11, 1992, hereby incor¬ porated by reference herein) or by in vi tro transcription (see Cech et al. , U.S. Patent 4,987,071) . Ribozymes are chemically synthesized with modifications that prevent their degradation by serum ribonucleases (as described in Example 13, above) . When synthesized in two parts, the fragments are ligated or otherwise juxtaposed as described (see original application and Mamone, supra) . The effects of the ribozymes on smooth muscle cell proliferation are assessed as in Examples 6 and 12, above. As the length of stems I and III can affect both hybridization to the target and the catalytic rate, the arm length of each ribozyme will be optimized for maximal inhibitory effect in cells. Similarly, the precise sequence of modified nucleotides in the stabilized ribozyme will affect the activity in cells. The nature of the stabilizing modifi¬ cations will be optimized for maximal inhibitory effect in cells. In each case, activity of the ribozyme that cleaves c-myb RNA will be compared to the activity of its catalytically inactive control (substitution of 2"-0- methyl U for G₅ and a 2'-0- methyl U for A₁₄) and to a ri¬ bozyme targeted to an irrelevant RNA (same catalytic core, with appropriate modifications, but different binding arm sequences) .

Sullivan, et al . , supra, describes the general methods for delivery of enzymatic RNA molecules. The data presented in Example 9 indicate that different cationic lipids can deliver active ribozymes to rat smooth muscle cells. In this example, 0.6 μM ribozyme delivered with Lipofectamine produced the same inhibitory effect as 0.3 μM ribozyme delivered with DMRIE. Thus, DMRIE is twice as efficacious as Lipofectamine at delivering active ribo¬ zymes to smooth muscle cells. There are a number of other cationic lipids known to those skilled in the art that can be used to deliver nucleic acid to cells, including but not limited to dioctadecylamidoglycylspermine (DOGS) , dioleoxltrimetylammonium propane (DOTAP) , N-[l-(2,3- dioleoyloxy) -propyl] -n,n,n-trimethylammoniumchloride (DOTMA) , N- [1- (2,3-dioleoyloxy) -propyl] -N,N-dimethyl-N- hydroxyethylammonium bromide (DORIE) , and N-[l-(2,3- dioleoyloxy)propyl] -N,N-dimethyl-N-hydroxypropylammonium bromide (DORIE-HP) . Experiments similar to those per¬ formed in Example 9 are used to determine which lipids give optimal delivery of ribozymes to smooth muscle cells. Other such delivery methods are known in the art and can be utilized in this invention.

The data described in Example 11 show that ribozyme delivery and efficacy may be augmented by agents that disrupt or alter cellular endosome metabolism. Chloroquine was shown to increase the ability of a ribozyme to inhibit smooth muscle cell proliferation by 2- to 3-fold. Experiments similar to those described in Example 11 can be performed to determine the optimal concentration of chloroquine to be used to augment deliv¬ ery of ribozymes alone (as in Example 13) , or delivery in the presence different cationic lipids (as in Example 9 and described above) or with other delivery agents (as described below) . Other agents that disrupt or alter endosomes known to those familiar with the art can be used to similarly augment ribozyme effects. These agents may include, but are not limited to, ammonium chloride, carbonyl cyanide p-trifluoromethoxy phenyl hydrazone

(FCCP) , chloroquine, monensin, colchicine, amphipathic peptides, viral proteins, and viral particles. Such compounds may be used in conjunction with ribozymes as described above, may be chemically conjugated directly to ribozymes may be chemically conjugated to liposomes, or may be incorporated with ribozymes in liposome particles

(see Sullivan, et al . , supra, incorporated by reference herein) . The data presented in Example 13 indicate that the proliferation of smooth muscle cells can be inhibited by the direct addition of chemically stabilized ribozymes. Presumably, uptake is mediated by passive diffusion of the anionic nucleic acid across the cell membrane. In this case, efficacy could be greatly enhanced by directly coupling a ligand to the ribozyme. The ribozymes are then delivered to the cells by receptor-mediated uptake. Using such conjugated adducts, cellular uptake can be increased by several orders of magnitude without having to alter the phosphodiester linkages necessary for ribozyme cleavage activity. Alternatively, 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, bio- degradable nanocapsules, and bioadhesive microspheres. The 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, intramuscular injection, aerosol inhalation, oral (tablet or pill form) , topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. More detailed descriptions of ribozyme delivery and administra¬ tion are provided in Sullivan, et al . , supra and Draper, et al . , supra which have been incorporated by reference herein.

Example 14 : Phosphorothioate linkages enhance the ability of ribozymes to inhibit smooth muscle cell proliferation.

As the applicant had shown in Example 13, the hammer- head (HH) ribozyme that cleaves c- yb RNA at site 575 can be modified to confer resistance to nucleases while main¬ taining catalytic activity (see also Usman et al. , supra) . To identify ribozymes with optimal activity in cells, several different chemically-modified ribozymes were directly compared for inhibition of rat smooth muscle cell proliferation. Non-limiting examples of chemically-modi¬ fied ribozymes used are diagrammed in Figure 9A. One ribozyme (designated "2'-O-methyl") contains ribonucleo- tide residues at all positions except the 5 terminal nucleotides of each target binding arm (Stems I and III) . The ribozyme designated "2'-O-methyl P=S" in addition con- tains five phosphorothioate linkages between the terminal nucleotides in each target binding arm. The ribozyme termed "2'-C-allyl iT" contains thirty 2'-O-methyl nucleo¬ tides as specified in Example 13. The ribozyme also con¬ tains 2'-C-allyl U (Usman et al. , 1994 Nucleic Acids Svma . Ser. 31, 163) at the U4 position and 2'-O-methyl U at the U7 position and a 3'-3'-linked inverted thymidine (Ortigao et al., 1992 Antisense Res . & Development 2 , 129; Seliger et al., Canadian Patent Application No. 2,106,819) at the 3' end of the molecule (referred to as 2'-C-allyl iT) . The fourth ribozyme contains the same 2'-O-methyl and 2'- C-allyl residues described above with the addition of 5 phosphorothioate linkages between the terminal nucleotides in each target binding arm (referred to as "2'-C-allyl P=S") . Ribozymes were delivered to smooth muscle cells as cationic lipid complexes (Sullivan et al. , supra) . In this example, the cationic lipid, Lipofectamine (GIBCO-

BRL) , was used at a charged lipid concentration of 3.6 μM

(see Examples 6 and 9) . Active versus inactive forms of each ribozyme were compared to determined whether inhibi¬ tion is mediated specifically by ribozyme cleavage. As shown in Figure 9B, the ribozyme synthesized with the 2'- C-allyl modification and the phosphorothioate linkages demonstrated enhanced inhibition of smooth muscle cell proliferation. The catalytically inactive form of the ribozyme had little effect on cell proliferation; thus, the inhibition observed requires the catalytic activity of the ribozyme. In contrast, ribozymes without the stable 2'-0-methyl- and 2'-C-allyl-modified catalytic core (2'-0- methyl and 2'-O-methyl P=S) at best showed only modest inhibition of smooth muscle cell proliferation. The stable core chemistry alone was not sufficient to greatly enhance ribozyme-mediated inhibition; without terminal P=S linkages, the 2'-C-allyl-modified ribozyme showed very little specific inhibition when compared to its inactive ribozyme control . These results demonstrate that certain chemical modifications greatly enhance the ability of exogenously-delivered ribozymes to cleave c-myb RNA and impact cell proliferation.

Example 15 : Dose response of the chemically modified ribozyme.

Varying doses of the 2'-C-allyl P=S-modified ribozyme were delivered to rat aortic smooth muscle cells as described above. As in previous examples, percent inhibi¬ tion was calculated by comparing the effects of the active ribozyme to the effects of the inactive ribozyme. As shown in Figure 10, the ribozyme concentration at which cell proliferation is inhibited by 50% (IC₅₀) is approxi¬ mately 70 nM. From day to day, the IC₅₀ varies between 25 and 100 nM.

Example 16 : Direct comparison of the effects of ribozymes and antisense DNA.

Ribozymes are thought to be more specific reagents for the inhibition of gene expression than antisense oligonucleotides due to their catalytic activity and strict sequence requirements around the site of cleavage (Castanotto et al., 1994 Adv. in Pharmacol . 25, 289) . To test this hypothesis, ribozyme activity was directly com¬ pared to the activity of phosphorothioate DNA oligonucleo- tides that target the same site in the c-myb mRNA. The ribozyme used was the 2'-C-allyl P=S-modified ribozyme described in Example 14, above. This ribozyme binds to a 15 nucleotide long region of the c-myb mRNA. Thus, a 15 nucleotide antisense phosphorothioate DNA molecule was prepared. A phosphorothioate DNA oligonucleotide with a randomly scrambled sequence of the same 15 nucleotides and a 2'-C-allyl P=S-modified ribozyme with randomly scrambled target binding arm sequences were synthesized as controls (by comparison to the murine c-myb cDNA sequence, the scrambled controls would not be expected to bind any region of the c-myb mRNA) . Since longer phosphorothioate DNA oligonucleotides are often utilized as antisense inhibitors (for a review see Wagner, 1994 Science 372, 333), a symmetrically placed, 25 nucleotide phosphoro¬ thioate DNA antisense oligonucleotide and its scrambled sequence control were also synthesized. The ribozymes and the antisense oligonucleotides were delivered to rat smooth muscle cells as complexes with the cationic lipid, Lipofectamine, and serum-stimulated smooth muscle cell proliferation was measured subsequently.

As shown in Figure 11, the 2'-C-allyl P=S-modified ribozyme demonstrated greater inhibition of smooth muscle cell proliferation than either of the antisense oligo¬ nucleotides. Furthermore, the scrambled arm ribozyme and inactive ribozyme controls demonstrated less non-specific inhibition than either of the scrambled sequence antisense control oligonucleotides. In fact, the non-specific inhi¬ bition demonstrated by the 25 nucleotide phosphorothioate molecule completely masked any specific effect of the antisense molecule. Similar results have been obtained with phosphorothioate DNA targeting other sites in the c- myb mRNA. Thus, a ribozyme that cleaves c-myb RNA is a more potent and more specific inhibitor of smooth muscle cell proliferation than phosphorothioate antisense DNA molecules.

Example 17: Chemically-modified ribozymes targeting different sites in the c-mvb mRNA specifically inhibit smooth muscle cell proliferation.

If the observed inhibition of smooth muscle cell proliferation is mediated by ribozyme cleavage of c-myb mRNA, then other ribozymes that target the same mRNA should have the same effect. Two other ribozymes target¬ ing two disparate sites in the c-myb mRNA (sites 549 and 1553, ribozyme Seq. ID Nos. 102 and 112) were synthesized with the 2'-C-allyl P=S modifications as described in Example 14. Inactive ribozyme controls also were synthe¬ sized corresponding to each new target sequence. Chemically-modified ribozymes targeting sites 549, 575, and 1553 were delivered to rat smooth muscle cells and their ability to inhibit serum-stimulated cell proliferation was assessed. Equivalent levels of inhibition are obtained with active ribozymes targeting sites 549, 575 and 1553 (see Figure 12) . None of the inactive ribozymes inhibited cell proliferation. Active ribozymes targeting other mRNA sequences not present in c- myb or ribozymes with scrambled arm sequences also fail to inhibit smooth muscle cell proliferation (see Figure 12) . Thus, inhibition of cell proliferation requires a catalytically active ribozyme that can bind to accessible c-myb mRNA sequences and is likely due to the reduction of c-myb mRNA levels by ribozyme cleavage.

Examples 18 and 19 describe experiments designed to determine the position and minimum number of phosphoro¬ thioate residues required for efficacy.

Example 18: Effect of position of phosphorothioate linkages on ribozyme inhibition. Ribozymes targeting c-myb site 575 were synthesized with the 2'-C-allyl modification and with phosphorothioate linkages between various nucleotides in the ribozyme. One ribozyme contained a total of 10 phosphorothioate linkages, 5 in Stem I and 5 in Stem III, identical to the ribozyme described in Examples 14 through 17 above (referred to as 10 P=S 5' and 3' in Figure 13A) . One ribozyme contained only 5 phosphorothioate linkages in Stem III (5 P=S 3' in Figure 13A) . Another ribozyme contained 5 phosphorothioate linkages between the 6 nucleotides comprising the last base pair of stem II and the GAAA loop (5 P=S loop in Figure 13A) . The fourth ribozyme contained 5 phosphorothioate linkages in stem I (5 P=S 5' in Figure 13A) . The latter two ribozymes also were synthesized with the 3'-3' thymidine at the 3' end to help protect the ribozyme from 3' exonucleases (Ortigao et al., 1992 Antisense Res . & Development 2, 129; Seliger et al., Canadian Patent Application No. 2,106,819) . The structure of these four different ribozymes is diagrammed in Figure 13A. Inactive ribozyme controls were synthe¬ sized for each individual ribozyme. The active and inactive ribozymes were applied to rat smooth muscle cells as RNA/Lipofectamine complexes and their effects on cell proliferation were measured.

Referring to Figure 13B, the ribozyme containing 5 phosphorothioate linkages in Stem I and the 3' inverted thymidine inhibited smooth muscle cell proliferation as well as the parent ribozyme with 10 total phosphorothioate linkages. None of the other ribozymes demonstrated significant differences between active and inactive controls. Therefore, the 3' inverted T can effectively substitute for the 5 phosphorothioate linkages in Stem III. Phosphorothioate linkages in the loop position lead to non-specific inhibition of smooth muscle cell proliferation, while phosphorothioate linkages in Stem I are necessary for enhanced efficacy in cells. Addition¬ ally, these results suggest that 3'-end modifications, such as iT, is desirable to minimize the amount of phosphorothioate contained in the ribozymes in order to minimize toxicity and facilitate chemical synthesis, while maintaining protection from endogenous 3' -exonuclease digestion.

Example 19: Minimizing phosphorothioate linkages in Stem I.

Fewer phosphorothioate linkages in the ribozyme will reduce the complexity and cost of chemical synthesis. Furthermore, phosphorothioate DNA molecules are known to have some undesirable and non-specific effects on cellular functions (for a review see Wagner, supra) ; reducing the phosphorothioate linkages in these RNA molecules is expected to enhance their specificity. A series of ribo¬ zymes targeting c-myb were synthesized to determine how many phosphorothioate linkages in Stem I are required for optimal ribozyme activity. The ribozymes contained 5, 4, 3, 2, or 1 phosphorothioate linkage (s) in Stem I, beginning with the phosphodiester bond between the first and second nucleotides and proceeding 3'. Each ribozyme contained the 2'-O-methyl modifications, the U₄ 2'-C-allyl nucleotide, and the inverted T nucleotide at the 3' end as described above. Activity of each of these ribozymes was compared to the activity of the ribozyme with 10 phosphorothioate linkages, 5 each in Stems I and III

(referred to as 10 P=S in Figure 14) . Active and inactive ribozymes were applied to rat smooth muscle cells as complexes with Lipofectamine and their effects on smooth muscle cell proliferation were measured in two separate experiments. The results are diagrammed in Figure 14. Ribozymes with 10, 5, and 4 phosphorothioate linkages showed equivalent efficacy. Ribozymes with fewer than four phosphorothioate linkages also showed efficacy, but the level of inhibition of smooth muscle cell proliferation was modestly reduced.

Example 20: Varying the length of Stems I and III

Ribozymes that cleave c-myb RNA at position 575 were synthesized with varying arm lengths. Each ribozyme contained 4 phosphorothioate linkages at the 5' end, 2'-0- methyl and 2'-C-allyl modifications and an inverted thymidine nucleotide at the 3' end as described above. Figure 15 shows the effects of these ribozymes upon rat smooth muscle cell proliferation. Ribozymes were deliv¬ ered at 100 nM with cationic lipid. Ribozymes with 6/6, 7/7 and 5/10 arms (where x/y denotes the nucleotides in Stem I/nucleotides in Stem III; see Figure 2) all showed comparable efficacy. As shown in Figure 15, ribozymes with longer arm lengths tended to demonstrate more non- specific inhibition (the inactive ribozyme controls with longer binding arms inhibited smooth muscle cell prolifer¬ ation) when compared to ribozymes with shorter binding arms. From these data, it appears that ribozymes with 6/6, 7/7, 5/10, 10/5, 8/8 and 10/10 nucleotide arms all specifically inhibit smooth muscle cell proliferation, optimal inhibition, however, is observed with 6/6, 7/7 and 5/10 nucleotide arms.

Example 21 : Ribozymes with different modified nucleotides inhibit smooth muscle cell proliferation.

Ribozymes containing seven nucleotides in both Stems I and III, four phosphorothioate residues at the 5' end and a 3'-3' inverted thymidine at the 3' end, were synthesized with various modified nucleotides at the U₄ and U₇ positions within the core of a HH ribozyme. All of the modified catalytic core chemistries retained ribozyme activity and demonstrated enhanced stability to serum nucleases (Usman et al., 1994 supra) . The ribozyme termed U4 2'-C-allyl contains a 2'-C-allyl uridine at the U₄ position and a 2'-O-methyl nucleotide at the U₇ position. The ribozyme termed U4,U7 2'-amino contains a 2'-amino nucleotide at both U4 and U7. The ribozyme termed U4 2'- fluoro contains a 2'-fluoro-modified nucleotide at U4 and 2'-O-methyl at U7. The ribozyme termed U4 6-methyl contains a 6-methyl uridine nucleotide at U4 and 2'-0- methyl at U7. The ribozyme termed U4 deoxyabasic contains a deoxyribose moeity and lacks a base at U4 (Beigelman et al., 1994 Bioorcranic & Med. Chem. Letters 4, 1715) and 2'- O-methyl at U7. Active and inactive versions of each of the chemically-modified ribozymes were applied to rat smooth muscle cells using Lipofectamine as described above. As diagrammed in Figure 16, all of the nuclease- stable, chemically-modified ribozymes demonstrated signif- icant inhibition of rat smooth muscle cell proliferation. Thus, the requirements for ribozyme activity in smooth muscle cells appear to be a catalytically core that is modified to minimize endonucleolytic degradation and modifications at the 5' and 3' ends which may prevent exonucleolytic degradation.

Chemical modifications described in this invention are meant to be non-limiting examples, and those skilled in the art will recognize that other modifications (base, sugar and phosphate modifications) to enhance nuclease stability of a ribozyme can be readily generated using standard techniques and are hence within the scope of this invention.

Example 22 : Ribozyme inhibition of pig smooth muscle cell proliferation.

Of the commonly used animal models of intimal hyper- plasia after balloon angioplasty, the pig model is believed to be most predictive of human disease (Steele et al., 1985 Circ. Res . 57, 105; Ohno et al. , 1994 Science 265, 781; Baringa, 1994 Science 265, 738) . Therefore, we wished to assess the ability of c-myb ribozymes to inhibit pig smooth muscle cell proliferation. Yucatan pig smooth muscle cells (YSM) were obtained from Dr. Elizabeth Nabel

(University of Michigan Medical Center) and were grown in

Dulbecco's modified Eagle's medium as described (see

Example 6) . The YSM cells were starved for 72 hours in DMEM with 0.1% FBS. Active and inactive ribozymes (four phosphorothioate linkages at the 5' end, 2'-C-allyl- modified core and 3'-3' inverted thymidine at the 3' end) were applied as RNA/Lipofectamine complexes as described in the above examples. Proliferation was stimulated with serum and assessed by BrdU incorporation. Figure 17 shows that a ribozyme dose of as low as 75 nM can inhibit pig smooth muscle cell proliferation by as much as 60%. The same chemical modifications of the ribozymes (2'-modified, stable core, 5' phosphorothioate linkages and 3' inverted thymidine) are required to obtain significant and repro¬ ducible inhibition of pig smooth muscle cell proliferation as were shown to be required for inhibition of rat cells in the above Examples.

Example 23 : Ribozyme inhibition of human smooth muscle cell proliferation.

In Example 12, we demonstrated that a minimally modi¬ fied ribozyme directed against c-myb site 549 could significantly inhibit human smooth muscle cell pro¬ liferation. The 2'-C-allyl and phosphorothioate-modified ribozyme targeting c-myb site 575 characterized above was applied to human smooth muscle cells as RNA/Lipofectamine complexes. Inactive ribozyme and inactive, scrambled arm ribozymes were applied as controls. At 200 nM, the active ribozyme inhibits human smooth muscle proliferation by greater than 75% while the inactive ribozyme inhibits proliferation by only 38%. The ribozyme with scrambled binding arm sequences fails to inhibit. At 100 nM, the active ribozyme still demonstrates significant inhibition while neither the inactive or scramble controls inhibit cell proliferation (see Figure 18) . Thus, the active ribozyme identified in these studies mediates significant inhibition of human smooth muscle cell proliferation and represents a novel therapeutic for restenosis and/or vascular disease.

Example 24: Delivery of c-mvb ribozymes to vessels in vivo.

The ribozyme that cleaves c-myb RNA at site 575 was synthesized in two parts (Mamone, supra) , the internal 5' end was labeled with ³³P using polynucleotide kinase and the two fragments were ligated with RNA ligase. The resulting RNA was an intact ribozyme with an internal ³³P label. This internally-labeled ribozyme was delivered to balloon injured rat carotid arteries as described (Simons et al., 1992 Nature 359, 67) . Rats were anesthetized and the carotid artery was surgically exposed. The external carotid was dissected and a 2F Fogarty balloon catheter was inserted and directed into the carotid artery. Injury was caused by repeated (3 times) inflation and retraction of the balloon. The injured region was isolated by ligatures and a cannula was inserted in the external carotid. Ribozymes alone (two rat vessels) or ribozyme/Lipofectamine complexes (two rat vessels) were applied to the injured vessel through the cannula and were left in the vessel for twenty minutes. After application, blood flow was restored by removal of the ligatures for five minutes and the vessels were harvested and processed as described below.

Half of the vessel was frozen in liquid nitrogen, crushed into a fine powder, and RNA was extracted using standard protocols. The extracted RNA was applied to a denaturing polyacrylamide gels and subjected to electrophoresis. Autoradiography of the gel permitted detection of the ³³P label; the amount of radioactivity in each band was quantitated using a Phosphor-imaging system. The amount of extracted and intact ribozyme was calculated by direct comparison to labeled ribozyme controls run on the same gel. The percentage of the ribozyme delivered intact could be estimated by quantifying the percentage of label that co-migrates with the intact ribozyme controls. After delivery of ribozymes in phosphate-buffered saline (PBS) , 3% of the ³³P label was recovered from the rat vessels and >90% of the label was present in the form of intact ribozyme. After delivery of ribozyme in RNA/Lipofectamine complexes, 10 to 11% of the ³³P label was recovered from the rat vessels and 20 to 90% of the label was present in the form of intact ribozyme. The signifi¬ cant uptake of the intact ribozyme demonstrates that local delivery of modified ribozymes to arterial walls is feasible.

The other half of each vessel was fixed in PBS- buffered 2% glutaraldehyde, sectioned onto slides and coated with emulsion. After autoradiography for four days, the emulsion was developed and the sections were stained with hematoxylin and eosin by standard techniques (Simons et al., 1992 supra) . Inspection of the sections showed a majority of the grains present over the medial smooth muscle cells after application of the ribozyme. Some ³³P label could be detected in the underlying adven- titia as well. Similar density and distribution of grains was observed when the ribozyme was delivered with or without Lipofectamine. These data demonstrate that ribo¬ zyme can penetrate the injured vessel wall and is in close apposition or within the underlying medial smooth muscle cells. Thus, therapeutic ribozymes can be locally deliv¬ ered to vessels for the treatment of vascular disease.

Similar experiments were performed in pig iliofemoral vessels. After balloon injury, a ribozyme, internally labeled with ³³P as described above, was delivered with a double balloon catheter device (Nabel and Nabel, supra; Ohno et al., 1994 supra) . After 20 minutes, blood flow was restored by deflating the balloons. The vessels were harvested after an additional hour or the surgical injuries were sutured and the vessels harvested one day later. Harvested vessels were sectioned, subjected to autoradiography and stained. One hour after delivery, the majority of the ³³P label could be detected in the media, overlying or within smooth muscle cells. Some label was also detected at the luminal surface of the vessel and in the adventitial tissue. One day after delivery, grains could be still be detected associated with remaining medial smooth muscle cells. No major differences in density or distribution was observed between ribozymes delivered with or without Lipofectamine . These data demon¬ strate that ribozymes can be locally delivered to smooth muscle cells of injured vessels in a large animal model that is clinically relevant to human vascular disease. Example 25: Ribozvme-mediated decrease in the level of c- mvb RNA in rat smooth muscle cells.

To determine whether a ribozyme catalyzes the cleav¬ age of c-myb RNA in a mammalian cell, applicant has used a sensitive quantitative competitive polymerase chain reaction (QCPCR) to assay the level of c-myb RNA in rat smooth muscle cells treated with either catalytically active or inactive ribozyme.

Rat smooth muscle cells (RASMC) were treated with ribozymes as described above. Following the ribozyme treatment for 4h, cells were stimulated with 10% serum (in the presence or absence of BrdU) . After 24h, cells were harvested for further analysis. Cells, that were treated with BrdU, were assayed for proliferation as described above. Cells, that were not treated with BrdU, were used for the QCPCR assay.

The following is a brief description of the QCPCR technique used to quantitate levels of c-myb mRNA from RASMC, normalizing to the housekeeping gene, GAPDH. This method was adapted from Thompson et al, Blood 79:1692, 1992. Briefly, total RNA was isolated from RASMC using the Guanidinium isothiocyanate technique of Chomczynski and Sacchi (Analytical Biochemistry. 162:156, 1987) . In order to construct a deletion competitor and control wild- type RNA, a cDNA clone of the rat c-myb message, referred to as pcδmyb, was used. The competitor RNA comprises a deletion of 50 bases, making it smaller than the wild-type cellular RNA, and spansfrom nucleotide 428 to nucleotide 753. A house-keeping gene, GAPDH, that is constitutively expressed by the RASMC, was used as an internal control for QCPCR assay. A deletion competitor and wild-type controls for GAPDH were made the same way as for c-myb. GAPDH-containing plasmid (pTri-GAPDH) was purchased from Ambion. The GAPDH competitor is also a deletion mutant, lacking 50 bases. The GAPDH competitor was used to quantitate the amount of this housekeeping gene in each sample, thus allowing for a confirmation of cellular RNA's integrity and for the efficiency of RNA isolation. All quantitations for the level of c-myb expression were normalized to the level of GAPDH expression in the same sample of cells.

Referring to Fig. 19, RASMC that were treated with a stabilized catalytically active 575 HH ribozyme did not proliferate well. There was greater than 70 % inhibition of RASMC proliferation when compared with approximately 25% inhibition of cell proliferation by a catalytically inactive version of the 575 HH ribozyme. The level of inhibition of RASMC proliferation correlates very well with the greater than 70 % decrease in the level of c-myb RNA. This shows that the inhibition of smooth muscle cell proliferation is directly mediated by the cleavage of c- myb RNA by a ribozyme in RASMC.

Figure 20 shows what Applicant presently believes is an optimal ribozyme configuration.

Example 26: Inhibition of smooth muscle cell proliferation by 2-5A antisense chimera.

By "2-5A antisense chimera" is meant, an antisense oligonucleotide containing a 5' phosphorylated 2' -5'- linked adenylate residues. These chimeras bind to target RNA in a sequence-specific manner and activate a cellular 2-5A-dependent ribonuclease which in turn cleaves the target RNA (Torrence et al. , 1993 Proc. Natl . Acad . Sci . USA 90, 1300) .

RNAs containing 2' -5' Adenosine with a terminal 5' phosphate has been shown to activate RNAse L (Torrence et al., 1993 Proc. Natl . Acad. Sci . USA 90, 1300) . The terminal phosphate is required for efficient activation of RNAse L. Ribozymes targeting c-myb site 575 were synthesized with 2-5A moieties on the 5' end, with and without the terminal 5' phosphate. The ribozyme-2-5A chimera was complexed with LipofectAMINE and assayed on rat aortic smooth muscle cells (RASMC) as described above. As shown in Figure 21, when no terminal phosphate is present, the active ribozyme [575 inactive Rz+ inactive (A) 4] functions similarly to a normal active ribozyme lacking a 2-5A modification (575 active Rz) . An inactive ribozyme core with 5' phosphate-2-5A [575 inactive Rz+ active P(A)4] shows significant inhibition relative to the controls, but has significantly lower activity when compared with an active ribozyme. A molecule that contains both an active ribozyme core and 5' phosphate- contining 2-5A [575 active Rz+active P (A) 4] shows even greater inhibition than that obtained by either mechanism individually, inhibiting the smooth muscle cell prolifera¬ tion to baseline levels (0% FBS) . Thus the ribozyme and 2-5A anitisense chimera together show an additive effect in inhibiting RASMC proliferation.

Use of Ribozymes That Cleave c-mvb RNA to Treat Restenosis.

The above discussion demonstrates, by way of example, how ribozymes that inhibit smooth muscle cell prolifera¬ tion are delivered directly, or through the use of expression vectors, to vessels. Preferably, ribozymes cleaving c-myb RNA are delivered to vessels at the time of coronary angioplasty. Local delivery during intervention can be achieved through the use of double balloon catheters , porous balloon catheters , balloon catheters coated with polymers (Riessen, R. , et al. , 1993, Human Gene Therapy. 4, 749-758), or biopolymer stents (Slepian and Schindler, U.S. Patent # 5,213,580) . In the above examples, ribozymes were identified that could inhibit roughly half of the smooth muscle cells in culture from proliferating in response to the growth factors present in serum. A corresponding 50% (or even lower) reduction in intimal thickening will significantly improve the outcome of patients undergoing coronary angioplasty. Use of Ribozymes Targeting c-mvb to Treat Cancer

Overexpression of the c-myb oncogene has been reported in a number of cancers, including leukemias, neuroblastomas, and lung, colon, and breast carcinomas (Torelli, G. , et al. , 1987, Cancer Res . . 47, 5266-5269 Slamon, D. J. , et al. , 1986, Science. 233, 203-206 Slamon, D. J., et al., 1984, Science, 224, 256-262 Thiele, C. J., et al . , 1988, Mol . Cell . Biol .. 8, 1677- 1683; Griffin, C. A. and Baylin, S. B., 1985, Cancer Res . . 45, 272-275; Alitalo, K. , et al. , 1984, Proc. Natl . Acad. Sci . USA. 81, 4534-4538) . Thus, inhibition of c-myb expression can reduce cell proliferation of a number of cancers. Indeed, in tissue culture, treatment of colon adenocarcinoma, neurectodermal, and myeloid leukemia cell lines with antisense c-myb oligonucleotides inhibits their proliferation (Melani, C, et al., 1991, Cancer Res . , 51, 2897-2901; Raschella, F., et al. , 1992, Cancer Res . , 52, 4221-4226; Anfossi, G. , et al. , 1989, Proc . Natl . Acad. Sci . USA, 86, 3379-3383) . Furthermore, myeloid cells from patients with chronic myelogenous leukemia and acute myelogenous leukemia are differentially sensitive to c-myb antisense oligonucleotides (Calabretta, B., et al. , 1991, Proc. Natl . Acad. Sci . USA. 88, 2351-2355) . Ratajczak, et al. (1992, Proc. Natl . Acad. Sci . USA. 89, 11823-11827) treated mice bearing human leukemia cells with c-myb antisense oligonucleotides and significantly prolonged their survival and reduced their tumor burden. Thus, reduction of c-myb expression in leukemic cells in tissue culture and in vivo can reduce their proliferative potential.

While the above studies demonstrated that antisense oligonucleotides can efficiently reduce the expression of c-myb in cancer cells and reduce their ability to proliferate and spread, this invention describes enzymatic RNAs, or ribozymes, shown to cleave c-myb RNA. Such ribozymes, with their catalytic activity and increased site specificity (see above) , are likely to represent more potent and safe therapeutic molecules than antisense oligonucleotides for the treatment of cancer as well as restenosis. In the present invention, ribozymes are shown to inhibit smooth muscle cell proliferation. From those practiced in the art, it is clear from the examples described, that the same ribozymes may be delivered in a similar fashion to cancer cells to block their proliferation.

In a preferred embodiment, autologous bone marrow from patients suffering with acute myelogenous leukemia or chronic myelogenous leukemia are treated with ribozymes that cleave c-myb RNA. Ribozymes will be delivered to the autologous bone marrow cells ex vivo at 0.1 to 50 μM with or without forming complexes of the ribozymes with cationic lipids, encapsulating in liposomes or alternative delivery agents. After several days, the proliferative capacity of the leukemic cells in the patients bone marrow will be reduced. The patient's endogenous bone marrow cells will be depleted by chemical or radiation treatments and their bone marrow reconstituted with the ex vivo treated cells. In such autologous bone marrow reconsti- tution treatments of leukemic patients, recurrence of the disease can be caused by proliferation of leukemic cells present in the transplanted bone marrow. Significantly reducing the proliferative potential of the leukemic cells by treating with ribozymes that cleave c-myb RNA will reduce the risk of recurrent leukemia.

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

RNAseP RNA (Ml 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 are all 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 (Figure 1)

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 II: Ribozyme catalyzed cleavage of c-mvb RNA

Hammerhead Sites % Cleavage

Cleavage Sequence Target Sequence Mouse Human

Site ID No. c-myb c-mγb RNA RNA

310 79 CGUCACU U GGGGAAA 28.5 0.1

549 80 GUCUGUU A UUGCCAA 87.4 91.6

551 81 CUGUUAU U GCCAAGC 56.8 82.4

575 82 GGAGAAU U GGAAAAC 93.9 91.3

634 83 AAAACCU C CUGGACA 68.4 87.1

738 84 UAAUGCU A UCAAGAA 78.1 0.01

839 85 CAAGCUU C CAGAAGA 27.2 0.01

936 86 UUCCUAU U ACCACAU 61.8 60.6

1017 87 UGUCCCU C AGCCAGC 40.3 0.1

1082 88 AGCGAAU A AAGGAAU 55.2 89.2

1363 89 UUAGAAU U UGCAGAA 11.6 0.1

1553 90 CAGCUAU C AAAAGGU 87.1 92.5

1597 91 ACACCAU U CAAACAU 71.2 62.7

1598 92 CACCAUU C AAACAUG 79.6 85.5 Hammerhead Sites Cleavage

Cleavage Sequence Target Sequence Mouse Human

Site ID No. c-myb c-myb

RNA RNA

1635 93 AUACGGU C CCCUGAA 8 844..44 82.3

1721 94 CUGGAAU U GUUGCUG 62.1 79.3

1724 95 GAAUUGU U GCUGAGU 65.6 86

1895 96 AUAUUCU U ACAAGCU 79.1 66.2

1909 97 UCCGUUU U AAUGGCA 31.1 0.1

1943 98 ACAAUGU U CUCAAAG 66.1 80

Hairpin Ribozymes

1632 99 ACG GUCC CCUGAAG 92.8 84.6

2231 100 ACA GUUG AGAGCAG 0.1 0.1

^a The nucleotide numbers given correspond to the nucleotide just 5' of the ribozyme cleavage site in the human c-myb sequence taken from Westin, et al., supra (GenBank Accession No. X52125) . All but two of the sequences (310; I.D. No. 79 and 2231; I. D. No. 100) overlap sequences in Table I .

Table III: Sequences of ribozymes used in these studies. Target Sequence Ribozyme Sequence Site ID No.

Hammerhead ribozymes with 7 nucleotide binding arms

310 101 UUUCCCCCUGAUGAGGCCGAAAGGCCGAAAGUGACG

549 102 UUGGCAACUGAUGAGGCCGAAAGGCCGAAAACAGAC 5 55511 1 10033 GCUUGGCCUGAUGAGGCCGAAAGGCCGAAAUAACAG

575 104 GCUUUCCCUGAUGAGGCCGAAAGGCCGAAAUUCUCC

634 105 UGUCCAGCUGAUGAGGCCGAAAGGCCGAAAGGUUUU

738 106 UUCUUGACUGAUGAGGCCGAAAGGCCGAAAGCAUUA

839 107 UCUUCUGCUGAUGAGGCCGAAAGGCCGAAAAGCUCG 9 93366 1 10088 AUGUGGUCUGAUGAGGCCGAAAGGCCGAAAUAGGAA

1017 109 GCCGGCUCUGAUGAGCGCGAAAGCGCGAAAGGGACG

1082 110 GCUCCUUCUGAUGAGGCCGAAAGGCCGAAAUUCGCU

1363 111 UUCUGCACUGAUGAGGCCGAAAGGCCGAAAUUCUAA 1553 112 ACCUUUUCUGAUGAGGCCGAAAGGCCGAAAUAGCUG

1597 113 AUGUUUGCUGAUGAGGCCGAAAGGCCGAAAUGGUGU

1598 114 CAUGUUUCUGAUGAGGCCGAAAGGCCGAAAAUGGUG

1635 115 UUCAGGGCUGAUGAGGCCGAAAGGCCGAAACCGUAU

1721 116 CAGCAACCUGAUGAGGCCGAAAGGCCGAAAUUCCAG

1724 117 ACUCAGCCUGAUGAGGCCGAAAGGCCGAAACAAUUC

1895 118 AGCUUGUCUGAUGAGGCCGAAAGGCCGAAAGAAUAU

1909 119 UGUCAUUCUGAUGAGGCCGAAAGGCCGAAAAACAGA

1943 120 CUUUGAGCUGAUGAGGCCGAAAGGCCGAAACAUUGU Bimolecular Hairpin Ribozymes

1632^a 121 5' Fragment:

UCAGGGAGAAGUAUACCAGAGAAACACACGCG 3' Fragment: CGCGUGGUACAUUACCUGGUA

2231^a 122 5' Fragment:

GCUCUCAGAAGUUGACCAGAGAAACACACGCG 3' Fragment: CGCGUGGUACAUUACCUGGUA

Hammerhead ribovzτnes with 6, 8, 9, 10, and 12 nucleotide binding arms 575 123 CUUUCCCUGAUGAGGCCGAAAGGCCGAA AUUCUC

6/6^b

575 124 UGCUUUCCCUGAUGAGGCCGAAAGGCCGAA

8/8 AUUCUCCC

575 125 CUGCUUUCCCUGAUGAGGCCGAAAGGCCGAA 9/9 AUUCUCCCU

575 126 ACUGCUUUCCCUGAUGAGGCCGAAAGGCCGAA

10/10 AUUCUCCCUU

575 127 ACACUGCUUUCCCUGAUGAGGCCGAAAGGCCGAA

12/12 AUUCUCCCUUUU 549 128 AGUGCUUGGCAACUGAUGAGGCCGAAAGGCCGAA

12/12 AACAGACCAACG

1553 129 GAUUGACCUUUUCUGAUGAGGCCGAAAGGCCGAA

12/12 AUAGCUGGAGUU

^aThe hairpin ribozymes were synthesized in two pieces as indicated. The two oligonucleotides were annealed and tested for activity against the c-myb RNA as described above. See Mamone, Ribozyme synthesis, filed May 11, 1992, U.S.S.N. 07/882,689, hereby incorporated by reference herein. designation of the ribozymes with different arm lengths is a/b where (a) represents the nucleotides in stem I and (b) represents the nucleotides in stem III (see Figure 1) .

Table IV: Comparison of the effects six hammerhead ribozymes, that cleave c-mvb RNA, on smooth muscle cell proliferation

Inactive Active % Inhibition Ribozyme Ribozyme

Ribozyme % Cell % Cell (Active vs. Site Proliferation Proliferation Inactive)

549 68 ± 1 59.5 + 1.5 14 ± 4

575 66.5 ± 0.5 54.5 ± 1.5 21 ± 3

1553 68.5 + 0.5 52 ± 1 28 ± 1

1597 66 ± 1 57 ± 3 16 ± 7

1598 67 ± 1 58.5 ± 0.5 15 ± 1

1635 62.5 ± 2.5 64 ± 1 0

Table V: Dose Response of c-mvb Hairpin Ribozyme 1632

Control Ribozyme 1632 Ribozyme

Ribozyme % % % Inhibition Dose (μM) Proliferation Proliferation (vs. control)

0.05 86.5 ± 1.5 88 ± 5 0

0.15 89.5 ± 1.5 78.5 + 2.5 10 ± 5

0.45 87.5 ± 1 66.5 ± 1.5 25 + 4 Table VI: Dose Response of c-myb Hammerhead Ribozymes 575 and 549

Control Ribozyme 575 Ribozyme 549

Ribozyme

Ribo¬ % cells % cells % % cells % zyme in S in S Inhibi¬ in S Inhibi¬

Dose phase phase tion phase tion

(μM) (vs. (vs. con¬ con¬ trol) trol)

0.05 89±5 77.5±1.5 14±8 92±1 0

0.15 90+1 68.5±1.5 26+2 84+2 9+4

0.45 91.5±0.5 59±5 38±7 76.5±2.5 18±5

Table VII: Delivery of c-myb Ribozyme 575 by Two

Different Cationic Lipids

Delivery with DMRIE/DOPE

Inactive Active Ribozyme 575 Ribozyme 575

Ribozyme % cells in S % cells in S % Inhibition Dose (μM) phase phase (vs. inactive)

0.075 79 ± 6 74.5 + 1.5 6 ± 6

0.15 79.5 ± 0.5 67 ± 1 17 + 4

0.30 77 ± 1 57 ± 2 28 + 5

Delivery with Lipofectamine

Inactive Active Ribozyme 575 Ribozyme 575

0.075 81 ± 1 83 ± 1 0

0.15 79 ± 3 71 ± 1 11 ± 4

0.30 82 + 1 68.5 ± 1.5 18 + 4

0.60 75 ± 1 59.5 ± 3.5 22 ± 7 Table VIII: Arm Length Variations of c-myb Hammerhead Ribozyme 575

Arm Length (base % cells in S % Inhibition (vs . pairs) phase Inactive 7/7)

6/6 62 ± 1 4 ± 4

7/7 60 ± 1 7 ± 3

8/8 60.5 ± 0.5 6 ± 2

9/9 53.5 ± 0.5 18 ± 2

10/10 55 ± 1 16 ± 4

12/12 48 ± 1 28 ± 3

Table IX: Hammerhead ribozymes with 7 vs. 12-nucleotide binding arms targeting three different sites

Ribozyme Length of Inactive Active %

Target Binding Ribozyme Ribozyme Inhibition

Site Arms (% Cell (% Cell (Active Prolifera¬ Prolifera¬ vs. tion) tion) Inactive)

575 7/7 51.5 ± 0.5 43 ± 0.5 24 ± 5

575 12/12 50.5 ± 3.5 37 ± 0.5 37 ± 4

549 7/7 49.5 ± 0.5 44.5 ± 1.5 21 ± 7

549 12/12 48.5 ± 1.5 35 ± 2 41 + 7

1553 7/7 49.5 ± 0.5 43.5 + 2.5 23 ± 9

1553 12/12 49 ± 1 33.5 ± 1.5 45 ± 6

Table X: Effect of chloroquine on ribozyme inhibition of smooth muscle cell proliferation

Ribozyme Chloro¬ Inactive Active % Inhibi¬ quine Ribozyme Ribozyme tion

(μM) (% Cell (% Cell (Active

Prolifera¬ Prolifera¬ vs. tion) tion) inactive)

575, 12/12 0 81.8 ± 0.5 74 ± 1 10 ± 2

575, 12/12 10 83 ± 4 62.5 + 0.5 28 ± 6 Table XI: Inhibition of Human Aortic Smooth Muscle Cells by c-mvb Ribozyme 549

Inactive Active % Inhibition Ribozyme Ribozyme

Ribozyme % Prolifera¬ % Prolifera¬ (active vs. Dose (μM) tion tion inactive)

0.075 55 ± 2 40.5 + 4.5 30 ± 13

0.15 53 ± 10 42 ± 1 23 ± 23

0.30 53 + 7 32.5 + 4.5 44 ± 22

Table XII: Inhibition of Rat Smooth Muscle Cell

Proliferation bv Direct Addition of a Chemically-Modified c-mvb Ribozyme 575

Inactive Active % Inhibition Ribozyme Ribozyme

Ribozyme % Prolifera¬ % Prolifera¬ (active vs. Dose (μM) tion tion inactive)

0.22 42 + 3 36 ± 0.5 15 ± 8

0.67 48 + 3 35 ± 2 28 ± 9

2.0 52 ± 5 25 ± 1 54 ± 7

Table XIII: Human c-mvb Hairpin Ribozyme and Target Sequences

Posi¬ Ribozyme Sequence Target tion

104 CCCUCCCC AGAA GCGC GCGCA GCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GGGGAGGG

148 ACCGACCG AGAA GCCG CGGCA GCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CGGUCGGU

185 GCGCGGCG AGAA GCGG CCGCC GCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CGCCGCGC

528 ACGUUUCG AGAA GUAU AUACG GUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CGAAACGU Posi¬ Ribozyme Sequence Target tion

715 UUCGUCCA AGAA GUAG CUACU GCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UGGACGAA

1025 AUGGCUGC AGAA GCUG CAGCU GCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GCAGCCAU

1187 CUGGUGUG AGAA GCAA UUGCC GAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CACACCAG

1532 GUUCUAAA AGAA GUAU AUACU GUU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UUUAGAAC

1632 CUUCAGGG AGAA GUAU AUACG GUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CCCUGAAG

1836 GGUAUUCA AGAA GUCC GGACA GUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UGAAUACC

1852 UCUGCGUG AGAA GUUG CAACU GUU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CACGCAGA

1861 CAGGCGAG AGAA GCGU ACGCA GAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CUCGCCUG

1993 UGCUACAA AGAA GCAA UUGCA GCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UUGUAGCA

2231 CUGCUCUC AGAA GUUG CAACA GUU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GAGAGCAG

2316 UUAGGUAA AGAA GUUA UAACA GUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UUACCUAA

3068 AAUUAUAA AGAA GUCA UGACU GUU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UUAUAAUU

3138 AUCCAUGC AGAA GUUC GAACU GUU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GCAUGGAU

3199 GUUCUUAA AGAA GUGA UCACU GCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UUAAGAAC

3264 UGCUACAA AGAA GUAA UUACU GCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UUGUAGCA Table XIV : Human c -myb Hammerhead Ribozyme and Target Sequence nt . Target Sequence Ribozyme Sequence

Posi - tion

14 CAACCUGU U UCCUCCUC GAGGAGGA CUGAUGA X GAA ACAGGUUG

15 AACCUGUU U CCUCCUCC GGAGGAGG CUGAUGA X GAA AACAGGUU

16 ACCUGUUU C CUCCUCCU AGGAGGAG CUGAUGA X GAA AAACAGGU 19 UGUUUCCU C CUCCUCCU AGGAGGAG CUGAUGA X GAA AGGAAACA 22 UUCCUCCU C CUCCUUCU AGAAGGAG CUGAUGA X GAA AGGAGGAA

25 CUCCUCCU C CUUCUCCU AGGAGAAG CUGAUGA X GAA AGGAGGAG

28 CUCCUCCU U CUCCUCCU AGGAGGAG CUGAUGA X GAA AGGAGGAG

29 UCCUCCUU C UCCUCCUC GAGGAGGA CUGAUGA X GAA AAGGAGGA 31 CUCCUUCU C CUCCUCCU AGGAGGAG CUGAUGA X GAA AGAAGGAG 34 CUUCUCCU C CUCCUCCG CGGAGGAG CUGAUGA X GAA AGGAGAAG

37 CUCCUCCU C CUCCGUGA UCACGGAG CUGAUGA X GAA AGGAGGAG 40 CUCCUCCU C CGUGACCU AGGUCACG CUGAUGA X GAA AGGAGGAG 49 CGUGACCU C CUCCUCCU AGGAGGAG CUGAUGA X GAA AGGUCACG 52 GACCUCCU C CUCCUCUU AAGAGGAG CUGAUGA X GAA AGGAGGUC 55 CUCCUCCU C CUCUUUCU AGAAAGAG CUGAUGA X GAA AGGAGGAG

58 CUCCUCCU C UUUCUCCU AGGAGAAA CUGAUGA X GAA AGGAGGAG

60 CCUCCUCU U UCUCCUGA UCAGGAGA CUGAUGA X GAA AGAGGAGG

61 CUCCUCUU U CUCCUGAG CUCAGGAG CUGAUGA X GAA AAGAGGAG

62 UCCUCUUU C UCCUGAGA UCUCAGGA CUGAUGA X GAA AAAGAGGA 64 CUCUUUCU C CUGAGAAA UUUCUCAG CUGAUGA X GAA AGAAAGAG

75 GAGAAACU U CGCCCCAG CUGGGGCG CUGAUGA X GAA AGUUUCUC

76 AGAAACUU C GCCCCAGC GCUGGGGC CUGAUGA X GAA AAGUUUCU 156 AGCCCGGU C GGUCCCCG CGGGGACC CUGAUGA X GAA ACCGGGCU 160 CGGUCGGU C CCCGCGGC GCCGCGGG CUGAUGA X GAA ACCGACCG 170 CCGCGGCU C UCGCGGAG CUCCGCGA CUGAUGA X GAA AGCCGCGG

172 GCGGCUCU C GCGGAGCC GGCUCCGC CUGAUGA X GAA AGAGCCGC

224 CACAGCAU A UAUAGCAG CUGCUAUA CUGAUGA X GAA AUGCUGUG

226 CAGCAUAU A UAGCAGUG CACUGCUA CUGAUGA X GAA AUAUGCUG

228 GCAUAUAU A GCAGUGAC GUCACUGC CUGAUGA X GAA AUAUAUGC 253 UGAGGACU U UGAGAUGU ACAUCUCA CUGAUGA X GAA AGUCCUCA

254 GAGGACUU U GAGAUGUG CACAUCUC CUGAUGA X GAA AAGUCCUC

274 CCAUGACU A UGAUGGGC GCCCAUCA CUGAUGA X GAA AGUCAUGG

287 GGGCUGCU U CCCAAGUC GACUUGGG CUGAUGA X GAA AGCAGCCC

288 GGCUGCUU C CCAAGUCU AGACUUGG CUGAUGA X GAA AAGCAGCC n . Target Sequence Ribozyme Sequence Posi- tion

295 UCCCAAGU C UGGAAAGC GCUUUCCA CUGAUGA X GAA ACUUGGGA

306 GAAAGCGU C ACUUGGGG CCCCAAGU CUGAUGA X GAA ACGCUUUC 310 GCGUCACU U GGGGAAAA UUUUCCCC CUGAUGA X GAA AGUGACGC 392 UGGAAAGU U AUUGCCAA UUGGCAAU CUGAUGA X GAA ACUUUCCA 393 GGAAAGUU A UUGCCAAU AUUGGCAA CUGAUGA X GAA AACUUUCC

395 AAAGUUAU U GCCAAUUA UAAUUGGC CUGAUGA X GAA AUAACUUU

402 UUGCCAAU U AUCUCCCG CGGGAGAU CUGAUGA X GAA AUUGGCAA

403 UGCCAAUU A UCUCCCGA UCGGGAGA CUGAUGA X GAA AAUUGGCA 405 CCAAUUAU C UCCCGAAU AUUCGGGA CUGAUGA X GAA AUAAUUGG 414 UCCCGAAU C GAACAGAU AUCUGUUC CUGAUGA X GAA AUUCGGGA

452 CAGAAAGU A CUAAACCC GGGUUUAG CUGAUGA X GAA ACUUUCUG

455 AAAGUACU A AACCCUGA UCAGGGUU CUGAUGA X GAA AGUACUUU

467 CCUGAGCU C AUCAAGGG CCCUUGAU CUGAUGA X GAA AGCUCAGG

470 GAGCUCAU C AAGGGUCC GGACCCUU CUGAUGA X GAA AUGAGCUC 477 UCAAGGGU C CUUGGACC GGUCCAAG CUGAUGA X GAA ACCCUUGA

480 AGGGUCCU U GGACCAAA UUUGGUCC CUGAUGA X GAA AGGACCCU

498 AAGAAGAU C AGAGAGUG CACUCUCU CUGAUGA X GAA AUCUUCUU

509 AGAGUGAU A GAGCUUGU ACAAGCUC CUGAUGA X GAA AUCACUCU

515 AUAGAGCU U GUACAGAA UUCUGUAC CUGAUGA X GAA AGCUCUAU 518 GAGCUUGU A CAGAAAUA UAUUUCUG CUGAUGA X GAA ACAAGCUC

526 ACAGAAAU A CGGUCCGA UCGGACCG CUGAUGA X GAA AUUUCUGU

531 AAUACGGU C CGAAACGU ACGUUUCG CUGAUGA X GAA ACCGUAUU

540 CGAAACGU U GGUCUGUU AACAGACC CUGAUGA X GAA ACGUUUCG

544 ACGUUGGU C UGUUAUUG CAAUAACA CUGAUGA X GAA ACCAACGU 548 UGGUCUGU U AUUGCCAA UUGGCAAU CUGAUGA X GAA ACAGACCA

549 GGUCUGUU A UUGCCAAG CUUGGCAA CUGAUGA X GAA AACAGACC

551 UCUGUUAU U GCCAAGCA UGCUUGGC CUGAUGA X GAA AUAACAGA

562 CAAGCACU U AAAGGGGA UCCCCUUU CUGAUGA X GAA AGUGCUUG

563 AAGCACUU A AAGGGGAG CUCCCCUU CUGAUGA X GAA AAGUGCUU 575 GGGAGAAU U GGAAAACA UGUUUUCC CUGAUGA X GAA AUUCUCCC

588 AACAAUGU A GGGAGAGG CCUCUCCC CUGAUGA X GAA ACAUUGUU

603 GGUGGCAU A ACCACUUG CAAGUGGU CUGAUGA X GAA AUGCCACC

615 ACUUGAAU C CAGAAGUU AACUUCUG CUGAUGA X GAA AUUCAAGU

623 CCAGAAGU U AAGAAAAC GUUUUCUU CUGAUGA X GAA ACUUCUGG 624 CAGAAGUU A AGAAAACC GGUUUUCU CUGAUGA X GAA AACUUCUG

634 GAAAACCU C CUGGACAG CUGUCCAG CUGAUGA X GAA AGGUUUUC nt. Target Sequence Ribozyme Sequence Posi- tion

659 GACAGAAU U AUUUACCA UGGUAAAU CUGAUGA X GAA AUUCUGUC

660 ACAGAAUU A UUUACCAG CUGGUAAA CUGAUGA X GAA AAUUCUGU

662 AGAAUUAU U UACCAGGC GCCUGGUA CUGAUGA X GAA AUAAUUCU

663 GAAUUAUU U ACCAGGCA UGCCUGGU CUGAUGA X GAA AAUAAUUC 664 AAUUAUUU A CCAGGCAC GUGCCUGG CUGAUGA X GAA AAAUAAUU

704 GCAGAAAU C GCAAAGCU AGCUUUGC CUGAUGA X GAA AUUUCUGC

713 GCAAAGCU A CUGCCUGG CCAGGCAG CUGAUGA X GAA AGCUUUGC

732 GAACUGAU A AUGCUAUC GAUAGCAU CUGAUGA X GAA AUCAGUUC

738 AUAAUGCU A UCAAGAAC GUUCUUGA CUGAUGA X GAA AGCAUUAU 740 AAUGCUAU C AAGAACCA UGGUUCUU CUGAUGA X GAA AUAGCAUU

756 ACUGGAAU U CUACAAUG CAUUGUAG CUGAUGA X GAA AUUCCAGU

757 CUGGAAUU C UACAAUGC GCAUUGUA CUGAUGA X GAA AAUUCCAG 759 GGAAUUCU A CAAUGCGU ACGCAUUG CUGAUGA X GAA AGAAUUCC 768 CAAUGCGU C GGAAGGUC GACCUUCC CUGAUGA X GAA ACGCAUUG 776 CGGAAGGU C GAACAGGA UCCUGUUC CUGAUGA X GAA ACCUUCCG

789 AGGAAGGU U AUCUGCAG CUGCAGAU CUGAUGA X GAA ACCUUCCU

790 GGAAGGUU A UCUGCAGG CCUGCAGA CUGAUGA X GAA AACCUUCC 792 AAGGUUAU C UGCAGGAG CUCCUGCA CUGAUGA X GAA AUAACCUU 802 GCAGGAGU C UUCAAAAG CUUUUGAA CUGAUGA X GAA ACUCCUGC 804 AGGAGUCU U CAAAAGCC GGCUUUUG CUGAUGA X GAA AGACUCCU

805 GGAGUCUU C AAAAGCCA UGGCUUUU CUGAUGA X GAA AAGACUCC

838 CACAAGCU U CCAGAAGA UCUUCUGG CUGAUGA X GAA AGCUUGUG

839 ACAAGCUU C CAGAAGAA UUCUUCUG CUGAUGA X GAA AAGCUUGU

852 AGAACAGU C AUUUGAUG CAUCAAAU CUGAUGA X GAA ACUGUUCU 855 ACAGUCAU U UGAUGGGU ACCCAUCA CUGAUGA X GAA AUGACUGU

856 CAGUCAUU U GAUGGGUU AACCCAUC CUGAUGA X GAA AAUGACUG

864 UGAUGGGU U UUGCUCAG CUGAGCAA CUGAUGA X GAA ACCCAUCA

865 GAUGGGUU U UGCUCAGG CCUGAGCA CUGAUGA X GAA AACCCAUC

866 AUGGGUUU U GCUCAGGC GCCUGAGC CUGAUGA X GAA AAACCCAU 870 GUUUUGCU C AGGCUCCG CGGAGCCU CUGAUGA X GAA AGCAAAAC

876 CUCAGGCU C CGCCUACA UGUAGGCG CUGAUGA X GAA AGCCUGAG

882 CUCCGCCU A CAGCUCAA UUGAGCUG CUGAUGA X GAA AGGCGGAG

888 CUACAGCU C AACUCCCU AGGGAGUU CUGAUGA X GAA AGCUGUAG

893 GCUCAACU C CCUGCCAC GUGGCAGG CUGAUGA X GAA AGUUGAGC 917 CCCACUGU U AACAACGA UCGUUGUU CUGAUGA X GAA ACAGUGGG

928 CAACGACU A UUCCUAUU AAUAGGAA CUGAUGA X GAA AGUCGUUG nt . Target Sequence Ribozyme Sequence

Posi¬ tion

930 ACGACUAU U CCUAUUAC GUAAUAGG CUGAUGA X GAA AUAGUCGU

931 CGACUAUU C CUAUUACC GGUAAUAG CUGAUGA X GAA AAUAGUCG

934 CUAUUCCU A UUACCACA UGUGGUAA CUGAUGA X GAA AGGAAUAG

936 AUUCCUAU U ACCACAUU AAUGUGGU CUGAUGA X GAA AUAGGAAU

937 UUCCUAUU A CCACAUUU AAAUGUGG CUGAUGA X GAA AAUAGGAA

944 UACCACAU U UCUGAAGC GCUUCAGA CUGAUGA X GAA AUGUGGUA

945 ACCACAUU U CUGAAGCA UGCUUCAG CUGAUGA X GAA AAUGUGGU

946 CCACAUUU C UGAAGCAC GUGCUUCA CUGAUGA X GAA AAAUGUGG

962 CAAAAUGU C UCCAGUCA UGACUGGA CUGAUGA X GAA ACAUUUUG 9 96644 AAAUGUCU C CAGUCAUG CAUGACUG CUGAUGA X GAA AGACAUUU

969 UCUCCAGU C AUGUUCCA UGGAACAU CUGAUGA X GAA ACUGGAGA

974 AGUCAUGU U CCAUACCC GGGUAUGG CUGAUGA X GAA ACAUGACU

975 GUCAUGUU C CAUACCCU AGGGUAUG CUGAUGA X GAA AACAUGAC

979 UGUUCCAU A CCCUGUAG CUACAGGG CUGAUGA X GAA AUGGAACA 9 98866 UACCCUGU A GCGUUACA UGUAACGC CUGAUGA X GAA ACAGGGUA

991 UGUAGCGU U ACAUGUAA UUACAUGU CUGAUGA X GAA ACGCUACA

992 GUAGCGUU A CAUGUAAA UUUACAUG CUGAUGA X GAA AACGCUAC

1002 AUGUAAAU A UAGUCAAU AUUGACUA CUGAUGA X GAA AUUUACAU

1004 GUAAAUAU A GUCAAUGU ACAUUGAC CUGAUGA X GAA AUAUUUAC 1 1000077 AAUAUAGU C AAUGUCCC GGACAUU CUGAUGA X GAA ACUAUAUU

1013 GUCAAUGU C CCUCAGCC GGCUGAGG CUGAUGA X GAA ACAUUGAC

1017 AUGUCCCU C AGCCAGCU AGCUGGCU CUGAUGA X GAA AGGGACAU

1037 GCAGCCAU U CAGAGACA UGUCUCUG CUGAUGA X GAA AUGGCUGC

1048 GAGACACU A UAAUGAUG CAUCAUUA CUGAUGA X GAA AGUGUCUC 1 1005500 GACACUAU A AUGAUGAA UUCAUCAU CUGAUGA X GAA AUAGUGUC

1082 AAGCGAAU A AAGGAAUU AAUUCCUU CUGAUGA X GAA AUUCGCUU

1090 AAAGGAAU U AGAAUUGC GCAAUUCU CUGAUGA X GAA AUUCCUUU

1091 AAGGAAUU A GAAUUGCU AGCAAUUC CUGAUGA X GAA AAUUCCUU

1096 AUUAGAAU U GCUCCUAA UUAGGAGC CUGAUGA X GAA AUUCUAAU 1 1110000 GAAUUGCU C CUAAUGUC GACAUUAG CUGAUGA X GAA AGCAAUUC

1103 UUGCUCCU A AUGUCAAC GUUGACAU CUGAUGA X GAA AGGAGCAA

1108 CCUAAUGU C AACCGAGA UCUCGGUU CUGAUGA X GAA ACAUUAGG

1124 AAUGAGCU A AAAGGACA UGUCCUUU CUGAUGA X GAA AGCUCAUU

1184 ACCACCAU U GCCGACCA UGGUCGGC CUGAUGA X GAA AUGGUGGU 1 1220033 CCAGACCU C AUGGAGAC GUCUCCAU CUGAUGA X GAA AGGUCUGG

1223 GCACCUGU U UCCUGUUU AAACAGGA CUGAUGA X GAA ACAGGUGC nt . Target Sequence Ribozyme Sequence

Posi¬ tion

1224 CACCUGUU U CCUGUUUG CAAACAGG CUGAUGA X GAA AACAGGUG

1225 ACCUGUUU C CUGUUUGG CCAAACAG CUGAUGA X GAA AAACAGGU

1230 UUUCCUGU U UGGGAGAA UUCUCCCA CUGAUGA X GAA ACAGGAAA

1231 UUCCUGUU U GGGAGAAC GUUCUCCC CUGAUGA X GAA AACAGGAA

1246 ACACCACU C CACUCCAU AUGGAGUG CUGAUGA X GAA AGUGGUGU

1251 ACUCCACU C CAUCUCUG CAGAGAUG CUGAUGA X GAA AGUGGAGU

1255 CACUCCAU C UCUGCCAG CUGGCAGA CUGAUGA X GAA AUGGAGUG

1257 CUCCAUCU C UGCCAGCG CGCUGGCA CUGAUGA X GAA AGAUGGAG

1269 CAGCGGAU C CUGGCUCC GGAGCCAG CUGAUGA X GAA AUCCGCUG 1 1227766 UCCUGGCU C CCUACCUG CAGGUAGG CUGAUGA X GAA AGCCAGGA

1280 GGCUCCCU A CCUGAAGA UCUUCAGG CUGAUGA X GAA AGGGAGCC

1297 AAGCGCCU C GCCAGCAA UUGCUGGC CUGAUGA X GAA AGGCGCUU

1316 UGCAUGAU C GUCCACCA UGGUGGAC CUGAUGA X GAA AUCAUGCA

1319 AUGAUCGU C CACCAGGG CCCUGGUG CUGAUGA X GAA ACGAUCAU 1 1333344 GGCACCAU U CUGGAUAA UUAUCCAG CUGAUGA X GAA AUGGUGCC

1335 GCACCAUU C UGGAUAAU AUUAUCCA CUGAUGA X GAA AAUGGUGC

1341 UUCUGGAU A AUGUUAAG CUUAACAU CUGAUGA X GAA AUCCAGAA

1346 GAUAAUGU U AAGAACCU AGGUUCUU CUGAUGA X GAA ACAUUAUC

1347 AUAAUGUU A AGAACCUC GAGGUUCU CUGAUGA X GAA AACAUUAU 1 1335555 AAGAACCU C UUAGAAUU AAUUCUAA CUGAUGA X GAA AGGUUCUU

1357 GAACCUCU U AGAAUUUG CAAAUUCU CUGAUGA X GAA AGAGGUUC

1358 AACCUCUU A GAAUUUGC GCAAAUUC CUGAUGA X GAA AAGAGGUU

1363 CUUAGAAU U UGCAGAAA UUUCUGCA CUGAUGA X GAA AUUCUAAG

1364 UUAGAAUU U GCAGAAAC GUUUCUGC CUGAUGA X GAA AAUUCUAA 1 1337766 GAAACACU C CAAUUUAU AUAAAUUG CUGAUGA X GAA AGUGUUUC

1381 ACUCCAAU U UAUAGAUU AAUCUAUA CUGAUGA X GAA AUUGGAGU

1382 CUCCAAUU U AUAGAUUC GAAUCUAU CUGAUGA X GAA AAUUGGAG

1383 UCCAAUUU A UAGAUUCU AGAAUCUA CUGAUGA X GAA AAAUUGGA

1385 CAAUUUAU A GAUUCUUU AAAGAAUC CUGAUGA X GAA AUAAAUUG 1 1338899 UUAUAGAU U CUUUCUUA UAAGAAAG CUGAUGA X GAA AUCUAUAA

1390 UAUAGAUU C UUUCUUAA UUAAGAAA CUGAUGA X GAA AAUCUAUA

1392 UAGAUUCU U UCUUAAAC GUUUAAGA CUGAUGA X GAA AGAAUCUA

1393 AGAUUCUU U CUUAAACA UGUUUAAG CUGAUGA X GAA AAGAAUCU

1394 GAUUCUUU C UUAAACAC GUGUUUAA CUGAUGA X GAA AAAGAAUC 1 1339966 UUCUUUCU U AAACACUU AAGUGUUU CUGAUGA X GAA AGAAAGAA

1397 UCUUUCUU A AACACUUC GAAGUGUU CUGAUGA X GAA AAGAAAGA nt. Target Sequence Ribozyme Sequence

Posi¬ tion

1404 UAAACACU U CCAGUAAC GUUACUGG CUGAUGA X GAA AGUGUUUA

1405 AAACACUU C CAGUAACC GGUUACUG CUGAUGA X GAA AAGUGUUU

1410 CUUCCAGU A ACCAUGAA UUCAUGGU CUGAUGA X GAA ACUGGAAG

1423 UGAAAACU C AGACUUGG CCAAGUCU CUGAUGA X GAA AGUUUUCA

1429 CUCAGACU U GGAAAUGC GCAUUUCC CUGAUGA X GAA AGUCUGAG

1440 AAAUGCCU U CUUUAACU AGUUAAAG CUGAUGA X GAA AGGCAUUU

1441 AAUGCCUU C UUUAACUU AAGUUAAA CUGAUGA X GAA AAGGCAUU

1443 UGCCUUCU U UAACUUCC GGAAGUUA CUGAUGA X GAA AGAAGGCA

1444 GCCUUCUU U AACUUCCA UGGAAGUU CUGAUGA X GAA AAGAAGGC 1 1444455 CCUUCUUU A ACUUCCAC GUGGAAGU CUGAUGA X GAA AAAGAAGG

1449 CUUUAACU U CCACCCCC GGGGGUGG CUGAUGA X GAA AGUUAAAG

1450 UUUAACUU C CACCCCCC GGGGGGUG CUGAUGA X GAA AAGUUAAA

1460 ACCCCCCU C AUUGGUCA UGACCAAU CUGAUGA X GAA AGGGGGGU

1463 CCCCUCAU U GGUCACAA UUGUGACC CUGAUGA X GAA AUGAGGGG 1 1446677 UCAUUGGU C ACAAAUUG CAAUUUGU CUGAUGA X GAA ACCAAUGA

1474 UCACAAAU U GACUGUUA UAACAGUC CUGAUGA X GAA AUUUGUGA

1481 UUGACUGU U ACAACACC GGUGUUGU CUGAUGA X GAA ACAGUCAA

1482 UGACUGUU A CAACACCA UGGUGUUG CUGAUGA X GAA AACAGUCA

1492 AACACCAU U UCAUAGAG CUCUAUGA CUGAUGA X GAA AUGGUGUU 1 1449933 ACACCAUU U CAUAGAGA UCUCUAUG CUGAUGA X GAA AAUGGUGU

1494 CACCAUUU C AUAGAGAC GUCUCUAU CUGAUGA X GAA AAAUGGUG

1497 CAUUUCAU A GAGACCAG CUGGUCUC CUGAUGA X GAA AUGAAAUG

1518 UGAAAACU C AAAAGGAA UUCCUUUU CUGAUGA X GAA AGUUUUCA

1530 AGGAAAAU A CUGUUUUU AAAAACAG CUGAUGA X GAA AUUUUCCU 1 1553355 AAUACUGU U UUUAGAAC GUUCUAAA CUGAUGA X GAA ACAGUAUU

1536 AUACUGUU U UUAGAACC GGUUCUAA CUGAUGA X GAA AACAGUAU

1537 UACUGUUU U UAGAACCC GGGUUCUA CUGAUGA X GAA AAACAGUA

1538 ACUGUUUU U AGAACCCC GGGGUUCU CUGAUGA X GAA AAAACAGU

1539 CUGUUUUU A GAACCCCA UGGGGUUC CUGAUGA X GAA AAAAACAG 1 1555511 CCCCAGCU A UCAAAAGG CCUUUUGA CUGAUGA X GAA AGCUGGGG

1553 CCAGCUAU C AAAAGGUC GACCUUUU CUGAUGA X GAA AUAGCUGG

1561 CAAAAGGU C AAUCUUAG CUAAGAUU CUGAUGA X GAA ACCUUUUG

1565 AGGUCAAU C UUAGAAAG CUUUCUAA CUGAUGA X GAA AUUGACCU

1567 GUCAAUCU U AGAAAGCU AGCUUUCU CUGAUGA X GAA AGAUUGAC 1 1556688 UCAAUCUU A GAAAGCUC GAGCUUUC CUGAUGA X GAA AAGAUUGA

1578 AAAGCUCU C CAAGAACU AGUUCUUG CUGAUGA X GAA AGAGCUUU nt. Target Sequence Ribozyme Sequence

Posi¬ tion

1587 CAAGAACU C CUACACCA UGGUGUAG CUGAUGA X GAA AGUUCUUG

1590 GAACUCCU A CACCAUUC GAAUGGUG CUGAUGA X GAA AGGAGUUC

1597 UACACCAU U CAAACAUG CAUGUUUG CUGAUGA X GAA AUGGUGUA

1598 ACACCAUU C AAACAUGC GCAUGUUU CUGAUGA X GAA AAUGGUGU

1610 CAUGCACU U GCAGCUCA UGAGCUGC CUGAUGA X GAA AGUGCAUG

1617 UUGCAGCU C AAGAAAUU AAUUUCUU CUGAUGA X GAA AGCUGCAA

1625 CAAGAAAU U AAAUACGG CCGUAUUU CUGAUGA X GAA AUUUCUUG

1626 AAGAAAUU A AAUACGGU ACCGUAUU CUGAUGA X GAA AAUUUCUU

1635 AAUACGGU C CCCUGAAG CUUCAGGG CUGAUGA X GAA ACCGUAUU 1 1664499 AAGAUGCU A CCUCAGAC GUCUGAGG CUGAUGA X GAA AGCAUCUU

1653 UGCUACCU C AGACACCC GGGUGUCU CUGAUGA X GAA AGGUAGCA

1663 GACACCCU C UCAUCUAG CUAGAUGA CUGAUGA X GAA AGGGUGUC

1665 CACCCUCU C AUCUAGUA UACUAGAU CUGAUGA X GAA AGAGGGUG

1668 CCUCUCAU C UAGUAGAA UUCUACUA CUGAUGA X GAA AUGAGAGG 1 1667700 UCUCAUCU A GUAGAAGA UCUUCUAC CUGAUGA X GAA AGAUGAGA

1673 CAUCUAGU A GAAGAUCU AGAUCUUC CUGAUGA X GAA ACUAGAUG

1680 UAGAAGAU C UGCAGGAU AUCCUGCA CUGAUGA X GAA AUCUUCUA

1694 GAUGUGAU C AAACAGGA UCCUGUUU CUGAUGA X GAA AUCACAUC

1705 ACAGGAAU C UGAUGAAU AUUCAUCA CUGAUGA X GAA AUUCCUGU 1 1771144 UGAUGAAU C UGGAAUUG CAAUUCCA CUGAUGA X GAA AUUCAUCA

1721 UCUGGAAU U GUUGCUGA UCAGCAAC CUGAUGA X GAA AUUCCAGA

1724 GGAAUUGU U GCUGAGUU AACUCAGC CUGAUGA X GAA ACAAUUCC

1732 UGCUGAGU U UCAAGAAA UUUCUUGA CUGAUGA X GAA ACUCAGCA

1733 GCUGAGUU U CAAGAAAA UUUUCUUG CUGAUGA X GAA AACUCAGC 1 1775533 ACCACCCU U ACUGAAGA UCUUCAGU CUGAUGA X GAA AGGGUGGU

1754 CCACCCUU A CUGAAGAA UUCUUCAG CUGAUGA X GAA AAGGGUGG

1766 AAGAAAAU C AAACAAGA UCUUGUUU CUGAUGA X GAA AUUUUCUU

1783 GGUGGAAU C UCCAACUG CAGUUGGA CUGAUGA X GAA AUUCCACC

1785 UGGAAUCU C CAACUGAU AUCAGUUG CUGAUGA X GAA AGAUUCCA 1 1779944 CAACUGAU A AAUCAGGA UCCUGAUU CUGAUGA X GAA AUCAGUUG

1798 UGAUAAAU C AGGAAACU AGUUUCCU CUGAUGA X GAA AUUUAUCA

1807 AGGAAACU U CUUCUGCU AGCAGAAG CUGAUGA X GAA AGUUUCCU

1808 GGAAACUU C UUCUGCUC GAGCAGAA CUGAUGA X GAA AAGUUUCC

1810 AAACUUCU U CUGCUCAC GUGAGCAG CUGAUGA X GAA AGAAGUUU 1 1881111 AACUUCUU C UGCUCACA UGUGAGCA CUGAUGA X GAA AAGAAGUU

1816 CUUCUGCU C ACACCACU AGUGGUGU CUGAUGA X GAA AGCAGAAG nt. Target Sequence Ribozyme Sequence Posi- tion

1839 GGGACAGU C UGAAUACC GGUAUUCA CUGAUGA X GAA ACUGUCCC

1845 GUCUGAAU A CCCAACUG CAGUUGGG CUGAUGA X GAA AUUCAGAC

1855 CCAACUGU U CACGCAGA UCUGCGUG CUGAUGA X GAA ACAGUUGG

1856 CAACUGUU C ACGCAGAC GUCUGCGU CUGAUGA X GAA AACAGUUG 1867 GCAGACCU C GCCUGUGG CCACAGGC CUGAUGA X GAA AGGUCUGC

1890 CACCGAAU A UUCUUACA UGUAAGAA CUGAUGA X GAA AUUCGGUG

1892 CCGAAUAU U CUUACAAG CUUGUAAG CUGAUGA X GAA AUAUUCGG

1893 CGAAUAUU C UUACAAGC GCUUGUAA CUGAUGA X GAA AAUAUUCG 1895 AAUAUUCU U ACAAGCUC GAGCUUGU CUGAUGA X GAA AGAAUAUU 1896 AUAUUCUU A CAAGCUCC GGAGCUUG CUGAUGA X GAA AAGAAUAU

1903 UACAAGCU C CGUUUUAA UUAAAACG CUGAUGA X GAA AGCUUGUA

1907 AGCUCCGU U UUAAUGGC GCCAUUAA CUGAUGA X GAA ACGGAGCU

1908 GCUCCGUU U UAAUGGCA UGCCAUUA CUGAUGA X GAA AACGGAGC

1909 CUCCGUUU U AAUGGCAC GUGCCAUU CUGAUGA X GAA AAACGGAG 1910 UCCGUUUU A AUGGCACC GGUGCCAU CUGAUGA X GAA AAAACGGA

1924 ACCAGCAU C AGAAGAUG CAUCUUCU CUGAUGA X GAA AUGCUGGU

1943 GACAAUGU U CUCAAAGC GCUUUGAG CUGAUGA X GAA ACAUUGUC

1944 ACAAUGUU C UCAAAGCA UGCUUUGA CUGAUGA X GAA AACAUUGU 1946 AAUGUUCU C AAAGCAUU AAUGCUUU CUGAUGA X GAA AGAACAUU 1954 CAAAGCAU U UACAGUAC GUACUGUA CUGAUGA X GAA AUGCUUUG

1955 AAAGCAUU U ACAGUACC GGUACUGU CUGAUGA X GAA AAUGCUUU

1956 AAGCAUUU A CAGUACCU AGGUACUG CUGAUGA X GAA AAAUGCUU 1961 UUUACAGU A CCUAAAAA UUUUUAGG CUGAUGA X GAA ACUGUAAA 1965 CAGUACCU A AAAACAGG CCUGUUUU CUGAUGA X GAA AGGUACUG 1975 AAACAGGU C CCUGGCGA UCGCCAGG CUGAUGA X GAA ACCUGUUU

1990 GAGCCCCU U GCAGCCUU AAGGCUGC CUGAUGA X GAA AGGGGCUC

1998 UGCAGCCU U GUAGCAGU ACUGCUAC CUGAUGA X GAA AGGCUGCA

2001 AGCCUUGU A GCAGUACC GGUACUGC CUGAUGA X GAA ACAAGGCU

2007 GUAGCAGU A CCUGGGAA UUCCCAGG CUGAUGA X GAA ACUGCUAC 2023 ACCUGCAU C CUGUGGAA UUCCACAG CUGAUGA X GAA AUGCAGGU

2053 GAUGACAU C UUCCAGUC GACUGGAA CUGAUGA X GAA AUGUCAUC

2055 UGACAUCU U CCAGUCAA UUGACUGG CUGAUGA X GAA AGAUGUCA

2056 GACAUCUU C CAGUCAAG CUUGACUG CUGAUGA X GAA AAGAUGUC 2061 CUUCCAGU C AAGCUCGU ACGAGCUU CUGAUGA X GAA ACUGGAAG 2067 GUCAAGCU C GUAAAUAC GUAUUUAC CUGAUGA X GAA AGCUUGAC

2070 AAGCUCGU A AAUACGUG CACGUAUU CUGAUGA X GAA ACGAGCUU nt. Target Sequence Ribozyme Sequence

Posi¬ tion

2074 UCGUAAAU A CGUGAAUG CAUUCACG CUGAUGA X GAA AUUUACGA

2086 GAAUGCAU U CUCAGCCC GGGCUGAG CUGAUGA X GAA AUGCAUUC

2087 AAUGCAUU C UCAGCCCG CGGGCUGA CUGAUGA X GAA AAUGCAUU

2089 UGCAUUCU C AGCCCGGA UCCGGGCU CUGAUGA X GAA AGAAUGCA

2105 ACGCUGGU C AUGUGAGA UCUCACAU CUGAUGA X GAA ACCAGCGU

2117 UGAGACAU U UCCAGAAA UUUCUGGA CUGAUGA X GAA AUGUCUCA

2118 GAGACAUU U CCAGAAAA UUUUCUGG CUGAUGA X GAA AAUGUCUC

2119 AGACAUUU C CAGAAAAG CUUUUCUG CUGAUGA X GAA AAAUGUCU

2131 AAAAGCAU U AUGGUUUU AAAACCAU CUGAUGA X GAA AUGCUUUU 2 2113322 AAAGCAUU A UGGUUUUC GAAAACCA CUGAUGA X GAA AAUGCUUU

2137 AUUAUGGU U UUCAGAAC GUUCUGAA CUGAUGA X GAA ACCAUAAU

2138 UUAUGGUU U UCAGAACA UGUUCUGA CUGAUGA X GAA AACCAUAA

2139 UAUGGUUU U CAGAACAC GUGUUCUG CUGAUGA X GAA AAACCAUA

2140 AUGGUUUU C AGAACACU AGUGUUCU CUGAUGA X GAA AAAACCAU 2 2114499 AGAACACU U CAAGUUGA UCAACUUG CUGAUGA X GAA AGUGUUCU

2150 GAACACUU C AAGUUGAC GUCAACUU CUGAUGA X GAA AAGUGUUC

2155 CUUCAAGU U GACUUGGG CCCAAGUC CUGAUGA X GAA ACUUGAAG

2160 AGUUGACU U GGGAUAUA UAUAUCCC CUGAUGA X GAA AGUCAACU

2166 CUUGGGAU A UAUCAUUC GAAUGAUA CUGAUGA X GAA AUCCCAAG 2 2116688 UGGGAUAU A UCAUUCCU AGGAAUGA CUGAUGA X GAA AUAUCCCA

2170 GGAUAUAU C AUUCCUCA UGAGGAAU CUGAUGA X GAA AUAUAUCC

2173 UAUAUCAU U CCUCAACA UGUUGAGG CUGAUGA X GAA AUGAUAUA

2174 AUAUCAUU C CUCAACAU AUGUUGAG CUGAUGA X GAA AAUGAUAU

2177 UCAUUCCU C AACAUGAA UUCAUGUU CUGAUGA X GAA AGGAAUGA 2 2118899 AUGAAACU U UUCAUGAA UUCAUGAA CUGAUGA X GAA AGUUUCAU

2190 UGAAACUU U UCAUGAAU AUUCAUGA CUGAUGA X GAA AAGUUUCA

2191 GAAACUUU U CAUGAAUG CAUUCAUG CUGAUGA X GAA AAAGUUUC

2192 AAACUUUU C AUGAAUGG CCAUUCAU CUGAUGA X GAA AAAAGUUU

2212 AAGAACCU A UUUUUGUU AACAAAAA CUGAUGA X GAA AGGUUCUU 2 2221144 GAACCUAU U UUUGUUGU ACAACAAA CUGAUGA X GAA AUAGGUUC

2215 AACCUAUU U UUGUUGUG CACAACAA CUGAUGA X GAA AAUAGGUU

2216 ACCUAUUU U UGUUGUGG CCACAACA CUGAUGA X GAA AAAUAGGU

2217 CCUAUUUU U GUUGUGGU ACCACAAC CUGAUGA X GAA AAAAUAGG

2220 AUUUUUGU U GUGGUACA UGUACCAC CUGAUGA X GAA ACAAAAAU 2 2222266 GUUGUGGU A CAACAGUU AACUGUUG CUGAUGA X GAA ACCACAAC

2234 ACAACAGU U GAGAGCAG CUGCUCUC CUGAUGA X GAA ACUGUUGU n . Target Seguence Ribozyme Sequence

Posi¬ tion

2255 AAGUGCAU U UAGUUGAA UUCAACUA CUGAUGA X GAA AUGCACUU

2256 AGUGCAUU U AGUUGAAU AUUCAACU CUGAUGA X GAA AAUGCACU

2257 GUGCAUUU A GUUGAAUG CAUUCAAC CUGAUGA X GAA AAAUGCAC

2260 CAUUUAGU U GAAUGAAG CUUCAUUC CUGAUGA X GAA ACUAAAUG

2270 AAUGAAGU C UUCUUGGA UCCAAGAA CUGAUGA X GAA ACUUCAUU

2272 UGAAGUCU U CUUGGAUU AAUCCAAG CUGAUGA X GAA AGACUUCA

2273 GAAGUCUU C UUGGAUUU AAAUCCAA CUGAUGA X GAA AAGACUUC

2275 AGUCUUCU U GGAUUUCA UGAAAUCC CUGAUGA X GAA AGAAGACU

2280 UCUUGGAU U UCACCCAA UUGGGUGA CUGAUGA X GAA AUCCAAGA 2 2228811 CUUGGAUU U CACCCAAC GUUGGGUG CUGAUGA X GAA AAUCCAAG

2282 UUGGAUUU C ACCCAACU AGUUGGGU CUGAUGA X GAA AAAUCCAA

2291 ACCCAACU A AAAGGAUU AAUCCUUU CUGAUGA X GAA AGUUGGGU

2299 AAAAGGAU U UUUAAAAA UUUUUAAA CUGAUGA X GAA AUCCUUUU

2300 AAAGGAUU U UUAAAAAU AUUUUUAA CUGAUGA X GAA AAUCCUUU 2 2330011 AAGGAUUU U UAAAAAUA UAUUUUUA CUGAUGA X GAA AAAUCCUU

2302 AGGAUUUU U AAAAAUAA UUAUUUUU CUGAUGA X GAA AAAAUCCU

2309 UUAAAAAU A AAUAACAG CUGUUAUU CUGAUGA X GAA AUUUUUAA

2313 AAAUAAAU A ACAGUCUU AAGACUGU CUGAUGA X GAA AUUUAUUU

2319 AUAACAGU C UUACCUAA UUAGGUAA CUGAUGA X GAA ACUGUUAU 2 2332211 AACAGUCU U ACCUAAAU AUUUAGGU CUGAUGA X GAA AGACUGUU

2322 ACAGUCUU A CCUAAAUU AAUUUAGG CUGAUGA X GAA AAGACUGU

2326 UCUUACCU A AAUUAUUA UAAUAAUU CUGAUGA X GAA AGGUAAGA

2330 ACCUAAAU U AUUAGGUA UACCUAAU CUGAUGA X GAA AUUUAGGU

2331 CCUAAAUU A UUAGGUAA UUACCUAA CUGAUGA X GAA AAUUUAGG 2 2333333 UAAAUUAU U AGGUAAUG CAUUACCU CUGAUGA X GAA AUAAUUUA

2334 AAAUUAUU A GGUAAUGA UCAUUACC CUGAUGA X GAA AAUAAUUU

2338 UAUUAGGU A AUGAAUUG CAAUUCAU CUGAUGA X GAA ACCUAAUA

2345 UAAUGAAU U GUAGCCAG CUGGCUAC CUGAUGA X GAA AUUCAUUA

2348 UGAAUUGU A GCCAGUUG CAACUGGC CUGAUGA X GAA ACAAUUCA 2 2335555 UAGCCAGU U GUUAAUAU AUAUUAAC CUGAUGA X GAA ACUGGCUA

2358 CCAGUUGU U AAUAUCUU AAGAUAUU CUGAUGA X GAA ACAACUGG

2359 CAGUUGUU A AUAUCUUA UAAGAUAU CUGAUGA X GAA AACAACUG

2362 UUGUUAAU A UCUUAAUG CAUUAAGA CUGAUGA X GAA AUUAACAA

2364 GUUAAUAU C UUAAUGCA UGCAUUAA CUGAUGA X GAA AUAUUAAC 2 2336666 UAAUAUCU U AAUGCAGA UCUGCAUU CUGAUGA X GAA AGAUAUUA

2367 AAUAUCUU A AUGCAGAU AUCUGCAU CUGAUGA X GAA AAGAUAUU nt. Target Sequence Ribozyme Sequence Posi- tion

2376 AUGCAGAU U UUUUUAAA UUUAAAAA CUGAUGA X GAA AUCUGCAU

2377 UGCAGAUU U UUUUAAAA UUUUAAAA CUGAUGA X GAA AAUCUGCA

2378 GCAGAUUU U UUUAAAAA UUUUUAAA CUGAUGA X GAA AAAUCUGC

2379 CAGAUUUU U UUAAAAAA UUUUUUAA CUGAUGA X GAA AAAAUCUG 2380 AGAUUUUU U UAAAAAAA UUUUUUUA CUGAUGA X GAA AAAAAUCU

2381 GAUUUUUU U AAAAAAAA UUUUUUUU CUGAUGA X GAA AAAAAAUC

2382 AUUUUUUU A AAAAAAAC GUUUUUUU CUGAUGA X GAA AAAAAAAU

2393 AAAAACAU A AAAUGAUU AAUCAUUU CUGAUGA X GAA AUGUUUUU

2401 AAAAUGAU U UAUCUGUA UACAGAUA CUGAUGA X GAA AUCAUUUU 2402 AAAUGAUU U AUCUGUAU AUACAGAU CUGAUGA X GAA AAUCAUUU

2403 AAUGAUUU A UCUGUAUU AAUACAGA CUGAUGA X GAA AAAUCAUU

2405 UGAUUUAU C UGUAUUUU AAAAUACA CUGAUGA X GAA AUAAAUCA

2409 UUAUCUGU A UUUUAAAG CUUUAAAA CUGAUGA X GAA ACAGAUAA

2411 AUCUGUAU U UUAAAGGA UCCUUUAA CUGAUGA X GAA AUACAGAU 2412 UCUGUAUU U UAAAGGAU AUCCUUUA CUGAUGA X GAA AAUACAGA

2413 CUGUAUUU U AAAGGAUC GAUCCUUU CUGAUGA X GAA AAAUACAG

2414 UGUAUUUU A AAGGAUCC GGAUCCUU CUGAUGA X GAA AAAAUACA 2421 UAAAGGAU C CAACAGAU AUCUGUUG CUGAUGA X GAA AUCCUUUA 2430 CAACAGAU C AGUAUUUU AAAAUACU CUGAUGA X GAA AUCUGUUG 2434 AGAUCAGU A UUUUUUCC GGAAAAAA CUGAUGA X GAA ACUGAUCU

2436 AUCAGUAU U UUUUCCUG CAGGAAAA CUGAUGA X GAA AUACUGAU

2437 UCAGUAUU U UUUCCUGU ACAGGAAA CUGAUGA X GAA AAUACUGA

2438 CAGUAUUU U UUCCUGUG CACAGGAA CUGAUGA X GAA AAAUACUG 2439 AGUAUUUU U UCCUGUGA UCACAGGA CUGAUGA X GAA AAAAUACU 2440 GUAUUUUU U CCUGUGAU AUCACAGG CUGAUGA X GAA AAAAAUAC

2441 UAUUUUUU C CUGUGAUG CAUCACAG CUGAUGA X GAA AAAAAAUA

2453 UGAUGGGU U UUUUGAAA UUUCAAAA CUGAUGA X GAA ACCCAUCA

2454 GAUGGGUU U UUUGAAAU AUUUCAAA CUGAUGA X GAA AACCCAUC

2455 AUGGGUUU U UUGAAAUU AAUUUCAA CUGAUGA X GAA AAACCCAU 2456 UGGGUUUU U UGAAAUUU AAAUUUCA CUGAUGA X GAA AAAACCCA

2457 GGGUUUUU U GAAAUUUG CAAAUUUC CUGAUGA X GAA AAAAACCC

2463 UUUGAAAU U UGACACAU AUGUGUCA CUGAUGA X GAA AUUUCAAA

2464 UUGAAAUU U GACACAUU AAUGUGUC CUGAUGA X GAA AAUUUCAA 2472 UGACACAU U AAAAGGUA UACCUUUU CUGAUGA X GAA AUGUGUCA 2473 GACACAUU A AAAGGUAC GUACCUUU CUGAUGA X GAA AAUGUGUC

2480 UAAAAGGU A CUCCAGUA UACUGGAG CUGAUGA X GAA ACCUUUUA n . Target Sequence Ribozyme Sequence

Posi¬ tion

2488 ACUCCAGU A UUUCACUU AAGUGAAA CUGAUGA X GAA ACUGGAGU

2490 UCCAGUAU U UCACUUUU AAAAGUGA CUGAUGA X GAA AUACUGGA

2491 CCAGUAUU U CACUUUUC GAAAAGUG CUGAUGA X GAA AAUACUGG

2492 CAGUAUUU C ACUUUUCU AGAAAAGU CUGAUGA X GAA AAAUACUG

2496 AUUUCACU U UUCUCGAU AUCGAGAA CUGAUGA X GAA AGUGAAAU

2497 UUUCACUU U UCUCGAUC GAUCGAGA CUGAUGA X GAA AAGUGAAA

2498 UUCACUUU U CUCGAUCA UGAUCGAG CUGAUGA X GAA AAAGUGAA

2501 ACUUUUCU C GAUCACUA UAGUGAUC CUGAUGA X GAA AGAAAAGU

2505 UUCUCGAU C ACUAAACA UGUUUAGU CUGAUGA X GAA AUCGAGAA 2 2550099 CGAUCACU A AACAUAUG CAUAUGUU CUGAUGA X GAA AGUGAUCG

2515 CUAAACAU A UGCAUAUA UAUAUGCA CUGAUGA X GAA AUGUUUAG

2521 AUAUGCAU A UAUUUUUA UAAAAAUA CUGAUGA X GAA AUGCAUAU

2523 AUGCAUAU A UUUUUAAA UUUAAAAA CUGAUGA X GAA AUAUGCAU

2525 GCAUAUAU U UUUAAAAA UUUUUAAA CUGAUGA X GAA AUAUAUGC 2 2552266 CAUAUAUU U UUAAAAAU AUUUUUAA CUGAUGA X GAA AAUAUAUG

2527 AUAUAUUU U UAAAAAUC GAUUUUUA CUGAUGA X GAA AAAUAUAU

2528 UAUAUUUU U AAAAAUCA UGAUUUUU CUGAUGA X GAA AAAAUAUA

2529 AUAUUUUU A AAAAUCAG CUGAUUUU CUGAUGA X GAA AAAAAUAU

2535 UUAAAAAU C AGUAAAAG CUUUUACU CUGAUGA X GAA AUUUUUAA 2 2553399 AAAUCAGU A AAAGCAUU AAUGCUUU CUGAUGA X GAA ACUGAUUU

2547 AAAAGCAU U ACUCUAAG CUUAGAGU CUGAUGA X GAA AUGCUUUU

2548 AAAGCAUU A CUCUAAGU ACUUAGAG CUGAUGA X GAA AAUGCUUU

2551 GCAUUACU C UAAGUGUA UACACUUA CUGAUGA X GAA AGUAAUGC

2553 AUUACUCU A AGUGUAGA UCUACACU CUGAUGA X GAA AGAGUAAU 2 2555599 CUAAGUGU A GACUUAAU AUUAAGUC CUGAUGA X GAA ACACUUAG

2564 UGUAGACU U AAUACCAU AUGGUAUU CUGAUGA X GAA AGUCUACA

2565 GUAGACUU A AUACCAUG CAUGGUAU CUGAUGA X GAA AAGUCUAC

2568 GACUUAAU A CCAUGUGA UCACAUGG CUGAUGA X GAA AUUAAGUC

2580 UGUGACAU U UAAUCCAG CUGGAUUA CUGAUGA X GAA AUGUCACA 2 2558811 GUGACAUU U AAUCCAGA UCUGGAUU CUGAUGA X GAA AAUGUCAC

2582 UGACAUUU A AUCCAGAU AUCUGGAU CUGAUGA X GAA AAAUGUCA

2585 CAUUUAAU C CAGAUUGU ACAAUCUG CUGAUGA X GAA AUUAAAUG

2591 AUCCAGAU U GUAAAUGC GCAUUUAC CUGAUGA X GAA AUCUGGAU

2594 CAGAUUGU A AAUGCUCA UGAGCAUU CUGAUGA X GAA ACAAUCUG 2 2660011 UAAAUGCU C AUUUAUGG CCAUAAAU CUGAUGA X GAA AGCAUUUA

2604 AUGCUCAU U UAUGGUUA UAACCAUA CUGAUGA X GAA AUGAGCAU n . Target Sequence Ribozyme Sequence Posi- tion

2605 UGCUCAUU U AUGGUUAA UUAACCAU CUGAUGA X GAA AAUGAGCA

2606 GCUCAUUU A UGGUUAAU AUUAACCA CUGAUGA X GAA AAAUGAGC 2611 UUUAUGGU U AAUGACAU AUGUCAUU CUGAUGA X GAA ACCAUAAA 2612 UUAUGGUU A AUGACAUU AAUGUCAU CUGAUGA X GAA AACCAUAA 2620 AAUGACAU U GAAGGUAC GUACCUUC CUGAUGA X GAA AUGUCAUU

2627 UUGAAGGU A CAUUUAUU AAUAAAUG CUGAUGA X GAA ACCUUCAA

2631 AGGUACAU U UAUUGUAC GUACAAUA CUGAUGA X GAA AUGUACCU

2632 GGUACAUU U AUUGUACC GGUACAAU CUGAUGA X GAA AAUGUACC

2633 GUACAUUU A UUGUACCA UGGUACAA CUGAUGA X GAA AAAUGUAC 2635 ACAUUUAU U GUACCAAA UUUGGUAC CUGAUGA X GAA AUAAAUGU

2638 UUUAUUGU A CCAAACCA UGGUUUGG CUGAUGA X GAA ACAAUAAA

2648 CAAACCAU U UUAUGAGU ACUCAUAA CUGAUGA X GAA AUGGUUUG

2649 AAACCAUU U UAUGAGUU AACUCAUA CUGAUGA X GAA AAUGGUUU

2650 AACCAUUU U AUGAGUUU AAACUCAU CUGAUGA X GAA AAAUGGUU 2651 ACCAUUUU A UGAGUUUU AAAACUCA CUGAUGA X GAA AAAAUGGU

2657 UUAUGAGU U UUCUGUUA UAACAGAA CUGAUGA X GAA ACUCAUAA

2658 UAUGAGUU U UCUGUUAG CUAACAGA CUGAUGA X GAA AACUCAUA

2659 AUGAGUUU U CUGUUAGC GCUAACAG CUGAUGA X GAA AAACUCAU

2660 UGAGUUUU C UGUUAGCU AGCUAACA CUGAUGA X GAA AAAACUCA 2664 UUUUCUGU U AGCUUGCU AGCAAGCU CUGAUGA X GAA ACAGAAAA

2665 UUUCUGUU A GCUUGCUU AAGCAAGC CUGAUGA X GAA AACAGAAA

2669 UGUUAGCU U GCUUUAAA UUUAAAGC CUGAUGA X GAA AGCUAACA

2673 AGCUUGCU U UAAAAAUU AAUUUUUA CUGAUGA X GAA AGCAAGCU

2674 GCUUGCUU U AAAAAUUA UAAUUUUU CUGAUGA X GAA AAGCAAGC 2675 CUUGCUUU A AAAAUUAU AUAAUUUU CUGAUGA X GAA AAAGCAAG

2681 UUAAAAAU U AUUACUGU ACAGUAAU CUGAUGA X GAA AUUUUUAA

2682 UAAAAAUU A UUACUGUA UACAGUAA CUGAUGA X GAA AAUUUUUA

2684 AAAAUUAU U ACUGUAAG CUUACAGU CUGAUGA X GAA AUAAUUUU

2685 AAAUUAUU A CUGUAAGA UCUUACAG CUGAUGA X GAA AAUAAUUU 2690 AUUACUGU A AGAAAUAG CUAUUUCU CUGAUGA X GAA ACAGUAAU

2697 UAAGAAAU A GUUUUAUA UAUAAAAC CUGAUGA X GAA AUUUCUUA

2700 GAAAUAGU U UUAUAAAA UUUUAUAA CUGAUGA X GAA ACUAUUUC

2701 AAAUAGUU U UAUAAAAA UUUUUAUA CUGAUGA X GAA AACUAUUU

2702 AAUAGUUU U AUAAAAAA UUUUUUAU CUGAUGA X GAA AAACUAUU 2703 AUAGUUUU A UAAAAAAU AUUUUUUA CUGAUGA X GAA AAAACUAU

2705 AGUUUUAU A AAAAAUUA UAAUUUUU CUGAUGA X GAA AUAAAACU n . Target Seguence Ribozyme Seguence

Posi¬ tion

2712 UAAAAAAU U AUAUUUUU AAAAAUAU CUGAUGA X GAA AUUUUUUA

2713 AAAAAAUU A UAUUUUUA UAAAAAUA CUGAUGA X GAA AAUUUUUU

2715 AAAAUUAU A UUUUUAUU AAUAAAAA CUGAUGA X GAA AUAAUUUU

2717 AAUUAUAU U UUUAUUCA UGAAUAAA CUGAUGA X GAA AUAUAAUU

2718 AUUAUAUU U UUAUUCAG CUGAAUAA CUGAUGA X GAA AAUAUAAU

2719 UUAUAUUU U UAUUCAGU ACUGAAUA CUGAUGA X GAA AAAUAUAA

2720 UAUAUUUU U AUUCAGUA UACUGAAU CUGAUGA X GAA AAAAUAUA

2721 AUAUUUUU A UUCAGUAA UUACUGAA CUGAUGA X GAA AAAAAUAU

2723 AUUUUUAU U CAGUAAUU AAUUACUG CUGAUGA X GAA AUAAAAAU 2 2772244 UUUUUAUU C AGUAAUUU AAAUUACU CUGAUGA X GAA AAUAAAAA

2728 UAUUCAGU A AUUUAAUU AAUUAAAU CUGAUGA X GAA ACUGAAUA

2731 UCAGUAAU U UAAUUUUG CAAAAUUA CUGAUGA X GAA AUUACUGA

2732 CAGUAAUU U AAUUUUGU ACAAAAUU CUGAUGA X GAA AAUUACUG

2733 AGUAAUUU A AUUUUGUA UACAAAAU CUGAUGA X GAA AAAUUACU 2 2773366 AAUUUAAU U UUGUAAAU AUUUACAA CUGAUGA X GAA AUUAAAUU

2737 AUUUAAUU U UGUAAAUG CAUUUACA CUGAUGA X GAA AAUUAAAU

2738 UUUAAUUU U GUAAAUGC GCAUUUAC CUGAUGA X GAA AAAUUAAA

2741 AAUUUUGU A AAUGCCAA UUGGCAUU CUGAUGA X GAA ACAAAAUU

2761 AAAAACGU U UUUUGCUG CAGCAAAA CUGAUGA X GAA ACGUUUUU 2 2776622 AAAACGUU U UUUGCUGC GCAGCAAA CUGAUGA X GAA AACGUUUU

2763 AAACGUUU U UUGCUGCU AGCAGCAA CUGAUGA X GAA AAACGUUU

2764 AACGUUUU U UGCUGCUA UAGCAGCA CUGAUGA X GAA AAAACGUU

2765 ACGUUUUU U GCUGCUAU AUAGCAGC CUGAUGA X GAA AAAAACGU

2772 UUGCUGCU A UGGUCUUA UAAGACCA CUGAUGA X GAA AGCAGCAA 2 2777777 GCUAUGGU C UUAGCCUG CAGGCUAA CUGAUGA X GAA ACCAUAGC

2779 UAUGGUCU U AGCCUGUA UACAGGCU CUGAUGA X GAA AGACCAUA

2780 AUGGUCUU A GCCUGUAG CUACAGGC CUGAUGA X GAA AAGACCAU

2787 UAGCCUGU A GACAUGCU AGCAUGUC CUGAUGA X GAA ACAGGCUA

2802 CUGCUAGU A UCAGAGGG CCCUCUGA CUGAUGA X GAA ACUAGCAG 2 2880044 GCUAGUAU C AGAGGGGC GCCCCUCU CUGAUGA X GAA AUACUAGC

2816 GGGGCAGU A GAGCUUGG CCAAGCUC CUGAUGA X GAA ACUGCCCC

2822 GUAGAGCU U GGACAGAA UUCUGUCC CUGAUGA X GAA AGCUCUAC

2843 AAGAAACU U GGUGUUAG CUAACACC CUGAUGA X GAA AGUUUCUU

2849 CUUGGUGU U AGGUAAUU AAUUACCU CUGAUGA X GAA ACACCAAG 2 2885500 UUGGUGUU A GGUAAUUG CAAUUACC CUGAUGA X GAA AACACCAA

2854 UGUUAGGU A AUUGACUA UAGUCAAU CUGAUGA X GAA ACCUAACA n . Target Sequence Ribozyme Sequence

Posi¬ tion

2857 UAGGUAAU U GACUAUGC GCAUAGUC CUGAUGA X GAA AUUACCUA

2862 AAUUGACU A UGCACUAG CUAGUGCA CUGAUGA X GAA AGUCAAUU

2869 UAUGCACU A GUAUUUCA UGAAAUAC CUGAUGA X GAA AGUGCAUA

2872 GCACUAGU A UUUCAGAC GUCUGAAA CUGAUGA X GAA ACUAGUGC

2874 ACUAGUAU U UCAGACUU AAGUCUGA CUGAUGA X GAA AUACUAGU

2875 CUAGUAUU U CAGACUUU AAAGUCUG CUGAUGA X GAA AAUACUAG

2876 UAGUAUUU C AGACUUUU AAAAGUCU CUGAUGA X GAA AAAUACUA

2882 UUCAGACU U UUUAAUUU AAAUUAAA CUGAUGA X GAA AGUCUGAA

2883 UCAGACUU U UUAAUUUU AAAAUUAA CUGAUGA X GAA AAGUCUGA 2 2888844 CAGACUUU U UAAUUUUA UAAAAUUA CUGAUGA X GAA AAAGUCUG

2885 AGACUUUU U AAUUUUAU AUAAAAUU CUGAUGA X GAA AAAAGUCU

2886 GACUUUUU A AUUUUAUA UAUAAAAU CUGAUGA X GAA AAAAAGUC

2889 UUUUUAAU U UUAUAUAU AUAUAUAA CUGAUGA X GAA AUUAAAAA

2890 UUUUAAUU U UAUAUAUA UAUAUAUA CUGAUGA X GAA AAUUAAAA 2 2889911 UUUAAUUU U AUAUAUAU AUAUAUAU CUGAUGA X GAA AAAUUAAA

2892 UUAAUUUU A UAUAUAUA UAUAUAUA CUGAUGA X GAA AAAAUUAA

2894 AAUUUUAU A UAUAUAUA UAUAUAUA CUGAUGA X GAA AUAAAAUU

2896 UUUUAUAU A UAUAUACA UGUAUAUA CUGAUGA X GAA AUAUAAAA

2898 UUAUAUAU A UAUACAUU AAUGUAUA CUGAUGA X GAA AUAUAUAA 2 2990000 AUAUAUAU A UACAUUUU AAAAUGUA CUGAUGA X GAA AUAUAUAU

2902 AUAUAUAU A CAUUUUUU AAAAAAUG CUGAUGA X GAA AUAUAUAU

2906 AUAUACAU U UUUUUUCC GGAAAAAA CUGAUGA X GAA AUGUAUAU

2907 UAUACAUU U UUUUUCCU AGGAAAAA CUGAUGA X GAA AAUGUAUA

2908 AUACAUUU U UUUUCCUU AAGGAAAA CUGAUGA X GAA AAAUGUAU 2 2990099 UACAUUUU U UUUCCUUC GAAGGAAA CUGAUGA X GAA AAAAUGUA

2910 ACAUUUUU U UUCCUUCU AGAAGGAA CUGAUGA X GAA AAAAAUGU

2911 CAUUUUUU U UCCUUCUG CAGAAGGA CUGAUGA X GAA AAAAAAUG

2912 AUUUUUUU U CCUUCUGC GCAGAAGG CUGAUGA X GAA AAAAAAAU

2913 UUUUUUUU C CUUCUGCA UGCAGAAG CUGAUGA X GAA AAAAAAAA 2 2991166 UUUUUCCU U CUGCAAUA UAUUGCAG CUGAUGA X GAA AGGAAAAA

2917 UUUUCCUU C UGCAAUAC GUAUUGCA CUGAUGA X GAA AAGGAAAA

2924 UCUGCAAU A CAUUUGAA UUCAAAUG CUGAUGA X GAA AUUGCAGA

2928 CAAUACAU U UGAAAACU AGUUUUCA CUGAUGA X GAA AUGUAUUG

2929 AAUACAUU U GAAAACUU AAGUUUUC CUGAUGA X GAA AAUGUAUU 2 2993377 UGAAAACU U GUUUGGGA UCCCAAAC CUGAUGA X GAA AGUUUUCA

2940 AAACUUGU U UGGGAGAC GUCUCCCA CUGAUGA X GAA ACAAGUUU nt . Target Seguence Ribozyme Sequence Posi- tion

2941 AACUUGUU U GGGAGACU AGUCUCCC CUGAUGA X GAA AACAAGUU

2950 GGGAGACU C UGCAUUUU AAAAUGCA CUGAUGA X GAA AGUCUCCC

2956 CUCUGCAU U UUUUAUUG CAAUAAAA CUGAUGA X GAA AUGCAGAG

2957 UCUGCAUU U UUUAUUGU ACAAUAAA CUGAUGA X GAA AAUGCAGA 2958 CUGCAUUU U UUAUUGUG CACAAUAA CUGAUGA X GAA AAAUGCAG

2959 UGCAUUUU U UAUUGUGG CCACAAUA CUGAUGA X GAA AAAAUGCA

2960 GCAUUUUU U AUUGUGGU ACCACAAU CUGAUGA X GAA AAAAAUGC

2961 CAUUUUUU A UUGUGGUU AACCACAA CUGAUGA X GAA AAAAAAUG 2969 AUUGUGGU U UUUUUGUU AACAAAAA CUGAUGA X GAA ACCACAAU 2970 UUGUGGUU U UUUUGUUA UAACAAAA CUGAUGA X GAA AACCACAA

2971 UGUGGUUU U UUUGUUAU AUAACAAA CUGAUGA X GAA AAACCACA

2972 GUGGUUUU U UUGUUAUU AAUAACAA CUGAUGA X GAA AAAACCAC

2973 UGGUUUUU U UGUUAUUG CAAUAACA CUGAUGA X GAA AAAAACCA

2974 GGUUUUUU U GUUAUUGU ACAAUAAC CUGAUGA X GAA AAAAAACC 2977 UUUUUUGU U AUUGUUGG CCAACAAU CUGAUGA X GAA ACAAAAAA

2978 UUUUUGUU A UUGUUGGU ACCAACAA CUGAUGA X GAA AACAAAAA

2980 UUUGUUAU U GUUGGUUU AAACCAAC CUGAUGA X GAA AUAACAAA

2983 GUUAUUGU U GGUUUAUA UAUAAACC CUGAUGA X GAA ACAAUAAC

2987 UUGUUGGU U UAUACAAG CUUGUAUA CUGAUGA X GAA ACCAACAA 2988 UGUUGGUU U AUACAAGC GCUUGUAU CUGAUGA X GAA AACCAACA

2989 GUUGGUUU A UACAAGCA UGCUUGUA CUGAUGA X GAA AAACCAAC

2991 UGGUUUAU A CAAGCAUG CAUGCUUG CUGAUGA X GAA AUAAACCA

3003 GCAUGCGU U GCACUUCU AGAAGUGC CUGAUGA X GAA ACGCAUGC 3009 GUUGCACU U CUUUUUUG CAAAAAAG CUGAUGA X GAA AGUGCAAC 3010 UUGCACUU C UUUUUUGG CCAAAAAA CUGAUGA X GAA AAGUGCAA

3012 GCACUUCU U UUUUGGGA UCCCAAAA CUGAUGA X GAA AGAAGUGC

3013 CACUUCUU U UUUGGGAG CUCCCAAA CUGAUGA X GAA AAGAAGUG

3014 ACUUCUUU U UUGGGAGA UCUCCCAA CUGAUGA X GAA AAAGAAGU

3015 CUUCUUUU U UGGGAGAU AUCUCCCA CUGAUGA X GAA AAAAGAAG 3016 UUCUUUUU U GGGAGAUG CAUCUCCC CUGAUGA X GAA AAAAAGAA

3030 AUGUGUGU U GUUGAUGU ACAUCAAC CUGAUGA X GAA ACACACAU

3033 UGUGUUGU U GAUGUUCU AGAACAUC CUGAUGA X GAA ACAACACA

3039 GUUGAUGU U CUAUGUUU AAACAUAG CUGAUGA X GAA ACAUCAAC

3042 GAUGUUCU A UGUUUUGU ACAAAACA CUGAUGA X GAA AGAACAUC 30 6 UUCUAUGU U UUGUUUUG CAAAACAA CUGAUGA X GAA ACAUAGAA

3047 UCUAUGUU U UGUUUUGA UCAAAACA CUGAUGA X GAA AACAUAGA nt. Target Sequence Ribozyme Sequence Posi- tion

3048 CUAUGUUU U GUUUUGAG CUCAAAAC CUGAUGA X GAA AAACAUAG

3051 UGUUUUGU U UUGAGUGU ACACUCAA CUGAUGA X GAA ACAAAACA

3052 GUUUUGUU U UGAGUGUA UACACUCA CUGAUGA X GAA AACAAAAC

3053 UUUUGUUU U GAGUGUAG CUACACUC CUGAUGA X GAA AAACAAAA 3060 UUGAGUGU A GCCUGACU AGUCAGGC CUGAUGA X GAA ACACUCAA

3071 CUGACUGU U UUAUAAUU AAUUAUAA CUGAUGA X GAA ACAGUCAG

3072 UGACUGUU U UAUAAUUU AAAUUAUA CUGAUGA X GAA AACAGUCA

3073 GACUGUUU U AUAAUUUG CAAAUUAU CUGAUGA X GAA AAACAGUC

3074 ACUGUUUU A UAAUUUGG CCAAAUUA CUGAUGA X GAA AAAACAGU 3076 UGUUUUAU A AUUUGGGA UCCCAAAU CUGAUGA X GAA AUAAAACA

3079 UUUAUAAU U UGGGAGUU AACUCCCA CUGAUGA X GAA AUUAUAAA

3080 UUAUAAUU U GGGAGUUC GAACUCCC CUGAUGA X GAA AAUUAUAA

3087 UUGGGAGU U CUGCAUUU AAAUGCAG CUGAUGA X GAA ACUCCCAA

3094 UUCUGCAU U UGAUCCGC GCGGAUCA CUGAUGA X GAA AUGCAGAA 3095 UCUGCAUU U GAUCCGCA UGCGGAUC CUGAUGA X GAA AAUGCAGA

3099 CAUUUGAU C CGCAUCCC GGGAUGCG CUGAUGA X GAA AUCAAAUG

3105 AUCCGCAU C CCCUGUGG CCACAGGG CUGAUGA X GAA AUGCGGAU

3115 CCUGUGGU U UCUAAGUG CACUUAGA CUGAUGA X GAA ACCACAGG

3116 CUGUGGUU U CUAAGUGU ACACUUAG CUGAUGA X GAA AACCACAG 3117 UGUGGUUU C UAAGUGUA UACACUUA CUGAUGA X GAA AAACCACA

3119 UGGUUUCU A AGUGUAUG CAUACACU CUGAUGA X GAA AGAAACCA

3125 CUAAGUGU A UGGUCUCA UGAGACCA CUGAUGA X GAA ACACUUAG

3130 UGUAUGGU C UCAGAACU AGUUCUGA CUGAUGA X GAA ACCAUACA

3132 UAUGGUCU C AGAACUGU ACAGUUCU CUGAUGA X GAA AGACCAUA 3141 AGAACUGU U GCAUGGAU AUCCAUGC CUGAUGA X GAA ACAGUUCU

3150 GCAUGGAU C CUGUGUUU AAACACAG CUGAUGA X GAA AUCCAUGC

3157 UCCUGUGU U UGCAACUG CAGUUGCA CUGAUGA X GAA ACACAGGA

3158 CCUGUGUU U GCAACUGG CCAGUUGC CUGAUGA X GAA AACACAGG 3185 ACUGUGGU U GAUAGCCA UGGCUAUC CUGAUGA X GAA ACCACAGU 3189 UGGUUGAU A GCCAGUCA UGACUGGC CUGAUGA X GAA AUCAACCA

3196 UAGCCAGU C ACUGCCUU AAGGCAGU CUGAUGA X GAA ACUGGCUA

3204 CACUGCCU U AAGAACAU AUGUUCUU CUGAUGA X GAA AGGCAGUG

3205 ACUGCCUU A AGAACAUU AAUGUUCU CUGAUGA X GAA AAGGCAGU 3213 AAGAACAU U UGAUGCAA UUGCAUCA CUGAUGA X GAA AUGUUCUU 3214 AGAACAUU U GAUGCAAG CUUGCAUC CUGAUGA X GAA AAUGUUCU

3240 ACUGAACU U UUGAGAUA UAUCUCAA CUGAUGA X GAA AGUUCAGU nt. Target Seguence Ribozyme Seguence

Posi¬ tion

3241 CUGAACUU U UGAGAUAU AUAUCUCA CUGAUGA X GAA AAGUUCAG

3242 UGAACUUU U GAGAUAUG CAUAUCUC CUGAUGA X GAA AAAGUUCA

3248 UUUGAGAU A UGACGGUG CACCGUCA CUGAUGA X GAA AUCUCAAA

3258 GACGGUGU A CUUACUGC GCAGUAAG CUGAUGA X GAA ACACCGUC

3261 GGUGUACU U ACUGCCUU AAGGCAGU CUGAUGA X GAA AGUACACC

3262 GUGUACUU A CUGCCUUG CAAGGCAG CUGAUGA X GAA AAGUACAC

3269 UACUGCCU U GUAGCAAA UUUGCUAC CUGAUGA X GAA AGGCAGUA

3272 UGCCUUGU A GCAAAAUA UAUUUUGC CUGAUGA X GAA ACAAGGCA

3280 AGCAAAAU A AAGAUGUG CACAUCUU CUGAUGA X GAA AUUUUGCU

3293 UGUGCCCU U AUUUUACC GGUAAAAU CUGAUGA X GAA AGGGCACA

3294 GUGCCCUU A UUUUACCU AGGUAAAA CUGAUGA X GAA AAGGGCAC

Where "X" represents stem II region of a HH ribozyme (Hertel et al . , 1992 Nucleic Acids Res. 20 3252) . The length of stem II may be ≥ 2 base-pairs.

Table XV: Mouse c-myb Hammerhead Ribozyme and Target Sequence nt. Target Seguence Ribozyme Sequence Posi¬ tion

10 CCGGGGCUC UUGGCGGA UCCGCCAA CUGAUGA X GAA AGCCCCGG

12 GGGGCUCUU GGCGGAGC GCUCCGCC CUGAUGA X GAA AGAGCCCC

33 GCCCGCCUC GCCAUGGC GCCAUGGC CUGAUGA X GAA AGGCGGGC 63 CACAGCAUC UACAGUAG CUACUGUA CUGAUGA X GAA AUGCUGUG

65 CAGCAUCUA CAGUAGCG CGCUACUG CUGAUGA X GAA AGAUGCUG

70 UCUACAGUA GCGAUGAA UUCAUCGC CUGAUGA X GAA ACUGUAGA

93 GAAGACAUU GAGAUGUG CACAUCUC CUGAUGA X GAA AUGUCUUC

113 CCAUGACUA CGAUGGGC GCCCAUCG CUGAUGA X GAA AGUCAUGG 134 GCCCAAAUC UGGAAAGC GCUUUCCA CUGAUGA X GAA AUUUGGGC

145 GAAAGCGUC ACUUGGGG CCCCAAGU CUGAUGA X GAA ACGCUUUC

149 GCGUCACUU GGGGAAAA UUUUCCCC CUGAUGA X GAA AGUGACGC

160 GGAAAACUA GGUGGACA UGUCCACC CUGAUGA X GAA AGUUUUCC

231 UGGAAAGUC AUUGCCAA UUGGCAAU CUGAUGA X GAA ACUUUCCA 234 AAAGUCAUU GCCAAUUA UAAUUGGC CUGAUGA X GAA AUGACUUU

241 UUGCCAAUU AUCUGCCC GGGCAGAU CUGAUGA X GAA AUUGGCAA nt. Target Sequence Ribozyme Sequence Posi¬ tion

242 UGCCAAUUA UCUGCCCA UGGGCAGA CUGAUGA X GAA AAUUGGCA

244 CCAAUUAUC UGCCCAAC GUUGGGCA CUGAUGA X GAA AUAAUUGG

264 ACAGAUGUA CAGUGCCA UGGCACUG CUGAUGA X GAA ACAUCUGU

306 CCUGAACUC AUCAAAGG CCUUUGAU CUGAUGA X GAA AGUUCAGG

309 GAACUCAUC AAAGGUCC GGACCUUU CUGAUGA X GAA AUGAGUUC

316 UCAAAGGUC CCUGGACC GGUCCAGG CUGAUGA X GAA ACCUUUGA

337 AAGAAGAUC AGAGAGUC GACUCUCU CUGAUGA X GAA AUCUUCUU

345 CAGAGAGUC AUAAAGCU AGCUUUAU CUGAUGA X GAA ACUCUCUG

348 AGAGUCAUA AAGCUUGU ACAAGCUU CUGAUGA X GAA AUGACUCU

354 AUAAAGCUU GUCCAGAA UUCUGGAC CUGAUGA X GAA AGCUUUAU

357 AAGCUUGUC CAGAAAUA UAUUUCUG CUGAUGA X GAA ACAAGCUU

365 CCAGAAAUA UGGUCCGA UCGGACCA CUGAUGA X GAA AUUUCUGG

370 AAUAUGGUC CGAAGCGU ACGCUUCG CUGAUGA X GAA ACCAUAUU

379 CGAAGCGUU GGUCUGUU AACAGACC CUGAUGA X GAA ACGCUUCG

383 GCGUUGGUC UGUUAUUG CAAUAACA CUGAUGA X GAA ACCAACGC

387 UGGUCUGUU AUUGCCAA UUGGCAAU CUGAUGA X GAA ACAGACCA

388 GGUCUGUUA UUGCCAAG CUUGGCAA CUGAUGA X GAA AACAGACC

390 UCUGUUAUU GCCAAGCA UGCUUGGC CUGAUGA X GAA AUAACAGA

401 CAAGCACUU AAAAGGGA UCCCUUUU CUGAUGA X GAA AGUGCUUG

402 AAGCACUUA AAAGGGAG CUCCCUUU CUGAUGA X GAA AAGUGCUU

414 GGGAGAAUU GGAAAGCA UGCUUUCC CUGAUGA X GAA AUUCUCCC

427 AGCAGUGUC GGGAGAGG ccucuccc CUGAUGA X GAA ACACUGCU

448 ACAACCAUU UGAAUCCA UGGAUUCA CUGAUGA X GAA AUGGUUGU

449 CAACCAUUU GAAUCCAG CUGGAUUC CUGAUGA X GAA AAUGGUUG

454 AUUUGAAUC CAGAAGUU AACUUCUG CUGAUGA X GAA AUUCAAAU

462 CCAGAAGUU AAGAAAAC GUUUUCUU CUGAUGA X GAA ACUUCUGG

463 CAGAAGUUA AGAAAACC GGUUUUCU CUGAUGA X GAA AACUUCUG

473 GAAAACCUC CUGGACAG CUGUCCAG CUGAUGA X GAA AGGUUUUC

498 GACAGAAUC AUUUACCA UGGUAAAU CUGAUGA X GAA AUUCUGUC

501 AGAAUCAUU UACCAGGC GCCUGGUA CUGAUGA X GAA AUGAUUCU

502 GAAUCAUUU ACCAGGCA UGCCUGGU CUGAUGA X GAA AAUGAUUC

503 AAUCAUUUA CCAGGCAC GUGCCUGG CUGAUGA X GAA AAAUGAUU

520 ACAAGCGUC UGGGGAAC GUUCCCCA CUGAUGA X GAA ACGCUUGU

543 GCAGAGAUC GCAAAGCU AGCUUUGC CUGAUGA X GAA AUCUCUGC

571 GGACUGAUA AUGCUAUC GAUAGCAU CUGAUGA X GAA AUCAGUCC

577 AUAAUGCUA UCAAGAAC GUUCUUGA CUGAUGA X GAA AGCAUUAU nt. Target Seguence Ribozyme Seguence Posi¬ tion

579 AAUGCUAUC AAGAACCA UGGUUCUU CUGAUGA X GAA AUAGCAUU

595 ACUGGAAUU CCACCAUG CAUGGUGG CUGAUGA X GAA AUUCCAGU

596 CUGGAAUUC CACCAUGC GCAUGGUG CUGAUGA X GAA AAUUCCAG

607 CCAUGCGUC GCAAGGUG CACCUUGC CUGAUGA X GAA ACGCAUGG

629 GGAAGGCUA CCUGCAGA UCUGCAGG CUGAUGA X GAA AGCCUUCC

643 AGAAGCCUU CCAAAGCC GGCUUUGG CUGAUGA X GAA AGGCUUCU

644 GAAGCCUUC CAAAGCCA UGGCUUUG CUGAUGA X GAA AAGGCUUC

677 CACGAGCUU CCAGAAGA UCUUCUGG CUGAUGA X GAA AGCUCGUG

678 ACGAGCUUC CAGAAGAA UUCUUCUG CUGAUGA X GAA AAGCUCGU

691 AGAACAAUC AUUUGAUG CAUCAAAU CUGAUGA X GAA AUUGUUCU

694 ACAAUCAUU UGAUGGGG CCCCAUCA CUGAUGA X GAA AUGAUUGU

695 CAAUCAUUU GAUGGGGU ACCCCAUC CUGAUGA X GAA AAUGAUUG

704 GAUGGGGUU UGGGCAUG CAUGCCCA CUGAUGA X GAA ACCCCAUC

705 AUGGGGUUU GGGCAUGC GCAUGCCC CUGAUGA X GAA AACCCCAU

716 GCAUGCCUC ACCUCCAU AUGGAGGU CUGAUGA X GAA AGGCAUGC

721 CCUCACCUC CAUCUCAG CUGAGAUG CUGAUGA X GAA AGGUGAGG

725 ACCUCCAUC UCAGCUCU AGAGCUGA CUGAUGA X GAA AUGGAGGU

727 CUCCAUCUC AGCUCUCU AGAGAGCU CUGAUGA X GAA AGAUGGAG

732 UCUCAGCUC UCUCCAAG CUUGGAGA CUGAUGA X GAA AGCUGAGA

734 UCAGCUCUC UCCAAGUG CACUUGGA CUGAUGA X GAA AGAGCUGA

736 AGCUCUCUC CAAGUGGC GCCACUUG CUGAUGA X GAA AGAGAGCU

749 UGGCCAGUC CUCCGUCA UGACGGAG CUGAUGA X GAA ACUGGCCA

752 CCAGUCCUC CGUCAACA UGUUGACG CUGAUGA X GAA AGGACUGG

756 UCCUCCGUC AACAGCGA UCGCUGUU CUGAUGA X GAA ACGGAGGA

767 CAGCGAAUA UCCCUAUU AAUAGGGA CUGAUGA X GAA AUUCGCUG

769 GCGAAUAUC CCUAUUAC GUAAUAGG CUGAUGA X GAA AUAUUCGC

773 AUAUCCCUA UUACCACA UGUGGUAA CUGAUGA X GAA AGGGAUAU

775 AUCCCUAUU ACCACAUC GAUGUGGU CUGAUGA X GAA AUAGGGAU

776 UCCCUAUUA CCACAUCG CGAUGUGG CUGAUGA X GAA AAUAGGGA

783 UACCACAUC GCCGAAGC GCUUCGGC CUGAUGA X GAA AUGUGGUA

801 CAAAACAUC UCCAGUCA UGACUGGA CUGAUGA X GAA AUGUUUUG

803 AAACAUCUC CAGUCACG CGUGACUG CUGAUGA X GAA AGAUGUUU

808 UCUCCAGUC ACGUUCCC GGGAACGU CUGAUGA X GAA ACUGGAGA

813 AGUCACGUU CCCUAUCC GGAUAGGG CUGAUGA X GAA ACGUGACU

814 GUCACGUUC CCUAUCCU AGGAUAGG CUGAUGA X GAA AACGUGAC

818 CGUUCCCUA UCCUGUCG CGACAGGA CUGAUGA X GAA AGGGAACG nt Target Seguence Ribozyme Sequence

Posi¬ tion

820 UUCCCUAUC CUGUCGCA UGCGACAG CUGAUGA X GAA AUAGGGAA

825 UAUCCUGUC GCAUUGCA UGCAAUGC CUGAUGA X GAA ACAGGAUA

830 UGUCGCAUU GCAUGUUA UAACAUGC CUGAUGA X GAA AUGCGACA

837 UUGCAUGUU AAUAUAGU ACUAUAUU CUGAUGA X GAA ACAUGCAA

838 UGCAUGUUA AUAUAGUC GACUAUAU CUGAUGA X GAA AACAUGCA

841 AUGUUAAUA UAGUCAAC GUUGACUA CUGAUGA X GAA AUUAACAU

843 GUUAAUAUA GUCAACGU ACGUUGAC CUGAUGA X GAA AUAUUAAC

846 AAUAUAGUC AACGUCCC GGGACGUU CUGAUGA X GAA ACUAUAUU

852 GUCAACGUC CCUCAGCC GGCUGAGG CUGAUGA X GAA ACGUUGAC

856 ACGUCCCUC AGCCGGCU AGCCGGCU CUGAUGA X GAA AGGGACGU

876 GCAGCCAUC CAGAGACA UGUCUCUG CUGAUGA X GAA AUGGCUGC

887 GAGACACUA UAACGACG CGUCGUUA CUGAUGA X GAA AGUGUCUC

889 GACACUAUA ACGACGAA UUCGUCGU CUGAUGA X GAA AUAGUGUC

921 AAGCGAAUA AAGGAGCU AGCUCCUU CUGAUGA X GAA AUUCGCUU

935 GCUGGAGUU GCUCCUGA UCAGGAGC CUGAUGA X GAA ACUCCAGC

939 GAGUUGCUC CUGAUGUC GACAUCAG CUGAUGA X GAA AGCAACUC

947 CCUGAUGUC AACAGAGA UCUCUGUU CUGAUGA X GAA ACAUCAGG

980 GCAGGCAUU ACCAACAC GUGUUGGU CUGAUGA X GAA AUGCCUGC

981 CAGGCAUUA CCAACACA UGUGUUGG CUGAUGA X GAA AAUGCCUG

IOOO ACCACACUU GCAGCUAC GUAGCUGC CUGAUGA X GAA AGUGUGGU

1007 UUGCAGCUA CCCCGGGU ACCCGGGG CUGAUGA X GAA AGCUGCAA

1028 CAGCACCUC CAUUGUGG CCACAAUG CUGAUGA X GAA AGGUGCUG

1032 ACCUCCAUU GUGGACCA UGGUCCAC CUGAUGA X GAA AUGGAGGU

1051 CCAGACCUC AUGGGGAU AUCCCCAU CUGAUGA X GAA AGGUCUGG

1060 AUGGGGAUA GUGCACCU AGGUGCAC CUGAUGA X GAA AUCCCCAU

1071 GCACCUGUU UCCUGUUU AAACAGGA CUGAUGA X GAA ACAGGUGC

1072 CACCUGUUU CCUGUUUG CAAACAGG CUGAUGA X GAA AACAGGUG

1073 ACCUGUUUC CUGUUUGG CCAAACAG CUGAUGA X GAA AAACAGGU

1078 UUUCCUGUU UGGGAGAA UUCUCCCA CUGAUGA X GAA ACAGGAAA

1079 UUCCUGUUU GGGAGAAC GUUCUCCC CUGAUGA X GAA AACAGGAA

1103 CACCCCAUC UCUGCCUG CAGGCAGA CUGAUGA X GAA AUGGGGUG

1105 CCCCAUCUC UGCCUGCA UGCAGGCA CUGAUGA X GAA AGAUGGGG

1117 CUGCAGAUC CCGGCUCC GGAGCCGG CUGAUGA X GAA AUCUGCAG

1124 UCCCGGCUC CCUACCUG CAGGUAGG CUGAUGA X GAA AGCCGGGA

1128 GGCUCCCUA CCUGAAGA UCUUCAGG CUGAUGA X GAA AGGGAGCC

1145 AAGUGCCUC ACCAGCAA UUGCUGGU CUGAUGA X GAA AGGCACUU nt. Target Sequence Ribozyme Sequence Posi¬ tion

1164 UGCAUGAUC GUCCACCA UGGUGGAC CUGAUGA X GAA AUCAUGCA

1167 AUGAUCGUC CACCAGGG CCCUGGUG CUGAUGA X GAA ACGAUCAU

1182 GGCACCAUU CUGGACAA UUGUCCAG CUGAUGA X GAA AUGGUGCC

1183 GCACCAUUC UGGACAAU AUUGUCCA CUGAUGA X GAA AAUGGUGC

1194 GACAAUGUU AAGAACCU AGGUUCUU CUGAUGA X GAA ACAUUGUC

1195 ACAAUGUUA AGAACCUC GAGGUUCU CUGAUGA X GAA AACAUUGU

1203 AAGAACCUC UUAGAAUU AAUUCUAA CUGAUGA X GAA AGGUUCUU

1205 GAACCUCUU AGAAUUUG CAAAUUCU CUGAUGA X GAA AGAGGUUC

1206 AACCUCUUA GAAUUUGC GCAAAUUC CUGAUGA X GAA AAGAGGUU

1211 CUUAGAAUU UGCAGAAA UUUCUGCA CUGAUGA X GAA AUUCUAAG

1212 UUAGAAUUU GCAGAAAC GUUUCUGC CUGAUGA X GAA AAUUCUAA

1224 GAAACACUC CAGUUUAU AUAAACUG CUGAUGA X GAA AGUGUUUC

1229 ACUCCAGUU UAUAGAUU AAUCUAUA CUGAUGA X GAA ACUGGAGU

1230 CUCCAGUUU AUAGAUUC GAAUCUAU CUGAUGA X GAA AACUGGAG

1231 UCCAGUUUA UAGAUUCU AGAAUCUA CUGAUGA X GAA AAACUGGA

1233 CAGUUUAUA GAUUCUUU AAAGAAUC CUGAUGA X GAA AUAAACUG

1237 UUAUAGAUU CUUUCUUG CAAGAAAG CUGAUGA X GAA AUCUAUAA

1238 UAUAGAUUC UUUCUUGA UCAAGAAA CUGAUGA X GAA AAUCUAUA

1240 UAGAUUCUU UCUUGAAC GUUCAAGA CUGAUGA X GAA AGAAUCUA

1241 AGAUUCUUU CUUGAACA UGUUCAAG CUGAUGA X GAA AAGAAUCU

1242 GAUUCUUUC UUGAACAC GUGUUCAA CUGAUGA X GAA AAAGAAUC

1244 UUCUUUCUU GAACACUU AAGUGUUC CUGAUGA X GAA AGAAAGAA

1252 UGAACACUU CCAGCAAC GUUGCUGG CUGAUGA X GAA AGUGUUCA

1253 GAACACUUC CAGCAACC GGUUGCUG CUGAUGA X GAA AAGUGUUC

1271 UGAAAACUC GGGCUUAG CUAAGCCC CUGAUGA X GAA AGUUUUCA

1277 CUCGGGCUU AGAUGCAC GUGCAUCU CUGAUGA X GAA AGCCCGAG

1278 UCGGGCUUA GAUGCACC GGUGCAUC CUGAUGA X GAA AAGCCCGA

1288 AUGCACCUA CCUUACCC GGGUAAGG CUGAUGA X GAA AGGUGCAU

1292 ACCUACCUU ACCCUCCA UGGAGGGU CUGAUGA X GAA AGGUAGGU

1293 CCUACCUUA CCCUCCAC GUGGAGGG CUGAUGA X GAA AAGGUAGG

1298 CUUACCCUC CACUCCUC GAGGAGUG CUGAUGA X GAA AGGGUAAG

1303 CCUCCACUC CUCUCAUU AAUGAGAG CUGAUGA X GAA AGUGGAGG

1306 CCACUCCUC UCAUUGGU ACCAAUGA CUGAUGA X GAA AGGAGUGG

1308 ACUCCUCUC AUUGGUCA UGACCAAU CUGAUGA X GAA AGAGGAGU

1311 CCUCUCAUU GGUCACAA UUGUGACC CUGAUGA X GAA AUGAGAGG

1315 UCAUUGGUC ACAAACUG CAGUUUGU CUGAUGA X GAA ACCAAUGA nt. Target Sequence Ribozyme Sequence Posi¬ tion

1333 CACCAUGUC GAGACCAG CUGGUCUC CUGAUGA X GAA ACAUGGUG

1366 AGGAAAAUU CCAUCUUU AAAGAUGG CUGAUGA X GAA AUUUUCCU

1367 GGAAAAUUC CAUCUUUA UAAAGAUG CUGAUGA X GAA AAUUUUCC

1371 AAUUCCAUC UUUAGAAC GUUCUAAA CUGAUGA X GAA AUGGAAUU

1373 UUCCAUCUU UAGAACUC GAGUUCUA CUGAUGA X GAA AGAUGGAA

1374 UCCAUCUUU AGAACUCC GGAGUUCU CUGAUGA X GAA AAGAUGGA

1375 CCAUCUUUA GAACUCCA UGGAGUUC CUGAUGA X GAA AAAGAUGG

1381 UUAGAACUC CAGCUAUC GAUAGCUG CUGAUGA X GAA AGUUCUAA

1387 CUCCAGCUA UCAAAAGG CCUUUUGA CUGAUGA X GAA AGCUGGAG

1389 CCAGCUAUC AAAAGGUC GACCUUUU CUGAUGA X GAA AUAGCUGG

1397 CAAAAGGUC AAUCCUCG CGAGGAUU CUGAUGA X GAA ACCUUUUG

1401 AGGUCAAUC CUCGAAAG CUUUCGAG CUGAUGA X GAA AUUGACCU

1404 UCAAUCCUC GAAAGCUC GAGCUUUC CUGAUGA X GAA AGGAUUGA

1412 CGAAAGCUC UCCUCGAA UUCGAGGA CUGAUGA X GAA AGCUUUCG

1414 AAAGCUCUC CUCGAACU AGUUCGAG CUGAUGA X GAA AGAGCUUU

1417 GCUCUCCUC GAACUCCC GGGAGUUC CUGAUGA X GAA AGGAGAGC

1423 CUCGAACUC CCACACCA UGGUGUGG CUGAUGA X GAA AGUUCGAG

1433 CACACCAUU CAAACAUG CAUGUUUG CUGAUGA X GAA AUGGUGUG

1434 ACACCAUUC AAACAUGC GCAUGUUU CUGAUGA X GAA AAUGGUGU

1446 CAUGCCCUU GCAGCUCA UGAGCUGC CUGAUGA X GAA AGGGCAUG

1453 UUGCAGCUC AAGAAAUU AAUUUCUU CUGAUGA X GAA AGCUGCAA

1461 CAAGAAAUU AAAUACGG CCGUAUUU CUGAUGA X GAA AUUUCUUG

1462 AAGAAAUUA AAUACGGU ACCGUAUU CUGAUGA X GAA AAUUUCUU

1466 AAUUAAAUA CGGUCCCC GGGGACCG CUGAUGA X GAA AUUUAAUU

1471 AAUACGGUC CCCUGAAG CUUCAGGG CUGAUGA X GAA ACCGUAUU

1485 AAGAUGCUA CCUCAGAC GUCUGAGG CUGAUGA X GAA AGCAUCUU

1489 UGCUACCUC AGACCCCC GGGGGUCU CUGAUGA X GAA AGGUAGCA

1499 GACCCCCUC CCAUGCAG CUGCAUGG CUGAUGA X GAA AGGGGGUC

1518 GAGGACCUA CAAGAUGU ACAUCUUG CUGAUGA X GAA AGGUCCUC

1530 GAUGUGAUU AAGCGGGA UCCCGCUU CUGAUGA X GAA AUCACAUC

1531 AUGUGAUUA AGCGGGAA UUCCCGCU CUGAUGA X GAA AAUCACAU

1541 GCGGGAAUC GGAUGAAU AUUCAUCC CUGAUGA X GAA AUUCCCGC

1550 GGAUGAAUC UGGAAUUG CAAUUCCA CUGAUGA X GAA AUUCAUCC

1557 UCUGGAAUU GUUGCUGA UCAGCAAC CUGAUGA X GAA AUUCCAGA

1560 GGAAUUGUU GCUGAGUU AACUCAGC CUGAUGA X GAA ACAAUUCC

1568 UGCUGAGUU UCAAGAGA UCUCUUGA CUGAUGA X GAA ACUCAGCA nt. Target Sequence Ribozyme Sequence Posi¬ tion

1569 GCUGAGUUU CAAGAGAG CUCUCUUG CUGAUGA X GAA AACUCAGC

1570 CUGAGUUUC AAGAGAGU ACUCUCUU CUGAUGA X GAA AAACUCAG

1589 ACCACCGUU ACUGAAAA UUUUCAGU CUGAUGA X GAA ACGGUGGU

1590 CCACCGUUA CUGAAAAA UUUUUCAG CUGAUGA X GAA AACGGUGG

1602 AAAAAAAUC AAGCAGGC GCCUGCUU CUGAUGA X GAA AUUUUUUU

1619 GGUGGAGUC GCCAACUG CAGUUGGC CUGAUGA X GAA ACUCCACC

1634 UGAGAAAUC GGGAAACU AGUUUCCC CUGAUGA X GAA AUUUCUCA

1643 GGGAAACUU CUUCUGCU AGCAGAAG CUGAUGA X GAA AGUUUCCC

1644 GGAAACUUC UUCUGCUC GAGCAGAA CUGAUGA X GAA AAGUUUCC

1646 AAACUUCUU CUGCUCAA UUGAGCAG CUGAUGA X GAA AGAAGUUU

1647 AACUUCUUC UGCUCAAA UUUGAGCA CUGAUGA X GAA AAGAAGUU

1652 CUUCUGCUC AAACCACU AGUGGUUU CUGAUGA X GAA AGCAGAAG

1691 CCAACUGUU CUCGCAGG CCUGCGAG CUGAUGA X GAA ACAGUUGG

1692 CAACUGUUC UCGCAGGC GCCUGCGA CUGAUGA X GAA AACAGUUG

1694 ACUGUUCUC GCAGGCGU ACGCCUGC CUGAUGA X GAA AGAACAGU

1703 GCAGGCGUC UCCUGUGG CCACAGGA CUGAUGA X GAA ACGCCUGC

1705 AGGCGUCUC CUGUGGCA UGCCACAG CUGAUGA X GAA AGACGCCU

1726 CCCCAAAUA UUCUUACA UGUAAGAA CUGAUGA X GAA AUUUGGGG

1728 CCAAAUAUU CUUACAAG CUUGUAAG CUGAUGA X GAA AUAUUUGG

1729 CAAAUAUUC UUACAAGC GCUUGUAA CUGAUGA X GAA AAUAUUUG

1731 AAUAUUCUU ACAAGCUC GAGCUUGU CUGAUGA X GAA AGAAUAUU

1732 AUAUUCUUA CAAGCUCU AGAGCUUG CUGAUGA X GAA AAGAAUAU

1739 UACAAGCUC UGUUUUAA UUAAAACA CUGAUGA X GAA AGCUUGUA

1743 AGCUCUGUU UUAAUGAC GUCAUUAA CUGAUGA X GAA ACAGAGCU

1744 GCUCUGUUU UAAUGACA UGUCAUUA CUGAUGA X GAA AACAGAGC

1745 CUCUGUUUU AAUGACAC GUGUCAUU CUGAUGA X GAA AAACAGAG

1746 UCUGUUUUA AUGACACC GGUGUCAU CUGAUGA X GAA AAAACAGA

1758 ACACCUGUA UCAGAAGA UCUUCUGA CUGAUGA X GAA ACAGGUGU

1760 ACCUGUAUC AGAAGAUG CAUCUUCU CUGAUGA X GAA AUACAGGU

1779 GACAAUGUC CUCAAAGC GCUUUGAG CUGAUGA X GAA ACAUUGUC

1782 AAUGUCCUC AAAGCCUU AAGGCUUU CUGAUGA X GAA AGGACAUU

1790 CAAAGCCUU UACCGUAC GUACGGUA CUGAUGA X GAA AGGCUUUG

1791 AAAGCCUUU ACCGUACC GGUACGGU CUGAUGA X GAA AAGGCUUU

1792 AAGCCUUUA CCGUACCU AGGUACGG CUGAUGA X GAA AAAGGCUU

1797 UUUACCGUA CCUAAGAA UUCUUAGG CUGAUGA X GAA ACGGUAAA

1801 CCGUACCUA AGAACAGG CCUGUUCU CUGAUGA X GAA AGGUACGG nt. Target Secme ce Ribozyme Seguence

Posi¬ tion

1822 UGGUGGGUC CCUUGCAG CUGCAAGG CUGAUGA X GAA ACCCACCA

1826 GGGUCCCUU GCAGCCAU AUGGCUGC CUGAUGA X GAA AGGGACCC

1859 GCCAGCAUC CUGUGGGA UCCCACAG CUGAUGA X GAA AUGCUGGC

1892 GACGGCCUC CGGUCCGG CCGGACCG CUGAUGA X GAA AGGCCGUC

1897 CCUCCGGUC CGGCUCGG CCGAGCCG CUGAUGA X GAA ACCGGAGG

1903 GUCCGGCUC GGAAAUAC GUAUUUCC CUGAUGA X GAA AGCCGGAC

1910 UCGGAAAUA CGUGAACG CGUUCACG CUGAUGA X GAA AUUUCCGA

1922 GAACGCGUU CUCAGCUC GAGCUGAG CUGAUGA X GAA ACGCGUUC

1923 AACGCGUUC UCAGCUCG CGAGCUGA CUGAUGA X GAA AACGCGUU

1925 CGCGUUCUC AGCUCGAA UUCGAGCU CUGAUGA X GAA AGAACGCG

1930 UCUCAGCUC GAACUCUG CAGAGUUC CUGAUGA X GAA AGCUGAGA

1936 CUCGAACUC UGGUCAUG CAUGACCA CUGAUGA X GAA AGUUCGAG

1941 ACUCUGGUC AUGUGAGA UCUCACAU CUGAUGA X GAA ACCAGAGU

1953 UGAGACAUU UCCAGAAA UUUCUGGA CUGAUGA X GAA AUGUCUCA

1954 GAGACAUUU CCAGAAAA UUUUCUGG CUGAUGA X GAA AAUGUCUC

1955 AGACAUUUC CAGAAAAG CUUUUCUG CUGAUGA X GAA AAAUGUCU

1967 AAAAGCAUU AUGGUUUU AAAACCAU CUGAUGA X GAA AUGCUUUU

1968 AAAGCAUUA UGGUUUUC GAAAACCA CUGAUGA X GAA AAUGCUUU

1973 AUUAUGGUU UUCAGAAC GUUCUGAA CUGAUGA X GAA ACCAUAAU

1974 UUAUGGUUU UCAGAACA UGUUCUGA CUGAUGA X GAA AACCAUAA

1975 UAUGGUUUU CAGAACAC GUGUUCUG CUGAUGA X GAA AAACCAUA

1976 AUGGUUUUC AGAACACU AGUGUUCU CUGAUGA X GAA AAAACCAU

1985 AGAACACUU AAAAGUUG CAACUUUU CUGAUGA X GAA AGUGUUCU

1986 GAACACUUA AAAGUUGA UCAACUUU CUGAUGA X GAA AAGUGUUC

1992 UUAAAAGUU GACUUUCG CGAAAGUC CUGAUGA X GAA ACUUUUAA

1997 AGUUGACUU UCGACACA UGUGUCGA CUGAUGA X GAA AGUCAACU

1998 GUUGACUUU CGACACAU AUGUGUCG CUGAUGA X GAA AAGUCAAC

1999 UUGACUUUC GACACAUG CAUGUGUC CUGAUGA X GAA AAAGUCAA

2011 ACAUGGCUC CUCAGCGU ACGCUGAG CUGAUGA X GAA AGCCAUGU

2014 UGGCUCCUC AGCGUGGA UCCACGCU CUGAUGA X GAA AGGAGCCA

2028 GGAGCGCUC CAUGGCUG CAGCCAUG CUGAUGA X GAA AGCGCUCC

2052 AGCCUGAUU UUGUUGUG CACAACAA CUGAUGA X GAA AUCAGGCU

2053 GCCUGAUUU UGUUGUGG CCACAACA CUGAUGA X GAA AAUCAGGC

2054 CCUGAUUUU GUUGUGGU ACCACAAC CUGAUGA X GAA AAAUCAGG

2057 GAUUUUGUU GUGGUACA UGUACCAC CUGAUGA X GAA ACAAAAUC

2063 GUUGUGGUA CAACAGUU AACUGUUG CUGAUGA X GAA ACCACAAC nt, Target Seguence Ribozyme Seguence

Posi¬ tion

2071 ACAACAGUU GAGAGCAG CUGCUCUC CUGAUGA X GAA ACUGUUGU

2092 AAGUGCAUU UUUAGUUG CAACUAAA CUGAUGA X GAA AUGCACUU

2093 AGUGCAUUU UUAGUUGC GCAACUAA CUGAUGA X GAA AAUGCACU

2094 GUGCAUUUU UAGUUGCU AGCAACUA CUGAUGA X GAA AAAUGCAC

2095 UGCAUUUUU AGUUGCUU AAGCAACU CUGAUGA X GAA AAAAUGCA

2096 GCAUUUUUA GUUGCUUG CAAGCAAC CUGAUGA X GAA AAAAAUGC

2099 UUUUUAGUU GCUUGAGA UCUCAAGC CUGAUGA X GAA ACUAAAAA

2103 UAGUUGCUU GAGAUCUC GAGAUCUC CUGAUGA X GAA AGCAACUA

2109 CUUGAGAUC UCACUUGA UCAAGUGA CUGAUGA X GAA AUCUCAAG

2111 UGAGAUCUC ACUUGAUU AAUCAAGU CUGAUGA X GAA AGAUCUCA

2115 AUCUCACUU GAUUUCAC GUGAAAUC CUGAUGA X GAA AGUGAGAU

2119 CACUUGAUU UCACACAA UUGUGUGA CUGAUGA X GAA AUCAAGUG

2120 ACUUGAUUU CACACAAC GUUGUGUG CUGAUGA X GAA AAUCAAGU

2121 CUUGAUUUC ACACAACU AGUUGUGU CUGAUGA X GAA AAAUCAAG

2130 ACACAACUA AAAAGGAU AUCCUUUU CUGAUGA X GAA AGUUGUGU

2139 AAAAGGAUU UUUUUUUU AAAAAAAA CUGAUGA X GAA AUCCUUUU

2140 AAAGGAUUU UUUUUUUA UAAAAAAA CUGAUGA X GAA AAUCCUUU

2141 AAGGAUUUU UUUUUUAA UUAAAAAA CUGAUGA X GAA AAAUCCUU

2142 AGGAUUUUU UUUUUAAA UUUAAAAA CUGAUGA X GAA AAAAUCCU

2143 GGAUUUUUU UUUUAAAA UUUUAAAA CUGAUGA X GAA AAAAAUCC

2144 GAUUUUUUU UUUAAAAA UUUUUAAA CUGAUGA X GAA AAAAAAUC

2145 AUUUUUUUU UUAAAAAU AUUUUUAA CUGAUGA X GAA AAAAAAAU

2146 UUUUUUUUU UAAAAAUA UAUUUUUA CUGAUGA X GAA AAAAAAAA

2147 uuuuuuuuu AAAAAUAA UUAUUUUU CUGAUGA X GAA AAAAAAAA

2148 UUUUUUUUA AAAAUAAU AUUAUUUU CUGAUGA X GAA AAAAAAAA

2154 UUAAAAAUA AUAAUAAU AUUAUUAU CUGAUGA X GAA AUUUUUAA

2157 AAAAUAAUA AUAAUGAA UUCAUUAU CUGAUGA X GAA AUUAUUUU

2160 AUAAUAAUA AUGAAUAA UUAUUCAU CUGAUGA X GAA AUUAUUAU

2167 UAAUGAAUA ACAGUCUU AAGACUGU CUGAUGA X GAA AUUCAUUA

2173 AUAACAGUC UUACCUAA UUAGGUAA CUGAUGA X GAA ACUGUUAU

2175 AACAGUCUϋ ACCUAAAU AUUUAGGU CUGAUGA X GAA AGACUGUU

2176 ACAGUCUUA CCUAAAUU AAUUUAGG CUGAUGA X GAA AAGACUGU

2180 UCUUACCUA AAUUAUUA UAAUAAUU CUGAUGA X GAA AGGUAAGA

2184 ACCUAAAUU AUUAGGUA UACCUAAU CUGAUGA X GAA AUUUAGGU

2185 CCUAAAUUA UUAGGUAA UUACCUAA CUGAUGA X GAA AAUUUAGG

2187 UAAAUUAUU AGGUAAUG CAUUACCU CUGAUGA X GAA AUAAUUUA nt. Target Sequence Ribozyme Sequence

Posi¬ tion

2188 AAAUUAUUA GGUAAUGA UCAUUACC CUGAUGA X GAA AAUAAUUU

2192 UAUUAGGUA AUGAAUUG CAAUUCAU CUGAUGA X GAA ACCUAAUA

2199 UAAUGAAUU GUGACCAU AUGGUCAC CUGAUGA X GAA AUUCAUUA

2208 GUGACCAUU UGUUAAUA UAUUAACA CUGAUGA X GAA AUGGUCAC

2209 UGACCAUUU GUUAAUAU AUAUUAAC CUGAUGA X GAA AAUGGUCA

2212 CCAUUUGUU AAUAUCAU AUGAUAUU CUGAUGA X GAA ACAAAUGG

2213 CAUUUGUUA AUAUCAUA UAUGAUAU CUGAUGA X GAA AACAAAUG

2216 UUGUUAAUA UCAUAAUC GAUUAUGA CUGAUGA X GAA AUUAACAA

2218 GUUAAUAUC AUAAUCAG CUGAUUAU CUGAUGA X GAA AUAUUAAC

2221 AAUAUCAUA AUCAGAUU AAUCUGAU CUGAUGA X GAA AUGAUAUU

2224 AUCAUAAUC AGAUUUUU AAAAAUCU CUGAUGA X GAA AUUAUGAU

2229 AAUCAGAUU UUUUAAAA UUUUAAAA CUGAUGA X GAA AUCUGAUU

2230 AUCAGAUUU UUUAAAAA UUUUUAAA CUGAUGA X GAA AAUCUGAU

2231 UCAGAUUUU UUAAAAAA UUUUUUAA CUGAUGA X GAA AAAUCUGA

2232 CAGAUUUUU UAAAAAAA UUUUUUUA CUGAUGA X GAA AAAAUCUG

2233 AGAUUUUUU AAAAAAAA UUUUUUUU CUGAUGA X GAA AAAAAUCU

2234 GAUUUUUUA AAAAAAAU AUUUUUUU CUGAUGA X GAA AAAAAAUC

2243 AAAAAAAUA AAAUGAUU AAUCAUUU CUGAUGA X GAA AUUUUUUU

2251 AAAAUGAUU UAUUUGUA UACAAAUA CUGAUGA X GAA AUCAUUUU

2252 AAAUGAUUU AUUUGUAU AUACAAAU CUGAUGA X GAA AAUCAUUU

2253 AAUGAUUUA UUUGUAUU AAUACAAA CUGAUGA X GAA AAAUCAUU

2255 UGAUUUAUU UGUAUUUU AAAAUACA CUGAUGA X GAA AUAAAUCA

2256 GAUUUAUUU GUAUUUUA UAAAAUAC CUGAUGA X GAA AAUAAAUC

2259 UUAUUUGUA UUUUAGAG CUCUAAAA CUGAUGA X GAA ACAAAUAA

2261 AUUUGUAUU UUAGAGAA UUCUCUAA CUGAUGA X GAA AUACAAAU

2262 UUUGUAUUU UAGAGAAU AUUCUCUA CUGAUGA X GAA AAUACAAA

2263 UUGUAUUUU AGAGAAUA UAUUCUCU CUGAUGA X GAA AAAUACAA

2264 UGUAUUUUA GAGAAUAC GUAUUCUC CUGAUGA X GAA AAAAUACA

2271 UAGAGAAUA CAACAGAU AUCUGUUG CUGAUGA X GAA AUUCUCUA

2280 CAACAGAUC AGUAUUUU AAAAUACU CUGAUGA X GAA AUCUGUUG

2284 AGAUCAGUA UUUUUGAC GUCAAAAA CUGAUGA X GAA ACUGAUCU

2286 AUCAGUAUU UUUGACUG CAGUCAAA CUGAUGA X GAA AUACUGAU

2287 UCAGUAUUU UUGACUGU ACAGUCAA CUGAUGA X GAA AAUACUGA

2288 CAGUAUUUU UGACUGUG CACAGUCA CUGAUGA X GAA AAAUACUG

2289 AGUAUUUUU GACUGUGG CCACAGUC CUGAUGA X GAA AAAAUACU

2303 UGGUGAAUU UAAAAAAA UUUUUUUA CUGAUGA X GAA AUUCACCA nt, Target Seguence Ribozyme Seguence

Posi¬ tion

2304 GGUGAAUUU AAAAAAAA UUUUUUUU CUGAUGA X GAA AAUUCACC

2305 GUGAAUUUA AAAAAAAA UUUUUUUU CUGAUGA X GAA AAAUUCAC

2316 AAAAAAAUU UACACAAA UUUGUGUA CUGAUGA X GAA AUUUUUUU

2317 AAAAAAUUU ACACAAAG CUUUGUGU CUGAUGA X GAA AAUUUUUU

2318 AAAAAUUUA CACAAAGA UCUUUGUG CUGAUGA X GAA AAAUUUUU

2330 AAAGAAAUA UCCCAGUA UACUGGGA CUGAUGA X GAA AUUUCUUU

2332 AGAAAUAUC CCAGUAUU AAUACUGG CUGAUGA X GAA AUAUUUCU

2338 AUCCCAGUA UUCCAUGU ACAUGGAA CUGAUGA X GAA ACUGGGAU

2340 CCCAGUAUU CCAUGUAU AUACAUGG CUGAUGA X GAA AUACUGGG

2341 CCAGUAUUC CAUGUAUC GAUACAUG CUGAUGA X GAA AAUACUGG

2347 UUCCAUGUA UCUCAGUC GACUGAGA CUGAUGA X GAA ACAUGGAA

2349 CCAUGUAUC UCAGUCAC GUGACUGA CUGAUGA X GAA AUACAUGG

2351 AUGUAUCUC AGUCACUA UAGUGACU CUGAUGA X GAA AGAUACAU

2355 AUCUCAGUC ACUAAACA UGUUUAGU CUGAUGA X GAA ACUGAGAU

2359 CAGUCACUA AACAUACA UGUAUGUU CUGAUGA X GAA AGUGACUG

2365 CUAAACAUA CACAGAGA UCUCUGUG CUGAUGA X GAA AUGUUUAG

2377 AGAGAGAUU UUUAAAAA UUUUUAAA CUGAUGA X GAA AUCUCUCU

2378 GAGAGAUUU UUAAAAAC GUUUUUAA CUGAUGA X GAA AAUCUCUC

2379 AGAGAUUUU UAAAAACC GGUUUUUA CUGAUGA X GAA AAAUCUCU

2380 GAGAUUUUU AAAAACCA UGGUUUUU CUGAUGA X GAA AAAAUCUC

2381 AGAUUUUUA AAAACCAG CUGGUUUU CUGAUGA X GAA AAAAAUCU

2399 AGAAGCAUU AUUUUGAA UUCAAAAU CUGAUGA X GAA AUGCUUCU

2400 GAAGCAUUA UUUUGAAU AUUCAAAA CUGAUGA X GAA AAUGCUUC

2402 AGCAUUAUU UUGAAUGU ACAUUCAA CUGAUGA X GAA AUAAUGCU

2403 GCAUUAUUU UGAAUGUU AACAUUCA CUGAUGA X GAA AAUAAUGC

2404 CAUUAUUUU GAAUGUUA UAACAUUC CUGAUGA X GAA AAAUAAUG

2411 UUGAAUGUU AGCUAAAU AUUUAGCU CUGAUGA X GAA ACAUUCAA

2412 UGAAUGUUA GCUAAAUC GAUUUAGC CUGAUGA X GAA AACAUUCA

2416 UGUUAGCUA AAUCCCAA UUGGGAUU CUGAUGA X GAA AGCUAACA

2420 AGCUAAAUC CCAAGUAA UUACUUGG CUGAUGA X GAA AUUUAGCU

2427 UCCCAAGUA AUACUUAA UUAAGUAU CUGAUGA X GAA ACUUGGGA

2430 CAAGUAAUA CUUAAUGC GCAUUAAG CUGAUGA X GAA AUUACUUG

2433 GUAAUACUU AAUGCAAC GUUGCAUU CUGAUGA X GAA AGUAUUAC

2434 UAAUACUUA AUGCAACC GGUUGCAU CUGAUGA X GAA AAGUAUUA

2445 GCAACCCUC UAGGAGCU AGCUCCUA CUGAUGA X GAA AGGGUUGC

2447 AACCCUCUA GGAGCUCA UGAGCUCC CUGAUGA X GAA AGAGGGUU nt. Target Seguence Ribozyme Seguence

Posi¬ tion

2454 UAGGAGCUC AUUUGUGG CCACAAAU CUGAUGA X GAA AGCUCCUA

2457 GAGCUCAUU UGUGGCUA UAGCCACA CUGAUGA X GAA AUGAGCUC

2458 AGCUCAUUU GUGGCUAA UUAGCCAC CUGAUGA X GAA AAUGAGCU

2465 UUGUGGCUA AUAAUCUU AAGAUUAU CUGAUGA X GAA AGCCACAA

2468 UGGCUAAUA AUCUUGGA UCCAAGAU CUGAUGA X GAA AUUAGCCA

2471 CUAAUAAUC UUGGAAAU AUUUCCAA CUGAUGA X GAA AUUAUUAG

2473 AAUAAUCUU GGAAAUAU AUAUUUCC CUGAUGA X GAA AGAUUAUU

2480 UUGGAAAUA UCUUUAUU AAUAAAGA CUGAUGA X GAA AUUUCCAA

2482 GGAAAUAUC UUUAUUAU AUAAUAAA CUGAUGA X GAA AUAUUUCC

2484 AAAUAUCUU UAUUAUAU AUAUAAUA CUGAUGA X GAA AGAUAUUU

2485 AAUAUCUUU AUUAUAUA UAUAUAAU CUGAUGA X GAA AAGAUAUU

2486 AUAUCUUUA UUAUAUAG CUAUAUAA CUGAUGA X GAA AAAGAUAU

2488 AUCUUUAUU AUAUAGCA UGCUAUAU CUGAUGA X GAA AUAAAGAU

2489 UCUUUAUUA UAUAGCAU AUGCUAUA CUGAUGA X GAA AAUAAAGA

2491 UUUAUUAUA UAGCAUUU AAAUGCUA CUGAUGA X GAA AUAAUAAA

2493 UAUUAUAUA GCAUUUAU AUAAAUGC CUGAUGA X GAA AUAUAAUA

2498 UAUAGCAUU UAUGAGGA UCCUCAUA CUGAUGA X GAA AUGCUAUA

2499 AUAGCAUUU AUGAGGAG CUCCUCAU CUGAUGA X GAA AAUGCUAU

2500 UAGCAUUUA UGAGGAGA UCUCCUCA CUGAUGA X GAA AAAUGCUA

2510 GAGGAGAUU UUGUUGUC GACAACAA CUGAUGA X GAA AUCUCCUC

2511 AGGAGAUUU UGUUGUCA UGACAACA CUGAUGA X GAA AAUCUCCU

2512 GGAGAUUUU GUUGUCAG CUGACAAC CUGAUGA X GAA AAAUCUCC

2515 GAUUUUGUU GUCAGCUU AAGCUGAC CUGAUGA X GAA ACAAAAUC

2518 UUUGUUGUC AGCUUGCU AGCAAGCU CUGAUGA X GAA ACAACAAA

2523 UGUCAGCUU GCUUGAAA UUUCAAGC CUGAUGA X GAA AGCUGACA

2527 AGCUUGCUU GAAAGUUA UAACUUUC CUGAUGA X GAA AGCAAGCU

2534 UUGAAAGUU AUUAUGUA UACAUAAU CUGAUGA X GAA ACUUUCAA

2535 UGAAAGUUA UUAUGUAU AUACAUAA CUGAUGA X GAA AACUUUCA

2537 AAAGUUAUU AUGUAUGA UCAUACAU CUGAUGA X GAA AUAACUUU

2538 AAGUUAUUA UGUAUGAA UUCAUACA CUGAUGA X GAA AAUAACUU

2542 UAUUAUGUA UGAAUAGU ACUAUUCA CUGAUGA X GAA ACAUAAUA

2548 GUAUGAAUA GUUUUAUU AAUAAAAC CUGAUGA X GAA AUUCAUAC

2551 UGAAUAGUU UUAUUGAA UUCAAUAA CUGAUGA X GAA ACUAUUCA

2552 GAAUAGUUU UAUUGAAA UUUCAAUA CUGAUGA X GAA AACUAUUC

2553 AAUAGUUUU AUUGAAAA UUUUCAAU CUGAUGA X GAA AAACUAUU

2554 AUAGUUUUA UUGAAAAA UUUUUCAA CUGAUGA X GAA AAAACUAU nt. Target Sequence Ribozyme Sequence

Posi¬ tion

2556 AGUUUUAUU GAAAAAAU AUUUUUUC CUGAUGA X GAA AUAAAACU

2565 GAAAAAAUU AUAUUUUU AAAAAUAU CUGAUGA X GAA AUUUUUUC

2566 AAAAAAUUA UAUUUUUA UAAAAAUA CUGAUGA X GAA AAUUUUUU

2568 AAAAUUAUA UUUUUAUU AAUAAAAA CUGAUGA X GAA AUAAUUUU

2570 AAUUAUAUU UUUAUUCA UGAAUAAA CUGAUGA X GAA AUAUAAUU

2571 AUUAUAUUU UUAUUCAG CUGAAUAA CUGAUGA X GAA AAUAUAAU

2572 UUAUAUUUU UAUUCAGU ACUGAAUA CUGAUGA X GAA AAAUAUAA

2573 UAUAUUUUU AUUCAGUA UACUGAAU CUGAUGA X GAA AAAAUAUA

2574 AUAUUUUUA UUCAGUAA UUACUGAA CUGAUGA X GAA AAAAAUAU

2576 AUUUUUAUU CAGUAAUU AAUUACUG CUGAUGA X GAA AUAAAAAU

2577 UUUUUAUUC AGUAAUUU AAAUUACU CUGAUGA X GAA AAUAAAAA

2581 UAUUCAGUA AUUUAAUU AAUUAAAU CUGAUGA X GAA ACUGAAUA

2584 UCAGUAAUU UAAUUUUG CAAAAUUA CUGAUGA X GAA AUUACUGA

2585 CAGUAAUUU AAUUUUGU ACAAAAUU CUGAUGA X GAA AAUUACUG

2586 AGUAAUUUA AUUUUGUA UACAAAAU CUGAUGA X GAA AAAUUACU

2589 AAUUUAAUU UUGUAAAU AUUUACAA CUGAUGA X GAA AUUAAAUU

2590 AUUUAAUUU UGUAAAUG CAUUUACA CUGAUGA X GAA AAUUAAAU

2591 UUUAAUUUU GUAAAUGC GCAUUUAC CUGAUGA X GAA AAAUUAAA

2594 AAUUUUGUA AAUGCCAA UUGGCAUU CUGAUGA X GAA ACAAAAUU

2617 AAAUGUGUU CGCUGCUA UAGCAGCG CUGAUGA X GAA ACACAUUU

2618 AAUGUGUUC GCUGCUAU AUAGCAGC CUGAUGA X GAA AACACAUU

2625 UCGCUGCUA UGGUUUUA UAAAACCA CUGAUGA X GAA AGCAGCGA

2630 GCUAUGGUU UUAGCCUA UAGGCUAA CUGAUGA X GAA ACCAUAGC

2631 CUAUGGUUU UAGCCUAU AUAGGCUA CUGAUGA X GAA AACCAUAG

2632 UAUGGUUUU AGCCUAUA UAUAGGCU CUGAUGA X GAA AAACCAUA

2633 AUGGUUUUA GCCUAUAG CUAUAGGC CUGAUGA X GAA AAAACCAU

2638 UUUAGCCUA UAGUCAUG CAUGACUA CUGAUGA X GAA AGGCUAAA

2640 UAGCCUAUA GUCAUGCU AGCAUGAC CUGAUGA X GAA AUAGGCUA

2643 CCUAUAGUC AUGCUGCU AGCAGCAU CUGAUGA X GAA ACUAUAGG

2652 AUGCUGCUA GCUAGUGU ACACUAGC CUGAUGA X GAA AGCAGCAU

2656 UGCUAGCUA GUGUCAGG CCUGACAC CUGAUGA X GAA AGCUAGCA

2661 GCUAGUGUC AGGGGGCA UGCCCCCU CUGAUGA X GAA ACACUAGC

2672 GGGGCAAUA GAGCUUAG CUAAGCUC CUGAUGA X GAA AUUGCCCC

2678 AUAGAGCUU AGAUGGAA UUCCAUCU CUGAUGA X GAA AGCUCUAU

2679 UAGAGCUUA GAUGGAAA UUUCCAUC CUGAUGA X GAA AAGCUCUA

2703 AAGAGACUC GGUGUUAG CUAACACC CUGAUGA X GAA AGUCUCUU nt Target Sequence Ribozyme Seguence

Posi¬ tion

2709 CUCGGUGUU AGAUAACG CGUUAUCU CUGAUGA X GAA ACACCGAG

2710 UCGGUGUUA GAUAACGG CCGUUAUC CUGAUGA X GAA AACACCGA

2714 UGUUAGAUA ACGGACUA UAGUCCGU CUGAUGA X GAA AUCUAACA

2722 AACGGACUA UGCACUAG CUAGUGCA CUGAUGA X GAA AGUCCGUU

2729 UAUGCACUA GUAUUCCA UGGAAUAC CUGAUGA X GAA AGUGCAUA

2732 GCACUAGUA UUCCAGAC GUCUGGAA CUGAUGA X GAA ACUAGUGC

2734 ACUAGUAUU CCAGACUU AAGUCUGG CUGAUGA X GAA AUACUAGU

2735 CUAGUAUUC CAGACUUU AAAGUCUG CUGAUGA X GAA AAUACUAG

2742 UCCAGACUU UUUUAUUU AAAUAAAA CUGAUGA X GAA AGUCUGGA

2743 CCAGACUUU UUUAUUUU AAAAUAAA CUGAUGA X GAA AAGUCUGG

2744 CAGACUUUU UUAUUUUU AAAAAUAA CUGAUGA X GAA AAAGUCUG

2745 AGACUUUUU UAUUUUUU AAAAAAUA CUGAUGA X GAA AAAAGUCU

2746 GACUUUUUU AUUUUUUA UAAAAAAU CUGAUGA X GAA AAAAAGUC

2747 ACUUUUUUA UUUUUUAU AUAAAAAA CUGAUGA X GAA AAAAAAGU

2749 UUUUUUAUU UUUUAUAU AUAUAAAA CUGAUGA X GAA AUAAAAAA

2750 UUUUUAUUU UUUAUAUA UAUAUAAA CUGAUGA X GAA AAUAAAAA

2751 UUUUAUUUU UUAUAUAU AUAUAUAA CUGAUGA X GAA AAAUAAAA

2752 UUUAUUUUU UAUAUAUA UAUAUAUA CUGAUGA X GAA AAAAUAAA

2753 UUAUUUUUU AUAUAUAU AUAUAUAU CUGAUGA X GAA AAAAAUAA

2754 UAUUUUUUA UAUAUAUG CAUAUAUA CUGAUGA X GAA AAAAAAUA

2756 UUUUUUAUA UAUAUGUA UACAUAUA CUGAUGA X GAA AUAAAAAA

2758 UUUUAUAUA UAUGUACC GGUACAUA CUGAUGA X GAA AUAUAAAA

2760 UUAUAUAUA UGUACCUU AAGGUACA CUGAUGA X GAA AUAUAUAA

2764 AUAUAUGUA CCUUUUCC GGAAAAGG CUGAUGA X GAA ACAUAUAU

2768 AUGUACCUU UUCCUUUU AAAAGGAA CUGAUGA X GAA AGGUACAU

2769 UGUACCUUU UCCUUUUG CAAAAGGA CUGAUGA X GAA AAGGUACA

2770 GUACCUUUU CCUUUUGU ACAAAAGG CUGAUGA X GAA AAAGGUAC

2771 UACCUUUUC CUUUUGUC GACAAAAG CUGAUGA X GAA AAAAGGUA

2774 CUUUUCCUU UUGUCAAU AUUGACAA CUGAUGA X GAA AGGAAAAG

2775 UUUUCCUUU UGUCAAUU AAUUGACA CUGAUGA X GAA AAGGAAAA

2776 UUUCCUUUU GUCAAUUG CAAUUGAC CUGAUGA X GAA AAAGGAAA

Where "X" represents stem II region of a HH ribozyme (Hertel et al., 1992 Nucleic Acids Res. 20 3252) . The length of stem II may be ≥ 2 base-pairs. Table XVI: Mouse c-mvb Hairpin ribozyme and target sequences

Posi¬ RZ Substrate tion

24 GCGAGGCG AGAA GGGGCU AGCCCCG GCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CGCCUCGC

28 CAUGGCGA AGAA GGCCGG CCGGCCC GCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UCGCCAUG

122 AUUUGGGC AGAA GCCCAU AUGGGCU GCU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GCCCAAAU

125 CAGAUUUG AGAA GCAGCC GGCUGCU GCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CAAAUCUG

216 UUCCAGUC AGAA GUUCCG CGGAACA GAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GACUGGAA

245 UCCGGUUG AGAA GAUAAU AUUAUCU GCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CAACCGGA

258 CACUGUAC AGAA GUCCGG CCGGACA GAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GUACAGUG

529 CUCUGCCC AGAA GUUCCC GGGAACA GAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GGGCAGAG

551 GUCCGGGC AGAA GCUUUG CAAAGCU GCU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GCCCGGAC

554 UCCGUCCG AGAA GCAGCU AGCUGCU GCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CGGACGGA

559 AUCAGUCC AGAA GGGCAG CUGCCCG GAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GGACUGAU

563 CAUUAUCA AGAA GUCCGG CCGGACG GAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UGAUAAUG

656 CCACUGGC AGAA GGCUGG CCAGCCA GAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GCCAGUGG

728 UUGGAGAG AGAA GAGAUG CAUCUCA GCU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CUCUCCAA

746 UGACGGAG AGAA GGCCAC GUGGCCA GUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CUCCGUCA

822 UGCAAUGC AGAA GGAUAG CUAUCCU GUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GCAUUGCA 857 CCGCAGCC AGAA GAGGGA UCCCUCA GCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GGCUGCGG

861 GCUGCCGC AGAA GGCUGA UCAGCCG GCU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GCGGCAGC

941 CUGUUGAC AGAA GGAGCA UGCUCCU GAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GUCAACAG

1040 GAGGUCUG AGAA GGUCCA UGGACCA GAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CAGACCUC

1045 CCCAUGAG AGAA GGUCUG CAGACCA GAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CUCAUGGG

1068 AAACAGGA AGAA GGUGCA UGCACCU GUU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UCCUGUUU

1075 UUCUCCCA AGAA GGAAAC GUUUCCU GUU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UGGGAGAA

1106 GAUCUGCA AGAA GAGAUG CAUCUCU GCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UGCAGAUC

1113 GAGCCGGG AGAA GCAGGC GCCUGCA GAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CCCGGCUC

1120 AGGUAGGG AGAA GGGAUC GAUCCCG GCU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CCCUACCU

1226 AAUCUAUA AGAA GGAGUG CACUCCA GUU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UAUAGAUU

1340 UUUUCACA AGAA GGUCUC GAGACCA GAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UGUGAAAA

1449 AUUUCUUG AGAA GCAAGG CCUUGCA GCU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CAAGAAAU

1468 CUUCAGGG AGAA GUAUUU AAAUACG GUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CCCUGAAG

1490 GGGAGGGG AGAA GAGGUA UACCUCA GAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CCCCUCCC

1542 CCAGAUUC AGAA GAUUCC GGAAUCG GAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GAAUCUGG

1648 GUGGUUUG AGAA GAAGAA UUCUUCU GCU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CAAACCAC

1672 GGUGCUCA AGAA GUUCUC GAGAACA GCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UGAGCACC 1688 CCUGCGAG AGAA GUUGGG CCCAACU GUU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CUCGCAGG

1713 UUUGGGGC AGAA GCCACA UGUGGCA GAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GCCCCAAA

1740 GUCAUUAA AGAA GAGCUϋ AAGCUCU GUU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UUAAUGAC

1880 AGGCCGUC AGAA GGUCCU AGGACCA GAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GACGGCCU

1887 GGACCGGA AGAA GUCAUC GAUGACG GCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UCCGGUCC

1894 CCGAGCCG AGAA GGAGGC GCCUCCG GUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CGGCUCGG

1899 UAUUUCCG AGAA GGACCG CGGUCCG GCU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CGGAAAUA

1926 AGAGUUCG AGAA GAGAAC GUUCUCA GCU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CGAACUCU

2048 ACAACAAA AGAA GGCUCU AGAGCCU GAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UUUGUUGU

2068 CUGCUCUC AGAA GUUGUA UACAACA GUU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GAGAGCAG

2170 UUAGGUAA AGAA GUUAUU AAUAACA GUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UUACCUAA

2225 UUUAAAAA AGAA GAUUAU AUAAUCA GAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UUUUUAAA

2276 AAAUACUG AGAA GUUGUA UACAACA GAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CAGUAUUU

2519 UUCAAGCA AGAA GACAAC GUUGUCA GCU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UGCUUGAA

2717 AGUGCAUA AGAA GUUAUC •GAUAACG GAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UAUGCACU

2737 AUAAAAAA AGAA GGAAUA UAUUCCA GAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UUUUUUAU Table XVII: Rat c-myb (Region A) Hammerhead Ribozyme and

Target Sequences (282 bp; nt . 428 start; human c-myb numbering system) nt. Target Sequence Ribozyme Sequence Posi¬ tion

467 CCUGAGCUC AUCAAAGG CCUUUGAU CUGAUGA X GAA AGCUCAGG

470 GAGCUCAUC AAAGGUCC GGACCUUU CUGAUGA X GAA AUGAGCUC

477 UCAAAGGUC CCUGGACC GGUCCAGG CUGAUGA X GAA ACCUUUGA

498 AAGAAGAUC AAAGAGUG CACUCUUU CUGAUGA X GAA AUCUUCUU

509 AGAGUGAUA GAGCUUGU ACAAGCUC CUGAUGA X GAA AUCACUCU

515 AUAGAGCUU GUCCAGAA UUCUGGAC CUGAUGA X GAA AGCUCUAU

518 GAGCUUGUC CAGAAAUA UAUUUCUG CUGAUGA X GAA ACAAGCUC

526 CCAGAAAUA CGGUCCGA UCGGACCG CUGAUGA X GAA AUUUCUGG

531 AAUACGGUC CGAAGCGC GCGCUUCG CUGAUGA X GAA ACCGUAUU

544 GCGCUGGUC UGUUAUUG CAAUAACA CUGAUGA X GAA ACCAGCGC

548 UGGUCUGUU AUUGCCAA UUGGCAAU CUGAUGA X GAA ACAGACCA

549 GGUCUGUUA UUGCCAAG CUUGGCAA CUGAUGA X GAA AACAGACC 551 UCUGUUAUU GCCAAGCA UGCUUGGC CUGAUGA X GAA AUAACAGA

562 CAAGCACUU AAAAGGGA UCCCUUUU CUGAUGA X GAA AGUGCUUG

563 AAGCACUUA AAAGGGAG CUCCCUUU CUGAUGA X GAA AAGUGCUU 575 GGGAGAAUU GGAAAACA UGUUUUCC CUGAUGA X GAA AUUCUCCC 588 AACAAUGUC GGGAGAGG CCUCUCCC CUGAUGA X GAA ACAUUGUU

609 ACAACCAUU UGAAUCCA UGGAUUCA CUGAUGA X GAA AUGGUUGU

610 CAACCAUUU GAAUCCAG CUGGAUUC CUGAUGA X GAA AAUGGUUG 615 AUUUGAAUC CAGAAGUU AACUUCUG CUGAUGA X GAA AUUCAAAU

623 CCAGAAGUU AAGAAAAC GUUUUCUU CUGAUGA X GAA ACUUCUGG

624 CAGAAGUUA AGAAAACC GGUUUUCU CUGAUGA X GAA AACUUCUG 634 GAAAACCUC AUGGACAG CUGUCCAU CUGAUGA X GAA AGGUUUUC 659 GACAGAAUC AUUUAUCA UGAUAAAU CUGAUGA X GAA AUUCUGUC

662 AGAAUCAUU UAUCAGGC GCCUGAUA CUGAUGA X GAA AUGAUUCU

663 GAAUCAUUU AUCAGGCA UGCCUGAU CUGAUGA X GAA AAUGAUUC

664 AAUCAUUUA UCAGGCAC GUGCCUGA CUGAUGA X GAA AAAUGAUU 666 UCAUUUAUC AGGCACAC GUGUGCCU CUGAUGA X GAA AUAAAUGA

Where "X" represents stem II region of a HH ribozyme (Hertel et al., 1992 Nucleic Acids Res. 20 3252) . The length of stem II may be ≥ 2 base-pairs. Table XVIII : Rat c-myb (Region B) Hammerhead Ribozyme and Target Sequences ( 262 bp ; nt . 1421 start ; human c-myb numbering system)

nt. Target Seguence Ribozyme Seguence

Posi¬ tion

1429 CUCGGGCUU AGAUACGC GCGUAUCU CUGAUGA X GAA AGCCCGAG

1430 UCGGGCUUA GAUACGCC GGCGUAUC CUGAUGA X GAA AAGCCCGA

1434 GCUUAGAUA CGCCUACU AGUAGGCG CUGAUGA X GAA AUCUAAGC

1440 AUACGCCUA CUUUACCC GGGUAAAG CUGAUGA X GAA AGGCGUAU

1443 CGCCUACUU UACCCUCC GGAGGGUA CUGAUGA X GAA AGUAGGCG

1444 GCCUACUUU ACCCUCCA UGGAGGGU CUGAUGA X GAA AAGUAGGC

1445 CCUACUUUA CCCUCCAC GUGGAGGG CUGAUGA X GAA AAAGUAGG

1450 UUUACCCUC CACGCCUC GAGGCGUG CUGAUGA X GAA AGGGUAAA

1458 CCACGCCUC UCAUUGGU ACCAAUGA CUGAUGA X GAA AGGCGUGG

1460 ACGCCUCUC AUUGGUCA UGACCAAU CUGAUGA X GAA AGAGGCGU

1463 CCUCUCAUU GGUCACAA UUGUGACC CUGAUGA X GAA AUGAGAGG

1467 UCAUUGGUC ACAAACUG CAGUUUGU CUGAUGA X GAA ACCAAUGA

1485 CACCGUGUC ACCGAGAC GUCUCGGU CUGAUGA X GAA ACACGGUG

1509 UGAAAACUN AAAAGGAA UUCCUUUU CUGAUGA X GAA AGUUUUCA

1522 GGAAAACUC NAUCUUUA UAAAGAUN CUGAUGA X GAA AGUUUUCC

1526 AACUCNAUC UUUAGAAC GUUCUAAA CUGAUGA X GAA AUNGAGUU

1528 CUCNAUCUU UAGAACUC GAGUUCUA CUGAUGA X GAA AGAUNGAG

1529 UCNAUCUUU AGAACUCC GGAGUUCU CUGAUGA X GAA AAGAUNGA

1530 CNAUCUUUA GAACUCCA UGGAGUUC CUGAUGA X GAA AAAGAUNG

1536 UUAGAACUC CAGCUAUC GAUAGCUG CUGAUGA X GAA AGUUCUAA

1542 CUCCAGCUA UCAAAAGG CCUUUUGA CUGAUGA X GAA AGCUGGAG

1544 CCAGCUAUC AAAAGGUN NACCUUUU CUGAUGA X GAA AUAGCUGG

1552 CAAAAGGUN AAUCCUCG CGAGGAUU CUGAUGA X GAA ACCUUUUG

1556 AGGUNAAUC CUCGAAAG CUUUCGAG CUGAUGA X GAA AUUNACCU

1559 UNAAUCCUC GAAAGCUC GAGCUUUC CUGAUGA X GAA AGGAUUNA

1567 CGAAAGCUC UCCCAGAA UUCUGGGA CUGAUGA X GAA AGCUUUCG

1569 AAAGCUCUC CCAGAACU AGUUCUGG CUGAUGA X GAA AGAGCUUU

1578 CCAGAACUC CCACACCA UGGUGUGG CUGAUGA X GAA AGUUCUGG

1588 CACACCAUU CAAACAUG CAUGUUUG CUGAUGA X GAA AUGGUGUG

1589 ACACCAUUC AAACAUGC GCAUGUUU CUGAUGA X GAA AAUGGUGU

1608 UGGCAGCUC AAGAAAUU AAUUUCUU CUGAUGA X GAA AGCUGCCA

1616 CAAGAAAUU AAAUACGG CCGUAUUU CUGAUGA X GAA AUUUCUUG nt. Target Seguence Ribozyme Sequence Posi¬

1429 CUCGGGCUU AGAUACGC GCGUAUCU CUGAUGA X GAA AGCCCGAG

1617 AAGAAAUUA AAUACGGU ACCGUAUU CUGAUGA X GAA AAUUUCUU

1621 AAUUAAAUA CGGUCCCC GGGGACCG CUGAUGA X GAA AUUUAAUU

1626 AAUACGGUC CCCUGAAG CUUCAGGG CUGAUGA X GAA ACCGUAUU

1640 AAGAUGCUA CCUNAGAC GUCUNAGG CUGAUGA X GAA AGCAUCUU

1644 UGCUACCUN AGACCCCC GGGGGUCU CUGAUGA X GAA AGGUAGCA

1654 GACCCCCUN UNAUGUAG CUACAUNA CUGAUGA X GAA AGGGGGUC

1656 CCCCCUNUN AUGUAGUN NACUACAU CUGAUGA X GAA ANAGGGGG

1661 UNUNAUGUA GUNNNANA UNUNNNAC CUGAUGA X GAA ACAUNANA

1664 NAUGUAGU NNANACCU AGGUNUNN CUGAUGA X GAA ACUACAUN

1673 NNANACCUN CANGAUGU ACAUCNUG CUGAUGA X GAA AGGUNUNN

Where "X" represents stem II region of a HH ribozyme (Hertel et al., 1992 Nucleic Acids Res. 20 3252) . The length of stem II may be ≥ 2 base-pairs.

Table XIX: Rat c-mvb (Region A) Hairpin Ribozyme and Target Sequences (282 bp; nt. 428 start; human numbering system)

Posi¬ RZ Substrate tion

528 GCGCUUCG AGAA GUAUUU AAAUACG GUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CGAAGCGC

690 UUCUGCCC AGAA GUUUCC GGAAACA GAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GGGCAGAA

Table XX: Rat c-myb (Region B) Hairpin Ribozyme and Target Sequences (262 bp ; nt . 1421 start ; human numbering system)

Posi¬ RZ Substrate tion

1495 UUUUCACA AGAA GGUCUC GAGACCA GAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UGUGAAAA

1604 AUUUCUUG AGAA GCCAGG CCUGGCA GCU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CAAGAAAU

1623 CUUCAGGG AGAA GUAUUU AAAUACG GUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CCCUGAAG

Table XXI : Porcine c-mvb (Region A) Hammerhead Ribozyme and Target Sequence (266 bp ; nt . 458 start ; human c-mvb numbering system) nt. Target Sequence Ribozyme Sequence Posi¬ tion

467 CCUNAUCUC AUCAAGGG CCCUUGAU CUGAUGA X GAA AGAUNAGG

470 NAUCUCAUC AAGGGUCC GGACCCUU CUGAUGA X GAA AUGAGAUN

477 UCAAGGGUC CUUGGACC GGUCCAAG CUGAUGA X GAA ACCCUUGA

480 AGGGUCCUU GGACCAAA UUUGGUCC CUGAUGA X GAA AGGACCCU

498 AAGAAGAUC AGAGAGUG CACUCUCU CUGAUGA X GAA AUCUUCUU

509 AGAGUGAUA GAGCUUGU ACAAGCUC CUGAUGA X GAA AUCACUCU

515 AUAGAGCUU GUACAGAA UUCUGUAC CUGAUGA X GAA AGCUCUAU

518 GAGCUUGUA CAGAAAUA UAUUUCUG CUGAUGA X GAA ACAAGCUC

526 ACAGAAAUA CGGUCCGA UCGGACCG CUGAUGA X GAA AUUUCUGU

531 AAUACGGUC CGAAACGU ACGUUUCG CUGAUGA X GAA ACCGUAUU

540 CGAAACGUU GGUCUGUU AACAGACC CUGAUGA X GAA ACGUUUCG

544 ACGUUGGUC UGUUAUUG CAAUAACA CUGAUGA X GAA ACCAACGU

548 UGGUCUGUU AUUGCCAA UUGGCAAU CUGAUGA X GAA ACAGACCA

562 CAAGCACUU AAAGGGGA UCCCCUUU CUGAUGA X GAA AGUGCUUG

563 AAGCACUUA AAGGGGAG CUCCCCUU CUGAUGA X GAA AAGUGCUU 575 GGGAGAAUU GGAAAACA UGUUUUCC CUGAUGA X GAA AUUCUCCC 588 AACAAUGUA GGGAGAGG CCUCUCCC CUGAUGA X GAA ACAUUGUU 603 GGUGGCAUA ACCACUUG CAAGUGGU CUGAUGA X GAA AUGCCACC 610 UAACCACUU GAAUCCAG CUGGAUUC CUGAUGA X GAA AGUGGUUA nt. Target Secrruence Ribozyme Seguence

Posi¬ tion

615 ACUUGAAUC CAGAAGUU AACUUCUG CUGAUGA X GAA AUUCAAGU

623 CCAGAAGUU AAGAAAAC GUUUUCUU CUGAUGA X GAA ACUUCUGG

624 CAGAAGUUA AGAAAACC GGUUUUCU CUGAUGA X GAA AACUUCUG

634 GAAAACCUC CUGGACAG CUGUCCAG CUGAUGA X GAA AGGUUUUC

659 GACAGAAUU AUUUACCA UGGUAAAU CUGAUGA X GAA AUUCUGUC

660 ACAGAAUUA UUUACCAG CUGGUAAA CUGAUGA X GAA AAUUCUGU

662 AGAAUUAUU UACCAGGC GCCUGGUA CUGAUGA X GAA AUAAUUCU

663 GAAUUAUUU ACCAGGCA UGCCUGGU CUGAUGA X GAA AAUAAUUC

664 AAUUAUUUA CCAGGCAC GUGCCUGG CUGAUGA X GAA AAAUAAUU

704 GCGGAAAUC GCAAAGCU AGCUUUGC CUGAUGA X GAA AUUUCCGC

713 GCAAAGCUA CUGCCUGG CCAGGCAG CUGAUGA X GAA AGCUUUGC

Table XXII: Porcine c-mvb (Region B) Hammerhead Ribozyme and Target Sequence (308 bp; nt. 1386 start; human c-myb numbering system) nt. Target Seguence Ribozyme Seguence

Posi¬ tion 1394 GAUUCUUUC UUAAACAC GUGUUUAA CUGAUGA X GAA AAAGAAUC 1396 UUCUUUCUU AAACACUU AAGUGUUU CUGAUGA X GAA AGAAAGAA 1397 UCUUUCUUA AACACUUC GAAGUGUU CUGAUGA X GAA AAGAAAGA 1404 UAAACACUU CCAAUAAC GUUAUUGG CUGAUGA X GAA AGUGUUUA 1405 AAACACUUC CAAUAACC GGUUAUUG CUGAUGA X GAA AAGUGUUU 1410 CUUCCAAUA ACCAUGAA UUCAUGGU CUGAUGA X GAA AUUGGAAG 1423 UGAAAACUU AGACUUGG CCAAGUCU CUGAUGA X GAA AGUUUUCA 1424 GAAAACUUA GACUUGGA UCCAAGUC CUGAUGA X GAA AAGUUUUC 1429 CUUAGACUU GGAAAUGC GCAUUUCC CUGAUGA X GAA AGUCUAAG 1440 AAAUGCCUU CUUUAACG CGUUAAAG CUGAUGA X GAA AGGCAUUU 1441 AAUGCCUUC UUUAACGU ACGUUAAA CUGAUGA X GAA AAGGCAUU 1443 UGCCUUCUU UAACGUCC GGACGUUA CUGAUGA X GAA AGAAGGCA 1444 GCCUUCUUU AACGUCCA UGGACGUU CUGAUGA X GAA AAGAAGGC 1445 CCUUCUUUA ACGUCCAC GUGGACGU CUGAUGA X GAA AAAGAAGG 1450 UUUAACGUC CACGCCUC GAGGCGUG CUGAUGA X GAA ACGUUAAA

1458 CCACGCCUC UCAGUGGU ACCACUGA CUGAUGA X GAA AGGCGUGG

1460 ACGCCUCUC AGUGGUCA UGACCACU CUGAUGA X GAA AGAGGCGU

1467 UCAGUGGUC ACAAAUUG CAAUUUGU CUGAUGA X GAA ACCACUGA

1474 UCACAAAUU GACUGUUA UAACAGUC CUGAUGA X GAA AUUUGUGA

1481 UUGACUGUU ACAACACC GGUGUUGU CUGAUGA X GAA ACAGUCAA

1482 UGACUGUUA CAACACCA UGGUGUUG CUGAUGA X GAA AACAGUCA

1492 AACACCAUU UCAUAGAG CUCUAUGA CUGAUGA X GAA AUGGUGUU

1493 ACACCAUUU CAUAGAGA UCUCUAUG CUGAUGA X GAA AAUGGUGU

1494 CACCAUUUC AUAGAGAC GUCUCUAU CUGAUGA X GAA AAAUGGUG

1497 CAUUUCAUA GAGACCAG CUGGUCUC CUGAUGA X GAA AUGAAAUG

1530 AGGAAAAUA CAUAUUUU AAAAUAUG CUGAUGA X GAA AUUUUCCU

1534 AAAUACAUA UUUUUGAA UUCAAAAA CUGAUGA X GAA AUGUAUUU

1536 AUACAUAUU UUUGAACU AGUUCAAA CUGAUGA X GAA AUAUGUAU

1537 UACAUAUUU UUGAACUC GAGUUCAA CUGAUGA X GAA AAUAUGUA

1538 ACAUAUUUU UGAACUCC GGAGUUCA CUGAUGA X GAA AAAUAUGU

1539 CAUAUUUUU GAACUCCG CGGAGUUC CUGAUGA X GAA AAAAUAUG

1545 UUUGAACUC CGGCUAUC GAUAGCCG CUGAUGA X GAA AGUUCAAA

1551 CUCCGGCUA UCAAAAGG CCUUUUGA CUGAUGA X GAA AGCCGGAG

1553 CCGGCUAUC AAAAGGUC GACCUUUU CUGAUGA X GAA AUAGCCGG

1561 CAAAAGGUC AAUCCUGG CCAGGAUU CUGAUGA X GAA ACCUUUUG

1565 AGGUCAAUC CUGGAAAG CUUUCCAG CUGAUGA X GAA AUUGACCU

1576 GGAAAGCUC UCCAAGAA UUCUUGGA CUGAUGA X GAA AGCUUUCC

1578 AAAGCUCUC CAAGAACU AGUUCUUG CUGAUGA X GAA AGAGCUUU

1587 CAAGAACUC CUACACCG CGGUGUAG CUGAUGA X GAA AGUUCUUG

1590 GAACUCCUA CACCGUUC GAACGGUG CUGAUGA X GAA AGGAGUUC

1597 UACACCGUU CAAACAUG CAUGUUUG CUGAUGA X GAA ACGGUGUA

1598 ACACCGUUC AAACAUGC GCAUGUUU CUGAUGA X GAA AACGGUGU

1610 CAUGCACUC GCAGCUCA UGAGCUGC CUGAUGA X GAA AGUGCAUG

1617 UCGCAGCUC AAGAAAUU AAUUUCUU CUGAUGA X GAA AGCUGCGA

1625 CAAGAAAUU AAAUAUGG CCAUAUUU CUGAUGA X GAA AUUUCUUG

1626 AAGAAAUUA AAUAUGGU ACCAUAUU CUGAUGA X GAA AAUUUCUU

1630 AAUUAAAUA UGGUCCCC GGGGACCA CUGAUGA X GAA AUUUAAUU

1635 AAUAUGGUC CCCUGAAG CUUCAGGG CUGAUGA X GAA ACCAUAUU

1649 AAGAUGCUA CCUCAGAC GUCUGAGG CUGAUGA X GAA AGCAUCUU

1653 UGCUACCUC AGACACCA UGGUGUCU CUGAUGA X GAA AGGUAGCA

1663 GACACCAUC UCAUUUAG CUAAAUGA CUGAUGA X GAA AUGGUGUC

1665 CACCAUCUC AUUUAGUA UACUAAAU CUGAUGA X GAA AGAUGGUG

1668 CAUCUCAUU UAGUAGAA UUCUACUA CUGAUGA X GAA AUGAGAUG 1669 AUCUCAUUU AGUAGAAG CUUCUACU CUGAUGA X GAA AAUGAGAU

1670 UCUCAUUUA GUAGAAGA UCUUCUAC CUGAUGA X GAA AAAUGAGA 1673 CAUUUAGUA GAAGACCU AGGUCUUC CUGAUGA X GAA ACUAAAUG

Table XXIII: Porcine c-mvb (region A) Hairpin Ribozyme and Target Sequence (266bp; nt. 458 start; Human numbering system)

Posi¬ RZ Substrate tion

528 ACGUUUCG AGAA GUAUUU AAAUACG GUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CGAAACGU

690 UUCCGCCC AGAA GUUCCC GGGAACA GAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GGGCGGAA

Table XXIV: Porcine c-myb (region B) Hairpin Ribozyme and Target Sequence (308 bp; nt. 1386 start; Human numbering system)

Posi¬ Hairpin Ribozyme Substrate tion

1504 UUUUCACA AGAA GGUCUC GAGACCA GAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UGUGAAAA

1594 CAUGUUUG AGAA GUGUAG CUACACC GUU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CAAACAUG

1613 AUUUCUUG AGAA GCGAGU ACUCGCA GCU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CAAGAAAU

Claims

1. An enzymatic nucleic acid molecule which cleaves c-myb RNA, wherein the the binding arms of said nucleic acid contain sequences complementary to the sequences defined in Tables II, XII-XXIV.

2. An enzymatic nucleic acid molecule which cleaves RNA produced from a gene selected from one encoding c-fos, oct-1 , SRF, PDGF receptor, bFGF receptor, angiotensin II, and endothelium-derived relaxing factor.

3. The enzymatic nucleic acid molecule of claims 1 or 2 wherein said nucleic acid molecule is in a hammerhead motif.

4. The enzymatic nucleic acid molecule of claim 1 or 2, wherein said nucleic acid molecule is in a hairpin, hepatitis delta virus, VS nucleic acid, group I intron, or RNAseP nucleic acid motif.

5. The enzymatic nucleic acid molecule of claim 3 or 4, wherein said nucleic acid comprises between 12 and 100 bases complementary to said mRNA.

6. The enzymatic nucleic acid molecule of claim 5, wherein said nucleic acid comprises between 14 and 24 bases complementary to said mRNA.

7. Enzymatic nucleic acid molecule consisting essentially of any sequence selected from the group of sequences listed in Tables III, XII-XXIV.

8. A mammalian cell including an enzymatic nucleic acid molecule of any one of claims 1 or 2.

9. The cell of claim 8, wherein said cell is human cell.

10. An expression vector including nucleic acid encoding an enzymatic nucleic acid molecule or multiple enzymatic molecules of claims 1 or 2 in a manner which allows expression of that enzymatic RNA molecule (s) within a mammalian cell.

0 11. A mammalian cell including an expression vector of claim 10.

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

13. A method for treatment of a stenotic condition by administering to a patient an enzymatic nucleic acid molecule of claims 1 or 2, or an enzymatic nucleic acid molecule which cleaves RNA produced from the gene c-myb.

14. A method for treatment of a stenotic condition by administering to a patient an expression vector of claim 10.

15. The method of claims 13 or 14, wherein said patient is a human.

16. A method for treatment of cancer by administer¬ ing to a patient or a patient's cells an enzymatic nucleic acid molecule of claims 1 or 2.

17. A method for treatment of cancer by administer- ing to a patient or a patient's cells an expression vector of claim 10.

18. The method of claims 16 or 17, wherein said patient is a human.

19. Method for administration of an enzymatic nucleic acid by mixing said nucleic acid with a chemical selected from the group consisting of chloroquine, ammonium chloride, carbonyl cyanide p-trifluoromethoxy phenyl hydrazone (FCCP) , monensin, colchicine, amphipathic peptides, viral proteins, and viral particles.

20. The enzymatic nucleic acid of claim 3, wherein said nucleic acid comprises of at least five ribose residues, and wherein said nucleic acid comprises phosphorothioate linkages at at least three of the six 5' terminal nucleotides, and wherein said nucleic acid comprises a 2' -C-allyl modification at position No. 4 of said nucleic acid, and wherein said nucleic acid comprises at least ten 2'-0-methyl modifications, and wherein said nucleic acid comprises a 3'- end modification.

21. The enzymatic nucleic acid of claim 20, wherein said nucleic acid comprises a 3' -3' linked inverted ribose moeity at said 3' end.

22. The enzymatic nucleic acid of claim 3, wherein said nucleic acid comprises of at least five ribose residues, and wherein said nucleic acid comprises of phosphorothioate linkages at at least three of the six 5' terminal nucleotides, and wherein said nucleic acid comprises a 2'-amino modification at position No. 4 and/or at position No. 7 of said nucleic acid, wherein said nucleic acid comprises at least ten 2'-O-methyl modifica¬ tions, and wherein said nucleic acid comprises a 3' -3' linked inverted ribose or thymidine moeity at its 3' end.

23. The enzymatic nucleic acid of claim 3, wherein said nucleic acid comprises of at least five ribose residues, and wherein said nucleic acid comprises phos¬ phorothioate linkages at at least three of the six 5' terminal nucleotides, and wherein said nucleic acid comprises non-nucleotide substitution at position No. 4 and/or at position No. 7 of said nucleic acid molecule, wherein said nucleic acid comprises at least ten 2'-0- methyl modifications, and wherein said nucleic acid comprises a 3'-3' linked inverted ribose or thymidine moeity at its 3' end.

24. The enzymatic nucleic acid of claim 3, wherein said nucleic acid comprises of at least five ribose resi¬ dues, and wherein said nucleic acid comprises phospho¬ rothioate linkages at at least three of the six 5' terminal nucleotides, and wherein said nucleic acid comprises 6-methyl uridine substitutions at position No. 4 and/or at position No. 7 of the said nucleic acid molecule, wherein said nucleic acid comprises at least ten 2' -O-methyl modifications, and wherein said nucleic acid comprises a 3' -3' linked inverted ribose or thymidine moeity at its 3' end.

25. The enzymatic nucleic acid of claim 3, wherein said nucleic acid comprises of at least five ribose residues, and wherein said nucleic acid comprises phos¬ phorothioate linkages at at least three of thje six 5' terminal nucleotides, wherein said nucleic acid comprises 2' -C-allyl modification at position No. 4 of the said nucleic acid, wherein said nucleic acid comprises at least ten 2' -O-methyl modifications, and wherein said nucleic acid comprises a 2'-3' linked inverted ribose or thymidine moeity at its 3' end.

26. Oligonucleotide having complementarity to c-myb at at least 5 contiguous bases comprising a 2' -5' -linked adenylate residue having a 5' -phosphate.

27. The oligonucleotide of claim 26, having enzymatic activity on c-myb RNA.

28. The oligonucleotide of claim 26, comprising at least 20 bases able to form a hybrid with c-myb RNA.