CA2190513A1 - Methods and compositions for treatment of restenosis and cancer using ribozymes - Google Patents

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

WO 95131541 2 1 9 5 1 3 1 ~1/~
.

DESCRIPTION
Me~hn~ and Coml)ositions for Treatment of Restenosis and Cancer ~sinq Ribozvmes .3ackqround Of The Invention The present invention r~ln~Prn~ 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 uot an admission that any of the work described below is prior art to the claimed invention.
Coronary angioplasty is one of the major surgical Ll~- ~ for heart disease. Its use has been accelerat-ing rapidly; over 450, 000 procedures are performed in the IJ. S . annually . The short term success rate of angioplasty is 80 to 90g6. IIowever, in spite of a number of technical ,v~ q 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 signif icant restenosis within 6 months of the procedure. The rate of restenosis is even higher (50 to 60g6) in patients who have undergone multivessel angioplasty (Califf, R. M., et al., 1990, in ~e~hool~ of Interventional Cardioloal~., E.J.
Topol, ed., W. B. Saunders, Philadelphia, pp 363-394 . ) .
~istopathological 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. A~n. Coll. Cardio., 17, 442-428;
Austin, G. E., et al., 1985, J . Ann Coll. Cardiol., 6, 369-375). Smooth muscle cell proliferation could be an overly robust response to injury. Alternatively, the W095/31541 21 935l 3 I~I;U~ C-7~ ~

intimal smooth muscle cells within atherosclerotic lesigns 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-5 strated a positive correlation between the presence ofactivated smooth muscle cells in coronary lesions and the extent of subsequent luminal narrowing af ter atherectomy (Simons, M., et al., 1993, New Enql. J. Med., 328, 608-613) . In any case, slowing smooth muscle cell :prolifera-10 tion after angioplasty could prevent intimal thickeningand restenosis.
The presently preferred therapeutic treatment for restenosis is the use of streptokinase, urokinase or other thrombolytic compounds, such as ~ish oil, anticoagulants, 15 ACE (angiotensin converting enzyme) inhibitors, aspirin and cholesterol lowering compounds. Alternative treatment includes the surgical i~cQrporation of ~nrir~ min~l stents.
The occurrence of pharmacologic side-effects (particularly bleeding disorders a3sociated with anti-coagulants and 20 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. (Circula~ign, 84, 1426-1436, 1991) .
Recently, the results of a clinical trial of the 25 ef f icacy 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, gpIIb/gpIIIa. After six months, patients with~the 30 treatment showed a 23~ reduction in the occurre~ce of restenosis than patients receiving placebo (27 vs. 359~;
p=O . 001) .
A number oi growth factors have been shown to induce smooth muscle cell proliferation. Irl vitro, platelet-35 derived growth factor (PDGF) is a potent smooth musclecell mitogen (Ross, R ., et aI ., 1974, Proc . Na tl . Acad .
~7ci. USA, 71, 1207-1210) and a smooth muscle cell chemo-~ WO 9S~31541 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 af ter arterial injury (Ferns, G. A. A., et al., 1991, Science, 253, 1129-1132). Analysis of 3H-thymidine incorporation in the lesiQns indicates that the anti - PDGF antibodies primarily inhibit smooth muscle cell migration.
Basic fibroblast growth factor (bFGF) is another smooth muscle cell mitogen in vitro ~Klagsbrun, M. and Edelman, E. R., 1989, Arteriosclero8is, 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., l991, Proc. Natl. Acad. Sci. USA, 88, 3739-3743) . In ;~lA; ~ n to bFGF, heparin binding epidermal growth factor (HB-EGF) (~igashiyama, S., et al., 1991, Science, 251, 936-539. ), insulin-like growth factor I (IGF-I) (Banskota, N. K., et al., 1989, Molec. Endocrinol., 3, 1183-1190) and endothelin (Komuro, I., et al., 1988, FEBS ~etters, 238, 249-252) have been shown to induce smooth muscle cell pro-liferation. A ~umber of other factors (such as inter-leukin-1 and tumor necrosis factor-~) may indirectly affect smooth muscle cell proliferation by inducing the expression of PDGF (Haj jar, K. A., et al., 1987, J. Exc.
Med., 166, 235-245; Raines, E. W., et al., 1989, Science, 243, 393-396).
3 o When whole serum is added to serum- starved smooth muscle cells in vitro, the oncogenes, c-myc, c-l'os, and c-myb, are induced (Kindy, M. S. and ~nn~nFIhf~;n~ G. E., 1986, J. Biol. Chem., 261, 12865-12868; Brown, K. E., et al., 1992, J. 3iol. Chem., 267, 4625-4Ç30) 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-Wo 95131541 "') ~ ~

2~90513 4630. ) . Thus, c-myb is re(luired for the Gl to S transition after stimulation by the multitude of growth factors present in serum. In vivo, a c-myb antisense oligonucleo-tide inhibits restenosis when applied to rat arteries 5 after balloon angioplasty (Simons, M., et al., 1992, ~, 359, 67-70). Similarly, an antisen~e oligonucleo-tide ~i rf~ct~tl against mRNA of the oncogene e-mye was shown to inhibit human smooth muscle cell proliferation (Shi, Y., et al., 1993, Cireulatisn, 88, 1190-5) and migration (Biro, S., et al, 1993, Proe. Natl. Aead. Sei. ~I S A, 90, 654-8) .
Ohno et al., 1994 Seie~ee 265, 781, have shown that a cor.'bination of viral thymidine kinase enzyme expression (gene therapy) and treatment with anti-viral drug ganci-15 clovir i~hibits smooth muscle cell proliferation in pigb,following baloon angioplasty.
Epstein et al., "Inhibition of non-transformed cell proliferation using antisense oligonucleotides, " NTIS
publication 1992 discusses use of antisense oligonucleo-20 tides to c-mye, PCNA or cyclin B. Fung et al., PCT
WO91/15580, describes gene therapy for cell proliferative disease and r~ n~ administration of a ribozyme con-struct against a PGR element. Mention is made of inacti-vation of c-myb. Rosenberg et=al., W093/08845, Calabretta 25 et al., W092/20348 and Gewirtz W093/09789 concern c-myb antisense oligonucleotides for treatment of ~ n~ or colorectal cancer, and administratio~ locally. Sytkowski, PCT WO 93/02654, describe the uses of antisense oligo-mlcl.ont;~ to inhibit e-myb gene e~pression in red blood 30 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 W0 95/31~41 ~ r ~ 21905l3 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 fl~nk;ng 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 protei~s 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 agai~st c-myb RNA
to treat stenosis.
C ry of The Invention This invention relates to ribozymes, or enzymatic RNA
molecules, directed to cleave mRNA species that are required for n~ r 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 cell3 in numerous cancers. To block restenosis, a target molecule required for the inflllctinn 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- KB protein activates cellular transcription and induces increases in cellular synthetic pathways. In a resting cell, this protein is found in the cytoplasm, ~ _ lP~fl with its inhibitor, I-KB. Upon rhnsrhnrylation of the I-KB l~mll.-, the complex dissociates and NF-KB is released for tr~n~port to the nucleus, where it binds DNA

Wo 95~31541 P~
2 1 9~:~ 1 3 and induces transcriptional activity in (NF-KB)-responsive genes. One of the ~NF-KB~-responsive gënes is the NF-KB
gene itself. Thus, release of the NF-KB protein from the inhibitory complex results in a cascade of gene expression 5 which is auto-induced. Early inhibition of NF-KB can reduce expression of a number of genes re~uired ~or growth and proliferation, such as c-myb.
Two other transcription factors, oct-l and serum response factor (SRF~ have been shown to be expressed 10 selectively in dividing cells. Both oct-l and SRF are expressed ubi~auitously in cultured cells, including smooth muscle cells. IIowever, R. Majack and his colleagues have recently shown that these transcription factors are not expressed by the smooth muscle cells in intact vessels.
15 Both oct-l and SRF are rapidly expressed upon dispersal of tissue into single cell suspensions. Thus, these tran-scription f actors are thought to be regulated by their interactions with the extr~cr~ r matrix (Weiser, M. C.
M., et al., 1994, .J. Cell. Biochem., S18A, 282; Belknap, 20 J. K., et al., 1994, ,J. Cell. Biochem., S18A, 277). Upon injury during ~n~ pl ~Rty, the expression of oct-l and SRF
may be ~nh~nre~, leading to increased smooth muscle cell prolif eration . Treatment with ribozymes that block the expression of these transcription factors can alleviate 2~ 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 re~auired ~or smooth 30 muscle cell proliferation, enzymatic ~NAs, 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-35 cules than antisense oligonucleotides. In the presentinvention, ribozymes that cleave c-myb mRNA are= described.
Le-JveL, applicant shows that these ribozymes are able to WO 95131541 1 ~ . 'C '"' 2lqasl~

inhibit smooth muscle cell proliferation and that the catalytic activity of the ribozymes is required f or their inhibitory effect. From those of ordinary skill in the art, it is clear from the examples described, that other 5 ribozymes that cleave target mRNAE required for smooth muscle cell proliferation may be readily designed and are within the invention_ By "inhibit" is meant that the activity of c-myb or level of mRNAs encoded by c-myb is re uced below that 10 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 b~nd to the same site on the mRNA, but unable to cleave that R~A.
By "enzymatic nucleic acid molecule" it is meant a nucleic acid molecule which has complementarity in a sub-strate binding region to a Rp~;f;~l gene target, and also has an enzymatic activity which is active to specifically cleave RNA in that target. That is, the enzymatic nucleic 2 o acid molecule is able to intermolecularly cleave RNA and thereby inactivate a target RNA molecule. This complemen-tarity functions to allow sufficie~lt hybridization of the t;C nucleic acid l.oc~ to the target RNA to allow the cleavage to occur. One hundred percent complemen-25 tarity is preferred, but complementarity as low as 50-759~
may also be useful in this invention. By "equivalent" RNA
to c-myb is meant to include those naturally occurring RNA
molecules Aq~or;~ter~ with r~t~n~ and cancer in various animals, including human, rat and pig. Such a molecule 3 0 will ge~erally 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 .
gy ll~ mrl Arity" is meant a nucleic acid that can 35 form hydroge~ bond(s) with other R~A sequence by either trArl;t;cm~l Watson-CriCk or other non-traditional types (for example, ~Ioogsteen type) of base-paired interactions.

W0 95/31541 2 1 9 0 ~ 1 3 ~ Il'J s t -?'~.
Si~c basic varieties Qf naturally-occurring enzymatic RNAs are known prese~tly. 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 f irst 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 re~eased from that R~A to search for arother 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 tr~nRl~t;-~n) since the concentra-tion of ribozyme ni,c.-c~ry to affect a therapeutic tr~at-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 - -hAnl flm of binding to the target RNA, but also on the -h~n; ~m of target RNA cleavage. Single mis-matches, or base-substitutions, near the site o~ cleavage can completely f'1 ;m;n~tP catalytic activity of a ribozyme.
Similar m; r~n ~t(~hf~S in antisense molecules do not preYent their action (Woolf, T. M., et al., 1992, Proc. Natl.
Ar~, SCi. USA~, 89, 7305-7309). Thus, the=specificity of WO9~/31541 I~ 7~n ~ ~90513 action of a ribozyme is greater than that of an antisense oligonucleotide binding the same RNA site.
In preferred embodimentæ of this invention, the t;f~ nucleic acid molecule is formed in a hammerhead 5 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 Neuro~pora VS
RNA. Examples of such h: -rhl~iqtl motifs are described by Rossi et al., 1992, Aids Research and J~uman Retroviru3e~
8, 183, of hairpin motifs by Hampel et al., EP03602~7, ~ampel 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 Biochemi6try 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, l990 Cell 61, 685-696; Saville and Collins, l991 Proc. Natl. Acad. Sci. USA 88, 8826-8830; Collins and Olive, 1993 Biochemi~try 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 specif ic substrate binding site which is complementary to one or more of the target gene RNA reglons, and that it have nucleotide sequences within or ~u- . . ..."l in~ that substrate binding site which impart an RNA cleaving activity to the molecule.
In a preferred '~ nt the invention provides a 30 method for producing a class of enzymatic cleaving agents which exhibit a high degree of specif icity f or the RNA of a desired target. The enzymatic nucleic acid molecule is - pref erably targeted to a highly conserved sequence region of a target mRNAs encoding c-myb proteins such that 35 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 Wo 95/31541 r~"~ ~ '7' 219~51 exogenously to specific cells as required. Alternatively, the ribozymes can be expressed from DNA/RNA vectors ~that are delivered to specif ic celIs .
Synthesis of nucleic acids greater than 100 nucleo-5 tides in length is dif f icult using automated methods, andthe therapeutic cost of such molecules is prohibitive. In this invention, small e~2ymatic nucleic acid motifs (e.g., of the h -rhP~l or the hairpin structure) are used for exogenous delivery. The simple structure of tkese mole-10 cules increases the ability of ~ the enzymatic nucleic acidto 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 AntiErense Res. I~ev., 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 ri:bozymes can be ~ ntpd by their release from the primary transcript by a second ribozyme (Draper et al., PCT W093/23569, and Sullivan et al., PCT
W094/02595, both hereby incorporated in their totality by reference herein; Ohkawa et al., 1992 Nucleic Acids S~m~.
~, 27, 15-6; Taira et al., 1991, Nucleic Acids Res., 19, 5125-30; Ventura et al., 1993 ~ucleic Acids Res., 21, 3249-55; Chowrira et al., 1994 ~. 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 molesules also contain domains that catalyze the cleavage of RNA. The RNA mole-cules are preferably ribozymes of the hammerhead or WO 95/31541 1 ~1;~.. ' '' ~ 2~90513 hairpin motif. Upon binding, the ribozymes cleave the target mRNAs, preventing tr~nRl~t;nn and protein accumula-tion. In ~ the absence of the expression of the target gene, cell proliferation is inhibited.
In a pref erred 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-l, SRF, NF-KB, PDGF
receptor, bFGF receptor,~ angiotensin II, and endothelium-derived relaxing f actor 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 q.~ or n~nrl; t; nn~ 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 r~ t;nn in the level of protein activity will relieve to some extent the symptoms o~ the disease or ~nn~;t;nn.
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, WO 95131541 . ~l,l C~~'^
21~351s 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 5 expressed from transcription units inserted into D~A or R~A vectors The recombinant~ vector~ are preferably DNA
plasmids or viral vectors. Ribozyme expressing viral vectors could be constructed based on, but not limited to, adeno-a~sociated virus, retrovirus, adenovirus, or alpha-10 virus~ Preferably, the recombinant vectors capable ofexpressing 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 15 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 20 the patient, or by any other means that would allow for intro~ rtirn 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 25 arms which are complementary to the sequences in the tables Il, XII-XXIV. Examples of such ribozymes are shown as Seri. 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 30 or porcine mRNA target. Examples of such ribozymes con-sist essentially of seriuences defined in tables III, XII-XXIV. By "consists essentially of " is meant that the active ribozyme contains an ~~ i r center eriuivalent to those in the examples, and binding arms able to bind c-myb 35 mRNA such~that cleavage at the target site occurs. other ~e,quences may be present which do not interfer~with such cleavage .

WO 95131~4~
~ 2193513 In another aspect of the ;nvl~nt;~n, ribozymes that cleave target molecules and inhibit cell prolif eration are expressed from transcription units inserted into DNA, RNA, or viral ue~ors. Preferably, the recombinant vectors 5 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 prol; ~f r;lt; on of their host cells. The recombinant vectors are preferably DNA plas-10 mids or adenovirus vectors . However, ~other, l; ~n cellvectors 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-15 ferred embodiments thereof, and from the claimæ.
De8cri~tion Of The Preferred Fmbodiments The drawings will f irst brief ly be described.
2 o ~Drawinqs:
Figure 1 is a diagrammatic representation of the - ~ ribozyme domain known in the art. Stem II can be 2 2 base-pair lo~g.
Figure 2a is a diayL t;c repr~Rf~nt;lt;on of the 25 ~ 1 ribozyme domain known in the art; Figure 2b is a diagrammatic representation of the h rhf~;~rl ribozyme as divided by Uhlenbeck (1987, Nature, 327, 596-600) into a substrate and enzyme portion; Figure 2c is a similar diagram showing the h: ~rhPp~ divided by Haseloff and 30 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. AcidR. Re~., 17, 1371-1371) illto two portions.
Figure 3 is a diagrammatic representation of the 35 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 optio~ally provided of le~gth 2 or wo 9S/315~1 2 1 9 0 5 1 3 r~l;u sr ~ ~

more bases (preferably 3 - 20 baæes, ~.e., m iG from 1 -20 or more). Helix 2 an~ ~elix 5 may be covalently linked by one or more bases ~i.e., r i8 2 1 base). Helix 1, 4 or ~ may also be extended by 2 or more base pairs ( e . g ., 4 5 20 base pairs) to stabilize the ribo2yme structure, and preferably is a protein binding site. In each instance, each N and N' ;n~lrrPn~on~1y is any normal or modified base and each dash represents a potential base-pairing inter-action. These nucleotides may be modi~ied at the sugar, 10 ba~e or phosphate. Complete base-pairing is not rerluired in the helices, but is pref erred . Helix l and 4 can be of any size (i.e., o and p is each in~l~r.on~n~ly ~rom 0 to any number, e.g., 20) as long as some base-pairing is maintained. Essential bases are shown as specific bases 15 in the structure, but those in the art will recognize that one or more may be modified rhPm;r~1ly (abasic, base, sugar and/or phosphate .modiEications) or replaced with another base without significant effect. Helix 4 can be f ormed f rom two separate m~olecules, i . e ., without a con-2 0 necting loop . The connecti~g loop when present may be arih~n1lrle~tide with or without modifications to ita base, sugar or phosphate. "~" is 2 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 25 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 3~ of the self-cleaving VS RNA ribozyme domain.
Figure 6 is a schematic representation of an RNAseH
aCcoRs;hll;ty assay. Specifically, the left side of Figure 6 is a diagram of complementary DNA origonucleo-tides bound to accessible sites on the target RNA.
35 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 WO 9S/31541 r~ r 21qO513 lS
Figure 6: is a schematic of a gel separation of uncut target ~NA from a cleaved target RNA. Detection o target RNA is by autoradiography of body-labeled, T7 transcript.
The bands common to each lane represent uncleaved target 5 RNA; the bands unirlue to each lane represent the cleaved products .
Figure 7 is a graph of the results of an R~AseX
=rr~ ;h;l;ty assay of murine c-myb RNA. On the abscissa is the se~uence number of the D~A oligonucleotide that is 10 homologous to the ribozyme target site. The ordinate represents the percentage of the intact transcript that was cleaved by RNAse X.
Figure 8 is a graph of the outcome of an RNAseX
acc~ ; h; l; ty assay of human c-myb mRNA. The graphs are 15 labeled as in Figure 7.
Figure 9 shows the effect of rh-~m;r~l r~~~;f;r~t;ons on the catalytic activity of h rh~ ribozyme targeted to c-myb site 575. A) diagrammatic representation of 575 ribozyme-substrate complex. 2'-O-methyl ribo-20 zyme represents a hammerhead (XX) ribozyme r~nt=;n;n~ 2'-O-methyl substitutions at five nucleotides in the 5' and 3 ' termini . 2 ' -O-methyl P=S ribozyme represents a hammer-head (XX) ribozyme rt~ntA;n;n~ 2'-O-methyl and phosphoro-thioate substitutions at f ive nucleotides in the 5 ' and 3 ' 25 termini. 2'-C-allyl iT ribozyme represents a hammerhead rrnt=;n;nS ribose residues at five positions. The remain-ing 31 nucleotide positions contain 2'-hydroxyl group substitutions, wherein 30 nucleotides contain 2'-O-methyl substitutions and one nucleotide ~U4) r~nt~; n~ 2 ' -C-allyl 30 suhgt;t~t;~n 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 35 3 ' -end inverted T modification. B) shows the ability of ribozymes described in Fig. 9A to inhibit smooth muscle cell prolif eration .

Wo 95/31541 r~ c '' Figure 10 shows the e~fect of ~2'-C-allyl P=S 575 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 5 inactive ribozyme) as a function of ribozyme concentration is shown .
Figure ll shows a comparison of the effects of 2 ' -C-allyl P=S 575 H~I ribozyme and phosphorothioate antisense D~A on the proliferation of smooth muscle cells.
Figure 12 shows the inhibition of 3mooth muscle cell proliferation catalyzed by 2 ' -C-allyl P=S H~ ribozymes targeted to sites 549, 575, and 1533 within c-myb rnRNA.
Figure 13 shows the efect 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 2 o 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 f ive 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 f ive 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) show3 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 prolif erat ion .
Figure 15 shows the effect of varying the length of substrate binding arm of 575 H~ ribozyme on the inhibition of smooth muscle cell prolii~eration. - -WO 95/31~41 r~ C ~''~

Figure 16 shows the~ effect of various chemical modi-f; ~ t i ~nq, at U4 and/or U7 positions within 575 H~ ribozyme core, on the ability of the ribozyme to inhibit smooth muscle cell proliferation.
Figure 17 shows the inhibition of pig smooth muscle cell pr~ r~t i nn by active c-myb 575 HH ribozyme .
Figure 18 shows the inhibition of human smooth muscle cell proliferation by active c-myb 575 H~ ribozyme.
Figure l9 shows ribozyme-mediated inhibition of c-myb expression and cell proliferation.
Figure 20 is diyL t i c representation of an optimal c-myb ~H ribozyme that can be used to treat diseases like restenosis .
Figure 21 shows the inhibition of Rat smooth muscle cells by 2-5A ~t~nt~; nl ng nucleic acids .
Tarqet sites Targets for use~ul ribozymes can be determined as disclosed in Draper et al sul~ra. Sullivan et al., suDra, as well as by Draper et al., "Method and reagent for treatment of arthritic conditions PCT No. PCT/US94/13129, IJ.S.S.N. 08/152,487, filed 11/12,/93, and hereby incor-porated by reference herein in totality. Rather than repeat the quidance provided in those ~ here, below are provided specific examples of such methods, not limiting to those in the art. Ribozymes to such targets are designed as described in those applications and synthesized to be tested in vitro and in vivo, as also described. Such ribo2ymes 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.

Wo 95~31541 r~
21qO~t3 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 r~bo-zyme cleavage 8ites were identified These sites are 5 shown in Tables II and XII-XXIV (All ~equences 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 sequence8 can be screened and ribozymes thereafter designed, the lO human targeted sequences are of most utility. However, murine and pig targeted ribozymes may be useful to test ef f icacy 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 15 of ribo2yme.
~ T. rhf~ or hairpin ribozymes were de~igned that could bind and were individually analyzed by computer folding (Jaeger et ~7., 1989 Proc. Natl. Acad. Sci. USA, 86, 7706) to assess whether the ribozyme sequences fold 20 into the appropriate secondary structure. Those ribozyme~
with unfavorable intramolecular interactions between the binding arms and the catalytic core are eliminated from consideration. Varying binding arm lerlgths can be chosen to optimize actiyity. Generally, at least 5 bases on each 25 arm are able to bind to, or otherwise interact with, the target R~. ~ ~
The sequences of the ribozymes that are chemically 8ynthf~ e~, useful in this study, are shown in Table III
and XII-XXIV. Those in the art will recognize that these 30 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 1 -rho;~tl ribozymes li8ted in Table III (5'-GGCCGA~AGGCC-3') can be altered 35 (sub8titution, deletion, andlcr insertion) to contain any sequences provided a minimum of two base-paired 8tem structure can form. Similarly, stem-loop IV sequence of ~ WO 95/31541 1 ~ Sl'~'~
21905~3 hairpin ribozymes listed in Table III, XIII, XVI, XIX, XX, XXIII, XXIV (5' -CACGWGUG-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 I:~I and XII-XXIV may be formed of r;hrn-lrleotides or other nucleo-tides or non-nucleotides. Such ribozymes are equivalent to the ribozymes described specif ically in the Tables .
O~t;m; 7inq ~;hrzYme 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 Firure 2c), or chemically synthesizing ribozymes with modifica-tions that prevent their degradation by serum ribo-nucleases (see e.g., ~ckstein et al., International Publication No. WO 92/07065; Perrault et al., l990 Nature 344, 565; Pieken et al, 1991 Science 253, 314; Usman and Ct:de:L~L~:u, 19 92 Trends in ~3ioc~em . Sci . 17 , 334; Usman et al., International Publication No. WO 93/15187; and Rossi et al., Tnt~rn~t;rn~l Publication ~o. WO 91/03162, as well as Usman, N. et al . US Patent ~pl; r~t; nn 07/829, 729, and Sproat, US Patent No. 5, 334, 711 which describe various chemical modif ications that can be made to the sugar moieties of enzymatic R~A 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 f or 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 h; r~h~cive m1crospheres. For some indications, ribozymes Wo 95131541 r~ ?~
2~05t3 may be directly delivered ex vivo to cells or tissues with or without the afuL tioned vehicles. Alternatively, the RNA/vehicle combination 18 locally delivered by direct injection or by use of a catheter, infusio~ pump or stent.
5 Other routes of delivery include, but are not limited to, intravaacular, intramuscular, subcutaneous or joint injec-tion, aerosol ; nh~l ~t j on, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intra-thecal delivery. More detailed descriptions of ribozyme 10 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-15 encoding ser~uences into a D~A or R~IA expressiQn vector.Transcription of the ribozyme ser~uences are driven f rom a promoter for eukaryotic RNA polymerase I (pol I), RNA
polymeraae II (pol II), or R~A polymeraae III (pol III).
Tranacripta from pol II or pol III promo~ers will be 2 o 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 ser~uenceg (onh~nror8, ,q; 1 onrorf~, etc . ) present nearby. Prokaryotic RNA polymer-ase promoters are also uaed, providing that the prokary-25 otic RNA polymeraae enzyme ia expressed in the appropriatecel~s (Elroy-Stein and Moss, 1990 Proc. Natl. Acad. Sci.
U S A, 87, 6743-7; Gao and ~uang 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) .
3 0 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., lg92 Proc. Natl. Acad.
Sci. U S A, 89, 10802-6; Chen et al., 1992 Nucleic Acids 35 Res., 20, 4581-9; Yu et al., 1993 Proc. Natl. Acad. Sci.
U s A, 90, 6340-4; L'~uillier et al., 1992 EMBO J. 11, 4411-8 ; Lisziewicz et al ., 1993 Pro.c. Natl . Acad. Sci .

WO 95/31~41 r~."~ c -2l9~513 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 pre~erred embodiment of the invention, a tran-scription unit expressing a ribo2yme 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 ~LL 75, 207;
Carter, 1992 ~ rr. Ol~i. 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 rer~ ' inilnt adenovirus or AAU particles are locally administered to the site of treatment, e.q., through ;nrllh~t;nn or ;nh~ t;nn in vivo or by direct application to cells or tissues ex vivo.
In another preferred : ' - '; , the ribozyme is administered to the site of c-myb expression (e.g., smooth muscle cells) in an appropriate 1 ;ros,: l vesicle.
F: le8 Abilitv Of ExoqenouslY-Delivered RibozYmes Directed Aqainst c-mYb To Inhibit Vascular Smooth Muscle Cell Proli~eration 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 ~or cell division. Also provided is a description of how such ribozymes may be delivered to smooth muscle cells. The examples demonstrate that upon deliYery, the ribozymes inhibit cell proliferation in culture. Moreover, no Wo 95~3l54l ~ ;,J ,~ ,, 2 1 9~5 1 3 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 divisign assay used represents a 5 model system for smooth muscle cell hyperproliferatioll in restenotic lesions.
F - le 1: Identificatio~ of pgt~-nt;~l Ribozyme Cleavaqe ~ites in E~uman c-mvb 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 f orm secondary f olding structures and cr~nt~;n~-l potential hammerhead ribo2yme cleavage sites were ; ~f~nt ' f; ~cl . These sites are shown in 15 Table II and XII-XXIV Sites are numbered using the sequence numbers from (Westin, E. H., et al., 1990, Oncoqene. 5, 1117-1124) (Gen~3ank P~-c~ ;r7n No. X52125);
the sequence is derived f rom a longer c-myb cDNA isolate and thus is more representative of the full-length RNA.
E~ 21e 2: Selection of Rl h~zYme Cleavaqe Sites in Murine ;3n~ Human c-mYb mRNA.
To test whether the sites predicted by the computer-based RNA folding algorithm corresponded to accessIble 25 sites in c-myb RNA, 41 hammerhead sites were selected for analysis. Ribo2yme target sites were chosen by comparing cDNA sequences o~ mouse and human c-myb (Gen3ank Accession No. X02774 and Gen3ank Accession No. X52125, respectively) and prioritizing the sites on the basis of overall nucleo-30 tide sequence homology. TT. ~ ribozymes were n--~ that could bind each target (see Figure 2C) and were individually analyzed by computer folding ~Jaeger, J.
A., et al, 1989, ~roc. Natl. Acad. Sci. USA, 86, 7706-7710) to assess whether the ribozyme sequences fold into 35 the appropriate secondary structure. Those ribozymes with unfavorable intramolecular interactior,s between the binding arms and the catalytic core were eliminated from WO9~/31541 r~ c~c--~
2~9~513 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.
F le 3: Screeninq Ribozvme Cleavaqe Sites bv RNaseH
Protection Murine and human mRNA was screened for accessible cleavage æites by the method descrioed generally in Draper et al., International PCT publication W0 93/23569, hereby incorporated by reference herein. Briefly, DNA oligo-nucleotides representing 41 potential hammerhead ribozyme cleavage sites were synth~;7~d. A polymerase chain reac-tion was used to generate a substrate f or T7 RNA
polymerase transcription from human or murine c-myb cDNA
clones. Labeled RNA transcripts were synthesized in vitro 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. RP~-t;nnR were stopped and RNA separated on se~uencing ~olyacrylamide 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, 2 0 ' rl~ ribozyme sites were chosen as the most accessible (see Table III) .
E:xam~le 4: Chemical Svnthesis and Purification of Ribozvmes for Efficient Cleavaqe of c-mYb RNA
3 0 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 nn~ described above. The ribozymes were chemically synthesized. The method of synthesis used followed the procedure ~o~ normal RNA synthesis as described in Usman et al., 1987 ~T. Am. Chem. Soc., 109, 7845 and in Scaringe et al, 1990 Nucleic Acids Res., 18, 5433 and made use of WO 95131541 ~ C ~
2~90~3 - ~

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 5 a U for Gs and a U for A14 (numbering from Hertel et 1., 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-10 sized from DNA templates using bacteriophage T7 RNApolymerase (Milligan and TThl~nhsr-k, 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, Z'-15 O-methyl, 2'-H (for a review see Usman and Cedergren, 1992 TI35 17, 34). Ribozymes were purified by gel electro-phoresis using general methods or were purif ied by high pressure liquid chromatography (HPLC; See bsman et al., Synthesis, deprotection, analysis and purification of RNA
20 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 se~uences of the chemically synthesized ribozymes used in this study are shown below in Table III.
E~ le 5: Ribgzyme Cleavaqe of Lona Substrate RNA
CorresT~ondinq to c-mYb mRNA Tarqet T~ l-type ribozymes which were targeted to the murine c-myb mRNA were designed and synthesized to test 30 the cleavage activity at the 20 most accessible sites in in vi tro transcripts of both mouse and human c-myb RNAs .
The target se~uences and the nucleotide location within the c-myb mRNA are given i~ -Table =II~= All hammerhead ribozymes were synthP~l z-~d with binding arm ~Stems I and 35 III; see Figure 2~) lengths of seven nucleotides. Two hairpin ribozymes were synthesized to sites 1632 and 2231.
The relative abilities of these ribozymes to cleave both WO 95131541 r~ 5'~ '' 21~0513 murine and human RNAs is summarized in Table II.
Ribozymes (l /lM) were incubated with 32P-labeled substrate RNA (prepared as described in Example 3, approximately 20 nM) for 60 minutes at 37C using buffers described previ-5 ously. Intact RNA and cleavage products were separated byelectrophoresis 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 h: -rh~ 1 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 15 against sites 551, 634, 936, 1082, -1597, 1721, 1724, 1895, and 1943) were ;n~ t~ in activity, cleaving ~ 50~6 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 20 h: rh~ l ribozymes and one hairpin ribozyme showed low activity on at least one of the substrates. The observed differences in ;IC'C~R~;h; l; ty between the two species of c-myb RNA demonstrate the sensitivity of ribozyme action to RNA ~iLLu~:LuLe and suggest that even when homologous target 25 sequences exist, ribozymes may be excluded from cleaving that RNA by structural constraints. This level of 3pecificity minimizes non-specific toxicity of ribozymes within cells.
3 0 ~xam~le 6: Abilitv of Hammerhead Ribozvmes 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 35 isolated and cultured as follows. Aortas from adult 5prague-Dawley rats were dissected, r~nnf~ ; ve tissue was removed under a dissecting microscope, and 1 mm2 pieces of Wo 95/31541 r~

the vessel were placed, intimal side up, in a Petri dish in Modified Eagle's Medium (MEM) with the following additives: 109~ FBS, 296 tryptose phosphate broth, 1~6 penicillin/streptomycin and 2 mM L-Glutamine. The 3mooth 5 muscle cells were allowed to migrate and grow to conflu-ence over a 3-4 week ~eriod. These primary cells were frozen and subsequent passages were grown at 37~ C in 5 CO2 in Dulbecco's modified Eagle' 9 medium (DMEM), 10~ fetal bovine serum (FBS), and the following additives: 2 mM L-10 ~ tAmin.-, 196 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 15 prepared by coating the wells with 0 . 2~6 gelatin and washing once with phosphate-buffered saline (PBS). RASMC
were inoculated at lx104 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.
20 The cells were serum-starved by removing the medium, washing once with PBS, and incubating 48-72 hours in DMEM
-.,ntA;n;n~ 0.596 FBS plus additives.
In several other systems, cationic lipids have been shown to enhance the bioavA; l Ah; l; ty of oligonucleotides 25 to cells in culture (Bennet , C . F ., et al ., 1992 , Mol .
ph;~ coloqy, 41, 1023-1033). In many of the following experiments, ribozymes were complexed with cationic lipids. The cationic lipid, Lipofectamine (a 3:1 (w/w) f~ llAtif-n of DOSPA (2,3-dioleyloxy-N- [2 (sperminecarbox-30 amido)ethyl]-N,N-dimethyl-l-propAnAm;n;um trifluoroace-tate) and dioleoyl p~osphatidylethanolamine (DOPE) ), was purchased from Life Technologies, Inc. DMRIE (N- [1- (2,3-ditetradecyloxy) propyl] -N, N-dimethyl -N-hydroxyethyl -ammonium bromide) was obtained from VICAL. DMRIE was35 resuspended in CHCl3 and mixed at a 1:1 molar ratio with dioleoyl phosphatidylethanolamine (DOPE). The CHCl3 was evaporated, the lipid was resuspended in water, vortexed ~ WO95131541 2 1 ~ ~ 5 ~ 3 r~ l;l r for 1 minute and bath sonicated ior 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 20-5 25C), cationic lipid was added to the final desiredconcentration and the solution was vortexed briefly. RNA
oligonucleotides were added to the f inal desired concentration and the solution was again vortexed brief ly and 1n~-1lh~t~cl for 10 minutes at room temperature. In dose 10 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; nf llhated for ;~ hours at 37C. The medium was then 15 removed and DMEM c~ t;~;n;n~ 1096 FBS, additives and 10 ~M
bromodeoxyuridine (BrdU) was added. In some wells, FBS
was omitted to determine the b~ ; ne of unstimulated proliferation. The plates were incubated at 37C for 20-24 hours, fixed with 0.3~ H202 in 10096 methanol, and 20 stained for BrdU incorporation by standard methods. In this pLu~eduLt:, cells that have proliferated and inuu.~u- ~ted BrdU stain brown; non-proliferating cells are counter-stained a light purple. Both BrdlJ positive and BrdlJ negative cells were counted under the microscope.
25 300-600 total cells per well were counted. In the following experiments, the percentage of the total cells that have incorporated BrdU ~9~ cell proliferation) is presented. Errors represent the range of duplicate wells.
Percent inhibition then is calculated from the % cell 30 proliferation values as follows: ~ inhibition = 100 -100 ( (Ribozyme - 0~6 serum) / (Control - Q~ serum) ) .
Six hammerhead ribozymes, including the best five ribozymes from the in vitro RNA cleavage test (directed against sites 549, 575, 1553, 1598, and 1635) and one with 35 ;n~ te cleavage levels (directed against site 1597) and their catalytically inactive controls were synthesized and purif ied as described above . The ribozymes were Wo95/31541 2 1 9 a 5 1 3 r~

delivered at a rf~nC~Ant~ation gf o . 3 ,LM, complexed with DMRIE/D~PE such that the cationic lipid charges and the anionic RNA charges were at 1:1 molar ratio. The results, shown in Table IVr demonstrate a considerable range in the 5 efficacy of ribozymes directed against different Eites.
Five of the 8iX 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 10 alterations in their catalytic core se~uence fail to inhibit rat smooth muscle cell proliferation. Thus, inhibition of cell proliferation by these five hammerhead reriuences iS due to their ability to cleave c-myb RNA, and not because of any antisense activity. The sixth ribo2yme 15 (directed against site 1635~ fails to function in smooth muscle cells. This ribozyme cleaved c-myb RNA very efficiently in vitro. In this experiment, 1096 FBS (no ribozyme added) induced 64 t lg~ proliferation; 0~6 FBS
produced a background of 9 i 1~6 proliferation.
Rl~Arn- le 7: Abilit~ of exoqenouslY delivered hAi~in ribozvme aqainst c-mvb ts inhibit va-Dcular 8mooth muscle cell ~roliferation In addition to the l - '--~ ribozymes tested above, 25 a bipartite hairpin ribozyme (Chowrira, B. M., supra, 1992, Nucleic Acids Res., 20, 2835-2840) was identified that also cleaves c-myb RNA . The ef f ect of this ribozyme on smooth muscle cell proliferation was tested. Ribozymes were delivered at the indicated doses with I.ipofectamine 30 at a 1:1 charge ratio. In this experiment, 10~6 F8S (no ribozyme) induced 87 ~ lg6 proliferation; 09; FBS produced 5 ~ 196 prolif~ ;rn The results of a dose-response experiment are shown in Table V In this example, the control was an irrelevant hammerhead ribozyme. The 35 irrelevant ribozyme control contains the same catalytic core serluences, but has binding arms that are directed to a cellular rNA that is not rer~uired for smooth muscle cell ~ WO95/31541 2 1 9 1~5 ~ 3 r~

proliferation. This control failed to significantly inhibit cell proliferation, demonstrating the se~[uence specif icity of these ribozymes . Another control that could be run is an irrelevant catalytically active ribo-5 zyme having the same G: C content as the test ribozyme .
F le 8: Ribozvmes inhihit ~roliferation of rat 8mooth muscle cells in a dose-de~endent f~h;on.
If the inhibition of proliferation observed in lO 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 ~ r;n~ dogeg of two h --~ Aal'l ribo-zymes. The results shown in Table VI indicate that two 15 hA rh~ l ribozymes that cleave c-myb RNA at sites 575 and 549 inhibit SMC proliferation in a dose-dependent f ashion . Ribozymes were delivered with the cationic lipid, ~ipof ectamine at a l: l charge ratio . In this experiment, lO9~ FBS (no ribozyme) gave 92 i 1%
20 proliferation; 0~ FBS gave 6 i l~ proliferation. The con-trol is an active ribozyme directed again3t an irrelevant mRNA target and shows no inhibition over the dose range tested. The control ribozyme ~nnti~;nll the same catalytic core sf~ n- ~ as the active ribozymes but differs in its 25 binding arm se~uences ~stems I and III in Figure 2c).
Thus, ribozyme inhibition of smooth muscle cell prolifera-tion requires se~uence-specific binding by the hammerhead arms to c-myb mRNA.
30 E le 9: Deliverv of a c-mvb Ribozvme With Different Cationic Li~ids The experiment in Table VII shows the response of rat smooth muscle cells to a hi -rh~ l ribozyme that cleaves c-myb RNA at site 575 delivered with two different 35 cationic lipids, DMRIE and ~lpofectamine. Similar efficacy is observed with either lipid. lO~6 FBS (no Wo 95/31541 ~ 7~0 ribo2yme) induced 78 i 2% proliferation; 096 FBS produced a background of 6 i 196 proliferation. : :
~ rArrmle 10: Effect of varYInq arm-lenqths on ribozYme 5 activitY.
The exact configuration of ~ each ribozyme can be optimized by altering the length of the binding arms (stems I and II~, see Figure 2C). The length of the binding arms may have an effect OIl ~oth the binding and the catalytic cleavage step (~erschlag, D., 1991, Proc.
Natl. Acad. Sci. ~T S A, 88, 6921-5). For example, Table ~TIII shows the ability of arm length variants of c-myb hammerhead 575 to inhibit SMC proliferation.= Dlote :that the dose used in this experiment (0.1 ~lM) is 3-fold lower than in previous experiments. At this rnnA-n}rationr the 7/7 arm variant gives relatively little inhibition. In this case, the degree oi~ inhibition increases with concomitant increases in arm length. =~
The optimum arm length may be site-s~ecific and should be det~r~;n~ ;riAAlly for each ribozyme.
Towards this end, l~ d ribozymes target with 7 nucleotide binding arms (7/7) and ribozymes with 12 nucleotide binding arms (12/12) targeted to three dif ~erent cleavage sites were compared.
Riboz~Ymes were delivered at 0 . 2 ,LM 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 3 0 muscle cell proli~eration with twelve nucleotide bi~ding arms. Each ribozyme showed greater inhibition than its catalytically inactive control, again demonstrating that the ribozymes function via their ability to cleave c-myb R~A. In this experiment, 109~ stimulation resulted in 54 i 2 96 cell proli~eration j unstimulated cells showed 8 O . 5 96 cell prolif eration .

~ 21 905~ 3 Exam~le 11: Effect of ~-hl oroauine on ribozyme activitY.
A number of substances that effect the trafficking of macromolecules through the endosome have been shown to enhance the ef f icacy of D~A delivery to cells These 5 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, IJ. Virol., 67, 3777-3785; Cotten, M. et al.,l992, Proc. Natl. Acad.
Sci. USA, 89, 6094-6098; Cristiano, R. J. et al. ,1993, Proc. Natl. 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.,l984, E7~P. Cell Reg., 155, 9-16; Harris, 15 C. E. et al.,l993, Am. ~. Res~ir. Celi ~ol. BiQl., 9, 441-7; Seth, P. et al.,l994, IJ. Viro7., 68, 933-40;
Zenke, M. et al.,1990, Proc. Natl. Acad. Sci. USA, 87, 3655-3659). It is thought that DNA i8 taken up by cells by endocytosis, resulting in DNA ~ lAtion in endosomes 20 (Akhtar, S. and Juliano, R. ~. ,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 agent3 may augment the functional delivery of RNA and ribozymes to smooth muscle cells, the 25 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 ( O . 2 /lM ribozyme complexed with DMRIE/DOPE at a 30 1:1 charge ratïo). In some cases, 10 ,uM chloroquine was added upon stimulation of the cells. The addition of chloroquine had no effect on untreated cells (stimulation with 1096 serum in the presence or~absence of chloroquine resulted in 80.5 i 1.5 9~ and 83 i 2~ cell proliferation, 35 respectively; unstimulated cells with and without chloro-quine showed 7 i 0.596 and 7 i 1~ cell proliferation, respectively). As shown in Table X below, addition of WO 951315~
2~9~513 1 chloroquine augments ribozyme inhibition of smooth muscle cell proliferation two- to three-fold.
~ mT-le 12 Effect of a hammerhead ribozyme on human 5 8mooth muscle cell ~roliferation.
The 1 '^?C~ ribozyme that cleaves human c-myb RNA
at site 549 was tested fo~ its ability to inhibit human aortic smooth muscle cell proliferation. The binding site f or this ribozyme is completely conserved hetween the 10 mouse and human cDNA sequences. Human aortic smooth muscle cells (AOSMC) were obtained from Clonetics and were grown in SmGM [Clonetics ~ . Cells ~rom passage five or six were used for assays. Conditions for the proliferation assay were the same as for the rat cells (see Example 6), 15 except that the cells were plated in SmGM and starYed in SmBM plus 0 . 596 FBS . The ribozyme that cleaves site 549 was delivered at varying doses t n~nrl~d with the cationic lipid DMRIE at a 1:1 charge ratio. In this experiment, 1096 FBS (no ribozyme) induced 57 i 7~6 proliferation; the 20 lln;nllllc~cl background was 6 i 196 proliferation. The results in Table XI show that inhibition is observed over a similar cnn~ ~n~ation range as was seen with rat smooth muscle cells.
25 Exam~le 13; Inhibition bY direct addition of a modif ied r ~hilized r~hQzymç, A h ~ ribozyme that cleaves site 575 was chemically synthesized with 12 nucleotide binding arms (se~uence ID NO. 127, in Table III). rh~m;n;l11y modi~ied 30 nucleotides were in.:u,~,-dLed into this ribozyme that have been shown to enhance ribozyme stability in serum without greatly impacting catalytic activity. (See Eckstein et al., International P~b~ication No. WO g2/07065, Perrault et al., 1990, Nature, 344, 565-568, Pieken,W. et al.
35 1991, Science, 253, 314-317, ~sman,N.; Cedergren,R~.J., 1992, Trends in Biochem. Sci., 17, 334-339, Usman,N. et al. ~JS Patent Application 07/829, 729, and Sproat,B.

WO 95131541 ~ 't -~' 219~513 European Patent Application 9Z110298. g describe various chemical modif ications that can be made to the sugar moieties of enzymatic RWA molecules. All these publica-tions are hereby incorporated by reference herein. ) The 5 modifications used were as follows. All the nucleotides of the ribozyme contained 2'-O-methyl groups with the following exceptions: U4 and U7 contained 2'-amino substi-tutions; Gs1 A6, G8, Gl2, and Als l were 2 -OH ribonucleo-tides ~numbering as in Figure l). An inactive ribozyme l0 was chemically synthesized in which Gs and Al4 were substi-tuted with 2 ' - O-methyl U . Ribozymes were added to rat smooth muscle cells at the ;n~l;rfltf~rl concentrations as per Example 6 except that cationic lipids were omitted.
Proliferation was assessed by BrdU incorporation and 15 staining. Table XII shows that the modified ribozyme is capable of inhibiting rat smooth muscle cell proliferation without addition of cationic lipids. In this ~ r~ri - ~, 10% serum induced 45 2 ~ proliferation while llnin~lllr~l cells showed a background of 2 . 3 t O . l % proli~eration .

Optimizinq RibozYme ACtiVitY
As f~ ~lcLted in the above examples, ribozymes that cleave c-myb RNA are capable of inhibiting 50% of the smooth muscle cells f rom prolif erating in response to 25 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 op~;m;7;nrJ the 30 deliverY of the ribozyme to the cells is expected to increase the extent of inhibition.
Tables VIII and IX demonstrate o~e means of - op~;m;7;n~ ribozyme activity. By altering the length of the ribozyme binding arms (stems I and III, see Figure 35 2c), the ability of the ribozyme to i~hibit smooth muscle cell proli~eration is greatly ~nhi~nr~d, Ribozymes with increasing arm lengths will be synthesized either chemic-Wo 95131541 2 1 9 0 ~ 1 3 ~ ~?rn ally in one or two parts (see above and sçe Mamone, U.S.Serial No. 07/882,689, filed May 11, 1992, hereby incor-porated by reference herein) or by in vitro transcription (see Cech et al., U.S. Patent 4,987,071). Ribozymes are 5 chemically synthesized with modif ications that prevent their degradation by serum ribo~ucleases (as described in Example 13, above). When synth~Ri~ed in two parts, the fragments are ligated or otherwise juxtaposed as described (see original applir;~t;nn and Mamone, su~ra) . The effects 10 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 ef~ect 15 in cells . Similarly, the precise sequence of modif ied nucleotides in the stabilized ribozyme will affect the activity in cells . The nature of the stabilizing modif i -cations will be optimized for maximal inhibitory effect in cells. In each case, activity of the ribozyme that 20 cleaves c-myb RNA will be compared to the activity of its catalytically inactive control (substitution of 2'-O-methyl U for Gs and a 2'-O- methyl U for Al~,) and to a ri-bozyme targeted to an irrelevant RNA [same catalytic core, with appropriate modifirAtinna, but different binding arm 25 se5~uences).
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 30 cells. In this example, 0.6 IIM ribozyme delivered with Lipofectamine produced the same inhibitory effect as 0.3 ~lM ribozyme delivered with DMRIE. Thus, DMRIE is twice as ~ffi~ r~iou8 ag I,ipofert~min~ at delivering active ribo-zymes to smooth muscle cells. There are a number of other 35 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 (POGS), _ ~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _, _ _ _ _ _ _ _ _ _ WO 95131541 r~
2190~13 dioleoxltrimetylammonium propane (DOTAP), N- [1- (2, 3-dioleoyloxy) -propyl] -n, n, n-trimethylammoniumchloride ~DOT~), N- [1- (2,3-dioleoyloxy) -propyl] -N,N-dimethyl-N-hydroxyethylammonium bromide (DORIE), and N- [1- (2,3-dioleoyloxy ) propyl ] - N, N- dimethyl -N-hydroxypropylammonium bromide (DORIE-HP) . Experiments 3imilar to those per-formed in Example 9 are used to determine which lipids give optimal delivery of ribozymes to smooth muscle cells.
Other 3uch delivery methods are known in the art and can be ~;1i 7~1 in this invention.
The data described in Example 11 show that ribozyme delivery and efficacy may be ~ ,1 by agents that disrupt or alter cellular l~nrl~s~ 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 thoae 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 pre3ence diferent cationic lipids (as in Example 9 and described above) or with otEier delivery agents (as de3cribed 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 (FCC~), 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., su~ra, incorporated by reference herein) .
The data presented in Example 13 indicate that the prolif eration of smooth muscle cells can be inhibited by the direct addition of chemically sr~h; 1; 7.o~1 ribozymes .

Wo 95131541 P~~

Presumably, uptake i8 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 5 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 linlcages necessary f or ribozyme cleavage activity .
Alternatively, ribozymes may be administered to cells by a variety of methods known to those f amiliar to the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by i~corporation into other vehicles, such as hydrogels, cyclodextrins, bio-15 degradable nanocapsules, and b; o~ heqive microspheres .
The R~lA/vehicle ,- ' !n~t;~n 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, 20 oral ~tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. More .l~t~ .l degcriptions o~ ribozyme delivery and administra-tion are provided in Sullivan, et al., suPra and Draper, et al., ~ which have been incorporated by reference 2 5 herein .
r le 14: Phos~horothioate linkaqes enhance the abilitY
of ribozymee to inhibit sTnooth muscle cell ~roliferation.
As the applicant had shown in Example 13, the hammer-30 head (HH) ribozyme that cleaves c-myb RNA at site ~7~ can be modi~ied to confer resistance to nucleases while main-taining catalytic activity (see also Usman et al., su~ra).
To identify ribozymes with optimal activity in cells, several different chemically-modified ribozymes were 35 directly compared for inhibition of rat smooth muscle cell proliferation . Non-limiting, 1 ~c of chemically-modi-f ied ribozymes used are did~L - 1 in Figure 9A. One .

WO95/31541 r "" ^-. n ribozyme (designated `'2'-0-methyln ) contai~s ribonucleo-tide residues at all positions except the 5 t~rm; nill nucleotides of each target binding arm ~Stems I and III).
The ribozyme designated "2'-O-methyl P=S" in addition con-tains five rhnsrhnrothioate linkages between the tPrm;n~l 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 Svm~.
~ 31, 163) at the U4 position and 2'-O-methyl U at the IJ7 position and a 3'-3'-linked inverted thymidine (Ortigao et al., 1992 Antis~nqe Re3. ~ Develo~ment 2, 129; Seliger et al., (~n;3~;~n Patent Application No. 2,106,819) at the 3' end of the molecule (referred ~to as 2'-C-allyl iT) .
The fourth ribozyme rnn~zl;n~ the same 2'-O-methyl and 2'-C-allyl residues described above with the addition of 5 phosphorothioate linkages between the ~Prm;n:31 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., su~?ra). In this example, the cationic lipid, Lipofert~m;nP (GIBCO-BRI ), was used at a charged lipid concentration of 3 . 6 ~M
(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, fl;f;r~tion and the rhn~rhnrothioate linkages demonstrated ~nh~nr~tl ;nh;h;~;rn of smooth muscle cell proliferation. The catalytically inactive form of the ribozyme had little effect on cell proliferation; thus, the inhibition observed re~uires the catalytic activity of the ribozyme. In contrast, ribozymes without the stable 2'-O-methyl- and 2'-C-allyl-~n~l;f;~tl catalytic core (2'-O-methyl and 2'-O-methyl P=S) at best showed only modest inhibition of smooth muscle cell proliferation. The stable core chemistry alone wa~ not suf f icient to greatly Wo 9S/31541 21qO513 enhance ribozyme-r~ t~l 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 5 chemical modifications greatly enhance the ability of exogenously-delivered ribozymes to cleave c-myb RNA and lmpact cell pro1iferation le 15: Dose res~onse of the chemi- ~11 v mo~1 i f ied 10 rihozvme.
Varying do8es 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 15 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 IC~jo varies between 25 and 10 0 nM .
r le 16: Direct rnml~rison of the effects of ribozvmes an~l antisense DNA.
Ribozymes are thought to be more specific reagents for the inhibition of gene er~preæsion than antisense 25 oligonucleotides due to their catalytic activity and strict sequence requirements around the site of cleavage (Castanotto et al., 1994 Adv. in Pharmacol. 25, 2~9) To test this hypothesis, ribozyme activity was directly com-pared to the activity of phosphorothioate DNA oligonucleo-30 tide3 that target the same site in the c-my~ 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-my~ mRNA. _ ~hus, a 15 nucleotide antisense phosphorothioate DNA molecule was 35 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 rarldomly scrarnbled WO 9~/31541 P~
2l~13 target binding arm sequence6 were synthesized as controls (by comparison to the murine c-myb cDNA sea,uence, the scrambled controls would not be expected to bind any region 4f the c-myb mRNA). Since longer phosphorothioate 5 DNA oligonucleotides are often utilized as antisense inhibitors (for a review see Wagner, 1994 7cience 372, 333 ), a symmetrically placed, 25 nucleotide phosphoro-thioate DNA antisense oligonucleotide and its scrambled sequence control were also synt~G~i7~d. The ribozymes and lO 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 sho~n in Figure ll, the 2'-C-allyl P=S-modified 15 ribozyme demonstrated greater inhibition of smooth muscle cell proliferation than either of the ~nt; cl~n~e oligo-nucleotides. Furt~ , the scrambled arm ribozyme and inactive ribozyme controls demonstrated less non-specific inhibition than either of the scrambled sea.,uence antisense 20 control nl ;~nn11n1eotide5 In ~act, the non-specific inhi-bition demonstrated by the 25 nucleotide phosphorothioate molecule completely masked any specif ic ef f ect of the antisense molecule. Similar results have been obtained with phosphorothioate DNA targeting other sites in the c-25 myb mRNA. Thus, a ribozyme that cleaves c-myb RNA is a more potent and more specif ic inhibitor of smooth muscle cell proliferation than phosphorothioate antisense DNA
molecules .
30 E~r~r--le 17: Chemicallv-modified r;hn7vmes taraet1na different sites in the c-mvb mRNA s~ecificallv inhibit smooth mll~cle cell ~roliferation.
If the observed inhibition of smooth muscle cell proliferation is mediated by ribozyme cleavage of c-myb 35 mRNA, then other ribozymes that target the same mRNA
should have the same effect. ~wo other ribozymes target-ing two disparate sites in the c-myb mRNA (sites 549 and Wo 9S/31541 ~ ?~n 2~0~3 1553, ribozyme Sea,. ID Nos. 102 and 112) were 6ynthe6ized with the 2'-C-allyl P=S modifications as described in Example 14. Inactive ribozyme controls al60 were synthe-sized corresponding to each new target sea.,uence.
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. Ea~uivalent levels of inhibition are obtained with active ribozymes targeting sites 549, 575 and 1553 (see Figure 12). None o~ the inactive ribozymes inhibited cell proliferation. Active ribozymes targeting other mRNA RP~IllPnrPA not present in c-myb or ribozymes with scrarnbled arm SP~lf~nr~=~ also fail to inhibit smooth muscle cell proliferation (see Figure 12).
Thus, inhibition of cell proliferation reg,uires a catalytically active ribozyme that can bind to accessible c-myb mRNA seg,uences and is likely due to the reduction of c-myb mRNA levels by ribozyme cleavage.
r l P~ 18 and 19 describe experiments designed to determine the position and minimum number of phosphoro-thioate residues realuired f or ef f icacy .
E le 18: Effect of ~osition of r,hos~horothioate ~;nk,~aeg on rihozym~ ~n~;hition.
Ribo2ymes 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 cnnt~;n~d a total o~ 10 phosphorothioate linkages, 5 in Stem I and 5 in Stem III, identical to the 3 o ribozyme described in Examples 14 through 17 above (referred to as lO P=S 5 and 3 in Figure 13A). One ribozyme cnnt~;nPd 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 GA~A loop (5 P-S loop in Figure 13~l. The fourth ribozyme cnnt~;n.~d 5 phosphorothioate linkages in stem I

W095131541 r~ . 'c-?'o 2t q~

( 5 P=S 5 in Figure 13A) . The latter two ribozymes also were synthp~i7pd with the 3'-3 thymidine at the 3 end to help protect the ribozyme from 3' exonucleases (Ortigao et al., 1992 Antis~nqe Res. & Develo~ment 2, 129; Seliger et 5 al., t~n~ n Patent Application No. 2,106,819). The structure of these four different ribozymes is diay- 1 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 10 as RNA/Lipofectamine complexes and their effects on cell proliferation were measured.
Referring to Figure 13B, the ribozyme cr,nt;~;n;n~ 5 phosphorothioate linkages in Stem I and the 3 inverted thymidine inhibited smooth muscle cell prolif eration as 15 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 20 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 Pnh~nr-pd efficacy in cells. Addition-ally, these results suggest that 3'-end modifications, 25 such as iT, is desirable to minimize the amount of phosphorothioate cnnt=;nPd in the ribozymes in order to minimize toxicity and facilitate chemical synthesis, while nt~;n;nrJ protection from endogenous 3'-exonuclease digestion .
Exam~le 19: ~; nim; 7; nq PhogPhorothioate linkaqes in Stem I.
- Fewer phosphorothioate linkages in the ribozyme will reduce the complexity and cost o~: chemical synthesis.
35 Furthermore, phosphorothioate DNA molecules are known to have some undesirable and non-speclfic effects on cellular fllnrtjnnq (for a review see Wagner, Bllora); reducing the Wo 95131541 P~

phosphorothioate linkages in these RWA molecules i8 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 re~uired for 5 optimaI ribozyme activity. The ribozymes cnntA;nPd 5, 4, 3, 2, or l phosphorothioate linkage ( 5 ) in Stem I, beginning with the phosphodiester bond between the f irst and second nucleotides and proceeding 3'. Each ribozyme ~nnti~;n~l the 2'-O-methyl modifications, the U4 2'-C-allyl lO nucleotide, and the i~verted T nucleotide at the 3' end as described above. Activity of each of these ribozymes was compared to the activity of the ribozyme with lO
phosphorothioate linkages, 5 each in Stems I and III
(referred to as lO P=S in Figure 14). ~ctive and inactive 15 ribozymes were applied to rat smooth muscle cells as complexee with Lipof ectamine and their ef f ects o~ smooth muscle cell proliferation were measured in two separate experiments. The results are diagrammed in Figure 14.
Ribozymes with lO, 5, and 4 phosphorothioate linkages 20 showed equivalent efficacy. Ribozymes with fewer than four rl~n~r~nrothioate linkages also showed efficacy, but the level of inhibition of smooth muscle cell proliferation was modestly reduced.
25 ~ r-~le 20: Varvinq the lenqth Qf Stem5 I and III
Ribozymes that cleave c-myb RNA at position 575 were synthesized with varying arm lengths. Each ribQzyme .nnt~;n~d 4 phosphorothioate linkages at the 5' end, 2'-O-methyl and 2'-C-allyl modifications and an inverted 3 0 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 lO0 nM with catiQnic~ lipid. Ribozymes with 6/6, 7/7 and 5/lO arms (where x,~y denotes the nucleotides in 35 Stem I/nucleotides in Stem III; see Figure 2) all showed comparable ef ficacy . As shown in Flgure 15, ribozymes wi-:h longer arm lengths tended to demonstrate more non-WO 95/31541 . ~ ., C '~' 21 ~351 3 specific inhibition (the inactive ribozyme controls withlo~ger binding arms inhibited smooth muscle cell prolifer-ation) when compared to ribozymes with shorter binding arms. From Ehese data, it appears that ribozymes with 5 6/6, 7/7, 5/10, 10/5, 8/8 and 10/lO nucleotide arms all speciiically inhibit smooth muscle cell proliferation, optimal inhibition, however, is observed with 6/6, 7/7 and 5/10 nucleotide arms.
10 F le 21: Ribozvmes with different modified nucleotides nh;hit smooth muscle cell Proliferation.
Ribozymes ~nnt;3inin~ 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 15 synth~q;7~1 with various modiiied nucleotides at the U4 and U7 FnR1 t j nnq within the core of a ~H ribozyme . All of the modified catalytic core chemistries retained ribozyme activity and demonstrated Pnh~n~ stability to serum mlf~ Aq.~q (Usman et al., 1994 su,~?ra~. The ribozyme termed 20 U4 2'-C-allyl ,nnt~;nq 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 rnnt~inq a 2'-amino nucleotide at both U4 and U7. The ribozyme termed U4 2'-fluoro rnnt,~;nq a 2'-fluoro-modified nucleotide at U4 and 25 2 ' - O-methyl at U7 . The ribozyme termed IJ4 6 -methyl contains a 6-methyl uridine nucleotide at U4 and 2'-O-methyl at U7. The ribozyme termed U4 deoxyabasic cnnt~;nq a deoxyribose moeity and lacks a base at U4 (Beigelman et al., 1994 E~ioorqanic & Med. Cl~em. ~etters 4, 1715) and 2'-3 0 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. ~8 diagrammed in Figure 16, all of the nuclease-stable, chemically-r ';f;~l ribozymes demonstrated signif-35 icant inhibition of rat smooth muscle cell proliferation.
Thus, the requirements f or ribozyme activity in smooth muscle cells appear to be a catalytically core that is WO 95/31541 P~ e 2 1 ~ 3 modif ied to minimize .~nrlrnllrleolytic ~degradation and modifications at the 5' and 3' ends which :may prevent exonucleolytic degradation. : ~ ~
Chemical modifications described in this invention 5 are meant to be non-limiting examples, and those skilled in the art will recognize that other modifications (base, E)ugar and phosphate modifications) to enhance nuclease stability of a ribozyme can be readily generated using standard techniriues and are hence within the scope of this 10 invention.
r 1~ 22 Riboz~me inhibition of ~ia smooth muscle cell proliferation.
Of the commonly used animal models of intimal hyper-15 plasia after balloo~l angioplasty, the pig model isbelieved to be most predictive of human disease ~Steele et al, 1985 Circ. Res. 57, 105; Ohno et al., 1994 Sclence 265, 781; Baringa, 1994 Science 265, 738). Therefore, we wiRhed to assess the ability of c-myb ribozymes to inhibit 20 pig smooth muscle cell proliferation. Yucatan pig smooth muscle cells (YSM) were obtained from Dr. Elizabeth ~abel (University of Michigan Medical Centerl and were grown in Dulbecco' s modified Eagle' s medium as described ~see Example 6). The YSM cells were starved for 72 hours in 25 DMEM with 0.196 FBS. Active and inactive ribozymes (four phosphorothioate linkages at the 5' end, 2'-C-aIlyl-modified core and 3'-3' inverted thymidine at the 3' end) were applied as RNA/~ipof~rtAm;n~ as described in the above examples. Proliferation was stimulated with 3 0 serum and asses~ed by Brd~ incorporation . Figure 17 show~
that a ribozyme dose of a~ low as 75 nM can inhibit pig smooth muscle cell proliferation by as much as 6096. The same chemical modifications of the ribozymes (2'-modified, stable core, 5' phosphorothioate linkages and 3' inverted 35 thymidine) are reauired to obtain significant and repro-ducible inhibition of pig smooth muscle cell proliferation W095.l3l54l P~~
219~13 as were shown to be required for~inhibition of rat cells in the above Examples.
Elc~ le 23: Ribozvme inhibition of human smooth muscle 5 c~ roliferation, In Example 12, we demonstrated that a min;m-lly 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 10 ribozyme targeting c-myb site 575 characterized above was applied to human smooth muscle cells ag RNA/Lipofert:~m;nf~
A~ Inactive ribozyme and inactive, scrambled arm ribozymes were applied as controls. At 200 nM, the active ribozyme inhibits human smooth muscle proliferation by 15 greater than 75g6 while the inactive ribozyme inhibits proliferation by only 38~6. The ribozyme with scram.bled binding arm sequences fails to inhibit. At 100 nM, the active ribozyme still demonstrates significant inhibition while neither the inactive or scramble controls inhibit 20 cell proliferation (see Figure 18). Thus, the active ribozyme identified in these studies ~ t~q significant inhibition of human smooth muscle cell proliferation and represents a novel therapeutic for restenosis and/or vascular disease.
F le 24: Deliverv of c-mYb ribozvmes to vessels in vi VO .
The ribozyme that cleaves c-myb RNA at site 575 was 8yn~h~A; 7e~1 in two parts (Mamone, ~u~ra), the internal 5' 30 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 ;nt~rn;.l 33p - label . This internally- labeled ribozyme was delivered to balloon injured rat carotid arteries as described (Simons 35 et al., 1992 Nature 359, 67). Rats were anesthetized and the carotid artery was surgically exposed. The ~ t~rn:~l carotid was dissected and a 2F Fogarty balloon catheter W0 95131541 P~ r-~

was inserted and directed into the carotid artery. Irl~ury was caused by repeated (3 times) inflation an~ retraction of the balloon. The injured regio~ was isolated by ligatures and a cannula was inserted in the external 5 carotid. Ribozymes alone (two rat vessels) or ribozyme/~ipofectamine 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 f low was restored by removal of the ligatures f or 10 five minutes and the vessels were harvested and processed as described below.
Half of the vevsel was frozen in liquid nitrogen, crushed into a fine powder, and ~NA was extracted using standard protocols. The extracted RDIA was applied to a 15 denaturing polyacrylamide : gels and sub; ected to electrophoresis. Autoradiography of the gel permitted detection of the 31p label; the amount of radioactivity in each band was quantitated using a Phosphor-imaging system.
The amount of extracted and i~tact ribozyme was calculated 20 by direct comparison to labeled ribozyme controls run on the same gel. The percentage of the ribozyme delivered intact could be estimated by ~auantifying the percentage of label that co-migrates with the intact ribozyme controls.
After delivery of ribozymes in phosphate-buffered saline 25 (PBS), 39~ of the 33P label was recovered from the rat vessels and ~909~ of the label was present in the form of intact ribozyme . Af ter delivery of ribozyme in RNA/JJipofectamine complexes, 10 to 11% of the 33P label was recovered from the rat vessels and 20 to 90~ of the label 30 was present in the form of intact ribozyme. The signifi-cant uptake of the intact ribozyme demonstrates that local delivery of mn~ ribozymes to arterial walls is f easible . ::
The other half of each vessel was fixed in PBS-35 buffered 29~i glutaraldehyde, sectloned onto~ slides andcoated with emulsio~. After autoradiography for four days, the emulsion was developed and the sections were .

stained with hematoxylin and çosin by standard technir~ues (Simons et al., 1992 supra). Inspection of the sectione showed a majority of the grains present over the medial smooth muscle cells after application of the ribozyme.
5 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 ~ipofectamine. These data demonstrate that ribo-zyme can penetrate the injured vessel wall and is in close lO 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 15 labeled with 33P as described above, was delivered with a double balloon catheter device ~Nabel and Nabel, su~ra;
Ohno et al., 1994 supra) . After- 20 minutes, blood flow was restored by deflating the balloons. The vessels were harvested af ter an additional hour or the surgical 20 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 ~ rt~ in the media, overlying or within smooth muscle cells. Some label was 25 also detected at the luminal surface of the vessel and in the adventitial tissue. One day after delivery, grains could be still be ~ ert~ associated with I~ i ni ng medial smooth muscle cells. No major differences in density or distribution was observed between ribozyme3 30 delivered with or without I.ipofectamine . These data demon-strate that ribozymes can be locally delivered to smooth muscle cells of injured veseels in a large animal model that is clinically relevant to human vascular disease.

Wo 95~1541 2 ~ 9 0 5 ~ 3 PCT/US95/06368 4s r le 25: RibozYme-media~ed decxease in the level of c-mYb RNA in ~at smooth muscle cells.
To determine whether a ribozyme catalyzes the cleav-age of c-myb RNA in a ~ n cell, applicant has used a sensitive quantitative competitive polymerase chain reaction (QCPCR) to assay the level of c-myb RNA in rat smooth muscle sells 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 1096 serum ( in the presence or absence of BrdU). After 24h, cells were harvested for ~llrther 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, nor-~l;7;ng to the housekeeping gene, GAPDX. This method was adapted from Thompson et al, Blood 79:1692, 1992. Briefly, total RNA was isolated from RASMC using the ~ n;ri;n;llm isothiocyanate t~rhn;~In~ of Chomczynski and Sacchi (AnalYtical Bioch~m;~try, 162:156, 1987) . In order to construct a ~ t;nn competitor and control wild-type RNA, a cDNA clone of the rat c-myb message, referred to as pc8myb, was used. The competitor RNA comprises a ~l.olPt;nn of 50 bases, making it smaller than the wild-type r RNA, and spansfrom nucleotide 428 to nucleotide 753 .
A house-keeping gene, GAPDH, that is co~stitutively expressed by the RASMC, was used as an internal control for QCPCR assay. A deletion competitor and wild-type controls f or GAPDX were made the same way as f or c-myb .
GAPDE~-c~nt~;ning plasmid (pTri-GAPDH) was purchased from Ambion. The GAPDH competitor is also a deletio~ mutant, lackin~ 50 bases. The GAPDX compe~itor was used to quantitate the amount of this housekeeping gene in each WO 9SI31541 1 ~ C
2l9~513 sample, thus allowing for a confirmation of cellular RNA' 8 integrity and for the ~ff;r;~nry of RNA isolation. All quantitations for the level of c-myb expression were normalized to the level of GAPD~I expression in the same 5 sample of cells.
~ f.fPrr;nr to Fig. 19, RASMC that were treated with a stabilized catalytically active 575 HH ribozyme did not proliferate well. There was greater than 70 96 inhibition of RASMC proliferation when compared with approximately lO 25% inhibition of cell proliferation by a catalytically inactive version of the 575 ~H ribozyme. The level of inhibition of R~SMC proliferation correlates very well with the~greater than 70 96 decrease il3 the level of c-myb RNA. This shows that the inhibition of smooth muscle cell 15 proliferation is directly mediated by the cleavage of c-myb RNA by a ribozyme in R~SMC.
Figure 20 3hows what Applicant presently believes is an optimal ribozyme conf iguration .
20 ExamDle 26: Inhibition of smooth muscle cell ~roliferation bv 2-5A ~nti1~enRe chimera.
By "2-5A antisense chimera~ is meant, an antisense olignnl-rlf~rtir~ r-rnt~;n;nJ a 5' phosphorylated 2~-5~-linked adenylate residues. These chimeras bind to target 25 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 rr,nt;3;n;n~ 2'-5' Adenosine with a terminal 5' 30 phosphate has been shown to activate RNAse L (Torrence et al., 1993 Proc. ~atl. Acad. Sci. 1:JSA 90, 1300). The terminal rhrcrh~t~ is required for off;r;~nt activation of - RNAse ~. Ribozymes targetinq c-myb site 575 were synthesized with 2 -5A moieties on the 5 ' end, with and 35 without the terminal 5 ' phosphate . The ribozyme-2-5A
chimera was rr~ P~rl with Lipof ectAMINE and assayed on rat aortic smooth muscle cells (RASMC) as described above.

Wo95/31541 P~l;IJ ~ ~' As shown in Figure 21, when no terminal phosphate is present, the active ribozyme [575 inactive ~z+ inactive ~A) 4] functions similarly to a normal active ribozyme lacking a 2-5A modification (575 active l~z) . An inactive 5 ribozyme core with 5' phosphate-2-5A [575 inactive Rz+
active P (A) 4] shows Si~Jn; f; ~fm~ inhibition relative to the controls, but has significantly lower activity when compared with an active ribozyme. A molecule that containe both an active ribozyme core and 5 ' phosphate-10 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 (0~6 FBS). Thus the ribozyme and 2-5A anitisenee chimera together ehow an additive effect 15 in ;nh;h;t;n~ RASMC proliferatio~
Use of RibozYmes That Cleave c-mYb RNA to Treat Restenosis .
The above discussion ~' LL~tes, by way of example, 20 how ribozymes that inhibit smooth muscle cell proli_era-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 ~:uL~laLy angioplasty. Local delivery during intervention 25 can be achieved through the use of double balloon catheters, porous balloon catheters, balloon catheters coated with polymers (Riessen, R., et al ., 1993 , Hwnan Gerle ~heraDY, 4, 749-758), or biopolymer stents (Slepian and Schindler, U.S. Patent # 5,213,580). In the above 30 examples, ribozymes were idon~;fied that could inhibit roughly half of the smooth muscle cells i~ culture from proliferating in response to the growth factors present in serum. A corresponding 5096 (or even lower) reduction in intimal thickening will significantly improve the outcome 35 of patients undergoing coronary angioplasty.

~ WO95131541 219~513 ';' 7' ~Jse of Ribozvmes Tarqetinq 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 Re6., 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, ~ol. Cell. Biol., 8, 1677-1683; Griffin, C. A and Baylirl, S. B., 1985, Cancer 10 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 A~lPnnr;~rcinoma, neurectodermal, and myeloid leukemia cell 15 lines with ~nt ' ~n~e c-myb r~ nllr1 .~ntides 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 20 patients with chronic myelogenous leukemia and acute myPlc~nrll~ lP1lk~om;~ are differentially sensitive to c-myb Ant;~f~n~ oli~r~nllrll~ntides (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~
25 treated mice bearing human leukemia cells with c-myb antisense oligonucleotides and sir,n;flr~nt~y 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 3 0 potential .
While the above studies demon~trated that autisen~e oligonucleotideE: can ef f lciently reduce the expression of c-myb in cancer cells and reduce their ability to proli_erate and spread, this invention describes enzymatic 35 RNAs, or ribozymes, shown to cleave c-myb RNA. Such ribozymes, with their catalytic activity and increased site ~7per;f;r;ty (see above), are likely to represent more Wo 95/31541 P~
2~90513 potent and safe therapeutic_ molecules than antisen3e oligonucleotides for the treatment of cancer.as well as restenosis. In the present invention, ribozymes are shown to inhibit smooth muscle cell prolif eration . :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-myo RNA. Ribozymes will be delivered to the autologous bone marrow cells e;r vivo at 0.1 to 50 IlM with or without forming complexes of the ribozymes with cationic lipids, encapsulating in 1 lro, - 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 ~n~ln~nml~ bone marrow cells will be depleted by chemical or radiation treatments and their bone marrow recoIlstituted with the ex vlvo 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 . Signif icantly reducing the proliferative potential of the leukemic cells by treating with ribozymes that cleave c-myb RNA will reduce the risk of recurrent leukemia.
Dia~nostic USes Ribozymes of this invention may be used as dlagnostic tools to examine genecic drift and mutatio~s within diseased cells or to detect the presence of c-my~ RNA in a cell. The close relationship between ribozyme activity and the structure of the target R~A 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 ribozy~nes degcribed in tEis WO 95131541 r~ ?

inveution, one may map nucleotide changes which are important to ~IA structure and flln~tinn ~n vitro, as well as in cells and tissues. Cleavage of target RNAs with ribozymes may be used to inhibit gene expression and define the role (essentially) of sp~c1f;-~d 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 hi~ 1 molecules). Other in vitro uses of ribozymes of this invention are well known in the art, and include detection of the ~ st:llce o_ mRNAs associated with c-myb_related condition. Such R~A is ,l,ot~ t~ 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 R~A 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 rP~.t;rln~ and the absence of cleavage of the "non-targeted" ~NA species. The cleavage products from the synthetic substrates will also serve to generate size markers for the analysis of wild-type and mutant R~As 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 R~IA can be analyzed in orLe lane of a Wo 95131541 r~ r ~
2~90513 polyacrylamide gel~ ~ It is llot absolutely required to ~[uantify the reæults to gain insight into the ~expresæion of mutant RNAs and pu~ativ~ risk of the desired phenotypic changes in target cells. The expression of mRNA whose 5 protein product is implicated in the development of the phenotype ~i. e., c-myb) is adequate to establish risk . If probeæ of comparable specif ic activity are used f or both transcripts, then a qualitative comparison of RNA levels will be adequate and will decrease the cost of the initial 10 ~ gnn.q;~:. Higher mutant form to wild-type ratios will be correlated with higher risk whether RNA levels are compared ~aualitatively or quantitatively.
Other embodiments are within the $ollowing claims.

WO 951315~1 2 1 ~ 5 13 r~
Table I ~ rac~eristics of Ribozvmes ~rou~ I Intr,~n R
Si2e: ~200 to ~l000 nucleotides Requires a U in the target sequence i ';i~tely 5' of the 5 cleavage site Binds 4-6 nucleotides at 5' side of cleavage site.
Over 75 known members of this class. Found in Tetrahvmena th~L~ rRNA, fungal mitrr~nn~lr;:~, chloroplasts, phage T4, blue-gree algae, and others.
RN~eP RNA (Ml RNA) Size: -290 to 400 nucleotides RNA portion of a ribonucleoprotein enzyme. Cleaves tRNA
precursors to f orm mature tRNA .
15 Roughly l0 known members of this group are all bacterial in origin.
1 Ribozvme Size: -13 to 40 nucleotides.
20 Requires the target sequence UH~ tely 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 25 plant r~ r~n~ (virusoids) that use RNA as the infectious agent (Figure l) Hain~in Ribozvme Size: -50 nucleotides.
3 0 Requires the target sequence GUC immediately 3 ' of the cleavage site.
Binds 4-6 nucleotides at 5 ' side Qf 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 35 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) .

WO95131541 2 1 q~5 ~ 3 P 1;" '~`-7't`

He~atitis Delta Virus (HDV) Ribozvme Size: 50-6~ nucleotides (at present).
Cleavage of target RNAs recently demon3trated.
Sequence requirements not fully determined.
5 Binding sltes and 6tructural 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 ) .
N~ ros~ora VS RNA Ribozvme Size: -144 nucleotides (at present) Cleavage of target RNAs recently demonstrated.
Sequence requirements not fully determined.
15 Binding sites and structural requirements not fully detPrm; nP-l . Only 1 known member of this class _ Found in Nellros~:1ora VS RNA (Figure 5).
TAhl e II: Ribozvme catalvzed cleavaqe of c-mvb RNA
H: --' ^Ad ~ites ~6 Cleavaqe Cleava~e Seauence TAr~et Se~uence Mouse Human Site ID No. c-mvb c-myb ~ ~NA
310 7 9 CGUCACIJ ~ GGGGA~A 2 8 . 5 0 . 1 549 80 GUCUGW A WGCCAA 87.4 91.6 25 551 81 CUGWAIJ :U~GCCAAGC 56.8 82.4 575 82 GGAGAAU U GGA~AAC 93.9 91.3 634 83 AA~ACCU C CUGGACA 68.4 87.1 738 84 UAAUGCU A~CAAGAA 78.1 : û.01 839 85 CAAGCUU C Cl~GAAGA 27.2 Q.01 30 936 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 I~UAGA~IJ~[JGCAGAA 11.6 0.1 1553 90 QGCUAU C A~AAGGU 87.1 92.5 35 1597 91 ACACCAU IJ CAAACAU 71.2 62.7 1598 92 CACCA~ C AaACA~G 79.6 85.5 WO 9S/3lS41 2 1 9 0 5 1 3 20 Hammerhead Sites _ ~6 Cleavaqe Cleavaqe Seauence Tarqet Sequence Mouse Human Site ~ c-mvb c-mvb RNA R~A

1724 95 GA~WGU U GCUGAGU 65 6 86 5 19 0 9 9 7 UCCGWW U A~UGGCA 31 1 0 Xair~in Ribozymes 1632 99 ACG GUCC CCUGl~AG 92 8 84 6 10 2231 lQ0 _ ACA GWG AGAGCAG 0 1 0 1 a The nucleotide numbers given correspond to the nucleotide j ust 5 ' of the ribozyme cleavage site in the human c -myb sequence taken from Westin, et al, supra (GenBank 15 Accession No X52125) All but two of the sequences ~310; I D No 79 and 2231; ~ D No 100) overlap sequences in Table I
Table III: Seauences of ribozvmes used in these studies 2 0 ~g~ Sequence Ribozv;ne Sequence Site ID No Hammerhead ribozvmes with 7 nucleotide bindinq arms 310 1~1 WwccCccUGAUGAGc.rrr.~r.rrrr.~r.TTGACG
549 102 wGGcAAcur~rTr~r~Grrr~AA~r~Gcrr~A~Ar~r~r 551 103 GcwGGccuGAuGAGGcrr.~Ar.Grrr.~TTAArAr.
57 5 10 4 G~ u u u ~ u~AUGAGGCCGAaaGGCCGAAAWCUCC
634 105 uGlJccAGcuGAuGAGGrcr~AA~r~r~ccGAAAGGu~w 738 106 wcwGAcuGAur~r~r~rrr~ r~r~rrr~A~r~r~TTuA
839 1Q7 ucwcFGcuGAuGAGGcrr~ r~r~rrr~AA~r~cucG
936 108 Au~iu~ u~:u~A~TGAGGrrr~A~zr~r~rrr~AAATTAr~GAA
1017 109 GCCGGCUCUGAUGAGCGCGAAAGCGrr.AAAr.r.r.~rr.
1082 110 G~u~uuw~iAUGAGGCrr.AA~r.rCrr.AAATT~CGCU
1363 111 WCUGCACUGAUGAGGCrr.~r.r.CCGAaAWCUAA

WO 95/31541 r~,l,.l.. '''~ ~
2190513 ~

1553 112 A~ UUUU~U~iATJGAGGCCGAhAGGCCGAaArTAGC~G
1597 113 ATTGWWGCUGArJGAGr.rrr.~ r.r.rCr.PiZ~TTGGTTGU
1598 114 CAU~UUU~u~;ArJGAGGCCGA~GGrrr.~TTGGuG
1635 115 WcAGGGcuGAT-TGAGGrcr~ ~r~r~rcr~ rcGuAT-T
1721 116 CAGCAACCUGAUGAGGCCGAAAGGCCGA~AWCCAG
1724 117 ACUCAGCCUGATJGAGGCCGAAAGGrrrZ~r~TT~C
1895 113 l~G~:uu~iu~u~GAGGrrr.~r.r.rcr.~a~rz~TT~TT
1909 119 T,TGUCAuu~u~Au~jAGGccGA~AGGCcG~ r~r.~
1943 120 CT~WGAGCUGA~JGAGGCCGAZ~AGGCrr.~r~TT~TGTT
Bimolecular HairT~in RibozYmes 1632a 121 5 ' Fragme~t:
ucAGr~r~r~r~rT~TT~rr~r~r~ r~r~-rr~cG
3 ' Fraqment CGCG~GGUACAWACCUGGUA
2231a 122 5' Fr _ GcucucAGAAGwGAcr~r~r~h~r~r~rr~rr~
3 ' Fraqment: CGrr~r-TGr~TT~r~TluAccuGG
T-T~ 1 r;hoyzmes with 6, B, 9, 10. and 12 _ nucleotide bindinq arms 575 123 ( :uuu~ u(iAUGAGGCCGA~-AGGCCGAA AWCTTC

6/6b 575 124 UGWUU~ U(iAUGAGGCCGAAAGGCCGAA

575 125 CUGCUUu~cu~AUGAGGCCGAAAGGCCGAA
9/9 Auu~:uC~:u 575 126 ACUG~uuuCC~:u~AUGAGGrrr.~r.Gcrr.~ --10/10 Auu~u~ uu 575 127 ACACUG~UUU~C~U~iAUGAGGCCGAAAGGCCGAA
12/12 Auu~:uc~uuuu 549 128 AGUGcwr.Gr~rTJr.z~TTGAGGrrr.A~r.r.rrr.
12/12 ~r~r.~rr~rr.
1553 129 r.~rTrTr.~ uuu~u~iAuGAGGrcr~ r~rccGA

3 0 aThe hairpin ribozymes were synthesized in two pieces as indicated The two oligonucleotides were annealea and tested for activity against the c-myb RNA as described above See Mamone, Ribozyme synthesis, filed May 11, W095131541 r~ll-J.. _'G ~'~
21q~513 1992, U.S.S.N. 07/882,689, hereby incorporated by ref erence herein.
bDesignation of the ribozyme3 with different arm lengths iB a/b where (a) represents the nucleotides in stem I and 5 (b) L~Les~ s the nucleotides in stem III (see Figure 1).
T~hle IV: Com~ari30n of the effects six hammerhead ~ibozYmeæ, that cleave c-myb RNA, on smooth muscle cell ~roliferation Inactive Active 96 Inhibition Ribozyme Ribozyme Ribozyme ~ Cell 96 Cell (Active vs.
Site Proliferation Proliferation Inactive) 54968 i 1 59.5 i 1.5 14 i 4 57566 . 5 i 0 . 5 54 . 5 i 1 . 5 21 i 3 15 1553 68 . 5 i 5 52 i 1 28 i 1 1597 66 i 1 57 i 3 16 i 7 1598 67 i 1 58.5 i 0.5 15 i 1 1635 62 . 5 i 2 . 5 64 i 1 0 TAhl e V: Dose Re8~0nse of c-mYb HairPin Riboz~me 1632 Control Ribozyme 1632 Ribozyme Ribozyme 96 ~ ~ Inhibition Dose (IlM) Proliferation Proliferation (v3. control) 250 . 05 86 . 5 i 1 . 5 88 i 5 0 0.15 89.5 i 1.5 78.5 ~ 2.5 10 i 5 0.45 87.5 i 1 66.S i 1.5 25 i 4 Wo 9~ 41 P
219~)513 Table VI: Dose Res~onse of c-mYb TT '^'d Ribozvmes 575 and 549 ==
Control Ribozyme 575 Ribozyme 549 Ribozyme Ribo- 96 cells ~ cells 96 ~ cells 9~
zyrne in S in S Inhibi- in S Inhibi-Dose phase phase tion phase tion (IlM) (vs. (vs.
con- con-trol ) trol ) 0 . 05 89i5 77 . 5il . 5 14i8 92il o 0.15 90il 68.5il.5 26i2 84i2 9i4 10 0.45 91 5i0.5 59i5 38i7 76.5i2.5 18i5 Table VII: Delive~v of c-mYb ~ibozvme 575 bv Two Different Cationic ~ ids Delivery with DMRIE/DOPE
Inactive Active Ribozyme 575 Ribozyme 575 Ribozyme 96 cells in S 96 cells in S 96 Inhibition Dose (ILM) phase phase (vs. inactive) 0 075 79 i 6 74 . 5 i 1 . 5 6 i 6 0.15 79.5 i 0.5 67 i 1 17 i 4 0.30 77 i 1 57 i 2 28 i 5 Deliver,v with Lip~f~ct~m;n.o Inactive Active Ribozvme 575 Ribozyme 575 Ribozyme 96 cells in S 96 cells in S ~ Inhibition 25Dose (~LM) phase phase (vs. inactive) 0 . 075 81 i 1 83 i 1 o 0 . 15 79 i 3 71 i 1 11 i 4 0.30 82 i 1 68.5 i 1.5 18 i 4 0 . 60 75 i 1 59 5 i 3 . 5 22 i 7 WO95/31i541 r~"~
21~05l3 Table ~IIIIi. Arr~ I-ensth V;lriatinnq Qf c-mYk TTi rhead RibozYme S 7 5 ~
Arm ~ength (base ~6 cells in S 96 Inhibition (V8.
pairs) phase Inactive 7/7) 5 6/6 62 i 1 4 i 4 7/7 60 i 1 7 i 3 8/8 60 . 5 i 5 6 i 2 9/9 53 . 5 i 0 . 5 18 i 2 10/10 55 i 1 16 i 4 10 12/12 48 i 1 28 i 3 Table IX~ 1 ribozYmes with 7 V8. 12-nucleotide bindina arrnq ~Arqe~;n~^ three ~l;fferent site8 Ribozyme ~ength of Inactive Active 96 15Target 3inding Ribozyme Ribozyme Inhibition Site Arms ~96 Cell ~96 Cell ~Active Prolifera- Prolifera- vs.
tion~ tion) Inactive) 575 7/7 51.5 i 0.5 43 i 0-5 24 i 5 575 12/12 50-5 i 3.5 37 i 0-5 37 i 4 549 7/7 49-5 i 0-5 44-5 i 1.5 21 i 7 20 549 12/12 48.5 i 1 5 35 i 2 41 i 7 15537/7 49-5 i 0.5 43.5 i 2.5 23 i 9 155312/12 49 i 1 33.5 i 1.5 45 i 6 TAhle X: ~ffect of chloroquine on r;hozYme inhibition of 25 smooth rn~^cle cell ,,roliferation Ribozyme Chloro- Inactive Active 9. Inhibi-quine Ribozyme Ribozyme tion (~LM) (9~ Cell (~6 Cell (Active Prolifera- Proli~era- V9.
tiQn) tion) inactive) 575, 12/12 0 81 . 8 i 5 74 i 1 10 i 2 575, 12/12 10 83 i 4 62 . 5 i 0 5 28 i 6 WO95131541 21 905 ~ 3 r~l~u~

l~hl e XI: Inhibitign of Human Aortic Smooth Mugcle Cells bv c-mYb Ribozvme 549 Inactive Active ~6 Inhibition Ribozyme Ribozyme Ribozyme g6 Prolifera- ~6 Prolifera- (active V8.
5 Dose (/lM) tion tion inactive) 0 075 55 i 2 40 . 5 i 4 5 30 i 13 0 . 15 53 i 10 42 i 1 23 i 23 0-3053 i 7 32.5 i 4 5 44 i 22 10Table XII: Inhibition of Rat Smooth Muscle Cell Pr~l;feration bY Direct Addition of a Chemicallv-Mgdified ~-mYb R; hozvme 575 Inactive Active 96 Inhibition Ribozyme Ribozyme Ribozyme ~ Prolifera- 96 Prolifera- (active V8.
15Dose (llM) tion tion inactive) 0 . 22 42 i 3 3 6 i 0 . 5 15 i 8 0 . 67 48 i 3 35 i 2 28 i 9 2 . 0 52 i 5 25 i 1 54 i 7 20 ~able XIII: T7llr-n c-mvb HairPin Ribozvme and TarcTet 5ecuences Posi- Ribozyme Sequence Target tion 104 ~ U~ AGAA GCGC - GCGCA GCC
Arr~r~r~7~r~r~ u~iu~ uAcATJrT~rrTTr~r~rT~ GGGGAGGG

Arr~r~r~ r~r~ lu(iu~ uAcAwAccuGGuA C(~l iu~ u Arrhr~Plr~AAr~r~ u(iu/i~iuACAWACCUGGUA CGCCGCGC
5 2 8 ACGW~TCG AGAA GU~U : : AUACG GUC
Acr~r~r~ Ar~r~ u~u~iuAcAurT~rrrTGr~rT~ CGA~ACGU

WO 95/31541 T~~
2~ 9~51 3 Posi- RibozylTle Sequence Target tion 715 UUC~U~ 'A AGAA GUAG CUACU GCC
Acr~~r7~AAr~rA~ uu~iw~iuAcAwAccuGGuA UGGACGAA

Arr~r.Ar.AAAr~CA~iuu(iu~iliuAcAwAccuGGuA GCAGCCAU
1187 ~:u~i~u~iu~i AGAA GCA~ WGCC GAC
ACrAr.Ar.AAArArZ~('(;I)U(iU(;(;~ rATTTTArrTTr.r.UA r~r~rrAr.

Arr~r.Ar.AAArArA( '(,~JU~iu(i~uACAWACCUGGUA WWAGAAC

~rrAr.Ar.AAArAr~( (.uu~u~uACAWACCUGGUA CCCUGAAG

ArrAr.Ar.~rArAl ~,UU~iU~i~iUACAWACCUGGUA UGAAUACC
1852 u~:u~ ~u~ AGAA GWG CAACU GW
ArrAr.Ar.AA~r~rA~ il lu~iu-i~;uACAUUACCUGGUA CACGCAGA

ArrAr.Ar.AAArArA( sJuu~;u~uACAWACCUGG~A C~TCGCCUG
19 9 3 UGCUACAA AGAA GC~A WGCA GCC
ArrAr7Ar.AAArAr~ u~u~uAcAwAccuGGuA WGUAGCA
l o 2 2 31 CUGCUCUC AGAA GWG CAACA GW
AccAr~r~ArArA~l~;uu~iuli~uAcAwAccuGGuA GAGAGCAG

ArrAr.~r.A~ArArA~ '(;uu(iu~i~uACAWACCUGGUA WACCUAA

ArrAr~AAA~rArA( (~UU~U~(iUACAWACCUGGUA WAUAAW
3138 AUCCAUGC AGAA GWC ~ GAACU GW
AcrAr.Ar.~AArAr~ ~-uu~iu~(iuAC~WACCUGGUA GCAUGGAU

ArrAr.Ar.AAArArA( ~ .1 I U~U~i(iU~CAWACCUGGUA WAAGAAC

Arr~r.Ar.A~ArArA~ ~iuu~iu(iL~u~CAWACCUGGUA WGUAGCA

WO 95/31541 T ~~ .'t 2 ~ q O ~

T~hle XIV: Human c-mvh E~ammerhead R;~o~vme and Tarqet Seruence nt . Tarqet Seque~ce ~; hr~7vre Se~uenCe 5~Q~

15 AACCUGW U CCIJCC~CC GGAGGAGG CUGA~rGA X GAA AACAGGW
16 ACCUGWU C ~:u~u~:u AGGAGGAG CUGAUGA X GAA AAACAGG~
19 UGWUCCU C CUCCUCCU AGGAGGAG CUGA~GA X GAA Ar.r.~ArA
22 uu~ u~ u C CUCCWCU AGAAGGAG CUGAJGA X GAA AGGAGGAA
CUCCUCCU C CWCUCCIJ AGGAGAAG CUGAUGA X GAA AGGAGGAG

29 UCCUCCW C ucccrccuc GAGGAGGA CUGAIJGA X GAA AAGGAGGA
31 CUCCUUCU C CUCCUCCU AGGAGGAG CUGAIJGA X GAP~ AGAAGGAG
15 34 CWCUCCU C CUCCUCCG CGGAGGAG CIJGAIJGA X GA~ AGGAGAAG
3 7 CUCCUCCU C CUCCG~GA UQCGGAG CIJGAIJGA X GAP~ AGGAGGAG
4 0 Cl rCCUCCU C CGUGACCU AGGUCACG CIJGAUGA X GAA AGGAGGAG
49 CGUGACCU C CUCCUCCU AGGAGGAG C~GAIJGA X GAA AGGUCACG
52 GACCUCCU C C~CCUCW AAGAGGAG CIJGAJGA X GA~ AGGAGG~:rC
20 55 CUCCUCCU C C~CWUCU AGAAAGAG CUGA~GA X GA~ AGGAGGAG
58 CUCCUCC~ C UUUCUL:~U AGGAGA~A CUGAIJGA X GA~ AGGAGGAG
6 0 CC~CCUCU U UCUCWGA UCAGGAGA C~GAIJGA X GAA AGAGGAGG

62 UCCUCWW C UCCUGAGA IJC~CAGGA CUGA~GA X GAA AAArAr.r.~
64 CUCWUCU C CUGAGAPA UWC~CAG C~JGAl~GA X GAA AGAAAGAG
75 GAGA~ACU U CGCCCCAG CJGGGGCG CTJGAUGA X GAA AGWUCUC
76 AGA~ACW C GCCCCAGC GCUGGGGC C~GA~GA X GAA AAGUWC[J

30 170 CCGCGGCU C UCGCGGAG CUCCGCGA CUGA~GA X GAA AGCCGCGG
172 GCGGCUCU C GCGGAGCC GGCUCCGC C~GA~GA X GAA AGAGCCGC
224 CACAGCAU A UAUAGCAG CUGCUAUA C~GAUGA X GAA AUGCUG~G
226 CAGCAUAU A UAGCAGUG CACUGCUA CUGAUGA X GAA AUA~GCIIG
228 GCAUAUAU A GCAG~GAC GUCACUGC CrJGAUGA X GAA AUAUA~GC

254 GAGGACW U GAGAUGUG CACAUCUC CUGAUGA X GAA AAG~CCUC
274 CCAUGACU A UGAUGGGC GCCCAUCA C~GAJGA X GAA AGUCAUGG

288 GGCUGCW C CCA~G~CIJ AGACûlJGG CUGAUGA X GAA A~r.rAr.rr WO 95/31541 1 ~ ~

~ Tarqet Sequence Ribozvme se~Tuence tiQr~
295 TICCCAAGU C TTGGAAAGC ~CWWCCA CUGATTGA X GAA ACWGGGA

310 GCGUCACU U GGGGAAAA WWCCCC CUGA~TGA X GAA AGUGACGC
392 I~TGGAAAGIJ U AWGCCAA nTTr.r.r~TT CUGAUGA X GAA ACWWCCA
393 GGAAAGW A UUGCCAaU AWGGCAA CUGATTGA X GAA AACUWCC
395 AAAGWAU U GCCA~WA UAAWGGC CUGAUGA X GAA AUAACWW

403 UGCCA~W A tTCUCCCGA ~CGGGAGA CUGAUGA X GAA AAWGGCA

lO 414 UCCCGAAU C GAACAGAU AUCUGWC CUGAUGA X GAA AWCGGGA
452 CAGAAAGU A CUAAAC GGGUWAG C[rGAUGA X GAA ACWWCUG
455 AAAGUACU A AACCC~TGA UQGGGW CIJGA~TGA X GAA AGUACWW
467 CCUGAGCU C AUQAGGG CCCWGAU CUGA~TGA X GAA AGCUQGG
470 GAGCUQU C AAGGGTTCC GGACCCW CUGAUGA X GAA A~TGAGCUC

4 a o AGGGUCCU TJ GGACCAAA TJWGGUCC CUGAUGA X GAA AGGACCCU
4 9 8 AAGAAGAU C AGAGAGUG CACUCUCU C~GA X GAA AUCWCW

515 AUAGAGCU U r7TT~r~r.~ WCUGUAC CUGAUGA X GAA AGCUC~TAU
20 518 GAGCWGU A CAGAAAUA UAWWCUG C~TGA~GA X GAA ACAAGCUC
5 2 6 ACAGAaA~ A CGGUCCGA UCGGACCG CUGAUGA X GAA AWWCUGU
531 AAUACGGU C CGAAACGU ACGWWCG C~TGATTGA X GAA ACCGUAW
540 CGAAACGU U GGUCUGW AAQGACC C~TGATTGA X GAA ACGWWCG
544 AcGwGru C UGWAWG CAAUAACA CUGAUGA X GAA ACCAACGU
25 548 U~7~7U~.:U~7U U AWGCCAA WGGQATT CUGAUGA X GAA ~r~r.Arr~

551 IJCUGWAU U GCCAAGQ UGCWGGC CUGAUGA X GAA Z~TTZ~r~
562 CAAGCACU U ~.rrr.~ UCCCCUW CUGAUGA X GAA AGUGCWG

615 ACWGAAU C CAGAAGW AACWC~TG C~TGAUGA X GAA AWCAAGU
623 CCAGAAGU U AAGAAAAC GWWC~U CUGAIJGA X GAA ACWCUGG
35 624 CAGAAGW A ~r~ rr GGWI~UCU CUGAUGA X GAA AACWCUG
634 GAAAACCU C CUGGACAG CUG~TCCAG CUGATTGA X GAA AGGWWC

WO95/31541 21 ~ 0 5 1 3 r~

nt.Tarqet Sequence R~hoZYme Sequence Pos i - =
tiQn 659 Ga~AGAAlJ U AWWACCA UGGUAAAU CUGAUGA X GAA AWCUGUC
6 6 0 ACAGAAW A WUACCAG CIJGG~A~A CIJGAUGA X GAA AAWCIJGU
662 AGAAWAIJ U UACCAGGC GCCIJGGUA CUGA~JGA X GAA AUAAWCU
663 GAAWAW U ACCAGGCA U~ u~iu CUGA~JGA X GAA AAUAAWC
664 A~WAWW A CCAGGCAC GIJGCCUGG CUGAUGA X GAA AAAUAAW

73 8 AUAAUGCU A UCAAGAAC GWCtIUGA CUGA~GA X GAA AGCAWAU

757 CUGGAAW C UACAAUGC GCAWGUA CUGAbGA X GAA AAWCCAG

768 CAAUGCGIJ C GGAAGGUC GACCWCC CUGA~JGA X GAA ACGCAWG

792 AAGG~UAU C ~GC~GGAG CUCCU~GCA C~GAIJGA X GAA AUAACCW
802 GCAGGAGU C WCAAAAG CU~WGAA CUGA~GA X GAA ACUCCIJGC
2 0 8 0 4 AGGAG~CU IJ CAA~AGCC GGCWWG CUGAIJGA X GAA AGACUCCU

839 ACAAGCW C CAGAAGAA WCWCUG CUGA~GA X GAA AAGCWGU

25 855 ACAGUCAU U UGAUGGGU ACCCAIJCA CUGAI~GA X GAA AUGACUGU
8 5 6 CAGUCAW 1~ GAIJGGGW AACCCA~C C~GAIJGA X GAA AAUGACUG
864 UGAUGGGU IJ WGCIJCAG C~GAGCAa CUGAUGA X GAA ACCCAUCA
865 GAUGGGW U UGCUCAGG CCUGAGCA CUGA~GA X GAA AACCCAUC
866 A~GGGWW U GCUCAGGC GCCJGAGC CUGAIJGA: X GAA AAACCCAU
30 870 GWWGCU C AGGCUCCG CGGAGCCU CUGAUGA X GAA AGCAAaAc 876 CUCAGGCU C CGCCIJACA UGUAGGCG C[JGA~JGA X GAA AGCCUGAG
882 CUCCGCCU A CAGCUCAA WGAGCUG CUGaUGA X GAA AGGCGGAG
888 CUACAGCU C AACUCCCU AGGGAGW C~AIIGA X GA~ AGCUG~AG

35 917 CCCACUGU U ~ UCGWGW CUGAIJGA X GAA ACAGIJGGG
928 CAACGACU A WCCUAW AAUAGGAA CUGAI~GA X GAA AGUCGWG

WO 95/31541 r~ ?
21~0513 Tarqet Seauence Ribozy:ne Seqnence ,Po~ i -~, 93 0 ACGACUAU U CCUAWAC GUAAl~AGG CIJGAUGA X GA~ AUAGUCGU

934 C~TAWCCU A WACCACA UGUGGUAA CUGAUGA X GAA AGGA~IJAG

937 WCCUAW A CCACAWW A~AUGUGG CIJGAUGA X GA~ ~rT~rr.
944 TTT~rr~r~lT U UCUGAAGC GCUUCAGA CUQAUGA X GA~ ~TTr,urrTT~
945 ACCACAW U CUQi~AGCA IJGCWCAQ CUGAUGA X GA~ A1~UGUGGU
946 CCACAWW C UGAAGCAC GUQCUUCA CUGAUGA X GAA AaAUGUGG
962 CAZ~AAUGU C IJCCAGUCA IJGACUGGA CUQAUQA X GAA ACAWWG
964 AI~AUGUCU C CAGJCAUG CAUGACUG CUGAUGA X GAA AGACAWW
9 6 9 UCUCCAQU C AUGWCCA UGGA~CATT CUGAUGA X GAA ACUGGAGA

975 G~TCAUGW C CAUACCCIJ AGGGUArJG CUGAUGA X GA~ A~CAUGAC
9 7 9 UGWCCAU A CCCIJGUAG CUACAGGG CUGA~QA X GA~ AUGGAACA
986 UACCC~IGU A GCGWACA IJGU~ACGC CUGAUGA X GA~ ~r~rr.r,TT~
991 rTr,rT~r.rr.rT U ~r~TTr,rr~ WACAUGU CUGAUQA X GAA ACGCUACA
992 GUAGCGW A CAUGUAaA WWACAUG CUQAUGA X GAA AACGCUAC
1002 AUGUAhAU A UAQUQAIJ AWGACUA CUGA~GA X QAA AwwAQrT

1013 GUQArJGU C CCUQQCC QGCUGAGG CUGAUGA X GAA AQWGAC

10 4 8 GAGACACIJ A UAAUGAIJG QIJQWA CUQAUGA X GA~ AGUGUCUC
25 1050 GAQCUAU A ~TTr.~TTr.~ WQUQU CUGAUGA X GAA AUAGUGUC
1082 AAGCG~AU A AAGGAAW APWCCW CUGAUGA X GAA AWCGCW
1090 A~AGGAAU U AGAAWGC GCAAWCU CUGAIJGA X GA~ AWCCWW
1091 A~GGA~W A GAAWGCU AGCA~WC CUGA~GA X GAA AAWCCW
1096 AWAGAAU U GC~TCCU~ WAGGAGC CUGAUGA X GAA AWCUAAU
30 1100 GAAWGCU C CUAAUGUC GACAWAG ClrQA~TGA X GA~ AGCAAWC
1103 WGCUCCU A AUGUCAAC GWGACAII CUGAITGA X GAA ~rr.~rr~z~
1108 CCUAAUGU C ~rrr.~r.A UCUCGGW CUQAUGA X GAA ACAWAGG
1124 ~AUGAGCU A ~r~r.~r~ UGUCCWW CUGAJGA X GAA AGCUCAW
1184 ACQCQU TT GCCGACQ TTrrTTrr.r.r CUQAUGA X GAA ArJGGuGGu 1203 CCAGACCU C AUGGAGAC GUCUCCAU CUQAUGA X GA~ AGGUCUGG

WO 9~/31541 2 1 9 0 5 1 3 nt. Tar~et Seauence Ri~QzYrne Sequence PQ8 i -5 tion 1230 WWCCUGU U UGGGAGAA WCUCCCA CUGAIrGA X GAA ~ rr,~
1231 WCCUGW U GGGAGAAC GI~CUCCC C~GA~GA X GAA P~
5 1246 ACACCACU C CACUCCAU A~GGAGUG CUGAIJGA X GAA AGUGGUGU
12 51 ACUCCACU C CAUCUCUG CAGAGAJG Ci rGAJGA X GA~ AGIrGGAGu 1255 CACUCCAU C UCUGCCAG CUGGCAGA CUGAUGA X GAA A~GGAGIJG
1257 CUCCA~CU C IJGCCAGCG CGC~GGCA CUGA~GA X GAA AGAUGGAG

10 1276 ~CCUGGCU C CCUACCUG CAGGUAGG C~rGAUGA X GAA AGCCAGGA
12 8 0 GGCUCCCU A CCUGAAGA ~rCWCAGG CIJGA~GA X GAA AGGGAGCC
129~7 AAGCGCCU C GCCAGCAA WGCJGGC C~GA~GA X GAA AGGCGCW
1316 UGCAUGAU C GUCCACCA UGGIJGGAC C~GAUGA X GAA AUCAUGCA
1319 AUGAUCGU C CACCAGGG CCC~GGUG CUGAUGA X GAA ACGAUCAU
15 1334 GGCACCAU l:J CUGGAIJAA WAUCCAG CUGA~GA X GAA AUGGUGCC

1346 GAUAAUGU U AAGAACCU AGGWCW CUGAUGA X GAA ACAWA~C
1347 A~AAUGW A AG~ACCUC GAGGWCU C~GAUGA X GAA AACA~A~
20 1355 AAGAACC~ C WAGAAW AAWC~AA CUGAUGA X GA~ AGGWCW
1357 GAACCUCU U AGAAWWG CAAAWCU CIJGA~GA X GAA AGAGGWC
1358 AACCUCW A GAAIIWGC GCAAA~UC CJGAUGA X GA~ AAGAGGW
13 63 CWAGAAU U UGCAGA~A WWCUGCA CJGAUGA X GAA AWCUAAG
13 64 WAGAA~U U GCAGAAAC GWWCIJGC C~GAIJGA X GAA AAWCUAA
25 1376 GAAACACU C CAAUWAU AUAfiAWG CUGA~GA X GAA AGUGWWC
1381 ACUCCAAU ~ UAUAGAW AAIJCUAIJA CUGAUGA X GAA AWGGAGU
1382 CUCCAAW U A~AGAWC GAAUCUAU CUGAUGA X GAA AAWGGAG

1385 CAAUUUAII }~ GAWCUW A~AGAPUC CUGAUGA X GAA AUAAAWG
30 1389 WAIJAGAU IJ ~:uuuCWA UAAGAAAG CUGAUGA X GAA AUC~AUAA
13 9 0 ~AUAGAW C WWCWAA WA~cGAAA CUGAIJGA X GAA AA~CUAIJA
1392 UAGAWCU U UCWAAAC GW~AAGA CUGAUGA X GAA AGAA~CUA
1393 AGA~CW U CWAAACA UGWWAAG CUGAUGA X GAA AAGAA~CU
1394 GAWC~W C WAAACAC GIJGWWAA CUGA~GA X GAA A~AGA~C
3 5 13 9 6 WCWWCU U AAACACW AAG~GWW CJGA~GA X GAA AGAAAGAA
13 9 7 UCWWCW A AACACWC GAAG~GW CUGAUGA X GAA ~AGAAAGA

~ WO 95/31541 - 2190~13 nt TarcTe'c Secruence Ribozvme SecTuenc~e ~g~ ~ :
1404 UA~ACU U CCAGUAAC GWACUGG CUGAUGA X GAA AGUGWWA
1405 AAACACW C CAGUAACC GGWACIJG CUGAUGA X GAA AaGUGWW
1410 CWCCAGU A Arr~TTr.A~ WCAUGGU CUGAUGA X GAA ACUGGAAG
1423 UG~AAACU C AGACWGG CCAAGlJrU CUGAUGA X GA~ AGWWCA

1440 A~AUGCCU U CWWAACU AGWAAAG CUGAUGA X GAA AGGCAWW
1441 AAUGCCW C WWAACW AAr~TJrT~ CUGAUGA X GAA AAGGCAW
1443 U~ UU~:U U UAACWCC GGAAGWA CUGAUGA X GAA ~r.~Ar.~.rA
1444 GCCWCW U AACWCCA UGGAAGW CUGAUGA X GAA ~Ar~AA~Gc lû 1445 CCWCUW A ACWCCAC GUGGAAGU CUGAUGA X GAA AAAGAAGG
1449 CWUAACU U CCACCCCC GGGGGUGG CUGAUGA X GAA AGwAaAG
1450 WWAACW C CACCCCCC GGGGGGrrG CUGAUGA X GAA AaGwAhA
1460 ~rrrrrrrJ C AWGGIJCA UGACCAAU CUGAUGA X GAA AGGGGGGU
1463 ccccr~cAlJ U GGUCACA~ WGUGACC CUGAUGA X GAA AUGAGGGG
1467 UCAWGGU C ACaAAWG CAAWWGU CUGAUGA X GAA Arr~TTr~

1482 UGACUGW A CAACACCA UGGUGWG cuGAlrGA X GAA AACAGUCA
1492 A~CACCAU U UCAUAGAG CrJClJAlrGA CUGAIJGA X GAA AUGGUGW
1493 ACACcaW U CAUAGAGA IrCUC~AUG CUGAUGA X GAA AAUGGUGU
1494 cacc~ww C AlrAGAGAc GUClJCrrAlJ CUGArTGA X GAA AAAUGGUG
1497 CAWWCAU A GAGACCAG CUGGUCUC CUGAUGA X GA~ AUGAAAUG
1518 uGAaAAcu C AaAAGGAA WCCWW CUGAUGA X GAA AGWWCA
15 3 0 AGGAA AArJ A CUGWt~W AaaaACAG CUGAUGA X GAA AWWCCU
25 1535 AAUACUGU U WWAGAAC GWCUAaA CUGAUGA X GAA ACAGUAW
1536 ~CUGW U WAGAACC GGWCUAA CUGAUGA X GAA AACAGUAU
1537 UAClrGWW IJ IrAGAACCC GGGWCUA CUGA~GA X GAA AAA~r.TT~
1538 ACUGWW r~ AGAACCCC GGGGWCU CUGAUGA X GAA AaAACAGu 153 9 CUGWUW A GAACCCCA UGGGGWC CUGAUGA X GaA AaAAACAG
30 1551 CCCCAGCU A ucAAaAGG CCWUUGA CUGAUGA X GAA AGCUGGGG
1553 CCAGCITAU C AaAAGGuc GACCWW CUGAUGA X GAA AUAGCUGG
1561 caAAAGGu C AAUCUUAG clraAGAw CUGAITGA X GAA ACCWWG
1565 GGrTcAA~ C WAGAAAG CWWCUAA CUGAUGA X GAA AWGACCU
1567 GlJCAalJCU U AGAAAGCU AGC~WCU CUGAUGA X GAA AGAWGAC
35 l568 UC~AUCW A GAAAGCUC GAGCWWC CUGAUGA X GAA AaGAwGA
1578 A~AGCUCrr C CAAGAACU AGWCWG CUGAUGA X GAA AGAGCWU

WO 95131541 P~~ , c~
21~0~13 nt. Tarcret Sequence ~hr~7vre Sequence . .
Po6 i -5 tion 1587 CAAGAACU C C~ACACCA IJGGUGIJAG CUGAUGA X GAA AGWCWG
1590 GAACUCCU A CACCAWC GAAUGG~G CUGAUGA X GAA AGGAGWC
1597 UACACCAU U CAAACAUG CAUGWWG C~GA~GA X GAA AUGG~GUA

5 1610 CAUGCACU lJ GCAGCIJCA IJGAGCUGC CUGA~GA X GAA AG~GCA~G
1617 WGCAGCU C AAGAAAW AAI~WCW C~GA~GA X GAA AGCUGCAA
1625 CAAGAAAU U AaAuAcGG CCGUAWW CUGAUGA X GAA AWUCWG

1635 AAUACGGU C CCC~GAAG CWCAGGG C~GAUGA X GAA ACCGUAW
10 1649 AAGAUGCU A CCUCAGAC G~CUGAGG CUGAUGA X GAA AGCAUCW
1653 UGCUACCU C ArAr~rrr : GGG~GUCU CUGAUGA X GAA AGGUAGCA
1663 GACACCC~ C ~rCAUCUAG CIJAGA~GA CUGA~GA X GAA AGGGUGUC
1665 CACCCUCU C AUCUAGUA UACUAGAU CUGAUGA X GAA AGAGGG~G
1668 CCUCUCAU C UAGUAGAA WCUAC~A C~GAJGA X GAA AUGAGAGG

1673 CA~CUAGU A GAAGAUCU AGAUCWC CUGAUGA X GAA ACUAGA~G

1694 GAUGUGAU C AAACAGGA ~CCIJGWU CUGAUGA X GAA AUCACAIJC
1705 ACAGGAAU C UGAUGAAU AWCAUCA CUGAUGA X GAA AWCC~GU
20 1714 UGAUGAAU C UGGAAWG CAAWCCA C~GAUGA X GAA AWCAUCA
1721 UCUGGAAU U GWGCUGA UCAGCAAC C~GAIJGA X GAA AWCCAGA
1724 GGAAWG~r ~r GCUGAGW AACUCAGC CUGA~GA X GAA ACAAWCC
1732 IJGCUGAGU IJ UCAaGAAA WWCWGA CUGA~GA X GAA ACUCAGCA
1733 GCUGAGW U CAAGAAAA WWCWG CUGA~:rGA X GAA AACUCAGC
25 1753 ACCACCCU U ACUGAAGA ~CWCAGU CUGA~GA X GAA AGGGIJGGU
1754 CCACCCW A: rrTr~r.~ WCWCAG CUGAUGA X GAA AAGGGUGG
1766 AAGAaAAU C AAACAAGA IJCWGWW CUGAUGA X GAA AW~CW
1783 GGUGGAAU C uccaAcuG CAGWGGA CUGAUGA X GAA AWCCACC
1785 UGGAAUCU C CAaCUGAU AUCAGWG ClrGAUGA X GAA AGAWCCA

1798 UGAUAAAU C AGGAAACU AGWWCCU CUGAUGA X GAA AWWA~CA
1807 AGGAAACU IJ CWCUGCU AGC~GAAG CIJGAIJGA X GAA AGWIJCCU
1808 GGAAACW C ~:rCUGCuc GAGCAGAA C~GAUGA X GAA AAGW~rCC
1810 AAACWCIJ ~ CIJGCUCAC GUGAGCAG C~GA~GA X GAA AGAAGWW
35 1811 AACWCW C UGCUCACA TJGUGAGCA CUGA~GA X GAA AAGAAGW
1816 cwclrGcu C ACACCACU AG~GGIJGU CUGAUGA X GAA AGCAGAAG

~ WO 95/31541 ~ '?~1.

n~ Tarqet Sequence Riboz~fme Sequence Posi- :
tion 1867 GCAGACCU C GCCUGUGG rrArAr~Gr CUGAUGA X GAA AGGUCUGC
18 9 0 CACCGAAU A WCWACA UGUAAGAA CUGA~GA X GAA AWCGGUG

1893 CGAAUAW C WACAAGC GCWGUAA CUGA~GA X GAA AAUAWCG
1895 AAUAWCU U ACAAGCUC GAGCWGU C~GAUGA X GAA AGAAUAW

1907 AGCUCCGU IJ WAAUGGC GCCAWA~ CUGAUGA X GAA ACGGAGCU
1908 GCUCCGW U UAAUGGCA UGCCAWA CUGAUGA X GAA A~rr.rAr.r 1909 CUCCGWW U AATTrrr1~r GUGCCAW CUGAUGA X GAA AAACGGAG
15 1910 UCCGWW A AUGGCACC r.r,ur.rr~TT C~JGAUGA X GAA ~ rrrA
1924 ACCAGCA~r C AGAAGAUG CAUCWCU CUGAUGA X GAA AUGCUGGU
1943 GACAAUGIJ U CUCA~AGC GC~WGAG CtJGAUGA X GAA ACAWGUC
1944 ACAAIJGW C UCAAAGCA IJGCWWGA cuGAlrGA X GAA AACAWGU

20 1954 Cl~AAGCAU U UACAGUAC GUACUGUA CUGAIJGA X GAA AUGCUWG

1956 AAGCAT.W A CAGUACCU AGGUAC~7G CUGAUGA X GAA A~AUGCW
1961 WWACAGU A CCUAA~AA WUWAGG CUGAUGA X G~A ACUGUA~A
1965 C~GUACCU A AAAACAGG CCUGWW CUGAUGA X GAA AGGUACUG
25 1975 AAACAGGU C CCUGGCGA UCGCCAGG CrJGAUGA X GAA ACCUGWW
1990 GAGCCCCU U GCAGCCW AAGGCUGC CUGAUGA X GAA Ar~GGGcuc 1998 UGCAGCCU U GUAGCAGU ACUGC~AC CUGAUGA X GAA AGGCUGCA

2 0 53 GAUGACAU C WCCAGUC GACUGGAA C~GAUGA X GAA AUGUCAUC
2055 UGACAUCU U CCAGUCAA WGACUGG CU.GAUGA X GAA AGAUGUCA

2061 CWCCAGU C AAGCUCGU ACGAGCW C~GA~GA X GAA ACUGGAA~G
35 2067 GUCAAGCU C GUAAAUAC G~rAWWAC CUGAUGA X GAA AGCWGAC

WO 95/31~41 r~ ~7' nt . Tarqet Sequence Ri}: o.7~,rme Seauence ~osi -5 ~
2 0 7 4 UCGUAaAU A CGUGAAUG CAWCACG CUGAUGA X GAA AWWACGA

a 0 8 7 AAUGCAW C UCAGCCCG CGGGCUGA CUGAUGA X GAA AAUGCAW

5 2105 ACGCUGGU C Al,TGUGAGA UCUCACAU CUGAUGA X GAA ACCAGCGU
2117 UGAGACAU ~ UCCAGAaA WWCUGGA CUGAUGA X GAA AUGUCUCA
2118 GAGACAW U CCAGAaaA WWCUGG CUGAUGA X GAA AAUGUCUC
2119 AGACAWW C CAGAaAAG CWWCUG CUGA~TGA X GAA AAA~TG~TCU
2131 hAAAGCAU U AUGGWW AMACCA~ CUGAUGA X GAA AUGCWW
10 2132 AaAGC~W A UGGWWC GAMACCA CUGAUGA X GAA AAUGCWW
2137 AWAUGGU U WCAGAAC GWC~TGAA CUGATTGA X GAA ACCATJAAtT
2138 WA~TGGW U UCAGAACA UGWCUGA CUGAUGA X GAA A~rrAr 2139 TTAUGGWW U CAGAACAC GUGWCUG CUGAUGA X GAA ~rr~TT~
214 0 AUGGWW C AGAACAC~T AGUGWCU CUGA~TGA X GAA AaAACCAU
15 2149 ArAACACU U C~AGWGA UCAACWG CUGAUGA X GAA AG~TGWCU
215 0 GAACACW C AAGWGAC GUCAACW CUGA~TGA X GAA AAGUGWC
2155 CWCAAGU U GACWGGG CCCAAG~TC C[JGA~TGA X GAA ACWGAAG
216 0 AGWGACU U GGGA~TAUA UAUAUCCC CUGAUGA X GAA AGUCAACU
2166 CWGGGAU A UAUCAWC GAAUGAUA CUGAUGA X GAa AUCCCAAG
2 0 216 8 UGGGA~TAU A ~TCAWCCIJ AGGAAUGA CUGAUGA X GAA AUAUCCCA

2173 UAUAUCAU U CCUCA~CA UGWGAGG CUGAUGA X GAA AUGAUAUA
2174 AUAUCAW C C~TCAACAU ATTGWGAG CUGAUGA X GAA AAUGAUAU
2177 UCAWCCU C ~r~TTn~ WCAUGW CUGAUGA X GAA AGGAATTGA
2 5 218 9 AUGAAACU U WCAUGAA WQUGAA CUGAUGA X GAa AGWWCAU
219 0 UGA~ACW U UCAUGAAU AIJITCA~TGA CUGAUGA X GAA AAGWWCA
2191 GAAACWW U CAUGAAUG CAT~UCAUG CUGAUGA X GAl~ A~AGWWC
2192 A~ACWW C AUGAAUGG CCAUUCAU rTTnZ~TTr~ 2 GAA AaaAGWW
2212 AAGAACCU A WIJWGW AACA~AaA CUGAUGA X GAA AGGWCW
30 2214 GAa~CUA~T U UUUGWGU ACaACAAA C17GAUGA-X GAA AUAGGWC
2215 A~CCUAW U WGWGUG CACAACaA CIJGAUGA X GAA AAUAGGW
2216 ACCUAUW U UGWGUGG CCACAACA CUGATJGA X GAA AaAUAGGU
2217 CCUAWW U GWGUGGU ACCACAAr CUGAUGA X GAA A~aAUAGG

35 2226 GWGUGGU A CaACAGW AACUGWG CUGAUGA X GAA ~rrl~r~
2234 ACaACaGU ~T GAGAGCAG CUGCUCU~ CUGAUGA X GAA ACUGWGU

~ WO 95/31541 1 ~
21905l3 ~ Tarqet Seauence Ribozvrne Sequence 5 ~L
2255 ~UGCAU U~UAGWGAA WCAACUA CUGAUGA X GAA AUGCACW
2 2 5 6 AGUGCAI~T U AGWGAAU AWCAACU C~JGAUGA X GAA AAUGCACU
2257 GUGCAWW A GWGAAUG CAWCAAC CUGAUGA X GAA AaAUGCAC
2260 CAWWAG~ U GAAUGAAG CWCAWC WGAJGA X GAA ACUAAAUG

2273 GAAGUCW C WGGAWW AAAUCCAA CUGAI:JGA X GAA AAGACWC

2280 IJCWGGAU U UCACCCAA WGGGUGA W~ALTGA X GAA AIJCCAAGA
10 2281 CWGGAW U CACCCAAC GWGGGUG CUGA~TGA X GAA AAUCCAAG
2282 WGGAWW C ACCCAACU AGWGGGU CUGAIJGA X GAA A~TTCt'Z~
2291 ACCCAACU A AaAGGAW AAIJCCWW CUGAUGA X GAA AGWGGGU

2 3 0 0 AAAGGAW U WAAAAAU AWtlWA~ CUGA~GA X GAA AAUCCUW
15 2301 AAGGAWW U UAAAAAUA UAW~WA CUGAUGA X GAA AAAUCCW
2302 AGGADUW U D~ TT~ WAWUW CUGAUGA X GAA AAAAUCCU
2 3 0 9 WA~AAU A AAIJAA~G CUGWAW CUGAUGA X GAA AW~lWAA

2 0 2 3 21 ~C~GUCU U ACCUAAAU AWUAGGU CUGAUGA X GAA AGACUGW

23 31 CCIJAAAW A WAGG[IAA WACCUAA CUGAUGA X GAA AAWWAGG
2 5 2 3 3 3 IJAAAWAU U AGG~AAIJG CAWACCU CUGAIJGA X GAA AUAAWWA

30 2355 UAGCCAGU U GWAAUAU AUAWAAC CUGAUGA X GAP. ACUGGCUA
2 3 5 8 CCAGWGU U AAUAUCW AAGAUAW CUGAUGA X GAA AC~ACUGG
2359 C~GWGW A AUAUCWA UAAGAUAU CUGAUGA X GAA AACAACIJG

3 5 2 3 6 6 ~TAAIJAUCU U AAUGCAGA UCUGCAW CUGA~GA X GAA AGAUAWA
2367 AAUAUCW A AUGC~GAU AUCUGCAU CUGAUGA X GAA AAGAUAW

WO 95/31~41 2 1 9 0 5 1 3 1 ~ t ~

Tarqet SeCuence ~7;hozyme Sequence ,~OG i -5 tion 2376 AUGCAGATT TJ IJWWAAA WUAAAAA clJGAlrGA X GAA AUCUGCATT

2378 GCAGAWW ~ WI~AAA WUWAAA CUGAUGA X GAA AAAUCUGC
2 3 7 9 CAGAWW U WAAAAAA WWWAA CUGA~GA X GAA AAAAUCIJG

2382 AWWWW A AAAAAAAC GWWWW CUGAUGA X GAA AAAAAAA~T
2393 AAAAACAU A AAAUGAW AAUCAWW CUGA~GA X GAA AIJGWUW
2 4 01 AAAAUGAU U uAur-uGuA UACAGAUA CUGAUGA X GAA AUCAWW
10 2402 AAAUGAW TT AUCUGtTA~T A~ACAGAU CUGATTGA X GAA AAUCAWW
2403 AAUGAWW A T~TC~TGUAW AATTACAGA CUGA~GA X GAA AAAUCAW
2405 UGAWWAU C UGUAUU~lU AAAAIJACA C~TGA~GA X GAA 7~TTA~TTr~
2409 WAUCUGU A WWAAAG CUWAAAA CIJGAIJGA X GAA Ar~nATT~

15 2412 UCUGUAW U UAAAGGAU AUCCUWA CUGAIJGA X GAA A~rT~r~n~
2413 CUGUAWW U AAAGGAUC GAUCCUW ClJGAIJGA X GAA AAATJAC~G
2414 UGUAWUU A AAGGA~CC GGAUCCW CUGAUGA X GAA ~ TT~r~
2421 ITAAAGGAU C CAACAGAU AUCUGWG CUGA~GA X GAA AUCCWUA
243 0 CAACAGAU C ~GUAU[~W AAAAIJACU CUGAUGA X GAA A~CUGWG
20 2434 AGAUQGU A WWWCC GGA~AAAA CUGAUGA X GAA ACUGAIJCU

243 8 CAGUAUW U WCCUGUG CACAGGAA CUGAUGA X GAA AAAUACbG
2439 AGUAUUW U UCC~GIJGA I~CACAGGA CUGAUGA X GAA AAAAtJACU
25 2440 GUAW~W U CCUGUGAU AUCACAGG CUGAUGA X GAA AAAAAlJAC
2441 I~WWW C CUGUGAUG CAUCACAG C~TGAUGA X GAA AAAAAAUA
2453 UGAUGGGU U UUWGAAA WWCAAAA CIJGA~GA X GAA ~rr~TTr~

2455 AUGGGWW U WGAAAW AA~WCAA CUGA~GA X GAA AAACCCAIJ
30 2456 UGGGWW U UGAAAWW A~AWWCA CUGAUGA X GAA ~ Cr~
2457 GGGUWW U GAAAWWG CA~AW[JC CUGAUGA X GAA AAAAACCC
2463 WWGAAAU :U UGACACAU AUGUGUCA CUGAUGA X GA-A AWWCA~A
2464 WGAAAW U GACACAW AAUGUG[JC CIJGAIJGA X GAA AAWUCAA
2472 UGACACAU U AAAAGG~A IJACCWW CUGAUGA X GAA AUGIJGTJCA

2480 UAAAAGGU A CUCCAGUA UACUGGAG CIJGA~TGA X GAA ACCWWA

WO 9~/~1541 r~ ,s~

nt. Tarqet Se~Tuence Ribo_YTne Sequence ,Pos i -5 ~.~
2488 ACUCCAGU A WWCACW AaGuGAaA CUGAUGA x GAA ACUGGAGU
2490 ~TCCAGTTAU U UCACWW AaAAGUGA C~TGAUGA X GAA AUACUGGA
2491 CCAGUAW u CACWWC GAAAAGUG CUGAUGA X GAA AAUACUGG

5 2496 AWWCACU U WCUCGAU AuCGAGAa CUG~TGA X GAA AGUGAaAU

2498 WCACWW TJ CUCGA~CA TTGAUCGAG CUGAUGA X GAA AaAGuGAA
2501 ACWWCU C GAUCACUA UAGUGAUC CUGAUGA X GAA AGAaAAGu 2505 WCUCGAU C ~rTT~r~ UGWWAGU CUGAUGA X GAA AUCGAGAA
lO 2509 CGATTCACU A AACAUAUG CAUAUGW CUG~UGA X GAA AGUGAUCG
2 515 CUAAACAU A UGCAUAUA U UAUGCA C~TGATTGA X GAA AUGWWAG
2521 ATJA~GCAU A UAWUWA UAAAAAUA CUGAUGA x GAA AUGCAUAU
2523 AUGCA~A~T A W~WAaA WWAAAAA CUGAUGA x GAa AUAUGCAU
2525 GCAUAUAU U WWAAAAA W~WAAA CUGAUGA X GAA AUAUAUGC
15 252 6 CAIJ UAW U WAMMAU AWUWAA C~GA~GA X GAA AAUAUAUG

2528 UAUAWW U ~A~TTr~ UGAW[~W CUGAUGA X GAA AaA~TT~TTA
2529 ATTAW~W A AaAAT-TcAG CIJGAWOU CtTGAUGA X GAA AAAAAUAU
2535 WAAAAAU C AGUAA~AG CWWACU CUG~rGA X GAA AWWWAA
20 2539 AaAucAGu A A~AGCAW AAUGC~W CUGAUGA X GAA ACUGAWW
2547 AAaAGcAu U ACUCUAAG CWAGAGU CUGAUGA X GAA AUGCU~W
2548 AAAGCAW A CUCUA~GU ACWAGAG CUGAUGA X GAA AAUGCWW
2551 GCAWACU C ~AAGUGUA UACACWA CUGAUGA X GAa AGUAAUGC

2564 UGUAGACU u AAuAcrAu AUGGUAW CUGAUGA x GAA AGUCUACA
2 5 6 5 GUAGACW A AUACCATTG CAUGG~TAU C~TGAUGA X GAA AAGUCUAC
2568 GACWAAU A CC~UGTJGA TJCACAUGG CUGAUGA X GAA AWAAGUC

- 3 0 2 5 81 GUGACAW U AAUCCAGA UC~GGAT~U CUGAUGA X GAA AAUGUCAC
2582 UGACAWU A AUCCAGAU AUC~TGGAUCUGATJGA x GAA AAAUGUCA
2 5 8 5 CAT~WAAU C CAGAWGu ACAAUCUG C~GAUGA x GAA AWAAAUG
2591 AUCCAGAU U GUAaAuGc GCAWWAC CUGA~GA X GAA AUCUGGAU
2594 CAGAWGU A AAUGCUCA TJGAGC~W CUGAUGA X GAA AcaAuCUG
35 2601 TTAAAUGCU C AUWAUGG CCAUAAAU CIJGAUGA x GAA AGCAWWA
2604 AUGCUCAU U UAUGGWA UAACCAUA CUGA~TGA X GAA AUGAGCAU

WO 95/31541 1 ~I;L

r~t. ~arqet Sequence R;hnzvTne Sequence ~
2605 IJGCUCAW U AIJGGWAA WAACCATJ CUG~UGA X GAA AAUGAGCA
2 6 0 6 GCUCAWW A UGGWAAU AWAACCA CUGATJGA X GAA A~AUGAGC
2611 WWATJGGTJ ~T AAUGAQU ATTGUCAW CUGATTGA X GAA ACCATJAAA

2631 AGGUACAU U UAWGUAC GUACAAT~TA CUGAUGA X GAA AUGUACCU

2633 GUACAWU A WGUACCA TTGGITACAA CUÇAUGA X GAA AhAUG~AC
10 2635 ACAWWAU U GUACCAAA W~TGGUAC CUGAUGA X GA~ AUA~ATJGU
2638 ~WAWGU A CCA~ACCA UGGUWGG SUGATTGA X GAA ACAATJAAA

2649 A~ACCAW U UAUGAGW AACIJCAUA CUGATJGA X GAA AAUGGWW
2650 AACCAWW U AUGAGWW A~ACUCAU CUGA~GA X GAA A~AUGGW

2657 WAUGAGU U WCUGWA UAACAGAA C~GA~GA X GAA ACUCAUAA

2659 AUGAGWW U CUGWAGC GCUAACAG CUGATJGA X GAA A~ACUCATJ
2 6 6 0 UGAGWW C IJGWAGCU AGCIJAACA CIJGAUGA X GAA A~AAC~CA
20 2664 WWCUGU U AGCWGCU AGCAAGCU CUGAUGA X GAA ACAGA~AA
2665 WWCUGW A GCWGCW AAGCAAGC CIJGAUGA X GAA AACAGA~A
2 6 6 9 UGWAGCU U GCUWA~A WUA~AGC CUGAUGA X GAA AGCUAaSA
2673 AGCWGCU U UAAAAAW AA~lWA CTJGATJGA X GAA AGCAAGCU
2674 GCWGCW U AAA~AWA UAAWUW CUGAUGA X GAA AAGCAAGC

2682 UAa~AAW A WACUGUA UACAGUAA CTJGAUGA X GAA AAW~WA
2684 A~AWAU U ACUGUAAG CWACAGU CUGATJGA X GAA AUAAWW
2 6 8 5 A~AWAW A CUGUAAGA UCWACAG CUGAUGA X GAA AAUAAWW
30 2690. AWACUGU ~ T7~f~ CUAWWCU CUGAUGA X GAA ACAGUAAU
2697 TTAAGAAATT A GWWAUA UAUA~AAC CUGAUGA X GAA AWWCWA
2 7 0 0 GA~AUAGU U WAUAAAA WWAUAA CUGAUGA X GAA ACUAWWC
2 7 01 AAAUAGW U UAUAAAAA WOWAUA CUGAUGA X GA~ AACUAWW
2702 A~TJAGWW U ~TJAA~A WU~WAU CUGAUGA X GAA A~ACTTAW
35 2703 AUAGWW A UAAAAAAU AWDUWA CUGAUGA X GAA A~AACUATJ
2 7 0 5 AGWWAU A AAAAAWA TJAAWUW CUGAIJGA X GAA AUA~AACU

WO 95/31541 r~ 7r0 nt. TarCTe~ SecTuence ~h~7V~e secTuence tion ,.
2712 T~A~AATT TT AUAWIJW A~AAAUAU CUGAUGA X GA~ AWU~WA
2 713 AAAAAAW A UAWUWA UAl~AAAUA CUGAUGA X GAA AAUWU~U
2715 AA1~AWAU A WWUAW A,A~M.A~A CUGATTGA X GAA AUAAWW
2717 AAWAUAU U WWAWCA IJGAAUA,AA WGAUGA X GAA AUAUAAW
2718 ATJUATTAW U WAWCAG CUGAAUAA CUGAUGA X GAA AAUAUA~U
2719 WAUAWW U UAWCAGU ACUGAATJA WGAUGA X GAA PAATTATT~A
2720 UAUAWW U AWCAGTJA ITACUGAAU CUGAUGA X GAA AAAATT~TTP
2721 ATTAWUW A WCAG~TAA WAWGAA CUGATJGA X GAA AA~AAUAU
2723 AWWUAU U CAGUAAW AAWAWG CUGAUGA X GAA AUAPl~AU
0 2724 WW~TAW C AGUAAWW AAAWAW WGAIJGA X GA~ AATTAAAAA
2728 UAWCAGU A AW~TAAW AAWAAAU CUGAUGA X GAA ArUr.AATTA

2 7 3 2 CAGTJAAW U AAWWGU ACA~AAW WGAUGA X GAA AAWACUG

15 2736 A~TWAAU U WGUAAAU AWWACAA WGA~TGA X GAA AWAAAW
2 7 3 7 AWWAAW U UGUAAAUG CAWWACA CUGA~TGA X GAA AAWA~AU

2741 AAWWGU A AAUGCCAA WGGCAW WGAUGA X GA~ ACAAAAW
2761 AAA~ACGU U WWGWG CAGCA,P~A WGA~TGA X GAA ACGWIJW

2763 AAACGWW U WGCUGCU AGCAGCAA WGAUGA X GAA A,AACGWW
2 7 6 4 AACGWW U ITGCUGWA UAGC~GCA WGAUGA X GAA AAAACGW
2 7 6 5 ACGWUW U GCUGWAU AUAGCAGC WGAUGA X GAA A~A,ACG~T
2772 WGCUGCU A UGGUWWA IJAAGACCA WGAUGA X GA~ AGCAGCAA

2779 UAUGGUC~ TT AGCCUGTJA UACAGGCU WGAUGA X GA~ AGACCAUA

2 7 8 7 UAGCCUGU A GACATTGCU AGCAUGUC WGAUGA X GA~ ACAGGCUA
2802 CTTGC~TAG~T A TTCAGAGGG CCWWGA CUGAUGA X GA~ ACUAGCAG

2822 G~AGAGW TT GGACAGAA WCUGUCC CIJGATTGA X GAA AGCUWAC
2843 ~GAAAW TJ GGUGWAG CUA,A5ACC CUGAUGA X GAA AGWWCW

35 2850 WGGTJGW A GGUAADUG cAauL~cc CUGATJGA X GAA A~r~rrDz-2854 UGWAGGU A AWGAWA ~TAGUCAAU CUGAUGA X GAA ~rcTT~ArA

WO 95/31541 P~

~ ~arqet SeaUenCe Bi~QZ SeaUenCe 5 ~Qn 2857 UAGGUAAU U GACUAUGC GCAtTAGUC cuGAr-TGA X GAA AWACCUA

2869 UAIJGCACU A GliAWWCA ~GAAATJAC CUGAtTGA X GA~ AGTTGCATJA
2872 GCACUAGU A WWCAGAC GTTCIJGAAA C~TGAUGA X GAA ACUAGUGC

2 8 7 6 UAGUAWW C AGACWW AAAAGtTCU ClJGAtTGA X GAA AAAUACUA
2882 WCAGACU U WWAAWU AaAWAAA CUGAUGA X GAA AGUCUGAA

10 2884 CAGACWW U UAAWWA UAAAAWA CUGA~GA X GAA A~AGtTCUG
2885 AGACWW l:J AAWWAIJ AUAAAAW CUGAUGA X GAA AAAAGUCU
2 8 8 6 GACWUW A AWWA~TA JA~JAAAAU CUGAUGA X GAA AAAAAGJC
2889 WlJWAAU TT WAUAUAU ~rT~TT~rTZ~ C~GAtTGA X GAA AWAAAAA

2 8 9 6 UrvWAUAU A UAUAUACA UGUAUAUA CUGAtTGA X GAA AUAUAAAA
2 8 9 8 WAUAUAU A UAUACAW AAUGUAUA CUGA~GA X GAA AUAUAUAA
2 02 9 o o AUAUAUAU A UACAWW AAAAUGUA CUGAUGA X GAA AUAUAUAU
2 9 0 2 AUAUAUAU A CAW[JUW AAAAAAUG CUGAUGA X GAA AUAIJAUAU
2 9 0 6 AIJAUACAU U WUUWCC GGA~AAAA cuGAr~TGA X GAA ArJGuAuA~
2 9 0 7 UAUACAW U WUWCCU AGGAaAAA CUGAUGA X GAA AAUGt7A~A
2 9 0 8 AUACAWW U WWCCW AAGGAAAA CIJGAIJGA X GAA AAAUG~TAtT
252 9o9 UACAWW U WWCCWC GAAGGAAA-CUGA~A X GAA APAAIJGTTA
2 910 ACAWUW U WCCWCU AGAAGGAA CUGAtTGA X GAA A~AAAtTGtT
2 911 CAWU~vW U lJCCWCUG CAGAAGGA CtTGA~GA X GAA A~AAAATTG

2913 WWUUW C CWCUGCA UGCAGAAG CUGATJGA X GAA ~
302916 WUWCCU U CUGCAAUA UAWGCAG CUGAUGA X GAA AGGAl~AA
2917 WWCCW C UGCAAUAC GUAWGCA CUGAIJGA X GAA 2~
2924 ~CUGCAATT A CAUUUGAA WCAAAUG CUGAUGA X GAA AWGCAGA
2 9 2 8 CAAUACAU U ~GAa aACU AGWWCA CUGAIJGA X GA~ AUGUAWG
2929 AAUACAW U GAAAACW A~GWWC CUGAUGA X GAA AAUGUAW
352937 UGAAAACU U GWWGGGA UCCCAaAC CUGAUGA X GAA AGWWCA
2940 AaACWGU u UGGGAGAC GUCUCCCA Ct7GAUGA X GA1~ ACAAGT7W

~ WO 9~/31541 r~ 7''`

nt.Tarqet Seauence Ribozvme Sequence Po~
5 tiQn 2941AACWGW T3 GGGAGACU AGUCT3CCC CUÇAT3GA X GAA AACAAGW
2950GGGAGACU C UGCAUr~W ADAAUGCA CTJGAT3GA X GAA AGT3CbCCC
2956 CUCUGCAU U WWAWG CDAUAaAA CUGAUGA X GAA AUGCAGAG
2957 UCUGCAW U UWAWGU D(~ADTTDAD CUGAUGA X GAA AAUGCAGA
5 295~3 CUGC_UW U WAWGUG CACAAUAA CUGAUGA X GAA AAAUGCAG
2959 JGCAWW U UAWGUGG CCACA~UA CUGAUGA X GAD. ADAAUGCA
2 9 6 0 GCAWUW U AWGUGGU ACCACAAU Cl:JGAUGA X GAA ADAAAUGC
2961 ('ATTTTTlTTTT~J A UULU~-iUU AACCACaA C~GA~GA X GAA ADAAAAUG
2969 l~WGUGGU U WWUGW DDCDDDDD CUGAUGA X GAA ACCACAAU
1û 2970 UU~U~LUU U WWGWA UAACDaaA: CUGAIJGA X GAA DA~-rDrDA
2971 ULULI.~UUU U WWGWAU AUAACAAA CUGAUGA X GAA AAACCAC~

2973 u~uuuuu U UGWAWG CaAUAACA CT3GAIJGA X GAA DDADArcD
2974 GGWWW U GWAWGU ACAAUAAC CUGAbGA X GAA DDDDDDC~
15 2977 uuuuuu~iu U AWGWGG CCaACAAU CUGAT3GA X GAA ACAAAAAA
2978 WUWGW A WGWGGU ACCAACDA CUGAT3GA X GAA AArDADAD
2980 WWGWAU U GWGGWW AAACCAAC CUGAUGA X GAA DTT7~D('DDA
2983 GWAWGU U GGWUAUA UAUAAACC CUGAUGA X GAA AcAauAAc 2987 UU~iUU~i~3U U UAUACAAG CWGUAUA CUGA~GA X GAA D~ A~'AA
20 2988 U~UUliL-UU U AUACAAGC GCWGUAU CUGAUGA X GAA AAt'-'AD('A
2989 GWGGWW A UACAAGCA T3GCWGT3A Cr3GAr3GA X GAA AAACCAAC
2991 UGGUWAU A CAAGCA~G CATJGCWG CUGAUGA X GAA ArTAAAr~'A

3 0 0 9 GWGC~CU U CT3~WWG CAAAAaAG CUGAr3GA X GAA AGUGCAAC
25 3010 WGCACW C WWUIJGG CCAAAAaA CUGAUGA X GAA AAGUGCAA
3012 GCACWCU U WWGGGA UCCCAaAA CUGA~3G~ X GAA AGAAGUGC
3013 CACUr3CW U WWGGGAG CUCCC~ CUGAT3GA ~ GAA AAGAAGT3G

3015 CWCWW U UGGGAGAU AT3CUCCCA CUGAUGA X GAA Ai~AGADG
30 3016 UULUUUUU U GGGAGAUG CAUCUC CUGAUGA X GAA AAAAA.AA
3 0 3 0 AUGUGUGU U GWGAUGU ACAT3CDAC Cr3GAUGA X GAA ACACACAU
3033 U~3U~UUl,U U GAUGWCU AGAACAUC CT3GAUGA ~ GAA DCAAf'At'A
3 0 3 9 GwGAr3GU U CUAUGWW AAACAUAG cuGaT3GA X GAA ACAUCAAC
3 042 GAUGWCU A UGWWGU A-'DAAArA CUGAUGA X GAa AGAACAUC
3046 WCUAUGU T3 WGWWG CAAAACAA CUGAT3GA X GAA Dr~ATTA~AA
3047 UCUAT3GW U UGWWGA UCDAAACA CUGAUGA X GAA AA~'ATTA~'.A

~VO 95/31541 P~ '~

a~ Tarqet Sec:Uence ~;hl~7v~e Se~uence ~Q~i~ , ticn 3 0 4 8 C~AUGWW U GWWGAG CIJCA~AAC CUGAUGA X GAA AAACAUAG
3051 U~iUUUU~iU U WGAGUGU ACAC~CAA CUGA~GA X GAA At~P7~P~''A

3 053 WWGWW U GAG~G~AG CUACACUC CUGAIJGA X GAP. AAACAAAA

3072 UGACUGW ~ UAUAAWW AAAWAUA CUGAUGA X GAA AACAGUCA

3074 ACUGWW A UAAWtJGG CCAaAWA CUGAUGA X GAA AAAACAGU
10 3076 UGWWAU A AWWGGGA 17CCCAPA~ cuGAl:rGA X GAA AUAAAACA
3 0 7 9 WWAUAAU IJ ~GGGAGW ;~CUCCCA CUGAUGA X GAA AWAUAAA
3 0 8 0 WAUAAW U GGGAGWC GAAC~CCC CUGAUGA X GAA AAWAUAA

3099 CAWWGAU C CGCAUCCC GGGAUGCG CUGAUGA X GAA AUCAAA~G
310 5 A~CCGCAU C CCCUGUGG CCACAGGG CUGAJGA X GAA AJGCGGAU
3115 CCUGUGGU U UCUAAGUG CACWAGA c~rGAuGA g GAA ACCACAGG
3116 C~GUGGW U CUAAGUGU ACACWAG CUGAUGA X GAA AACCACAG
20 3117 UGUGGWW C UAAGUGUA UACACWA CUGAUGA X GAA P~ A~A
3119 UGGUWC~ A AGl:rG~AuG CAUACACU CUGAUGA X GAA Prr~A~A
3125 CUAAGUGU A ~GGUCUCA UGAGACCA CUGAUGA X GAA ACACWAG

3132 UAUGGUCU C AGAACUGIJ ACAGWC~ CUGAUGA X GAA A~'.A~rTA
25 3141 AGAACUGU U GCAUGGAU AUCCAUGC CUGAl:JGA X GAA ACAGWCU
3150 GCAUGGAU :C C~G~GWW AAACACAG CUGA~GA X GAA AUCCA~GC
3157 UCCUGUG~ U UGCAACUG CAGWGCA CUGAUGA X GAA ACACAGGA

3185 ACUGUGGU U GAUAGCCA ~GGCIJAUC ClJGAUGA X GAA ACCACAGU
30 3189 UGGWGA~ A GCCAG~CA IJGAC~GGC ~GAUGA X GAA AUCAACCA

3204 CACUGCCU U AAGAACA~ AIJGWCW CIJGAIJGA g GAA AGGCAGUG
3205 AC~GCCW A AGAACAW AAUGWCU CIJGA~GA X GAA AAGGCAGU

35 3214 AGAACAW ~ GAUGCAAG CWGCAIJC C~rGAlJGA X GAA AAUGWCU

Wo 95/31541 r~ J.. ,5.. ~~'^
219~513 ~t. Tarqet Sequence Ribozyme Sequence Posi -3241 CUGAACW U tTGAGAUAU AUAUCUCA CUGAUGA X GAA AAGWCAG
3242 ~ UGAAC~ U GAGAUATJG CAtTAucur rrTr.~TT~ X GAA AAAGUUCA

3262 GUGUACW A CUGCCWG CAAGGCAG C~GAUGA X GAA AAGUACAC
3269 UACUGCCU U GUAGCA~A U[~UGCUAC CUGAUGA X GAA AGGCAGUA
3272 UGCCTJUGU A GCAA~AIJA IJAWUUGC CUGAUGA X GAA ArAA~r~
3 2 8 0 AGCAaAAU A AAGAUGUG CACAUCW CUGAUGA X GAA AWUUGCU
3293 U~iU~L~U U AU~WACC GGUA~AAU CUGAUGA X GAA AGGGCACA
3294 GUGCCCI~U A WUUACCU ~r.r.TTAA~ CUGAUGA X GAA AAGGGCAC
Where "X" represents stem II region of a HH rlbozyme (Hertel et al., 1992 Nucleic Acids Res. 20 3252). The 15 length of stem II may be 2 2 base-pairs.
Table XV: Mo~l~e ~-mYb Hammerhead Ribozyme and Tarqet SecTuence ~t. T~rqet Sequence Ribozyme Sequence tion CCGGGGCUC WGGCGGA UCCGCCA~ CUGAUGA X GAA Ar.crrrr.r.
12 GGGGC~CW GGCGGAGC GCUCCGCC C TGAUGA X GAA ~rz~r.rrrc CAGCAUCUA CAGUAGCG CGCUACUG CJGAUGA X GAA AGAUGCUG
UCUACAGUA GCGAUGAA WCAUCGC CIJGAUGA X GAA ~rTTGTT~r.~
93 r.A~r.~rATT~T GAGA~GIJG CACAUCUC CUGAUGA X GAA AIJGUCWC
113 rrATTr.ArTT~ CGAUGGGC GCCCAUCG CUGAUGA X GAA AGUCAUGG
3 0 13 4 GCCCAAAUC IJGGAAAGC GCWWCCA CtTGAUGA X GAA AWWGGGC
14~ GAAAGCGIJC AC~UGGGG CCCCAAGIJ CUGAUGA X GAA ACGCWWC
149 GCGUCACW GGGGAAAA WWCCCC CT~TGAUGA X GAA AGUGACGC
160 GGAAAACUA GGuGGAr-A UGUCCACC C~GAUGA X GAA AGWWCC
231 ~GGAAAGUC AWGCCAA WGGCAAU C TGAUGA X GAA ACWWCCA
35 234 AAAGUCAW GCCAAWA IIAAWGGC CUGAUGA X GAA AUGACI~W
241 U~GCCAAW AUCUGCCC GGGCAGAU CUGAUGA X GAA AWGGCAA

WO 95131541 P~
219û513 ~ ~arqet Sequence R; hQ~;yme ~equence .
~n , 244 CCAaWAUC UGCCCAAC GWGGGCA CUGAUGA X GAA ATJAAWGG
2 6 4 ACAGAUGUA CAGTJGCCA UGGCACUG CUGAUGA X GAA ACAtTCUGU

309 GAACUCAUC AAAGGJCC GGACCWW CTJGATTGA X GAA A~TGAGWC
316 UCAAAGGl~C CCUGGACC GGIJCCAGG CUGATJGA X GAA ACCUWGA
337 AAGAAGAUC AGAGAGUC GAC~CUCU CUG~UGA X GAA ATTCWCW

10 354 AUAAAGCW GUCC~GAA WC~TGGAC CUGATTGA X GAA AGCWWATJ
3 5 7 AAGCWGUC CAGAAAIJA UAWWCUG C[JGATTGA X GAA ACAAGCUU
365 crArAAATTA tTGGTTCCGA UCGGACCA CUGA~TGA X GAA AWWCUGG
3 7 0 AAUAUGGUC CGAhGCGU ACGCWCG C~TGAUGA X GAA ACCAUAW
379 CGAAGCGW GGTTC~TGW A~CAGACC CUGAUGA X GAA ACGCWCG
15 383 GCGWGGUC UGWAWG CAATTAACA CUGAT~GA X GAA ACCAACGC
387 u~ U~:u~iuu AWGCCAA T.~TGGCAAU CTTGATTGA X GAA ArArArrA
388 G~jU~:UljUUA WGCQAG CWGGCAA CUGATJGA X GAA AACAGACC

4 01 CAAGCACW AAAAGGGA TJCCCWW CUGA~TGA X GAA AGUGCWG
20 402 AAn~'A~UTTA AAAGGGAG CUCCC~W CUGATTGA X GAA AAG~TGCW
414 GGGAGAAW GGA1~AGCA TTGCUWCC CUGATTGA X GAA AWCUCCC
427 AGCAGUGUC GGGAGAGG CCUCUCCC C~TGA~TGA X GAA ACACUGC~
448 AQACCAW UGAAUCCA UGGAWCA CUGAUGA X GAA A~TGGWGU
449 CAACCAWW GAATJCQG C~TGGAWC CUGAtTGA X GAA AA~TGGWG
2 5 4 5 4 AWWGAAUC QGAAGW AACWCUG CUGAUGA X GAA AWCAAAtT

463 CAGAAGUUA AGAAAACC GGWWCU CTTGA~GA X GAA AACWCUG
473 rAAAArrTTr CUGGAQG CUGTTCQG CTJGAUGA X GAA AGGWWC

502 GAATTCAWW ACCAGGCA TTGCC~GGU CUGAUGA X GAA AAUGAWC

520 ACAAGCGUC UGGGGAAC GWCCCCA C~TGAUGA X GAA ACGCWGTT
543 GCAGAGAUC GCAAAGCU AGCUWGC CUGA~TGA X GAA AlJCUCUGC
35 571 GGACU.'ATTA ATTr~rTTATTc GAUAGQU CUGAIJGA X GAA AUCAGTJCC
S77 AUAAUGCUA UCAAGAAC GWCWGA ClTGAUGA X GAA AGCAWAU

W095J31541 r~".l~.,s 21qO513 ~ Tarqe~ SecTuence Riboz~me SecTuence 20 Posi-579 AAUGCUAUC ~ ('A UGGWCW CUGAUGA X GAA A~AGCAW

596 CUGGAaWC QCCA~GC GCAIJGGUG CUGAUGA X GAA AAWCCAG

629 ~:~AAr~GrrTA CCUGQGA UCUGQGG C[JGAUGA X GAA AGCCWCC
643 AGAAGCCW CCAAAGCC GGCWWGG CUGA~GA X GAA AGGCWCU
644 GAAGCCWC QAAGCCA U~ UUU(i CUGAUGA X GAA AAGGCWC
677 QCGAGCW CQGAAGA UCWCUGG C~GAUGA X GAA AGCUCGUG

lO 691 AGAACAAUC AWWGAUG CAUCAAAU CUGAUGA X GAA AWGWCU
6 9 4 AQ~UQW UGAUGGGG CCCCAUCA CUGAUGA X GAA AUGAWGU

704 GAUGGGGW UGGGCA~G QIJGCCCA cl:rGAuGA X GAA ACCCCAUC
705 AUGGGGUW GGGQl:JGC GCAIJGCCC CUGAUGA X GAA AACCCCAU
15 716 GCAIJGCCUC ACCUCCAU AIJGGAGGU CUGAUGA X GAA AGGCA~GC
721 CCUCACCUC CAUCUQG CUGAGA~G-CUGAIJGA X GAA AGGUGAGG
725 ACCUCCAUC UQGCUCU A~ TtlA CUGAUGA X GAA AUGGAGGU
727 CUCCAUCUC AGCUCUCU AGAGAGCU CUGAl:JGA X GAA AGAUGGAG

749 IJGGCCAGUC CUCCG~CA UGACGGAG CIJGAIJGA X GAA ACUGGCCA

756 u~uc:~:~u~ AACAGCGA IJCGCUGW CUGAUGA X GAA A~ A

776 IJCCCUAWA CCAQ~CG CGAUGUGG CUGAUGA X GAA AATTAr.~"`
30 783 rT~ 'A~'ATTr GCCGAAGC GCWCGGC CUGAUGA X GAA AUGUGGUA

803 A~'ArT~TJ:' CAGUCACG CGUGACUG CrJGAUGA X GAA AGAUGWW
808 IJCUCCAGUC ACGWCCC GGGAACGU C~GAUGA X GAA A~Ur~Ar.A
813 AGUCACGW CCCUAUCC GGAUAGGG ~CUGAUGA X GAA ACGUG~CU

818 ~:~UU~ ~UA UCCUGUCG CGACAGGA CUGAUGA X GAA AGGGAACG

WO95/31541 l'~ .'C6. 5 84 =
nt. TArcTet SecTuence R~hozvme Segu-çnce ---2 0 ~2&i.
tirn 820 WCCCUAUC CtTGUCGCA TT&CGACAG CUGAJGA x GAA AUAGGGAA
825 UAUCCUGUC GCAWGCA TTGCAAUGC CUGAUGA x GAA ACAGGAUA
8 3 0 UGUCGCAW GCAUGWA UAA~Ar7GC CtTGAUGA X GAA AUGCGACA
8 3 7 WGCAUGW AAUAUAGU ACUAtTAW CUGAUGA x GAA ACAUGCAA
83 8 UGCAUGWA AtTAUAGUC GACUAUAU CUGAUGA x GAA AACAUGCA
841 AUGWAAUA UAGUCAAC GWGACUA C~GAUGA x GAA AWAACAtT
843 GWAAUAUA GtTCAACGTJ ACGWGAC CUGAUGA x GAA AUAWA~C
846 AAr7AUAGUC AACGUCCC GGGACGW CUGAUGA x GAA ACUAUAW
852 GUCAACGUC CCUCAGCC GGCUGAGG CUGAtTGA x GAA ACGWGAC
10 856 ACGUCCCUC AGCCGGCtT AGCCGGCU CUGATTGA x GAA AGGGACGIT
8 7 6 GCAGCCAUC CAGAGACA UGUCUCUG CUGAtTGA x GAA AUGGCUGC
887 r~Ar.~rDrrT~ UAACGACG CGUCGWA CUGAtTGA x GAA AGTTGUCUC
889 r~rArTTATTA ACGACGAA WCGUCGU cnGATJGA x GAA AUAGUGUC
921 AAGCGAAUA AAGGAGCU AGCUCCW CUGAtTGA x GAA AWCGCW
15 935 GCUGGAGW Gcuccr7GA ncAGGAGc CUGAUGA x GAA ACUCCAGC
939 GAGWGCUC CUGAtTGTJC GACATTCAG CUGAUGA x GAA AGCAACtTC
947 CcuGA~TGuc AACAGAGA UCUCUGW CUGAUGA x GAA ACAUCAGG
930 GCAGGCAW ACCAACAC GTTGWGGtT CUGATTGA x GAA AUGCCUGC
981 C~GGCAWA CCAACACA UGUGWGG CUGAtTGA x GAA AAUGCCUG
20 1000 ACCACACW GCAGCUAC GtTAGCUGC cnGAuGA x GAA AGUGUGGU
10 0 7 WGCAGCUA CCCCGGGU ACCCGGGG cuGAnGA x GAA AGcnGcAa 1028 CAGCACCUC CAWGUGG CCACAAUG Cr7GAuGA x GAA AGGUGCUG
1032 ACCnCCAW Gr7GGAccA UGGUCCAC CUGAUGA x GAA AuGGAGGn 1051 CCAGACCUC AUGGGGAn AUCCCCAU CUGAUGA x GAA AGGUCUGG
25 1060 AUGGGGAUA GUGCACCU AGGnGcAc ctTGAnGA x GAA AUCCCCATT
1071 GCACCUGW UCCUGWW AAACAGGA cnGAtTGA x GAA ACAGGUGC
1072 CACCUGWW CCUGWWG CAAACAGG CtTGA~TGA x GAA AACAGGUG
1073 AccnGWWC CUGWWGG CCA~AG ctTGAnGA x GAA AAACAGGTT
1078 UUU~ U~iUU tTGGGAGAA WCUCCCA CUGAnGA X GAA ACAGGAAA
30 1079 UU(:~:U~iUUU GGGAGAAC GWCUCCC CUGAtTGA X GAA AACAGGAA
1103 CACCCCAUC UCUGCCUG CAGGCAGA ctTGAnGA x GAA AUGGGGUG
1105 CCCCAUCUC UGCCUGCA ITGCAGGCA ctTGAnGA x GAA AGAUGGGG
1117 CIJGCAGAUC CCGGCUCC GGAGCCGG CUGAUGA x GAA AncuGcAG
1124 UCCCGGCUC.CCUACCUG CAGGUAGG cr7GAnGA x GAA AGCCGGGA
35 1128 r~Grr7r-rrrTA CCUGAAGA UCWCAGG CtTGAUGA x GAA AGGGAGCC
1145 AAGUGCCUC ACCAGCAA WGCUGGU cnGAuGA x GAA AGGCACW

WO 95131541 P~
- 2~9~5l3 nt. Ti~rqet Seruen~e Riboz~me Sec~uence Fosi- .
tion 1167 AUGAIJCGUC CACCAGGG CCC~TGGUG CUGAUGA X GAA ACGAUCAU

1194 GAC~AUGW AAGAACCU AGGWCW CUGAUGA X GAA ACAWGUC

1203 AAGhACCUC WAGAAW AAWCUAA CUGAUGA X GAA AGGWCW
1205 GAACCIJCW AGAAWWG CA~AWCU=CUGAUGA X GAA AGAGGWC

1211 CW~AAW IJGCAGAAA WWCUGCA C~TGAUGA X GAA AWCUAAG
1212 WAGAAWW GCAGAAAC liUUU~:UUI~ CUGAUGA X GAA AAWCUAA

1233 CAGWWAUA GAWCWW AAAGAAUC C~TGAUGA X GAA AUA~ACUG
1237 WAUAGAW CUWCWG CAAGA~AG CUGAUGA X GAA AUCUAUAA
1238 IJAUAGAWC WWCWGA UCAAGAAA CUGAIJGA X GAA AAUCUAl:JA

1241 AGAWC~lW CWGAACA UGWCAAG CUGAUGA X GAA AAGAAUCU

1244 uu: :uuu~uu GAACACW AAGIJGWC C~GAIJGA X GAA ~r.~r.~

1277 CIJCGGGCW AGAUGCAC GuGcAIJrTT rnr.~TTr.~ X GAA AGCCCGAG
1278 U~ 3L~UU~ GAIJGCACC GGIJGCAIJC CUGAUGA X GAA AAGCCCGA
1288 AUGCACCUA CCWACCC GGGUAAGG CUGAIJGA X GAA AGG~TGCAU
1292 ~rrTTArrT~T ACCCUCCA UGGAGGGU CUGAUGA X GAA AGGUAGGU

1298 CWACCCUC CACUCCUC GAGGAGUG CUGAUGA X GAA AGGGUA~G
13 03 CC~TCCACUC CUCUCAW AAUGAGAG CUGAUGA X GAA AGUGGAGG
1306 CCACUCCUC UCAWGGU ~rr~r.~ CUGAUGA X GAA AGGAG~GG

1315 UCAWGGUC ACAAACUG CAGWUGU CUGA~GA X GAA ACCAAUGA

WOg5/31541 r~ ,.c-~

nt. Tarqet Sequence RibozYme Sequence 20 ~
~Qn ' 13 6 6 AGGAAAAW CCAUCUW AaAGAUGG CUGAUGA X GAA AW~rCCU

1371 AAWCCAUC WWAGAAC GWCUAaA CUGAUGA X GAA AUGGAAW
5 1373 WCCAUCW UAGAACUC GAGWCUA CUGAUGA X GAA AGAUGGAZ~
1374 UCCAUCWW AGAACUCC GGAGWCU CUGAUGA X GAA AP,GAUGGA
1375 CCAIJCWWA GAACUCCA UGGAGWC CUGAtTGA X GAa AaAGAUGG

1387 CUCCAGCUA T-rcAaAAGG CCWUUGA CUGA~rGA X GAA AGCUGGAG
10 1389 CCAGCUAUC AAAAGGUC GACCWW CUGAUGA X GA~ AUAGCUGG
1397 rA~r.rUC AAUCCJCG CGAGGAW CUGATTGA X GAA ACCWCJ~G
1401 AGGUCAAUC CUCGAaAG CWUCGAG CUGAUGA X GA1~ AWGACCU

1412 CGAAAGCUC UCCUCGAA WCGAGGA CUGAl:JGA X GAA AGCWWCG
15 1414 AAAGCIJCUC C~TCGAPCU AGWCGAG CUGAUGA X GAA AGAGCUW
1417 G~:u~:u~ u~ GAACUCCC GGGAGWC CUGAUGA X GAA AGGAGAGC
1423 CUCGAACUC CCACACCA ~GGUGIJGG CUGA~TGA X GAA -AGWCGAG
1433 r~r~rr~rTTT CAhACAUG CA~GUWG CUGAUGA X GAA AUGGUGUG
1434 ACACCAWC AAACAl:rGC GCAUGWW CUGAUGA X GAA AA~TGG~GU
20 1446 CAUGCCCW GCAGCUCA UGAGCUGC CIJGA~JGA X GAA AGGGCAUG
1453 WGCAGCUC AAGA~AW AAWWCW CUGAUGA X GAA AGCUGCI~A
1461 CAAGA~AW ~AAUACGG CCGUAWW CUGAUGA X GAA AWWCWG
1462 AAGAAAWA AAUACGGU ACCG~TAW CUGAUGA X GAA AAWWCW

25 1471 AAUACGGUC CCCUGAAG CWCAGGG CUGA~TGA X GAA ACCGUAW
1485 AAGAUGCUA CCUCAGAC GJCIJGAGG C~GAUGA X GAA AGCAUCW
1489 UGCUACCUC AGACCCCC GGGGG~JCU CUGAUGA X GAA AGGUAGCA
1499 GACCCCCUC CCAUGCAG CUGCAUGG CUGAUGA X GA~ ~i~u~:
1518 r.~r.r.~rrrJ~ CAAGAUGU ACATJCWG CUGAUGA X GAA AGGUCCUC

3 0 153 0 GAUGUGAW AAGCGGGA UCCCGCW CUGAUGA X GAA AuCACauc 1531 AUGUGAWA AGCGGGAA WCCCGCU CUGAUGA X GAA AAUCAC~U
1541 GCGGGAAUC GGAUG~AU Aurrr~rTrr rTlr.~TTGA X ~A:~WCCCGC
1550 GGAUGAAUC UGGAAWG CI~WCCA CUGAUGA X GAA AWCAUCC

35 1560 GGAAWGW GCUGAGW AACUCAGC CIJGAUGA X GAA AC~AWCC

WO 95/31541 r ~ s~
,~ 219~5~3 ~ Tarqet Sequence ~ibozvme Sequence 2 0 ~a~.
~ion 1589 ACCACCGW ArrTrAAAA WWCAGU CUGAUGA X GAA ACGGUGGU

1619 GGUGGAGUC GCCAACUG CAGWGGC CUGAIJGA X GAA ACUCCP~CC

1643 GGGAAA~DU CWCUGCU AGCI~GAAG .CUGAUGA X GAA AGWWCCC

1652 ~:uu~u~ DADrrArrl AGUGGWU CUGAUGA X GAA AGCAGAAG
1691 CCAACUGW CUCGCAGG CCtJGCGAG ClrGAUGA X GAA ACAGWGG

15 1694 ACUGWCUC GCAGGCGU ACGCCUGC CUGAl:JGA X GAA AG~ACAGU

1705 AGGCGUCUC CUGIJGGC~ UGCCACAG CUGAUGA X GAA AGACGCCU
1726 ('rrrAAATTA WCUUACA UGUAAGAA CUGAUGA X GAA AWWGGGG
1728 CCAaAUAW CWACAAG CWGUAAG CUGAUGA X GAA AUAWWGG

1732 AUAWCWA CAAGCUCU AGAGCWG C~GAUGA X GAA AAGAAUAU

25 1744 (x:u~:U~ iuuu UAAUGACA UGUCAWA CUGAUGA X GAA AACAGAGC
1745 ~:u~:uliuuuu AAUGACAC GUGUCAW CUGAUGA X GAA A~ACAGAG
1746 u~:u~uuuu~ AUGAC~CC GGbGUCAU .CIJGAUGA X GAA ~7~AArAr7~
1758 ArArrTTr.TTA UCAGAAGA UCWCUGA CIJGAIJGA X GAA ACAGGUGU
1760 ACCUGUAUC AGAAGA~TG CAUCUUCTJ CUGAUGA X GAA A~ACAGGU
30 1779 GACAAUG~C CUCAAAGC . GCUUUGAG_ CUGAUGA X GAA ACAWGUC
1782 AAUGUCCUC AAAGCCW AArr~rTTTTTT CUGAUGA X GAA AGGAC~UU

1791 AaAGCCUW ACCGUACC GGUACGGU CUGAUGA X GAA AAGGCUW
1792 AAGCC~WA CCGIJACCU AGGUACGG CUGAUGA X GAA AAAGGCW
35 1797 WUACCG~A CC~AAGAA WCWAGG CUGAUGA X GAA ArrrTTAAA
1801 rrr.TTArrTTA AGAACAGG CCUGWCU CUGAUGA X GAA AGGUACGG

nt. Tarqet Sequence Ribczvme Sequen~e tiQrl 1822 Uli~iU~i~U~: CCWGCAG CUGCAAGG C~TGAT~TGA X GAA ACCCACCA
1826 ~iu~:~uu GCAGCCAtT ATTGGCIJGC CUGATTGA X GAA AGGGACCC
1859 GCCAGCAUC CUGUGGGA UCCCACAG CUGA~TGA X GAA AUGCUGGC
1892 r~A~ crTTc cGGl:rccGG CCGGACCG CLTGAUGA X GAa AGGCCG~C
1897 ~:U~::3iiU~ CGGCLTCGG CCGAGCCG CUGAUGA X GAA ACCGGAGG
1903 ~iu~ iG~:u~: GGAAA~AC GLTAW~TCC CUGAT~TGA X GAA AGCCGGAC
1910 UCGGAAAUA CGUGAACG CGWCACG CUGAl:rGA X GAA AWWCCGA
1922 GAACGCGW CLTCAGCUC GAGCLTGAG CUGAUGA X GAA ACGCG~TC
1923 AaCGCGWC UCAGC~CG CGAGCUGA CtTGAT~TGA X GAA AACGCGW

1930 UCUCAGCUC GAACUCUG CAGAG~UC CUGAUGA X GAA AGCUGAGA
1936 CLTCGAACUC U~iiU~:AU(i CAUGACCA CUGAUGA X GAA AGWCGAG
1941 ACUC~TGGLTC AUGUGAGA UCUCACAU CUGAUGA X GAa ACCAGAGU

15 1954 GAGACAWW CCAGAAAA WWCUGG CUGAUGA X GAA ~AUGUCUC
1955 AGACAWUC CAGAAAAG CWWCUG CUGAT~TGA X GAA A~AUGUCU
1967 AaAAGcAw AUGGWW AaAAccAu CUGAUGA X GAA ALTGCWW
1968 A~AGCAWA UGGWWC GAAAACCA C~TGAUGA X GAA AA~TGC~W
1973 AWAUGGW WCAGAAC GWCUGAA C~TGAUGA X GAA ACCATTAAU
20 1974 WAUGGWW UCAGAACA UGWCCGA CUGAUGA X GAA ~A~'rl~TT~2 1975 UAU~i~UUUU CAGAPCAC GLTGWCUG CUGALTGA X GAA ~A~r~TT~
1976 AUGGWWC AGAACACU AGUGWCU C~TGAUGA X GAA A~AACCATT
1985 AGAACACW AhAAGWG CAACWW CUGATTGA X GAA AGUGWCU
19 8 6 GAACACWA AAAGWGA ~CAACUW CUGAUGA X GAA AAGUGWC
25 1992 wAaAAGw GACUWCG CGA~AGUC CLTGAUGA X GAA ACWWAA
1997 AGWGACW UCGACACA UGUGUCGA CUGA~GA X GAA~GIJCAACU
1998 GWGAC~W CGACACAU AUGIJGUCG CLTGA~TGA X GAA AAGLTCAAC
1999 WGACUWC GACACAUG CAUGUGUC C~TGA~TGA X GAA AAAGLTCAA

2053 GCCUGAWW UGWGLTGG CCACAACA CUGAITGA X GAA AA~TCAGGC
2054 CCUGAWW GWGUGGU ~rz~r~ CUGAUGA X GAA AAATTCAGG
35 2057 GAWWGW GUGGUACA UGUACCAC CUGAUGA X GAA ACAaaAUC
2063 ~iuu~iu~ CAACAGW AACUGWG CUGAUGA X GAA ACCACAaC

WO 95~ 41 r~u.. ~S '''^
219~513 Ilt. ~rqet SecTuence Ri~ozYme Sequence 2 0 ~Qa~
2071 ACAA~AGW GAGAGCAG ~GCUCiJC CUGAUGA X GAA ACUGWGU
2092 AAGIJGCAW WWAGWG CAAC~AAA~C~GAUGA X GAA AUGCACW
2093 AGUGCAWW WAGWGC GCAACJAA CUGA~TGA X GAA AAUGCACU
2094 GJGCAWW IJAGWGCU AGCAACI:7A CUGAUGA X GAA AAAUGCAC
5 2095 UGCAWUW AGWGCW AAGCAAC~ CUGAUGA X GAA AAAAUGCA
2096 GCAWI~WA GWGCWG CAAGCAAC C~GA~GA X GAA AAAAAUGC
2099 WUWAGW GCWGAGA ~CUChAGC ~GA~GA X GAA ACUAAAAA
2103 UAGWGCW GAGAUCUC GAGAIJCUC CUGA~:rGA X GAA AGCAACUA
210 9 cwGAGAlrc UCACWGA UCAAGIJGA CUGA~GA X GAA AUCUCAAG

2115 AUClrCACW GAWWCAC G~GAhAUC CUGAUGA X GAA AGUGAGAU
2119 CACWGAW UCACACAA WGUG~GA CUGAUGA X GAA AUCAAGUG
212 0 ACWGA~W CACACAAC awGuGuG CUGA~aA X GAA AAUCAAGU
2121 CWGAUWC ACACAACU AGWGUGU CUGAUGA X GAA A~AUCAAG
15 213 0 ~f Ar?iArrTA AAAAGGAu AIJCCWW CUGAUGA X GAA AGWG~GU
2139 A~AAGGAW UUUUUUUU AAAAAAAA CUGAIJGA X GAA A~:rccwW
2140 AAAGGAWW WW~WA UAAA~AAA CUGAUGA X GAA AAUCCWW
2141 AAGGAU~W WUUWAA WAAAAAA C~GAUGA X GAA AAAUCCW
2142 AGGAuuuuu WUWAAA WWAAAAA CUGA~GA X GAA AAAAUCC~
20 2143 GGAWt~W WWAAAA WWAAAA CUGAUGA X GAA AAAAAUCC
2144 GAWWDW WWAAAAA WUWA~A CUGA~GA X GAA AAAAAAUC
214 5 Au u u u u u u u WA~AAA~ AWUWAA CUGAUGA X GAA AAAAAAA~
2146 uuuuuuuuu UAAAAAUA UAW~WA CUGAUGA X GAA AAAAAAA~
2147 uuuuuuuuu AAAAAUAA WAUU~W CUGAUGA X GAA AA~AAAAA
25 2148 UUUUUUUUA AAAAUAAU AWAWW CUGAUGA X GAA AAA~A~AA
2154 rTrT7~T~T~TTA AUAAUAAU AWAWAU CUGAUGA X GAA AWUWAA
2157 AAAATTAATT~ AUAAUGAA WCAWAU CUGAUGA X GAA AWAWW
2160 DTTAATTA~TTA ATTr~ATTAA WAWCAU CUGAUGA X GAA AWAWAU
2167 TT~ATTr~A~TTA ACAGUCW AAGACUGU CtJGAUGA X GAA AWCAWA

2175 AACAGIJCW ACCUA~AU AwwAGGl:r ~GA~aA X GAA AGACUGW
2176 ACAG~CWA CCUAAAW AAIJWAGG CUGAUGA X GAA AAGACUGU
2180 UCWACCUA AAWAWA UAAUAAW CUGAUGA X GAA Ar.r,TTZ~.'A
2184 ACCIJAAAW AWAGGUA UACCUAA~ CUGAUGA X GAA AWWAGGU
35 2185 crTT~r~r~rTTTA WAGGUAA WACCUAA CUGAUGA X GAA AAWWAGG
218 7 UAAAWAW AGGUAAUG C~WACCU CUGAUGA X GA~ AUAAWWA

WO 95131541 r~,,u~ s -7'~
2~0513 ~t. Tarqet Sequence l~ibo~ylre Sequence 2 0 ~a;
tion 2192 UAWAGGUA AUGAAWG CAAWCAU CUGAUGA X GAA ArrTTzATTA

2212 CCAWWGW A~UAUCAU AUGAUAW CUGAUGA X GAA ACAAAUGG
2213 CAWWGWA ~UATTCAUA UAUGAUAU CUGAUGA X GAA AACA~AUG

10 2221 A~TTATTrATT~ ATTCAGA~TU Az~UCUGAU CUGAUGA X GAA A~TGAUAW
2224 AUCAUAAUC AGAWUW AAAAAUC~T CUGAUGA X GAA AWAUGATJ
2229 AAUCAGAW WWAAAA WWAZ~AA CUGAUGA X GAA AUCUGAW
223 0 AUCAGAWW WWAAAAA WUWAhA C~TGAUGA X GAA AAUC~TGAU

2234 GAWWWA A~AAAAU AWUUUW CUGAUGA X GAA AA~AAAUC
2243 AA~A~AATTA AAAUGAW AAUCAWW CUGATTGA X GAA AWWWW
2251 zzzzrTr,ZrT~T UAWWGUA UACAAAUA CUGAUGA X GAA AUCAWW
20 2252 AAAUGAWW AW~TGUAU AUACAZAU CUGAUGA X GAA AAUCAWW
2253 AAUGAWWA WWGUAW AAUA~aAA CUGAUGA X GAA AAAUCAW

2256 GAWWAWU r.TT~TnnnT~ UAZAAUAC CUGAUGA X GAA AALTAAAUC
2 2 5 9 WAWWGUA WWAGAG C~TCUAAAA CUGAUGA X GAA ACAAAUAA
25 2261 ALTWG~JAW WAGAGAA WCUCUAA CUGAUGA X GAA AUACAAAU
2262 WWGUAWW UAGAGAAU AWC~TC~TA CUGAUGA X GAA AAUACAAA
2263 WGUAWW Zr.Ar.zAT7~ UAWCUCU C~TGAUGA X GAA A~AUACAA

2271 rT~rZr.AATT~ CAZCAGAU AUCLTMlUG CUGAUGA X GAA AWCUCUA
30 2280 CAACAGAUC AGUAWW AA~ATJACU CUGAUGA X GAA AUCLTGWG
2284 AGAUCAGUA TTWWGAC GucAaAAA CUGAUGA X GAZ~ ACUGAUCU

2287 UCAGUAWW WGACUGU ArAr.TTrAA CLTGAUGA X GAA AAUACUGA
2288 CAGUAWW UGACLTGUG CAC}~G~TCA CUGAUGA X GAA AAAUACTTG
35 2289 Ar.TT~TnnnnT GACUGUGG CCACAGUC CUGAUGA X GAZ A~z-aAUAcU
2303 UGGUGAAW UAA~AAAA WWUWA CUGA~TGA X GAA AWCACCA

WO 95/31~41 r ~ ? jfl 2 1 9~5 ~ 3 ~Tarc7et SecTuence RibozYme Sec7ue~ce 2 0 ~Qai.~
tior~ :
2 3 0 4 GG~7U ~ u u u u u u u u CUGAUGA X GAA AAWCACC
2305 GUGAAUtJUA AAAAAAAA uuuuuuuu CUGAUGA X GAA AAAUUCAC
2316 AAaAAAA~U UACACAAA UUUGUGUA C~7GAUGA X GAA AUU7JUUUU

2318 AAAAAW~7A CACA~AGA UU:UUU~:iUb C~7GAUGA X GAA AAAuuuuu 2330 A~Ar.~T7~ UCCCAGUA 17ACUGGGA CIJGATJGA X GAA AUUUCUUU
2332 AGAAAL7A~7C CCAGUAW AAUACUGG CUGAUGA X GAA AUAUI~UCU
2338 AL7CCCAG77A UUCCA~GU ACAUGGAA CUGA~7GA X GAA AC~GGGAU
2340 CCCAG~7A~TL7 CCAL7G~AL7 AUACAUGG CUGAUGA X GAA AUACUGGG
10 2341 CCAG77AIJL7C CA~GUAUC GAUACA7JG C~7GA~TGA X GAA AAUACUGG
2347 uuu:~u~u~ UCUCAGUC GAC~7GAGA C~TGACrGA X GAA Ar~T7Gr.~

2 3 51 AL7GUA~7CUC AG~7CACUA 77AGUGACU CUGAUGA X GAA AGAUACAU
2355 A~7CUCAGUC ~r7T~hT~r~ UGWUAG~ C~GA~GA X GAA ACUGAGAU
15 2359 CAGUCACUA ~r~TT~r~ UGUAUGW CUGAUGA X GAA AGUGACUG
2365 rTT~r~TT~ CACAGAGA ~CUCUG~G CJGA~TGA X GAA A~TGWWAG
2377 ;cr.~r.~r.~TTU WWAAAAA WIlWAaA CUGAUGA X GAA AUCUCUCU
2378 r.~r.~r.~T~T WAAAAAC GWUWAA CUGAUGA X GAA AAUCUCUC
2 3 7 9 AGAGAWW UAAAAACC GGWUWA CIJGAUGA X GAA AaAUCUCU
2û 2380 GAGAWUW AAAAACCA UGGWLlW CUGAIJGA X GAA AAAA~TCUC

2400 r~A~r~rArTTT~ WWGAAU AWCAAAA CUGAJGA X GAA AAUGCWC

2411 WGAA~TGW AGCUAAAU AWWAGC~T CUGAUGA X GAA ACAWCAA

2416 ~TGWAGCUA AAUCCCAA WGGGAW CUGAUGA X GAA AGCJAACA
30 2420 AGCUAAAUC CCAAGUAA WACWGG C~GA~GA X GAA AWWAGCIJ

2433 c~TT~TT~rTTu AAUGC~AC GWGCAW CUGA~GA X GAA AGUAWAC
2434 IJAAUACWA AUGQACC GGWGCAU CUGAUG~ X GAA A~GUAWA
35 2445 GQACCCUC UAGGAGCU AGCUCCU~ CIJGA~GA X G}~ AGGGWGC
2447 AACCC~TCUA GGAGCUCA UGAGCUCC CUGAUGA X GAA AGAGGGW

WO 95/31~41 . ~ , '7' 2 1 9 ~ 3 n~ T~rqe~ Se~Tuence RibozYITe Se~Tuence ~osi-tion 2454 UAGGAGCUC AWWGUGG CCACAAAU CUGAUGA X GAA AGCTJCC~A
2457 GAGCUCAW UGUGGCUA UAGCCACA CUGAUGA X GAA AUGAGC~TC
2458 AGCUCAW~T GITGGCTJAA WAGCCAC CUGAUGA X GAA: AAUGAGCU

2468 UGGCUA~UA AUCWGGA UCCAAGAU CUGATJGA X GAA AWAGCCA

2480 WGGAAAUA UCT~WAW AAUA~AGA CUGAUGA X GAA AWUCCAA
2482 ~ ATT~TT~' WUAWAU AUAAUAA~ CTJGA~TGA X GAA ~TTATTnur~

2 4 8 5 AAUAUCUW AWA~AUA UAUAUAAU CUGAUGA X GAA AAGAUAW
2 4 8 6 AUAIJCUWA WAUAUAG CUAUAUAA C~TGA TGA X GAA Aa~aIJAU

2489 UCUWAWA UAUAGCAU AUGCUAUA CUGA~GA X GAA 1~1~TT~
2491 TTTTTT~TTTT~TTA TT/~('.'~'ATTl,TU AAa~cuA ~'TT(~ TT ~ TTA~TT~A1 2493 ~TAWAUATJA GCAWWAU AUAAAUGC CUGAUGA X GAA ATT~TT~TT~
2498 UAUAGCAW UAUGAGGA UCWCAUA CUGA~GA X GAA AUGCUAUA
2499 ATJAGCAWW AUGAGGAG CUCCUCALT ~UGAIJGA X GAA AAJGCUAU

2510 I'.Art.~ TT~T WGWGUC GACAACAA CUGAUGA X GAA AUCTTCCTTC

2512 GGAGAWW GWGUCAG CUGACAAC WGAUGA X GAA A~AUC~CC
2515 GAWUUGW GUCAGCW AAGWGAC CUGAUGA X GAA ACAA~AUC
2518 uuu~,uu~u~ AGCWGCU: AGCAAGCU CUÇATTGA X GAA ~ A~
2523 UGUCAGCUU GCWGA~A WWCAAGC CUGATTGA X GAA AGCUGACA
2527 AGCWGCW GAAAGWA UAACUt~UC CUGATJGA X GAA AGCAAGCU

2535 IT~ At~.TTTT~ UUAUGUAU AUACA~TAA CUGAUGA X GAA AACWWCA
2537 . AAAGWAW ~TTGTT~TTt~.~ UCAUACAU WGAUGA X GAA AUAACWW
2538 A~ÇWAWA UGUAUGAA W~AIJACA WGAIJGA X GAA AAUAACW
2542 TTI~TTTJ7~TT .TT~ TJGAAUAGU AWAWCA CUGATJGA X GAA A('ATT~TT~
2548 GUAUGAAUA GWWAW AAUA~AAC WÇAUGA X GAA AWCAUAC
2551 UGAAUAGW WAWGAA WC~AUAA CUGAUGA X GAA ACUAWCA
2552 GAATJAGWW UAWGAAA WWCAAUA CUGAUGA X GAa AACUAWC
2553 AAUAGWW AWGA~AA WWCAAU CUGAUGA X GAA A~ACUAW
2 5 5 4 AUAGWWA WÇAAAAA WUWCAA WGAUGA X GAA AAAACUAU

WO 95/3~541 r~
~ 21~05~3 -nt. TarcTet secTuenCe T~;hr-7yne Sequence 2 0 ~Q~
2556 AGUUWATTU GAAAAAATT AT~WWUC CUGAUGA X GAA AUAAAACU
2565 ~ rTIT AuAuWWU AAAAAUAU CUGATJGA X GAA AWWWC
2566 ~ TTAW~WA UAAAAAUA CUGAUGA X GAA A~WWW

2 5 7 0 AAWAUAW UWAWCA ~GAAUA~A CUGAUGA X GAA AUAUAAW
2571 AWAUAWW WAWCAG CTTGAAtTAA CUGATTGA X GAA AA~AUAAtT
2 5 7 2 WAUAWUU UAWCAGU ACUGAA~TA CtTGA~TGA X GAA AAAUAUAA
2 5 7 3 UAUAWWU AWCAGTJA ~TACtTGAAU CUGAUGA X GAA AA AAUAUA
2574 ATJAWUWA WCAGTTAA TJ~TACUGAA CUGATTGA X GAA AAAAAUAU
10 2576 AWUWAW CAGTJAAW AATJIJACUG CUGATTGA X GAA AtTA~AAU
2577 WUWAWC AGUAAWW AAAT~UACU CUGA~GA X GAA AAUAAAAA
2581 TJAWCAGUA AUWAAW AAWAAATJ CUGA~GA X GAA ~rTJr.~TT~
2 5 8 4 UCAGT TAAW UAAU~WG CAA AAUUA CUGAT TGA X GAA AWACUGA

15 2586 AGUAAWWA Auuu~JGUA UACAAAAU CUGAtTGA X GAA AAAWACU
2589 AAWUAA~tT WGUAAATT AWUACAA CUGATTGA X GAA ATJUAAAW
2 5 9 0 AWWAAWU UGUAAAUG CAWWACA CUGAl:JGA X GAA AAWAAAU
2591 T~WAAWW GUAAAUGC GCAT~WAC CTTGATTGA X GAA A~AWAAA
2594 AAWWGUA ~TTGrr~A WGGCAW C~TGATJGA X GAA ACAAAAW

2625 UL:G~:U~ UA UGGWUUA T~ACCA C[TGATTGA X GAA AGCAGCGA

25 2632 UAUGGUUW AGCCUAUA T~TATJAGGCU CUGAUGA X GAA AAACCATTA
2633 ATTGGWWA GCC~TAUAG CUAUAGGC CUGAUGA X GAA AAAACCAU
2638 WUAGCCUA UAGUCAUG CAUGACUA CUGAUGA X GAA AGGC~AAA
2640 TT~r.rrTT~rT~ GTTCATTGCU AGCATTGAC C~TGAUGA X GAA AUAGGCUA
2643 CCUATTAGUC A~TGCUGW AGCAGCAU CUGA~TGA X GAA ACUAUAGG

2656 TJGCUAGCUA G~TGUCAGG CCUGACAC CUGATTGA X GAA AGCUAGCA
2661 GCUAGUGUC ~r~r~r~Gr~r~ TTGCCCCCU CUGAUGA X GAA ACACUAGC
2672 GGGGCAAUA GAGCWAG CTTAAGC~TC CUGAUGA X GAA AWGCCCC

35 2679 TT2~r.~r.rT,TrT~ GA~TGGAAA TW~CCA~C CUGA~TGA X GAA AAGCUCUA
2703 AAGAGACtTC GGTTGWAG CUAACACC CUGAUGA X GAA AGUCUCW

WO 9~ 4l ~ '7~ ~
21qO513 nl~ T;~qet: Seauence Rlh.~zvme Se~Tuence :
2 0 ~2;
tion 2709 c u~ iu~iuu AGAUAACG CGWAIJCU CUGAUGA X GAA ACACCGAG
2710 U~ iU(lUU~ GAUAACGG CCGWAUC C~GAtTGA X GAA ~ rrr.A
2714 UGWAGATJA ACGGACUA UAGUCCGU CUGAUGA X GAZ~ AUCUAACA
2722 AArr~r~ArTTA UGCAC~AG CUAGUGCA CUGATJGA X GAA AGUCCGW
5 2729 UAUGCACUA GUAWCCA ~GGAAUAC CUGAIJGA X GAA AGUGCA~TA
2732 GCAC~AGUA WCCAGAC GUCUGGAA C~TGATTGA X GAA ACUAGUGC
2734 ArrTAr~TT~TTu CCAGACUU AAGUCUGG CUGA~GA X GAA A~ACUAGU
2735 CUAGUAWC CAGACWW A~AGTJCUG CITGAUGA X GAA AAUACUAG
2742 UCCAGACW WWAW~T AAAT~AAA CTJGATTGA X GAA AGUCUGGA

2744 CAGACWW WAWUW AAAAATJAA CUGA~TGA X GAA AAAGUCUG

2 7 4 6 GACWWWU AWUUWA UAAAAAAU CUGAUGA X GAA ~AAGUC
2747 ACWUUUUA WUUWATJ AUA~AA CUGA~GA X GAA AAAAAAGU
15 2749 WWWAW WUUAUAU AUAUAAAA CUGAUGA X GAA ~rTAAA~AA
2750 WUWAWU WWAUAUA UAUAUAAA CUGAUGA X GAA ~ATTAAAAA
2751 WUUAWW WAUAUAU AUAUAUAA CUGAUGA X GAA AAATTAA~
2 7 5 2 gWAW~w UAUAUAUA UAUAUAUA CUGAUGA X GAA AA AAUAAA
2 7 5 3 WAWWW AUA~AUAU AUAUAUAU CUGA~GA X GAA AAAAAUAA
2û 2754 ~AWWWA UAUAUAUG CAUAUAUA CUGAUGA X GAA AAAAAAlJA
2756 WWWAUA UAUAUGUA UACAUAUA CUGAUGA X GAA ATTAAA1~7~
2758 WUUAUAUA UAUGUACC GGUACAUA CUGAUGA X GAA AlrAuA~A
2760 WAUAUAIJA UGUACCW AAGGUACA CUGA~GA X GAA ATT~rT~TTA~
2764 ATTArTArTr,TTA CCWWCC GGAAAAGG CUGAUGA X GAA ACAUALTAIT

2769 UGUACCUW UCCWWG CAAAAGGA CUGA~GA X GAA AAGG~ACA
2770 G~ACCWW CCWWGU ACAAAAGG CUGAUGA X GAA AAAGGUAC
2771 UACCWUUC CWWGTJC GACAAAAG CUGA~GA X GAA AAAAGG~A
2 7 7 4 c u u u u c~: u u WGUCAAU AWGACAA CUGAITGA X GAA AGGAA AAG
30 2775 UUUUCLUUU UGTJCAAW AAWGACA CUGAlrGA X GAA AAGGA~AA
2776 TJIJUCCUUUU GUCAAWG rAATT~Tt~Ar CUGAUGA X GAA AAAGGAAA
Where "X" represent~ stem II reg1on of a HH ribozyme (Hertel et al., 1992 Nucleic Acids Re~. 20 3252). The 35 length of stem II may be 2 2 base-pairs.

WO 95/31541 P~l;u.,,., ~~' 2~05~3 Table XVI ~ouse c-mvb Hair~in ~ibQ~yme and tarqet serUence8 Posi- RZ Su~strate tion ~rr~r.~r.~z~Ar~r7~ .UU~iU~ Ar~TlTT~rCUGGUA CGCCUCGC

~rc~r-~n~hr~r~ IU~iU~ UACAWACCUGGUA UCGCCAUG
122 AU~TGGGC AGAA GCCCA~T ~ AUGGGCU GCU
~rrz~r.~n~r~r~ lu~ llLrliTTTTArc~uGGn~ GCCCAAAU
12 5 CAGAUU~TG AGAA GCAGCC GGCUGCU GCC
Arrzln?~r.z~ r;~ A~ ~iUutiuliliuAcAwACCUGGUA CAAAucuG
216 WCCAGUC AGAA GT~TCCG CGGAACA GAC
~rr~r~Ar~AcAcA~ uu(iu~ IL.r~n~rArrTTr7r,n~ GACUGGAA
lû 245 UCCGGWG AGAA GATTAAU AWAUCU GCC
~rrAn~r~ r~r~ uu~iu~ uAcAwAccuGGuA CAACCGGA

Arr~ r.~r~r~ uu~ l~r~rTTT~rrr-Tr~MT~ GUACAGUG
529 CUCUGCCC AGAA G~TCCC GGGAACA GAU
~rr~r~ r~ l u~iu~ r~nTTArr~TGG~TA GGGCAGAG
551 GUCCGGGC AGAA GCW~TG CAAAGCTT GCU
~rr~r.~r.~A~r~P~ JUliu~ ~r~TTTJ~rrTTr~r~TT~ GCCCGGAC
554 UCCG~TCCG AGAA GCAGCU AGCUGCU GCC
~rr1~r.~r.~rArT~ iUu~.u~ r~, lu~i~r:ou~ uA CGGACGGA

z~rr1~r~r~D~r7~r~ lu~iu~ uAcAwAcc~TGGu~ GGACUGAU

Arr~r~ Ar~r~ ~.l lU~ju~ TTTT~rr~Tr.GTT~ UGAUAAUG

~rr~n~r.3~r~r~ uu~iu~iiuAcAT~TT~rrTTGr~TT~ GCCAGUGG

~rr~r.~r.~r~r~, Iliuutiu~iiuAcAuTT~rrTTr~r~TT~ CUCUCCAA

z~rr7 r~ r~r~ .l luriu~ibuAcAuTT~rrTTGrTTA CUCCGrTCA
2 0 8 2 2 UGCAAUGC AGAA GGAtTAG ~ CUAUCCU GUC
~rr~r.~nP~r~r~ i,luriuiiu~LCAWACCUGGUA GCAWGCA

WO 95/31541 2 1 9 0 5 1 3 .--857 CCGCAGCC AGAA GAGGGA ~ UCCCUCA GCC
Arr~r.Ar.AAArArA~ lu-ju~ uACAWACCUGGUA GGCUGCGG

~rrAr~Ar~AAArArA( (il lurjurjrjuAcAuTTArrTlr~rjrTA GCGGCAGC

ArrAr.AriAAArArA~ u~ju~ uACAWACCT;~GGUA GUCAACAG
1040 GAGGUCUG AGAA GGUCCA . . UGGACCA GAC
ArrAr.Ar.AAArArA~ ,juu-ju~ uACAUrTArrrTr.r.TTA CAGACCUC

ArrAAr~T ArArA~ u(.-illArATTTTArCUGGUA CUCAUGGG
1068 AAArAr.r.A AGAA GGUGCA . . _ . UGCACCU GUU
ArrAr.Ar.AAArArAr ~illurjurirjUACA~ACClJGGUA UCCUGWW
1075 WCUCCCA AGAA GGAaAc : GWWCCU GW
ArrAr.Ar.AAArArA~ Iiuu~ju~ uACAWACCUGGUA UGGGAGAA
1106 GAUCUGCA AGAA GAGAUG . . CAUCUW GCC
Arr~r~Ar~DADr~rAll~illurju~ uAcAuTTArrTTr~r~TJA UGCAGAUC
1113 GAGCCGGG AGAA GCAGGC ._ GCCUGCA GArT
ArrAr.AriAAArArA~ u~ju~ uACAWACCUGGUA CCCGGCUC
lû1120 AGGUAGGG AGAA GGGAUC . . GAUCCCG GCU
ArrAr.Ar.PAArArA~ lu~ju~ uACAWACCUGG17A CCC~ACCU
1226 AAUCUAUA AGAA GGAGUG . CACUCCA GW
Arr~r~Ar~AAAr~rA~ lilJurjlJljrjllAr7`rTuAccuGGuA UAUAGAW
1340 WWCACA AGAA GGUCUC : GAGACCA GAC
Arr~r.Ar.PAArArD~ lu~ i[il ~ArArTTTArrT-TGGTTA UGUGA~AA

ArrAr.ArAAArArA- ~jlJu~ju~ uACAWAcclTGGUA CAAGAAAU

ArrAriAr.AAArArA( -il lu~ju~i[il l~rATTTTArrrTr.GUA CCCUGAAG

Arr1\r.Ar.AAArArA~'~iUU~jU~ UACAWACCIJGGUA CCCCUCCC
1542 CCAGAWC AGAA GAWCC ~ GGAAUCG GAU
ArrAriAr.AAArArA, ~(jl lu~ju~j:il ,ArATTTTArrrTr.r.rTA GAAUCUGG ~ ~
1648 GUGGWWG AGAA GAAGAA : WCWCU GCU
Arr~riAr~ADArArA~ ~iulllil~ lArATTTTArrTTGGuA CAAACCAC
1672 GGUGCUCA AGAA GWCUC . : ~ GAGAACA GCC
ArrAriAr.AAArArAI lil ~ iu~ lArATTTTArrTrGGuA UGAGCACC

WO 95/31!i41 r~ s~
2t90513 Arrl~r.Ar.AAArArA~ U~iU i~iUACAWACCUGGUA CUCGCAGG

ArrAr.Ar.AAArAr~ Juliu~ uAcAwAccuGGuA GCCCCAAA

ArrArArAAArArA~ lU~iUli~iUACAWACCUGGUA WAAUGAC

Arr~r.Ar.A7~ArArAI ~il lu~ rl~TTuAccuGGuA GACGGCCU

ArrAr.Ar.ADl\rArAI ~UUriU(i~iUACA7~T7LrrTJr~r~TTA UCCGGUCC
1894 CCGAGCCG AGAA GGAGGC : GCCUCCG GUC
ACrAriAriAAArArA' ~iUU~iu~i~iuACAWACCUGGUA CGGCUCGG

AcrAr~ArAAArArA~ (iuu~iu~ 5~rATTTlArruGGuA CGGAAAUA
19 2 6 AGAGWCG AGAA GAGAAC ~ GWCUCA GCU
ArrAr.Ar.AAAr~rA- -jUU~jU'j~jUACAWACCUGGUA CGAACUCU
2048 Arl~ArZS1~ AGAA GGr~Tr~7 AGAGCCU GAU
ArrAr.AriAAArArA- ~;UU~iU~i~iUACAWACCUGGUA WWGWGU

ArrAriAr~DAArArA~ lu~iu~ uAcAuTTArr-7-TGGT7A GAGAGCAG

ArrAr.Z~r.AAArArA~~;UU~ rATTrTArrTTr.r.r7A WACCUAA

ArrAr~ArAAArArA~ ~;uuriuri~iuAcAwAccuGGuA WUUUAAA
2276 AAAUACUG AGAA GWGlrA UACAACA GAU
ArrAr~AriAAArArA~ Iriu~ uA--rAwAccr~rGGuA CAGUAWW
2519 WCAAGCA AGAA GACAAC GWGUCA GCrr Arrpr~Ar7~AArArA~ ~iuu~iuri~iuAcAwAccuGGuA UGCWGAA

ArrAr.Ar.~AArArA~ ur,uri(iuAcAwAccuGGuA UAUGCACU
2737 ATTAAAAAA AGAA GGAA7rA UAWCCA GAC
ArrArAriAAAr~rA~ '5iUU~iU(~ rATTrTArrTTrr,rTA WUUWAU

WO 9S/31541 2 1 ~ ~ 5 1 3 i ~~ ?ro Table XVII: Rat c-mYb (Reqion A) Hammerhead R; hnzvme and T~rqet Seauences (282 b~: nt. 428 start: human c-mYb nllmherinq gYgtem) nt. Target Sequence Ribozyme Sequence 5 Po~i-tion 467 CCUGAGCUC A~CAAAGG CCUWGAU CUGAUGA X GAA AGCUCAGG

477 UCAAAGGUC CCUGGACC GG~CCAGG CUGAUGA X GAA ACCWWGA
l O 4 9 8 AAGAAGAUC AAAGAGUG CACUCWW CUGAUGA X GAA AUCWCW
509 AArUr~TT~ GAGCWGU ACAAGCUC CUGAIJGA X GAA A~CAC~CU

518 GAGCWGUC CAGAAAIJA TJAW~CUG CUGA~rGA X GAA ACAAGCUC
526 ('t'1~ ATTZ~ CGGIJCCGA UCGGACCG CUGAIJGA X GAA AWWCUGG

544 G~ u(iiiuu: UGWAWG CAAUAACA CUGAUGA X GAA ACCAGCGC
548 U~iU~:U~UU AWGCCAA WGGCAAU CUGAIJGA X GAA ~r~r.Arr!~
549 ~U~:U~iUU~ WGCCAAG CWGGCAA CUGAUGA X GAA AACAGACC

20 562 CAAGCACW AA~Ar.~ UCCCUUW CUGA~GA X GAA AGUGCWG
563 AAGCACWA AaAGGGAG CUCCCUW CUGAUGA X GAA AAGUGCW
575 GGGAGAPW GGAAAACA ~GWWCC CUGAIJGA X GAA AWCUCCC
588 AACAAUGUC GGGAGAGG CCUCUCCC Cl~GAUGA X GAA ACAWGW
609 ~rA~rrATTU UGAA~CCA IJGGAWCA CUGAUGA X GAA AUGGWGU
25 610 CAACCAWW GAAUCCAG C~GGAWC CUGA~GA X GAA AAUGGWG
615 AWWGAAUC CAGAAGW AAC~UCUG CUGAUGA X GAA AWCAAAU
623 CCA~AAGW ~r~D~r GWWCW CUGA~GA X GAA ACWCUGG
624 CAGAAGWA AGAAAACC GGr~uuuCU CUGAIJGA X GAA AACWCUG
634 GAAAACCUC ~UGGACAG CUGUCCAU CUGAUGA X GA~ AGGWWC
3 0 65 9 GACAGAAJC ~AIJCA UGAUAAAU CIJGAUGA X GAA AwcuGl:rc 662 AGAAUCAW IJAUCAGGC GCC~GAUA CUGAUGA X GAA AUGAWCU

664 AA~CAWWA UCAGGCAC GUGCCUGA C~GAUGA X GAA AAAUGAW
6 6 6 UCAWUAUC AGGCACAC GuGlrGcc~ CIJGAIJGA X GAA AUAAAUGA

Where "X" represents 3tem II reglon of a HH ribozYme (Hertel et al., 1992 Nucleic Acid3 Res. 20 3252). The length o~ 3tem II may be 2 2 base-pair3.

WO 95/31541 ~ ~,11~ ?p 2l9~513 ~Rhl e XVIII; Rat c-mYb (Reqion B) Xamm.erhead Ribozvme and Tarqet Se~uences (262 bP; nt. 1421 start: human c-mvb nllmhe~inq sv8tem) nt . Tarqet Seauence R i h~zvme Secruence poci -tion 1429 CUCGGG~U AGAUACGC GCGUAUCU CUGA TGA X GAA AGCCCGAG

10 1434 GC~UAGAJA CGCCUACU AGUAGGCG CUGAUGA X GAA AUCUAAGC
1440 ~TT~rr~rrTI~ CWUACCC GGGlJAaAG CUGA~GA X GAA AGGCGUAU
1443 CGCCUACUU UACCC~CC GGAGGG~A CUGA~GA X GAA AGIJAGGCG
1444 GC~ACI~lU ACCCUCCA UGGAGGGU CUGAIJGA X GAA AAGUAGGC
1445 CCUACUUUA CCCUCCAC GUGGAGGG CUGA~GA X GAA AAAGUAGG

1460 ACGCCUC~TC AWGG~CA IJGACCAAU CUGAUGA X GAA AGAGGCGU
1463 CCUCUCAW GGUCACAA WGUGACC cuGAl:rGA X GAA AUGAGAGG
1467 UCAWGGUC ACAAACUG CAGUUUGU CUGAUGA X GAA ~r~TT~'~
20 148~ CACCGUG~C ACCGAGAC GUCUCGGU CUGAUGA X GAA ACACGGUG
1509 UGAaAACUN ~A~ WCCUUUU :CUGAUGA X GAA AGT~UUUCA
1522 GGAAAACUC NAUCUWA uAaAGAuN CUGAUGA X GAA AGWUUCC
1526 AACUCNAUC WWAGAAC GWClrA~A CUGA~GA X GAA AUNGAGW

25 1529 UCNAUCWW AGAACIJCC GGAGWQr CIJGAUGA X GAA i~'.DT~r:A

1536 WAGAACUC CAGCUAIJC GAUAGCUG C~GAUGA X GAA AGWCUAA
1542 rTTcr~r:rTT~ UCA~AAGG CCIJUWGA CIJGAIJGA X GAA AGCUGGAG

3 0 1552 r~ ~TT~T AAUCCUCG CGAGGAT~U CUGAUGA X GAA ACCUUWG
1556 AGGU~AAUC CUCGAAAG CUUUCGAG CUGATTGA X GAA AWNACCIJ
1559 UNAAUCCUC GA~AGCUC GAGCUWC CUGAUGA X GAA AGGAWNA

35 1578 CCAGAACUC CCACACCA UGG TGUGG C~TGAUGA X GAA AGWCUGG

1589 ACACCAWC A~AQUGC GCAUGWW CJGAUGA X GAA AATJGGIJGU

1616 CAAGAAAW AAAUACGG CCGUAWU CUGA~GA X GAA AWWCWG

WO 95~31541 P~ r -~

n~ Tarqet Seq~lence ~ibszvme Se~Tuence Po~ i -1429 rrTrrr~rrTTTT AGA~ACGC GCGUA~rCU CUGAUGA X GAA AGCCCGAG
1617 AAGAAAWA AAUACGGU ACCGUAW C~rGAUGA X GAA AAUWCW
1621 A~WA~AUA CGGUCCCC GGGGACCG ~TGAUGA X GAA AUWAAW
1626 AAUACGGUC CCCUGAAG CWCAGGG crTGAuGA X GAA ACCGUAW
1640 AAGAUGCUA CCUNAGAC G~CUNAGG CUGAUGA X GAA AGCA~TCW
1644 ~GCUACCUN AGACCCCC GGGGG~rcu crrGAuGA X GAA AGGUAGCA
1654 rArrrcrTTI~T UNAIJGUAG CUACAU~A CUGA~GA X GAA AGGGGGUC
1656 ( r (,'~ AUGUAGUN NAcr-rAcAu cT-rGAuGA X GAA ANAGGGGG
1661 ~NAIJG~rA GUNNNANA U~VNNNAC CUGAUGA X GAA ~r~TTN~l~T~
1664 NAUGrrAGr~N ~NANACCU AGGUNUNN CUGAUGA X GAA ACUACAUN
10 1673 T~T~T~rrTTl\T CANGAUGU ACAUC~UG CUGAUGA X GAA AGGUNUNN
Where rX" represents stem II region of a HH ribozYme (Hertel et al., 1992 ~7ucleic Acids Res. 20 3252) . The length o~ stem II may be 2 2 base-pairs.
T;~hle ~Tl~ Rat c-mvb (Reqion A) Hairr~in Ribozyme and T~rqet Sequences ~282 bT~: nt. 428 start: human numberinq sYstem) Posi- RZ Sub~trate 20 tion 528 GCrCWCG At~AA GUAWW AAAUACG GUC
~rr~r.~r~r~r~ uui~u~ uAcA~TT~rrtTr~r~TT~ CGAAGCGC

~rr~r.~r.~r~r~ .U~ J(~ r~TTTT~rrrTGrrT~ GGGCAGAA

~ WO 95/31541 . ~~ S.C ~'^
21q~5~3 Table XX: Rat c-mYb (Reqion B) Hairpin Ribozvme and Tarqet Seauences (262 b~. nt. 1421 start: human numberinq svstem) Posi- RZ SubGtrate - tion ~rrAr.~r.~rArA-l-.u~ r~TTuAccuGGuA UGUGAAAA
1604 AUWCWG AGAA GCCAGG : : CCUGGCA GCU
ArrAr~Ar~ rAr~ uu~,u~i~,uAcAl~uAccuGGuA CAAGAAAU

~rr~r.~r.~r~rA- ~iuu~-u~-(-ll~r~TTuAccuGGuA CCCUGAAG
10 Table ~S~T: Por~ n~ c-mYb (Reqion A) Hammerhead RibozYme ~nfl Tarqet Se~uence (266 b~. nt. 458 start, human c-mvb numberinq svstem) ~, Tarqet Ser~ence Rlboz~me Se~uence ~Q~
~}
467 CCUNAUCUC AUCAAGGG CCC~UGA~ CUGAUGA X GAA AGAUNAGG
~70 NAUCUCAUC AArGGucc GGACCCW CUGAUGA X GAA AUGAGAUN
477 ur~r-r,rtTc CUUGGACC GGUCCAAG CU~aUGA X GAA ACCCWGA
4 8 0 prrr,TTrrTTrT GGACCAAA UCUGGUCC CUGAUGA X GAA ArGAcccu 498 D~a~i~ri~TTr AGAGAGUG CACUCUCU CUGAUGA X r,AA AIJC~CW
509 ~ri~rTTri~TT~ GAGC~nUGU ACAAGCUC CUGAUGA X GAA AUCACUCU
515 AlJAGAGCW GUACAGAA WC~GUAC CUGAIJGA X GAA AGCUCIJAIJ
518 ri~rrwrTT~ CAGAAAUA uAr~wcuG CUGAUGA X GAA ACAAGC~IC
526 ~r~r.A~TTII CGGUCCGA UCGGACCG cuGAIrGA X GAA AWWCUGU
531 AauAcGGuc CGA~ACGU ACGW~CG CrJGAUGA X GAA ACCGUAW
540 rr~rr,TTrT GGUCUGW Ah~GACC CUGAUGA X GAA ACGWWCG
544 ACGWGGUC UGWAWG C~AUAACA CUGAUGA X GAA ACCAACGU
548 u-~ u AWGCCAA WGGCA~U CUG~UGA X GAA ACAGACCA
549 GG~CtJGWA WGCCAAG CWGGCAA CUGAUGA X GAA AACAGACC
551 UrrTrTTTT~TT~T GCCAAGCA UGCWGGC CrJCAUGA X GAA AUAACAGA
562 r~ r~rTTrT AAAGGGGA UCCCCI~W crJGAuGA X GAA AGUGCWG
563 ~rr~rTTTTA AAGGGGAG CUCCCCW cuGAlrGA X GAA A~GUGCW
575 rr~r~ATr~T GGA~acA UGWWCC CUGAUGA X GAA AWWCCC
588 ~r7\~'Tr,TT~ GGGAGAGG CCUCUCCC CUG~UGA X GAA ACAWGW
603 rirTTrrr~TT~ ACCACWG Ca~GUGGU CUGAUGA X GAA AUGCCACC
610 UAACC~CW GAAUCCAG CUGGAWC cu~a x GAA AGUGGWA

WO 95131541 T~,l;l 2~90513 -TarcTet sequence Ribozyme seouence E'0 5 i -15~
615 ~CWGAAUC CAGAaGUU AACWCUG CUGAUGA X GAA AWCAAGU
623 CCAGAAGW AaGAMAC GUr~WCUU CUGArJGA X GAa ACWCUGG
624 r~r.~r.rTTT~ AGAMACC GGUUWCU CUGAUGA X GAA AACWCUG
634 GAMACCUC CUGGACAG . CUGUCCAG CUGAUGA X GAA AGGWWC
5659 GACAGAAW AWWACCA UGGUA~AJ CUGAUGA X GAA AWCUGUC
660 ~r~n~ TlTTZ~ WWACCAG CUGGUAi~A CUGAUGA X GAA AAWCUGU
662 ~r.l~TlTT~TTU UACCAGGC GcC[rGGUA CUGAUGA X GAA AUAAWCU
663 GAaWAWW ACCAGGCA UGrC~GGlJ CUGAUGA X GAA AAUAAWC
664 ~''TTTTATTUTTI~ CCAGGCAC GUGCCUGG C[TGAUGA X GAA AhAUA~W
10704 r7rr.r.~ TTC GCAaAGCU AGCUWGC CUGAUGA X GAA AWWCCGC
713 r,r~rrTTI~ CUGCC[TGG CC~G~CAG CUGAUGA X GAA AGCUWGC
Where "X" represents 3tem II region of a ~IE ribozyme ~Hertel-et al., 1992 Nucleic Acids Res. 20 3252) . The 15 leIlgth of stem II may be 2 2 base-pair3.
Table XxTI: Porcine c-mvb ~RecTion B) Hammerhead Ribozvme An~ Tarc~et Secruence t308 b~: nt. 1386 start: human c-mvb numbe~incr svstem) 2 0 ~, Tarqet Seruence RibozYme Seruence ~Q~ai~
tio~
1394 GAWCWWC WAaACAC GUG~WAA CUGAlrGA X GAA Al~AGAAUC
1396 UUL:UUU8UU AAACl~CW AAGUGWW CUGAUGA X GAA AGAAAGAA
251397 UC:UuU~:UUA AAQWUC GAAGUGW CUGAUGA X GAA ~z~r~hr~
1404 UAAACAWW CCAAUAAC GWAWGG WG~UGA X GAA AGUGUWA
1405 AAACACWC CAAUAACC GGWAWG CUGAUGA X GAa AAGUGWW
1410 WWCCAAUA ACCAUGAA WCI~rGGU CUGAUGA X GAA AWGGAAG

301424 r~ 7~z~rrTrT~ GACWGGA UCCaAGUC CUGAUGA X GAA AAGWWC
1429 CWAGACW GGAaAUGC GCAUWCC CUGAUGA X GAa AGUCUAAG
1440 AAAUGCCW CUWAACG CGWA~AG CUGAlrGA X GAa AGGCAUW
1441 l~AUGCWWC WUAaCGU ACGWA~iA CUGAUGA X GAA AAGGCAW
1443 UGCWWCW UAACGIJCC GGACGWA CUGAUGA X GAA ~r~ rr~
351444 G~uu~:uuu AaCGlJCCA UGGl~CGW CUGAUGA X GAA AaGAAGGC
1445 CC:UUc:uuuA ACGUCCAC GUGGACGU CUGAUGA X GAA A~AGAAGG

WO 9S/31541 r~
21'~0513 1450 W~AAC~TC CACGCCUC GAGGCGJG C~TGAUGA X GAA ACGWAAA
1453 CCACGCC~TC IJCAGUGGU ACCACUGA CTJGA~TGA X GAA AGGCGIJGG
1460 ACGCCIJCUC AGTJGGTJCA TJGACCACU C[TGAITGA X GAA AGAGGCGU
1467 UCAGTJGGUC ACAAAWG CAATJWG~T CITGAIJGA X GAA ACCACUGA

l4al WGACUGW ACAACACC GGIJGWGU CUGAUGA X GAA r~7~'.rTf~

1492 A~CACCAW TJCAUAGAG CTJCTJAUGA CUGAUGA X GAA AUGGUGW
1493 ACACCA1JUU CATJAGAGA ~TCTTCUAUG C~JGAUGA X GAA AAUGGUGU

153 0 p~r~ TTA CA~TAWW AAAATJAUG CUGATTGA X GAA AWUUCCU
1534 DA~TT~nZ~ WWUGA~ TLrOCAAAAA CTTGAUGA X GAA AUGUAWW
1536 ATJACAln.W WWGAACU AGWCAAA CUGAIJGA X GAA AUAUGUAU
15 1537 UACATTAWW WGAACUC GAG~JCAP~ CTJGAlrGA X GA~ AAUAUGUA
153 8 ACAUAW[lU TJGAACTJCC GGAGUCCA CTJGATTGA X GAA AAATTA~TGU

1545 WWGAACUC CGGCUAIJC GATJAGCCG C~GATTGA X GAA AGWCAAA
1551 CUCCGGCUA UCAAAAGG CCWUTTGA C~TGATTGA X GAA AGCCGGAG

1561 CAAAAGGTJC AATJCCUGG CCAGGAW CUGA~TGA X GAA ACCWUUG
1565 AGGUCAATTC CUGGA~AG CWUCCAG CUGAUGA X GAA AWGACCU

1578 AAz~ ~TcrTf~ CAAGAACtJ AGWCWG CUGAUGA X GAA AGAGCtlW
25 1587 CAAGAA~UC CUACACCG CGGTJGUAG CtJGAT~TGA X GAA AGWCWG
1590 t~ r~rCTJ;I~ CACCGWC GAACGGTJG CUGAUGA X GAA AGGAGWC
1597 UACACCGW CAAACA~TG CAUGUWG CUGA~TGA X GAA ACGGIJGUA
1598 ACACCGWC Ai~ACATTGC GCATTGUW CUGAUGA X GAA AACGGUGIJ
1610 CA~GCACtJC GCAGC~JCA UGAGCUGC CUGAUGA X GAA AGUGCAUG
3 0 1617 UCGCAGCUC AAGA~\AW AAWWCW CUGAUGA X GAA AGC[TGCGA
1625 CAAGAAAW AAATTATJGG CCAUAWW C~TGATJGA X GAA AWWCWG
1626 AAGAAATJUA AATJATJGGU ACCA~TAW CUGAUGA X GAA AAWWCW
1630 ~2~TTTTI~ TTZ~ UGGUCCCC GGGGACCA CtJGAlJGA X GAA AWWAAW
1635 AAUAUGGUC CCCUGAAG CWCAGGG C[TGAUGA X GAA ACCAUATJU
35 1649 ~ TTr::~~TTZ~ CCUCAGAC GUCUGAGG CTTGATTGA X GAA AGCAUCW
1653 UGCUACCUC ~ IJGGUGUCU CUGAUGA X GAA AGGTJAGCA
1663 GACACCAUC UCAWWAG CUA~ATJGA CUGAUGA X GAA ATJGGUGUC
1665 CACCA~CTTC AWWAGTJA TJACUAAATT C~JGATTGA X GAA AGATJGGUG

_ _ _ _ _ WO 95/31541 2 1 ~ 0 5 1 3 1669 AUCUCAWW AGUAGAAG c~:rcuAcu CUGAUGA X GAA A~UGAGAU
1670 UCUCAWWA GUAr~AGA UCWCUAC CUGAUGA X GAA A~TTr.~r~
1673 CAWWAGUA GAAGACCU AGGUCWC CUGAUGA X GAA ACUA~AUG
5 Where "X" represent5 stem II region of a HH ribo~yme ~Hertel et al., 1992 Nucleic Acid8 Re9 20 3252) The length of stem II may be 2 2 base-pairs Table XXIII: Porcine c-myb (reqion A) ~; rT~in RibozYme lO and T~rqet Seruence (266b~ nt 45~ start: Human nuTnberinq qYStem) Posi- RZ Substrate tion 528 ACGWWCG AGAA GUAWU ~ _ A~AUACG GUC
~rr~r.~r.~r~r~ Uu~iu~ uAcAuTT~rrTJGr7TTL CGA~ACGU

~rr~r.~r~r~[ ~ u~u~ uACA~TT~r~lTr.rTJ~ GGGCGGAA
Table :X-XTV: Porcine c-mYb (reqion B) Hair~in RibozYme and Tarc~et Seruence ~30~ b~: nt 1386 start Human numberinq 20 svstem) Posi- Hairpin Ribozyme Substrate tion 15û4 WWQCA AGAA GGUCUC GAGACCA GAC
z~rr~r.~r.~r~r~ .u~ 7~ rl~TTTT2~rrTTGGUA UGUGAhAA

~rr~r.~r.~A~r~r~ lJU~,U~ r~rTTT~rrTTGGUA CAAACAUG

~rr~r.z~r.~r~r~ ,u~iuAcAwAccuGGuA CAAGA~AU

Claims (28)

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 a 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.

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'-O-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' -O-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.