ENZYMATIC NUCLEIC ACID TREATMENT OF DISEASES OR CONDITIONS RELATED TO LEVELS OF EPIDERMAL GROWTH FACTOR RECEPTORS

Abstract: Enzymatic nucleic acid molecules which cleave EGFR RNA.

Description: DESCRIPTION Enzymatic Nucleic Acid Treatment Of Diseases Or Conditions Related To Levels Of Epidermal Growth Factor Receptors Background Of The Invention The present invention concerns therapeutic compositions and methods for the treatment of cancer. The present invention relates to therapeutic compositions and methods for the treatment or diagnosis of diseases or conditions related to EGFR expression levels, such as cancer. The following summary is not meant to be complete and is provided only for understanding of the invention that follows. This summary is not an admission that any of the work described below is prior art to the claimed invention. The epidermal growth factor receptor (EGFR) is a 170 kDa transmembrane glycoprotein consisting of an extracellular 'ligand' binding domain, a transmembrane region and an intracellular domain with tyrosine kinase activity (Kung et: al. , 1994) . The binding of growth factors to the EGFR results in down regulation of the ligand-receptor complex, autophosphorylation of the receptor and other protein substrates, leading ultimately to DNA synthesis and cell division. The external ligand binding domain is stimulated by EGF and also by TGFα, amphiregulin and some viral growth factors ( odj tahedi & Dean, 1994) . The EGFR gene (c-erbBl) , is located on chromosome 7, and is homologous to the avian erythroblastosis virus oncogene (v-erbB) , which induces malignancies in chickens. The v-erbB gene codes for a truncated product that lacks the extracellular ligand binding domain. The tyrosine kinase domain of the EGFR has been found to have 97% homology to the v-erbB transforming protein (Downward et al. , 1984) . EGFR is overexpressed in a number of malignant human tissues when compared to their normal tissue counterparts (for review see Khazaie et al. , 1993) . The gene for the receptor is both amplified and overexpressed in a number of cancer cells. Overexpression of the EGFR is often accompanied by the co-expression of the growth factors EGF and TGFα, suggesting that an autocrine pathway for control of growth may play a major part in the progression of tumors (Sporn & Roberts, 1985) . Growth factors and their receptors may play a role in the development of human brain tumors. A high incidence of overexpression, amplification, deletion and structural rearrangement of the gene coding for the EGFR has been found in biopsies of brain tumors (Ostrowski et. al . , 1994) . In fact the amplification of the EGFR gene in glioblastoma multiform tumors is one of the most consistent genetic alterations known, with the EGFR being overexpressed in approximately 40% of malignant gliomas (Black, 1991) . It has also been demonstrated that in 50% of glioblastomas, amplification of the EGFR gene is accompanied by the co-expression of mRNA for at least one or both of the growth factors EGF and TNFα (Ekstrand et al., 1991) . The amplified genes are frequently rearranged and associated with polymorphism leading to abnormal protein products (Wong et al. , 1994). The rearrangements that have been characterized usually show deletions of part of the extracellular domain, resulting in the production of an EGFR protein that is smaller in size. Three classes of deletion mutant EGF receptor genes have been identified in glioblastoma tumors. Type I mutants lack the majority of the external domain, including the ligand binding site, type II mutants have a deletion in the domain adjacent to the membrane but can still bind ligands and type III, which is the most common and found in 17% of glioblastomas, have a deletion of 267 amino acids spanning domains I and II of the EGFR. In addition to glioblastomas, abnormal EGFR expression has also been reported in a number of squamous epidermoid cancers and breast cancers (reviewed in Kung et. al, 1994; Modjtahedi & Dean, 1994) . Many patients with tumors that overexpress the EGFR have a poorer prognosis than those who do not (Khazaie et. al . , 1993) . Consequently, therapeutic strategies which can potentially inhibit or reduce the aberrant expression of the EGFR receptor are of great interest as potential anti-cancer agents . Summary Of The Invention This invention relates to ribozymes, or enzymatic nucleic acid molecules, directed to cleave RNA species that are required for cellular growth responses. In particular, applicant describes the selection and function of ribozymes capable of cleaving RNA encoded by the receptor of epidermal growth factor (EGFR) . Such ribozymes may be used to inhibit the hyper-proliferation of tumor cells in one or more cancers. In the present invention, ribozymes that cleave EGFR RNA are described. Those of ordinary skill in the art will understand that from the examples described that other ribozymes that cleave target RNAs required for cell proliferation may be readily designed and are within the invention. Such RNAs may have at least 90% homology to EGFR in humans with a normal EGFR gene. By "inhibit" is meant that the activity of EGFR or level of RNAs encoded by EGFR is reduced below that observed in the absence of the nucleic acid, particularly, inhibition with ribozymes preferably is below that level observed in the presence of an inactive RNA molecule able to bind to the same site on the mRNA, but unable to cleave that RNA. By "enzymatic nucleic acid molecule" it is meant a nucleic acid molecule which has complementarity in a substrate binding region to a specified gene target, and also has an enzymatic activity which is active to specifically cleave RNA in that target. That is, the enzymatic nucleic acid molecule is able to intermolecularly cleave RNA and thereby inactivate a target RNA molecule. This complementarity functions to allow sufficient hybridization of the enzymatic nucleic acid molecule to the target RNA to allow the cleavage to occur. One hundred percent complementarity is preferred, but complementarity as low as 50-75% may also be useful in this invention. The term enzymatic nucleic acid is used interchangeably with phrases such as ribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, nucleozyme, DNAzyme, RNA enzyme, endoribonuclease, minizyme, leadzyme, oligozyme or DNA enzyme, as used in the art. All of these terminologies describe nucleic acid molecules with enzymatic activity. By "equivalent" RNA to EGFR is meant to include those naturally occurring RNA molecules associated with cancer in various animals, including human. By "complementarity" is meant a nucleic acid that can form hydrogen bond(s) with another RNA sequence by either traditional Watson-Crick or other non-traditional types (for example, Hoogsteen type) of base-paired interactions. Seven basic varieties of naturally-occurring enzymatic RNAs are known presently. Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions. Table I summarizes some of the characteristics of these ribozymes. In general, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of an enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets. The enzymatic nature of a ribozyme is advantageous over other technologies, since the concentration of ribozyme necessary to affect a therapeutic treatment is lower. This advantage reflects the ability of the ribozyme to act enzymatically. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA. In addition, the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base-pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage. Single mismatches, or base-substitutions, near the site of cleavage can be chosen to completely eliminate catalytic activity of a ribozyme. Nucleic acid molecules having an endonuclease enzymatic activity are able to repeatedly cleave other separate RNA molecules in a nucleotide base sequence- specific manner. Such enzymatic RNA molecules can be targeted to virtually any RNA transcript, and efficient cleavage achieved iri vitro (Zaug et. al . , 324, Nature 429 1986 ; Uhlenbeck, 1987 Nature 328, 596; Kim et a ^, 84 Proc. Natl. Acad. Sci. USA 8788, 1987; Dreyfus, 1988, Einstein Quart. J. Bio. Med., 6, 92; Haseloff and Gerlach, 334 Nature 585, 1988; Cech, 260 JAMA 3030, 1988; and Jefferies et al., 17 Nucleic Acids Research 1371, 1989). Because of their sequence-specificity, trans-cleaving ribozymes show promise as therapeutic agents for human disease (Usman & McSwiggen, 1995 Ann. Rep. Med. Chem. 30, 285-294; Christoffersen and Marr, 1995 J. Med. Chem. 38, 2023-2037). Ribozymes can be designed to cleave specific RNA targets within the background of cellular RNA. Such a cleavage event renders the RNA non-functional and abrogates protein expression from that RNA. In this manner, synthesis of a protein associated with a disease state can be selectively inhibited. Ribozymes that cleave the specified sites in EGFR RNAs represent a novel therapeutic approach to treat diseases, such as cancer and other conditions. Applicant indicates that ribozymes are able to inhibit the activity of EGFR and that the catalytic activity of the ribozymes is required for their inhibitory effect. Those of ordinary skill in the art, will find that it is clear from the examples described that other ribozymes that cleave these sites in EGFR RNAs may be readily designed and are within the scope of this invention. In one of the preferred embodiments of the inventions herein, the enzymatic nucleic acid molecule is formed in a hammerhead or hairpin motif, but may also be formed in 1 the motif of a hepatitis δ virus, group I intron, group II intron or RNaseP RNA (in association with an RNA guide sequence) or Neurospora VS RNA. Examples of such hammerhead motifs are described by Dreyfus, supra, Rossi et al. , 1992, AIDS Research and Human Retroviruses 8, 183; of hairpin motifs by Hampel e j t al . , EP0360257, Hampel and Tritz, 1989 Biochemistry 28, 4929, Feldstein et al_^, 1989, Gene 82, 53, Haseloff and Gerlach, 1989, Gene, 82, 43, and Hampel et al. , 1990 Nucleic Acids Res. 18, 299; of the hepatitis δ virus motif is described by Perrotta and Been, 1992 Biochemistry 31, 16; of the RNaseP motif by Guerrier- Takada et al. , 1983 Cell 35, 849; Forster and Altman, 1990, Science 249, 783; Li and Altman, 1996, Nucleic Acids Res . 24, 835; Neurospora VS RNA ribozyme motif is described by Collins (Seville and Collins, 1990 Cell 61, 685-696; Saville and Collins, 1991 Proc. Natl. Acad. Sci. USA 88, 8826-8830; Collins and Olive, 1993 Biochemistry 32, 2795-2799; Guo and Collins, 1995, EMBO. J. 14, 363); Group II introns are described by Griffin et. al. , 1995, Chem. Biol. 2, 761; Michels and Pyle, 1995, Biochemistry 34, 2965; Pyle et al. , International PCT Publication No. WO 96/22689; 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 (or multiple fragments of such molecules) of this invention is that it has a specific substrate binding site or arm(s) which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule (enzymatic portion) . By "enzymatic portion" is meant that part of the ribozyme essential for cleavage of an RNA substrate. By "substrate binding arm" is meant that portion of a ribozyme which is complementary to (i.e., able to base- pair with) a portion of its substrate. Generally, such complementarity is 100%, but can be less if desired. For example, as few as 10 bases out of 14 may be base-paired. Such arms are shown generally in Figures 1-3 as discussed below. That is, these arms contain sequences within a ribozyme which are intended to bring ribozyme and target RNA together through complementary base-pairing interactions; e.g., ribozyme sequences within stems I and III of a standard hammerhead ribozyme make up the substrate-binding domain (see Figure 1). In a preferred embodiment the invention provides a method for producing a class of enzymatic cleaving agents which exhibit a high degree of specificity for the RNA of a desired target. The enzymatic nucleic acid molecule is preferably targeted to a highly conserved sequence region of a target mRNAs encoding EGFR proteins such that specific treatment of a disease or condition can be provided with either one or several enzymatic nucleic acids. Such enzymatic nucleic acid molecules can be delivered exogenously to specific cells as required. Alternatively, the ribozymes can be expressed from DNA/RNA vectors that are delivered to specific cells . Synthesis of nucleic acids greater than 100 nucleotides in length is difficult using automated methods, and the therapeutic cost of such molecules is prohibitive. In this invention, small nucleic acid motifs (e.g., antisense oligonucleotides, hammerhead or the hairpin ribozymes) are used for exogenous delivery. The simple structure of these molecules increases the ability of the nucleic acid to invade targeted regions of the mRNA structure. However, these nucleic acid molecules can also be expressed within cells from eukaryotic promoters (e.g., Izant and Weintraub, 1985 Science 229, 345; McGarry and Lindquist, 1986 Proc. Natl. Acad. Sci. USA 83, 399; SullengerScanlon et al . , 1991, Proc. Natl. Acad. Sci. USA, 88, 10591-5; Kashani-Sabet et al. , 1992 Antisense Res. Dev. , 2, 3-15; Dropulic et al. , 1992 J. Virol, 66, 1432- 41; Weerasinghe et a _, 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; Thompson et al. , 1995 Nucleic Acids Res. 23, 2259) . Those skilled in the art realize that any nucleic acid can be expressed in eukaryotic cells from the appropriate DNA/RNA vector. The activity of such nucleic acids can be augmented by their release from the primary transcript by a ribozyme (Draper et al. , PCT W093/23569, and Sullivan et al . , PCT WO94/02595, both hereby incorporated in their totality by reference herein; Ohkawa et al. , 1992 Nucleic Acids Symp. Ser. , 27, 15-6; Taira et aL_, 1991, Nucleic Acids Res. , 19, 5125-30; Ventura et a _, 1993 Nucleic Acids Res. , 21, 3249-55; Chowrira et _, 1994 J. Biol. Chem. 269, 25856) . Such ribozymes are useful for the prevention of the diseases and conditions discussed above, and any other diseases or conditions that are related to the levels of EGFR activity in a cell or tissue. By "related" is meant that the inhibition of EGFR RNAs and thus reduction in the level respective protein activity will relieve to some extent the symptoms of the disease or condition. Ribozymes are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells. The nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex vivo, or in vivo through injection, infusion pump or sent, with or without their incorporation in biopolymers. In preferred embodiments, the ribozymes have binding arms which are complementary to the sequences in Tables III and IV. Examples of such ribozymes are also shown in Tables III and IV. Examples of such ribozymes consist essentially of sequences defined in these Tables. By "consists essentially of" is meant that the active ribozyme contains an enzymatic center or core equivalent to those in the examples, and binding arms able to bind mRNA such that cleavage at the target site occurs. Other sequences may be present which do not interfere with such cleavage. Thus, in a first aspect, the invention features ribozymes that inhibit gene expression and/or cell proliferation via cleavage of RNA expressed from the EGFR gene. These chemically or enzymatically synthesized RNA molecules contain substrate binding domains that bind to accessible regions of their target mRNAs . The RNA molecules also contain domains that catalyze the cleavage of RNA. The RNA molecules are preferably ribozymes of the hammerhead or hairpin motif. Upon binding, the ribozymes cleave the target mRNAs, preventing translation and protein accumulation. In the absence of the expression of the target gene, cell proliferation is inhibited. In a preferred embodiment, the enzymatic RNA molecules cleave EGFR mRNA and inhibit cell proliferation. Such ribozymes are useful for the prevention and/or treatment of cancer. 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 sent, with or without their incorporation in biopolymers. The ribozymes, similarly delivered, also are useful for inhibiting proliferation of certain cancers associated with elevated levels of the EGFR, particularly glioblastoma multiform. Using the methods described herein, other enzymatic RNA molecules that cleave EGFR and thereby inhibit tumor cell proliferation may be derived and used as described above. Specific examples are provided below in the Tables and figures. In another aspect of the invention, ribozymes that cleave target molecules and inhibit EGFR activity are expressed from transcription units inserted into DNA or RNA vectors. The recombinant vectors are preferably DNA plasmids or viral vectors. Ribozyme expressing viral vectors could be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Preferably, the recombinant vectors capable of expressing the ribozymes are delivered as described above, and persist in target cells. Alternatively, viral vectors may be used that provide for transient expression of ribozymes. Such vectors might be repeatedly administered as necessary. Once expressed, the ribozymes cleave the target mRNA. Delivery of ribozyme expressing vectors could be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex- planted from the patient followed by reintroduction into the patient, or by any other means that would allow for introduction into the desired target cell (for a review see Couture and Stinchcomb, 1996, TIG. , 12, 510) . By "patient" is meant an organism which is a donor or recipient of explanted cells or the cells themselves. "Patient" also refers to an organism to which enzymatic nucleic acid molecules can be administered. Preferably, a patient is a mammal or mammalian cells. More preferably, a patient is a human or human cells. By "vectors" is meant any nucleic acid- and/or viral- based technique used to deliver a desired nucleic acid. These ribozymes, individually, or in combination or in conjunction with other drugs, can be used to treat diseases or conditions discussed above. For example, to treat a disease or condition associated with EGFR levels, the patient may be treated, or other appropriate cells may be treated, as is evident to those skilled in the art. In a further embodiment, the described ribozymes can be used in combination with other known treatments to treat conditions or diseases discussed above. For example, the described ribozymes could be used in combination with one or more known therapeutic agents to treat cancer. In preferred embodiments, the ribozymes have binding arms which are complementary to the sequences in the tables III and IV (Seq ID NOs . 1-823 and 1759-1870. Examples of such ribozymes are also shown in Tables III and IV (Seq. ID Nos. 824-1758). Other sequences may be present which do not interfere with such cleavage. Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Description Of The Preferred Embodiments The drawings will first briefly be described. Drawings : Figure 1 is a diagrammatic representation of the hammerhead ribozyme domain known in the art. Stem II can be > 2 base-pair long. Figure 2a is a diagrammatic representation of the hammerhead ribozyme domain known in the art; Figure 2b is a diagrammatic representation of the hammerhead ribozyme as divided by Uhlenbeck (1987, Nature, 327, 596-600) into a substrate and enzyme portion; Figure 2c is a similar diagram showing the hammerhead divided by Haseloff and Gerlach (1988, Nature, 334, 585-591) into two portions; and Figure 2d is a similar diagram showing the hammerhead divided by Jeffries and Symons (1989, Nucl . Acids. Res., 17, 1371-1371) into two portions. Figure 3 is a diagrammatic representation of the general structure of a hairpin ribozyme. Helix 2 (H2) is provided with a least 4 base pairs (i.e. , n is 1, 2, 3 or 4) and helix 5 can be optionally provided of length 2 or more bases (preferably 3 - 20 bases, i.e., m is from 1 - 20 or more) . Helix 2 and helix 5 may be covalently linked by one or more bases (i.e. , r is > 1 base) . Helix 1, 4 or 5 may also be extended by 2 or more base pairs (e.g. , 4 - 20 base pairs) to stabilize the ribozyme structure, and preferably is a protein binding site. In each instance, each N and N' independently is any normal or modified base and each dash represents a potential base-pairing interaction. These nucleotides may be modified at the sugar, base or phosphate. Complete base-pairing is not required in the helices, but is preferred. Helix 1 and 4 can be of any size (i.e., o and p is each independently from 0 to any number, e.g., 20) as long as some base- pairing is maintained. Essential bases are shown as specific bases in the structure, but those in the art will recognize that one or more may be modified chemically (abasic, base, sugar and/or phosphate modifications) or replaced with another base without significant effect. Helix 4 can be formed from two separate molecules, i.e., without a connecting loop. The connecting loop when present may be a ribonucleotide with or without modifications to its base, sugar or phosphate. "q" is > 2 bases. The connecting loop can also be replaced with a non-nucleotide linker molecule. H refers to bases A, U, or C. Y refers to pyrimidine bases. " " refers to a covalent bond. Figure 4 is a representation of the general structure of the hepatitis delta virus ribozyme domain known in the art . Figure 5 is a representation of the general structure of the self-cleaving VS RNA ribozyme domain. Figure 6 shows _in. vitro RNA cleavage activity of Amino ribozymes targeted against EGFR RNA. a Autoradiograph of the cleavage reaction. The reaction was performed in the presence of 50mM Tris.HCl (pH 7.5), lOmM MgCl 2 at 37°C as described below. Times of the reaction in minutes are given above the lanes. SO represents intact substrate in Tris.HCl buffer without the addition of ribozyme at time 0. SI represents intact substrate in Tris.HCl buffer at time 60min. +C represents a positive control of cleaved product only. Band S represents intact substrate, band P cleaved product and band D degradation; b Time course of cleavage. Bands from autoradiography were quantified by scanning densitometry and the fraction of substrate remaining plotted against time. inset. Semilog plots were used to determine the half life of the substrate (tι /2 = 0.693 / k) ; c Autoradiograph showing reaction of the EGFR ribozyme against a non complementary substrate RNA. 40nM ribozyme was added to InM substrate in the presence of 50mM Tris.HCl (pH 7.5), lOmM MgCl 2 at 37°C. Band S refers to intact substrate and band P is cleaved product. Reaction times are given in minutes (unless stated otherwise) . C represents intact substrate without the addition of ribozyme. +C represents cleaved product. Figure 7 Representative examples of autoradiographs depicting the time course of cleavage reactions exhibited by EGFR ribozyme against it's target substrate under multiple turnover reactions. a In. vitro activity of lOnM ribozyme with 300nM of 5' [32P] labeled substrate RNA; b In vitro activity of lOnM ribozyme with lμM of 5'[32P] labeled substrate RNA. Reactions were performed in the presence of 50mM Tris.HCl (pH 7.5), lOmM MgCl 2 at 37°C as described below. Reaction times, in minutes, are given above the lanes. C represents intact substrate in Tris.HCl buffer without the addition of ribozyme. Band S refers to intact substrate and band P refers to cleaved product, c Kinetics of hammerhead cleavage reactions exhibited by the EGFR ribozyme. The initial rate of reaction (Vo,nM / min) is plotted versus substrate concentration. Ribozyme concentration was lOnM while substrate concentration varied as indicated. inset Eadie-Hofstee plot of this data . Figure 8 shows a generic structure of chemically modified amino hammerhead ribozyme. Figure 9 shows a generic structure of chemically modified C-allyl hammerhead ribozyme. Target sites Targets for useful ribozymes can be determined as disclosed in Draper et al. , WO 93/23569; Sullivan et al. , WO 93/23057; Thompson et al^_, WO 94/02595; Draper et al. , WO 95/04818; McSwiggen et aL, US Patent No. 5,525,468 and hereby incorporated by reference herein in totality. Rather than repeat the guidance provided in those documents here, below are provided specific examples of such methods, not limiting to those in the art. Ribozymes to such targets are designed as described in those applications and synthesized to be tested in vitro and in vivo, as also described. Such ribozymes can also be optimized and delivered as described therein. The sequence of human EGFR RNAs were screened for optimal ribozyme target sites using a computer folding algorithm. Hammerhead or hairpin ribozyme cleavage sites were identified. These sites are shown in Tables III and IV (All sequences are 5' to 3' in the tables) The nucleotide base position is noted in the Tables as that site to be cleaved by the designated type of ribozyme. The nucleotide base position is noted in the Tables as that site to be cleaved by the designated type of ribozyme . Hammerhead or hairpin ribozymes were designed that could bind and were individually analyzed by computer folding (Jaeger et. al. , 1989 Proc. Natl. Acad. Sci. USA, 86, 7706) to assess whether the ribozyme sequences fold into the appropriate secondary structure. Those ribozymes with unfavorable intramolecular interactions between the binding arms and the catalytic core are eliminated from consideration. Varying binding arm lengths can be chosen to optimize activity. Generally, at least 5 bases on each arm are able to bind to, or otherwise interact with, the target RNA. Ribozymes of the hammerhead or hairpin motif were designed to anneal to various sites in the mRNA message. The binding arms are complementary to the target site sequences described above. The ribozymes were chemically synthesized. The method of synthesis used follows the procedure for normal RNA synthesis as described in Usman et al. , 1987 J. Am. Chem. Soc, 109, 7845; Scaringe et al. , 1990 Nucleic Acids Res., 18, 5433; and Wincott et al. , 1995 Nucleic Acids Res. 23, 2677-2684 and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5 '-end, and phosphoramidites at the 3 '-end. Small scale synthesis were conducted on a 394 Applied Biosystems, Inc. synthesizer using a modified 2.5 μmol scale protocol with a 5 min coupling step for alkylsilyl protected nucleotides and 2.5 min coupling step for 2'-0-methylated nucleotides. Table II outlines the amounts, and the contact times, of the reagents used in the synthesis cycle. A 6.5-fold excess (163 μL of 0.1 M = 16.3 μmol) of phosphoramidite and a 24-fold excess of S-ethyl tetrazole (238 μL of 0.25 M = 59.5 μmol) relative to polymer-bound 5 ' -hydroxyl was used in each coupling cycle. Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, were 97.5-99%. Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer : detritylation solution was 2% TCA in methylene chloride (ABI) ; capping was performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); oxidation solution was 16.9 mM I 2 , 49 mM pyridine, 9% water in THF (Millipore) . B & J Synthesis Grade acetonitrile was used directly from the reagent bottle. SXEthyl tetrazole solution (0.25 M in acetonitrile) was made up from the solid obtained from American International Chemical, Inc. Deprotection of the RNA was performed as follows . The polymer-bound oligoribonucleotide, trityl-off, was transferred from the synthesis column to a 4mL glass screw top vial and suspended in a solution of methylamine (MA) at 65 °C for 10 min. After cooling to -20 °C, the supernatant was removed from the polymer support. The support was washed three times with 1.0 mL of EtOH:MeCN:H 2 0/3: 1 : 1, vortexed and the supernatant was then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, were dried to a white powder . The base-deprotected oligoribonucleotide was resuspended in anhydrous TEA-HF/NMP solution (250 μL of a solution of 1.5mL N-methylpyrrolidinone, 750 μL TEA and 1.0 mL TEA-3HF to provide a 1.4M HF concentration) and heated to 65°C for 1.5 h. The resulting, fully deprotected, oligomer was quenched with 50 mM TEAB (9 mL) prior to anion exchange desalting. For anion exchange desalting of the deprotected oligomer, the TEAB solution was loaded onto a Qiagen 500® anion exchange cartridge (Qiagen Inc.) that was prewashed with 50 mM TEAB (10 mL) . After washing the loaded cartridge with 50 mM TEAB (10 mL) , the RNA was eluted with 2 M TEAB (10 mL) and dried down to a white powder. Inactive hammerhead ribozymes were synthesized by substituting a U for G 5 and a U for A i4 (numbering from Hertel, K. J. , et. al . , 1992, Nucleic Acids Res., 20, 3252) . The average stepwise coupling yields were >98% (Wincott et al^_, 1995 Nucleic Acids Res. 23, 2677-2684). Hairpin ribozymes are synthesized in two parts and annealed to reconstruct the active ribozyme (Chowrira and Burke, 1992 Nucleic Acids Res., 20, 2835-2840) . Ribozymes are also synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol. 180, 51) . Ribozymes are modified to enhance stability and/or enhance catalytic activity by modification with nuclease resistant groups, for example, 2 '-amino, 2'-C-allyl, 2'- flouro, 2 ' -O-methyl, 2'-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992 TIBS 17, 34; Usman et: al. , 1994 Nucleic Acids Symp. Ser. 31, 163; Burgin et. al. , 1996 Biochemistry 6, 14090) . Ribozymes are purified by gel electrophoresis using general methods or are purified by high pressure liquid chromatography (HPLC; See Wincott et al ■ , supra) the totality of which is hereby incorporated herein by reference) and are resuspended in water . The sequences of the ribozymes that are chemically synthesized, useful in this study, are shown in Tables III-IV. Those in the art will recognize that these sequences are representative only of many more such sequences where the enzymatic portion of the ribozyme (all but the binding arms) is altered to affect activity. For example, stem-loop II sequence of hammerhead ribozymes can be altered (substitution, deletion, and/or insertion) to contain any sequences provided a minimum of two base- paired stem structure can form. Similarly, stem-loop IV sequence of hairpin ribozymes listed in Tables IV (5'- CACGUUGUG-3' ) can be altered (substitution, deletion, and/or insertion) to contain any sequence, provided a minimum of two base-paired stem structure can form. Preferably, no more than 200 bases are inserted at these locations. The sequences listed in Tables III and IV may be formed of ribonucleotides or other nucleotides or non- nucleotides. Such ribozymes (which have enzymatic activity) are equivalent to the ribozymes described specifically in the Tables. Optimizing Ribozyme Activity Ribozyme activity can be optimized as described by Draper et. al. , supra. The details will not be repeated here, but include altering the length of the ribozyme binding arms (stems I and III, see Figure 2c) , or chemically synthesizing ribozymes with modifications (base, sugar and/or phosphate) that prevent their degradation by serum ribonucleases and/or enhance their enzymatic activity (see e.g., Eckstein e_t al . , International Publication No. WO 92/07065; Perrault et al. , 1990 Nature 344, 565; Pieken et al. , 1991 Science 253, 314; Usman and Cedergren, 1992 Trends in Biochem. Sci . 17, 334; Usman e_t al . , International Publication No. WO 93/15187; and Rossi et al . , International Publication No. WO 91/03162; Sproat, US Patent No. 5,334,711; and Burgin et. al . , supra; all of these describe various chemical modifications that can be made to the base, phosphate and/or sugar moieties of enzymatic RNA molecules) . Modifications which enhance their efficacy in cells, and removal of stem II bases to shorten RNA synthesis times and reduce chemical requirements are desired. (All these publications are hereby incorporated by reference herein.). By "enhanced enzymatic activity" is meant to include activity measured in cells and/or _in vivo where the activity is a reflection of both catalytic activity and ribozyme stability. In this invention, the product of these properties in increased or not significantly (less that 10 fold) decreased jLn vivo compared to an all RNA ribozyme . The enzymatic nucleic acid having chemical modifications which maintain or enhance enzymatic activity is provided. Such nucleic acid is also generally more resistant to nucleuses than unmodified nucleic acid. By "modified bases" in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at 1' position or their equivalents; such bases may be used within the catalytic core of the enzyme as well as in the substrate-binding regions. In particular, the invention features modified ribozymes having a base substitution selected from pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyluracil, dihydrouracil, naphthyl, 6-methyl-uracil and aminophenyl . As noted above, substitution in the core may decrease i_n vitro activity but enhances stability. Thus, in a cell and/or i_n vivo the activity may not be significantly lowered. As exemplified herein such ribozymes are useful in a cell and/or iri vivo even if activity over all is reduced 10 fold. Such ribozymes herein are said to "maintain" the enzymatic activity on all RNA ribozyme. Sullivan, et al. , supra, describes the general methods for delivery of enzymatic RNA molecules. Ribozymes may be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres . For some indications, ribozymes may be directly delivered ex vivo to cells or tissues with or without the aforementioned vehicles. Alternatively, the RNA/vehicle combination is locally delivered by direct injection or by use of a catheter, infusion pump or sent. Other routes of delivery include, but are not limited to, intravascular, intramuscular, subcutaneous or joint injection, aerosol inhalation, oral (tablet or pill form) , topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. More detailed descriptions of ribozyme delivery and administration are provided in Sullivan et al. , supra and Draper et al . , supra which have been incorporated by reference herein. Another means of accumulating high concentrations of a ribozyme (s) within cells is to incorporate the ribozyme- encoding sequences into a DNA or RNA expression vector. Transcription of the ribozyme sequences are driven from a promoter for eukaryotic RNA polymerase I (pol I) , RNA polymerase II (pol II) , or RNA polymerase III (pol III) . Transcripts from pol II or pol III promoters will be expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type will depend on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby. Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990 Proc. Natl. Acad. Sci. USA, 87, 6743-7; Gao and Huang 1993 Nucleic Acids Res., 21, 2867-72; Lieber et a , 1993 Methods Enzymol. , 217, 47-66; Zhou et al^, 1990 Mol . Cell. Biol., 10, 4529-37) . Several investigators have demonstrated that ribozymes expressed from such promoters can function in mammalian cells (e.g. Kashani-Sabet ____ al . , 1992 Antisense Res. Dev., 2, 3-15; Ojwang et al . , 1992 Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al . , 1992 Nucleic Acids Res. , 20, 4581-9; Yu et al. , 1993 Proc. Natl. Acad. Sci. U.S.A., 90, 6340-4; L'Huillier et al. , 1992 EMBO J. 11, 4411-8; Lisziewicz et al., 1993 Proc. Natl. Acad. Sci. U.S.A., 90, 8000- ; Thompson et al . , 1995 Nucleic Acids Res. 23, 2259; Sullenger & Cech, 1993, Science, 262, 1566) . 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) (for a review see Couture and Stinchcomb, 1996, supra) . In a preferred embodiment of the invention, a transcription unit expressing a ribozyme that cleaves mRNAs encoded by EGFR is inserted into a plasmid DNA vector or an adenovirus or adeno-associated virus DNA viral vector or a retroviral RNA vector. Viral vectors have been used to transfer genes and lead to either transient or long term gene expression (Zabner et_ al . , 1993 Cell 75, 207; Carter, 1992 Curr. Opi . Biotech. 3, 533) . The adenovirus vector is delivered as recombinant adenoviral particles. The DNA may be delivered alone or complexed with vehicles (as described for RNA above) . The recombinant adenovirus or AAV particles are locally administered to the site of treatment, e.g., through incubation or inhalation in. vivo or by direct application to cells or tissues ex vivo. Retroviral vectors have also been used to express ribozymes in mammalian cells (Ojwang et al . , 1992 supra; Thompson et al. , 1995 supra; Couture and Stinchcomb, 1996, supra) . In another preferred embodiment, the ribozyme is administered to the site of EGFR expression (e.g., tumor cells) in an appropriate liposomal vesicle. Examples Example 1: Identification of Potential Ribozyme Cleavage Sites in Human EGFR RNA The sequence of human EGFR RNA was screened for accessible sites using a computer folding algorithm. Regions of the mRNA that did not form secondary folding structures and potential hammerhead and/or hairpin ribozyme cleavage sites were identified. The sequences of these cleavage sites are shown in tables III and IV. Example 2: Selection of Ribozyme Cleavage Sites in Human EGFR RNA To test whether the sites predicted by the computer- based RNA folding algorithm corresponded to accessible sites in EGFR RNA, 20 hammerhead sites were selected for analysis. Ribozyme target sites were chosen by analyzing genomic sequences of human EGFR (GenBank Accession No. X00588) and prioritizing the sites on the basis of folding. Hammerhead ribozymes were designed that could bind each target (see Figure 2C) and were individually analyzed by computer folding (Christoffersen et al . , 1994 J. Mol. Struc. Theochem, 311, 273; Jaeger et al. , 1989, Proc. Natl. Acad. Sci. USA, 86, 7706) to assess whether the ribozyme sequences fold into the appropriate secondary structure. Those ribozymes with unfavorable intramolecular interactions between the binding arms and the catalytic core were eliminated from consideration. As noted below, varying binding arm lengths can be chosen to optimize activity. Generally, at least 5 bases on each arm are able to bind to, or otherwise interact with, the target RNA. Example 3: Chemical Synthesis and Purification of Ribozymes for Efficient Cleavage of EGFR RNA Ribozymes of the hammerhead or hairpin motif were designed to anneal to various sites in the RNA message. The binding arms are complementary to the target site sequences described above. The ribozymes were chemically synthesized. The method of synthesis used followed the procedure for normal RNA synthesis as described in Usman et al. , (1987 J. Am. Chem. Soc. , 109, 7845), Scaringe et al. , (1990 Nucleic Acids Res. , 18, 5433) and Wincott et al . , supra, and made use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'- end, and phosphoramidites at the 3 '-end. The average stepwise coupling yields were >98%. Inactive ribozymes were synthesized by substituting a U for G 5 and a U for A i4 (numbering from Hertel et al . , 1992 Nucleic Acids Res., 20, 3252). Hairpin ribozymes were synthesized in two parts and annealed to reconstruct the active ribozyme (Chowrira and Burke, 1992 Nucleic Acids Res. , 20, 2835- 2840) . Ribozymes were also synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol. 180, 51) . All ribozymes were modified to enhance stability by modification with nuclease resistant groups, for example, 2 '-amino, 2 ' -C- allyl, 2'-flouro, 2'-0-methyl, 2 ' -H (for a review see Usman and Cedergren, 1992 TIBS 17, 34) . Ribozymes were purified by gel electrophoresis using general methods or were purified by high pressure liquid chromatography (HPLC; See Wincott et al. , supra; the totality of which is hereby incorporated herein by reference) and were resuspended in water. The sequences of the chemically synthesized ribozymes used in this study are shown below in Table III and IV. Example 4: Ribozyme Cleavage of EGFR RNA Target Twenty hammerhead-type ribozymes targeted to the human EGFR RNA were designed and synthesized to test the cleavage activity .i vitro. The target sequences and the nucleotide location within the EGFR mRNA are given in Table III. All hammerhead ribozymes were synthesized with binding arm (Stems I and III; see Figure 2C) lengths of seven nucleotides. The relative abilities of a HH ribozyme to cleave human EGFR RNA is summarized in Figure 6 and 7. Full-length or partially full-length, internally- labeled target RNA for ribozyme cleavage assay was prepared by Ln vitro transcription in the presence of [a- 32 P ] CTP, passed over a G 50 Sephadex column by spin chromatography and used as substrate RNA without further o n purification. Alternately, substrates were 5'- P-end labeled using T4 polynucleotide kinase enzyme. Assays were performed by pre-warming a 2X concentration of purified ribozyme in ribozyme cleavage buffer (50 mM Tris- HC1, pH 7.5 at 37°C, 10 mM MgCl 2 ) and the cleavage reaction was initiated by adding the 2X ribozyme mix to an equal volume of substrate RNA (maximum of 1-5 nM) that was also pre-warmed in cleavage buffer. As an initial screen, assays were carried out for 1 hour at 37°C using a final concentration of either 40 nM or 1 mM ribozyme, i.e., ribozyme excess. The reaction was quenched by the addition of an equal volume of 95% formamide, 20 mM EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol after which the sample was heated to 95°C for 2 minutes, quick chilled and loaded onto a denaturing polyacrylamide gel. Substrate RNA and the specific RNA cleavage products generated by ribozyme cleavage were visualized on an autoradiograph of the gel. The percentage of cleavage was determined by Phosphor Imager® quantitation of bands representing the intact substrate and the cleavage products . Single Turnover Reaction: Alternately, Cleavage reactions were carried out in 50mM Tris.HCl, pH 7.5 and lOmM MgCl 2 at 37°C. In order to disrupt aggregates that can form during storage, unlabeled ribozyme and 5'end labeled substrate were denatured and renatured separately in standard cleavage buffer (50mM Tris.HCl, pH 7.5 ) by heating to 90°C for 2 minutes and allowed to equilibrate to the reaction temperature of 37°C for 15 minutes. Each RNA solution was then adjusted to a final concentration of lOmM MgCl 2 and incubated at 37°C for a further 15 minutes. Cleavage reactions were initiated by combining the ribozyme and the substrate samples to the required concentrations in a final volume of lOOμl. Ribozyme concentration was 40nM and substrate concentration was InM. The reaction was also repeated using double (2nM) and half (0.5nM) the concentration of substrate to verify that the reaction was indeed performed under single turnover conditions. Aliquots of lOμl were removed at appropriate time intervals between 0 and 120 minutes and quenched by adding an equal volume of formamide loading buffer ( 9:1 (v:v) formamide: lx TBE) and frozen on dry ice. Product and substrate were separated by denaturing 20% polyacrylamide (7M urea) gel electrophoresis. To determine the fraction of cleavage, substrate and product bands were located by autoradiography of wet gels and quantified by densitometry of these autoradiograms . Autorads were scanned using an AGFA focus scanner connected to a Macintosh computer and images were saved as TIFF files. The programme NIH Image 1.58 (Division of Computing and Research Technology, NIH, Bethesda, USA ) was used to plot and quantify the band intensities. In addition, the relevant bands were excised from the gel and quantified by scintillation counting of the slices cut from the gel (Packard Tricarb 2000 CA liquid scintillation analyser) . Reaction rate constants (k) were obtained from the slope of semilogarithmic plots of the amount of substrate remaining versus time. The activity half time tl/2 was calculated as 0.693/ k. Each rate constant was determined from duplicate experiments. In order to show the specificity of cleavage demonstrated under the above conditions, the experiment was repeated using a different substrate, relating to another site along the human EGFR mRNA. All conditions remained as described above except samples were taken over a longer time period i . e . at intervals spanning over 24 hours rather than over 2 hours. Multiple Turnover Reactions : The kinetic characteristics of ribozyme RPI.4782 were determined from Eadie - Hofstee plots obtained from initial velocities with multiple turnovers done with 5' 32P labeled substrate. Cleavage reactions were carried out in 50mM Tris.HCl, pH7.5 and lOmM MgCl 2 at 37°C. Stock solutions of lOOnM ribozyme and 500nM - 2uM substrate RNA were prepared in 50mM Tris.HCl, pH 7.5, preheated separately at 0°C for 2 minutes and cooled to 37°C for 15 minutes. After MgCl 2 was added to each of these solutions to a final volume of lOmM, a further incubation period of 15 minutes at 37°C took place. Cleavage reactions were performed in a final volume of lOOμl with a concentration of lOnM ribozyme and concentrations of substrate between lOOnM and lμM. Reactions were initiated by the addition of ribozyme stock solution to substrate. Aliquots of lOμl were taken at time intervals between 0 and 120 minutes, quenched by adding an equal volume of formamide loading buffer and frozen on dry ice. Intact substrate and products of cleavage were separated by electrophoresis on a 20% polyacrylamide / 7M urea denaturing gel and were detected by autoradiography. The degree of cleavage at each time point was quantified by scanning densitometry of the resulting autoradiogram. Initial rates of reaction were measured at eight substrate concentrations and values of Kcat and Km were determined using Eadie-Hofstee plots. As shown in Figure 6 and 7, Amino hammerhead ribozymes (RPI.4782) targeted against EGFR RNA cleaved their target RNAs in a sequence-specific manner the cleavage rates appeared to follow saturation kinetics with respect to concentration of substrate. Cleavage rates were first order at low substrate concentrations, however, as the concentration of substrate increased, the reaction rates leveled off suggesting that ribozymes were effectively saturated with substrate. These results indicate that the cleavage reactions were truly catalytic and were therefore amenable to analysis using Michaelis Menten rate equation. From a Eadie-Hofstee plot the kinetic parameters Km and Kcat were determined; ribozyme exhibited a Km value of 87nM and a Kcat value of 1.2 min -1 . Under single turnover conditions, ribozyme RPI.4782 exhibited rapid cleavage of it's target sequence, the half life of the substrate being only 7 minutes. The high activity of this ribozyme is in agreement with the findings of Beigelman et al. (1995c) . They reported that a ribozyme modified in the same manner as RPI.4782 exhibited almost wild type activity, with the half life of the substrate being only 3 minutes. Although cleavage was slightly slower than that demonstrated by Beigelman et al . (1995c) , these findings clearly demonstrate that ribozyme RPI.4782 is able to cleave it's target in a highly efficient manner. When the experiment was repeated using a different, non complementary, substrate sequence, no cleavage products were evident (figure 3.3), demonstrating the sequence specificity of this molecule. To assess more precisely the activity of ribozyme 7Λmino ribozyme (RPI.4782), the kinetic parameters K M and k cat were determined under multiple turnover conditions. The results indicate that the cleavage reaction was truly catalytic with a turnover rate (K cat ) of 1.2 min -1 and a K M value of 87nM (figure 6 and 7) . These results fall in line with typical values reported for the hammerhead ribozyme of 1-2 min "1 and 20-200nM for Kcat and Km respectively (Kumar et. al. , 1996) . Direct comparisons are difficult, however, since many factors including base sequence, length of substrate binding arms and varying chemical modifications can have an effect on these kinetic parameters (Fedor & Uhlenbeck, 1992) . Example 5: Stability of EGFR Ribozymes in Fetal Calf Serum. To assess the stability of the chemically modified ribozyme, a comparative stability study was carried out in 100% foetal calf serum (Gibco, Paisley, U. K. ) at 37°C. Degradation profiles of 5' and internally [ 32 P] labeled ribozyme were compared to those of 5'-end [ 32 P] labeled phosphoodiester (PO) , phosphorothioate (PS) oligodeoxynucleotides and unmodified RNA. Synthesis / labeling: 37mer PO and PS oligonucleotides were synthesized on an automated DNA synthesizer (model 392, Applied Biosystems, Warrington, U.K.) using standard phosphoramide chemistry (section 2.2.1). The chemically modified 37mer ribozyme (Amino Hammerhead Ribozyme; Figure 8) and the 15mer unmodified all RNA substrate were synthesized as described above. Ribozymes and oligonucleotides were radiolabelled with [ 32 P] ATP and purified on 20% polyacrylamide gel as previously described. Degradation study conditions : Radiolabelled ribozymes/ oligonucleotides were incubated in 100 μl of FCS at 37°C to give a final concentration of 200nM. lOul aliquots were removed at timed intervals, mixed with a loading buffer containing 80% formamide, lOmM EDTA ( pH8.0), 0.25% xylene cyanol, 0.25% bromophenol blue, and frozen at -20°C prior to gel loading. Degradation profiles were analyzed by 20% polyacrylamide (7M urea) gel electrophoresis and autoradiography. A comparative stability study was undertaken in 100% fetal calf serum (FCS) to compare the degradation profiles of 5' end labeled and internally labeled amino ribozyme to those of 5'end labeled unmodified RNA substrate, phosphodiester (PO) and phosphorothioate (PS) oligodeoxynucleotides . The chemical modifications of the amino ribozyme resulted in a substantial increase in nuclease resistance over that of the unmodified substrate. The half life (t 50 % ) of the internally labeled ribozyme was approximately 20 hours whereas the substrate was completely degraded within the time that it took to add the RNA to serum, mix and quench the reaction ( t 50% < lmin) . It was interesting to note that although the patterns of degradation were clearly different for the internally labeled ribozyme (figure 3.6a) and the 5' end labeled ribozyme, the kinetics of degradation were strikingly similar. ( t 5 0% of ~ 20 hours for both) . A comparison of ribozyme degradation and oligodeoxynucleotide degradation was also performed. The chemically modified ribozyme appeared to be more stable in FCS than either the PO oligonucleotide or the PS oligonucleotide; the approximate half lives being 10 minutes and 5 hours respectively. It must be noted, however, that the apparent degradation products migrated to the position of free phosphate. This suggests that dephosphorylation (removal of [ 32 P] label) occurred, resulting in a progressive increase in free phosphate concentration with time. There is no doubt, however, that the findings of this study show that the chemical modifications applied to ribozyme result in an extremely stable structure. Under the conditions of this experiment amino ribozyme proved to be the most stable to nuclease mediated degradation in fetal calf serum. Example 6: Ribozymes uptake studies Cell Culture Techniques U87-MG cell line was purchased from the European Cell Culture Collection, Porton Down, U.K. These human glioblastoma astrocytoma cells were originally derived from a grade 3 malignant glioma by explant technique (Poten et. al . , 1968) . A431 cells were derived from a vulval carcinoma and expresses the EGFR at levels 10 to 50 fold higher than seen in other cell lines (Ullrich et al. , 1984) . The cell lines U87-MG and Raw 264.7 were maintained in Dulbecco's modified Eagle's media (DMEM) supplemented by 10% v/v foetal bovine serum (FBS), 1% penicillin/streptomycin and 1% v/v L-glutamine (all supplied from Gibco, Paisley, U.K.). The same media, without the addition of the foetal bovine serum, was used in the stability and uptake studies. A431 cells were maintained under the same conditions except glutamine was added to a final concentration of 2% v/v. CaCo-2 cells were kindly cultured and plated by Vanessa Moore in DMEM, 10% FBS, 1% non essential amino acids, 1% penicillin/ streptomycin, and 1% L-glutamine. Cells were cultured in 75cm 3 plastic tissue culture flasks (Falcon, U.K.) with 25ml of the respective media. The cultures were incubated at 37°C in a humidified (95%) atmosphere of 5% C02 in air. Stock cultures were maintained by changing the media every 48 hours and passaged (1:5) when confluent (after approximately 4 days) . Passaging was carried out using the following procedure: The media was removed and the cells washed with 10ml of phosphate-buffered saline solution (PBS) . Following this, 5ml of 2x Trypsin/EDTA (0.25% w/v trypsin, 0.2% disodium ethylenediamine tetraacetate in PBS, pH 7.2) was added and the flasks incubated at 37°C for 5minutes. The flasks were tapped to dislodge the cell monolayer from the bottom and fresh media was added to neutralize the trypsin. The cells were split as required and media added to a final volume of 25ml. For long term storage, frozen stock cultures were prepared in the following manner: Stock cultures were trypsinised as described and neutralized with the addition of 10ml of DMEM media. The cell suspension was then transferred to a 15ml universal tube (Falcon, U.K.) and centrifuged for 3 minutes at 350 revolutions per minutes. The supernatant was decanted and the cell pellet was resuspended in 1ml of freezing media (10% DMSO, 90% heat inactivated foetal calf serum) and transferred to a 2ml screw capped cryovial (Costar, U.K.). The ampule was then placed in the freezing head of a liquid nitrogen freezer for 4-6 hours before being transferred into liquid nitrogen (-196°C) cell bank. When required, the cells were recovered by rapid thawing at 37°C and gradual dilution with DMEM media before seeding in 25cm 3 flasks (Falcon, U.K.). The viable cell density of stock cultures was measured by haemocytometry using a trypan blue exclusion test. lOOμl of trypan blue (4mg ml -1 ) was mixed with 400μl of cell suspension (1:1.25 dilution). A small amount of the trypan blue-cell suspension was transferred to the counting chamber of a Neubauer haemocytometer, with depth of 0.1mm and area l/400mm 2 (Weber Scientific International Ltd, U.K.). The cells were counted in the 5 large squares of the haemocytometer using a light microscope. Since live cells do not take up the trypan blue dye, while dead cells do, the number of viable (unstained) cells were counted. The cell density was calculated using the following equation: cells ml -1 = average count per square x 10 4 x 1.25 ( dilution factor of trypan blue) Cell Association Studies: A series of experiments were conducted to examine the mechanism of uptake of the ribozyme in the U87-MG glioblastoma cell line. The following general experimental procedure was used throughout these studies unless otherwise stated. Synthesis/ labelling: Prior to use in uptake studies, the 37mer ribozyme was internally labeled with 32P as previously described (section 2.3.2) and purified by 20% native polyacrylamaide gel electrophoresis. [14C] Mannitol (specific activity 56mCi / mol) was purchased from Amersham (Amersham, U.K.). Uptake study procedure: U87-MG cells were cultured on plastic 24-well plates (Falcon, U.K.). Confluent stock cultures were trypsinised and the cell density of the stock suspension diluted to 0.5 x 10 5 cells ml -1 with DMEM media. Each well was seeded with 2ml of the diluted cell suspension to give a final concentration of lxlO 5 well-1. The plates were incubated at 37°C in a humidified (95%) atmosphere of 5% C0 2 in air. After approximately 20 - 24 hours, the cell monolayers had reached confluency and were then ready for uptake experiments. The media was then removed and the monolayer carefully washed twice with PBS ( 2 x 1ml x 5min) to remove any traces of serum. The washing solution was aspirated and replaced with 200μl of serum free DMEM media containing the radiolabelled ribozyme. Both PBS and serum free media were equilibrated at 37°C for lhour prior to use. The plates were incubated at 37°C, unless otherwise stated, in a dry environment for the duration of the experiment. Once incubated for the desired period of time, the apical media was carefully collected and their radioactive content assessed by liquid scintillation counting (LSC) The cells were then washed 3 times * (3 x 0.5ml x 5min) with ice cold PBS/ sodium azide (0.05% w/v NaN 3 / PBS ) to inhibit any further cellular metabolism and remove any ribozyme loosely associated with the cell surface. The washings were collected and their radioactive content determined by LSC. Cell monolayers were solubilized by shaking with 0.5ml of 3% v/v Triton X100 (Aldrich Chemical Company, Gillingham, UK) in distilled water for 1 hour at room temperature. The wells were washed twice more (2 x 0.5ml) with Triton X-100 to ensure that all the cells had been harvested and the radioactivity content of the cellular fraction determined by LSC. Unless otherwise indicated, all experiments were performed at a final concentration of O.OlμM 32P internally labeled riboxyme and incubated for a period of 60 minutes. The uptake of Amino ribozymes were compared in different cell lines. The results show that cellular association of these ribozymes ranged from 0.325 ± 0.021 ng/10 5 cells in intestinal epithelial cells to 1.09 ± 0.207 ng/10 5 cells in the macrophage cell line. The ability of ribozymes to penetrate the cell membrane and the mechanism of entrance are important considerations in developing ribozymes as therapeutics. The mechanisms by which oligodeoxynucleotides enter cells has been well documented (for review see Akhtar & Juliano, 1991) and include the involvement of fluid phase, adsorptive and receptor mediated endocytosis. The mechanism and extent of uptake is dependent on many factors including oligonucleotide type and length and cell line studied. In contrast, however, no mechanism of cellular uptake has yet been described for ribozymes and ribonucleotides . In order to investigate the means of uptake of ribozyme RPI.4782 in glioma cells, a series of cellular association studies were performed in the human glioma derived cell line, U87-MG. The cellular association of ribozyme RPI.4782 to U87- MG cells appeared to be biphasic, with a rapid initial phase continuing for approximately two hours followed by a slower second phase. The cellular association of oligonucleotides has been shown to be a dynamic process representing both uptake and efflux processes (Jaroszewski & Cohen, 1990) . Consequently, the plateauing seen in the second phase could represent an equilibrium of both uptake and exocytosis of ribozyme. The uptake of ribozyme RPI.4782 was strongly dependent on temperature, suggesting that an active process is involved. In addition, the metabolic inhibitors, sodium azide and 2-deoxyglucose significantly inhibited cellular association by 66%, demonstrating that ribozyme uptake was also energy dependent. The energy and temperature dependency of cellular association of this ribozyme in U87-MG cells are characteristic of an active process, indicating that the mechanism of uptake is via endocytosis. These findings do not, however, distinguish whether fluid phase endocytosis or receptor mediated endocytosis is involved; since both mechanisms will be effected by these parameters (Beltinger et al . , 1994) . In order to evaluate the pathway of internalization, the uptake of a fluid phase marker, [14C] mannitol, was measured to determine the extent of pinocytosis in U87-MG cells. The basal rate of pinocytosis in these cells remained extremely low throughout the time period tested and it is unlikely, therefore, to account for a significant fraction of ribozyme uptake in this cell line. To investigate whether ribozyme RPI.4782 is taken up into U87-MG cells by receptor mediated endocytosis a self competition study was conducted. Ribozyme uptake was found to be significantly inhibited by competition with unlabeled ribozyme. This demonstrates that cellular association was concentration dependent and suggests that the dominant uptake mechanism is via receptor mediated endocytosis . Receptor mediated endocytosis involves the internalization of molecules via specific membrane protein, cell surface receptors. Consequently, a proteolytic enzyme such as trypsin or pronase® can be used to determine the extent to which membrane proteins mediate uptake (Beck et al . , 1996; Shoji et al. , 1991; Wu-pong et al . , 1994). In a study investigating the cellular association of oligonucleotides in intestinal CaCo-2 cells, Beck et_ al. (1996) reported a 50% reduction of uptake upon cell surface washing with pronase, while 60% of oligonucleotide uptake was reported to be trypsin sensitive in Rauscher Red 5-1.5 ertythroleukemai cells (Wu-Pong e_t al . , 1994) . To further characterize ribozyme uptake, the effects of the endocytosis inhibitor, phenylarsine oxide and the endosomal alkalinizers, chloroquine and monensin could be studied (Loke et al . , 1989; Wu-Pong et al. , 1994) . To determine whether specific binding sites are involved in the uptake of ribozyme RPI.4782 in U87-MG cells, competition studies are required to evaluate the effect on ribozyme uptake by competitors such as oligonucleotides, ATP and other polyanions, such as dextran sulphate and heparin. The cellular association of ribozyme RPI.4782 to U87-MG cells was also found to be pH dependent. In fact a decrease in pH from pH 8 to pH 5 resulted in a significant increase in cellular association. The effect of pH on ribozyme partition coefficients had not as yet been undertaken in order to determine whether the increase in cellular association was due to an increase in the partition coefficient of the ribozyme, at low pH conditions. The increase of cellular association at low pH is in agreement with the work of Goodarzi et al . (1991) and Kitajima et al . (1992) who found that cellular association of oligonucleotides also increased under acidic conditions. It has been postulated that enhanced binding could be due to the presence of a 34kDa membrane protein receptor that functions around pH 4.5 (Goodarzi e_t al . , 1991). In addition, the α amino group of lysine, the guanidium group of arginine and protonated imidazole of histidine have been suggested to be possible oligonucleotide binding sites (Blackburn et al. , 1990). Histidine, having a pKa of 6.5 is susceptible to protonation over a pH range of 7.2 to 5.0. Therefore, the enhanced affinity of ribozyme RPI.4782 to U87-MG cells at pH 5.0 could be due to protonation of histidine residues present at the binding site. In general these observations suggest that the pathway of cellular uptake of ribozyme involves an active cellular process; indications are that the predominant mechanism of uptake is via receptor mediated endocytosis. Example 7: Ribozyme stability in U87-MG Cells In order to ensure that the results obtained from the uptake studies represented cell association of intact 37mer ribozyme and not degraded ribozyme or free [ 32 P] label, the stability of this ribozyme, when incubated with U87 cells ,was examined. U87-MG cells were seeded onto 24 well-plates as previously described and used approximately 24 hours post seeding. Internally [ 32 P] labeled ribozyme RPI.4782 was added to 200μl of serum free media to give a final concentration of lOnM. lOμl aliquots of the apical solution were collected at variable time points over a period of 4 hours, mixed with an equal volume of formamide loading buffer ( 9:1 v/v formamide: lx TBE) and stored at -20C. Prior to gel loading, the samples were heated to 100°C for 5 minutes and separated on 7M urea / 20% acrylamide gels; bands were detected by autoradiography of wet gels. For comparative purposes, the stability profiles of 5' labeled ribozyme RPI.4782, 5' end labeled all RNA 15mer substrate, and 5' end labeled 37mer PO and PS oligodeoxynucleotides were also measured under the same conditions . To ensure that any findings obtained from uptake studies represented the cellular association of intact 37mer ribozyme and not that of shorter degraded fragments or free [ 32 P] label, the degradation of 5'-end and internally [ 32 P] labeled ribozyme was examined when exposed to U87-MG cells. For comparative purposes, the stability profile of an unmodified RNA substrate was also measured under the same conditions. The chemically modified ribozyme remained largely intact throughout a four hour incubation period. While no degradation was evident from the internally labeled sample, the 5'-end labeled ribozyme did exhibit some degradation after 120 minutes. This indicates that 5' dephosphorylation occurred in the latter case. In contrast, however, the unmodified RNA substrate was completely degraded within 10 minutes incubation with the U87-MG cell monolayer. The ribozyme was clearly protected from cellular nucleuses by the chemical modifications previously described. Optimizing Ribozyme Activity Sullivan, et. al . , supra, describes the general methods for delivery of enzymatic RNA molecules. The data presented in Examples above indicate that different cationic lipids can deliver active ribozymes to smooth muscle cells. Experiments similar to those performed in above-mentioned Examples are used to determine which lipids give optimal delivery of ribozymes to specific cells. Other such delivery methods are known in the art and can be utilized in this invention. The proliferation of smooth muscle cells can also be inhibited by the direct addition of chemically stabilized ribozymes. Presumably, uptake is mediated by passive diffusion of the anionic nucleic acid across the cell membrane. In this case, efficacy could be greatly enhanced by directly coupling a ligand to the ribozyme. The ribozymes are then delivered to the cells by receptor-mediated uptake. Using such conjugated abducts, cellular uptake can be increased by several orders of magnitude without having to alter the phosphodiester linkages necessary for ribozyme cleavage activity. Alternatively, ribozymes may be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres. The RNA/vehicle combination is locally delivered by direct injection or by use of a catheter, infusion pump or sent. Alternative routes of delivery include, but are not limited to, intramuscular injection, aerosol inhalation, oral (tablet or pill form) , topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. More detailed descriptions of ribozyme delivery and administration are provided in Sullivan, et. al. , supra and Draper, et al. , supra which have been incorporated by reference herein. Chemical modifications, ribozyme sequences and ribozyme motifs described in this invention are meant to be non-limiting examples, and those skilled in the art will recognize that other modifications (base, sugar and phosphate modifications) to enhance nuclease stability of a ribozyme can be readily generated using standard techniques and are hence within the scope of this invention. Use of Ribozymes Targeting EGFR Overexpression of the EGFR has been reported in a number of cancers (see above) . Thus, inhibition of EGFR expression (for example using ribozymes) can reduce cell proliferation of a number of cancers, _in vitro and _in vivo and can reduce their proliferative potential. Ribozymes, with their catalytic activity and increased site specificity (see above) , are likely to represent a potent and safe therapeutic molecule for the treatment of cancer. In the present invention, ribozymes are shown to inhibit smooth muscle cell proliferation and stromelysin gene expression. 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. These ribozymes can be used in conjunction with existing cancer therapies . Gliomas are the most common primary tumors arising from the brain, in fact each year malignant gliomas account for approximately 2.5% of the deaths from cancer (Bruner, 1994). These gliomas are morphologically and biologically heterogeneous and include neoplasms derived from several cell types. Astrocytomas form the largest single group among the primary tumors (75-90%) which also includes oligodendrogliomas, ependymomas and mixed gliomas (Bruner, 1994) . Distinct histological features allow astrocytomas to be graded into levels of anaplasia, the most widely used today involves a three tiered grading system (Ringertz, 1950) dividing astrocytomas into low grade astrocytomas, anaplastic astrocytomas and glioblastomas . The most malignant and frequently occurring form, glioblastoma multiform (GBM) , accounts for approximately one third of all primary brain tumors (Wong et al . , 1994) . This tumor is so undifferentiated that it cell of origin remains obscure, however most examples are generally thought to arise from astrocytes because glial fibrillary acidic protein (GFAP) , a histological marker for astrocytes, can be identified in the cell cytoplasm. The histological morphology of glioblastoma can be highly variable, confirming the name "multiforme". The characteristic features of glioblastoma multiform is tumor necrosis. The individual cells may be small with a high nuclear / cytoplasmic ratio or very large and bizarre with abundant eosinophilic cytoplasm. The small cells are the more proliferative ones and show a more aggressive course. In fact some glioblastomas are so highly cellular that the population of small anaplastic cells stimulates primitive neuroectodermal tumors such as medulloblastoma. These small cells often appear to condense around areas of tumor necrosis forming characteristic 'pseudopalisades" . They also have the propensity to infiltrate the brain extensively, giving the appearance of multifocal gliomas. Despite advances in many areas of cancer research and treatment, glioblastoma multiform almost always proves fatal, with a median survival rate of less than one year and a 5 year survival rate of 5.5% or less (Martuza et al. , 1991) . At present, no therapeutic modality has substantially changed the outcome of patients with glioblastoma. Characteristics of this type of tumor, including it's invasive nature, it's ability to spread locally and distantly while avoiding recognition by the immune system, it's relative resistance to radiation and a high local recurrence rate, limit the success of conventional therapy. The effective treatment of glioblastoma multiform, therefore, presents a tremendous challenge. The current methods of treatment used in the management of malignant gliomas are briefly reviewed. Surgery: The cornerstone of therapy for glioblastoma multiform tumors has been surgery. The use of microsurgical techniques, intraoperative ultrasonic aspiration, electrophysiologic monitoring and lasers make the surgical procedure safe and accurate (Kornblith et al. , 1993) . Although surgery does improve the survival of patients with glioblastoma multiform, the inability to surgically remove eloquent areas of cerebral cortex invaded by the tumor render such ablative technologies of only modest value. Radiotherapy: Malignant gliomas such as glioblastoma multiform exhibit an extraordinary resistance to radiotherapy and as a consequence the effectiveness of this form of treatment is limited. The sensitivity of the surrounding, unaffected, brain limits the dose that can safely be delivered to 60Gy (Leibel et. al. , 1994), which is well below the level required to completely eradicate the primary tumor in the majority of patients. In addition, whole brain radiotherapy does not prevent local tumor recurrence. The effective use of more localized forms of radiotherapy, such as radiosensitizers and radiosurgical techniques, are at present under review. Chemotherapy : Chemotherapy has been shown to be effective adjuncts to surgery and radiotherapy in the treatment of cancer. Unfortunately, however, chemotherapy has had a limited impact on survival in patients with high grade astrocytomas. A report published in 1993 determined that adding chemotherapy to surgery and radiation improved the median survival duration in these patients from 9.4 to 12 months (Fine et al^_, 1993) . Generally, the relatively lipid soluble and non ionized nitrosourea drugs; e.g. carmustine, lomustine, semustine and nimustine, have proved to be the most active single chemotherapy agents for treating malignant astrocytomas (Lesser & Grossman, 1994). New drugs continue to enter clinical trials in patients with glioblastoma; none so far, however, have substantially prolonged a patient's life span. A myriad of physiological and biological factors such as the blood brain barrier, heterogeneous and resistant tumor cell populations and unacceptable toxicities have limited the efficacy of these agents. Different routes of administration have been used to overcome the impenetrability of the blood brain barrier. A unique delivery system has been reported (Brem et. al . , 1991) which incorporates biodegradable polymers impregnated with chemotherapy agents. These polymers are placed topically at the resection site and slowly release the drugs as they degrade. Direct injection into tumors may also be useful as a means to deliver the highest dose to the tumor site without systemic exposure. Immunotherapy : Glioblastoma multiform is an appropriate target for immunological directed therapy. Studies have revealed that sera from patients with GBM stimulates little or no humoral response. A realistic approach, therefore, is to stimulate a stronger immune response in glioblastoma patients. Although this approach looks promising in theory, as yet no effective means of stimulating a clinically immune response has been identified. The most promising avenue, through the use of lymphokine activated killer (LAK) cells and interleukin - 2, has been limited by lack of tumor specific cell homing and difficulties with LAK cell delivery and toxicity. Advances in the understanding of the molecular basis of cancer has now made it possible to design molecules that specifically interact with cancer cells. The most promising modes of therapy for the treatment of GBM, therefore, may lie with molecular based technologies which employ genetic interventions to alter the properties or behaviour of specific cells. In fact , glioblastoma multiform tumors are ideal candidates for this type of therapy since they rarely metastasize, are accessible to direct delivery techniques and can be precisely monitored by MRI and CT scans. The tumor cells may also divide rapidly, which enables agents such as retroviruses to infect the cells and synthesize genes leading to tumor cell destruction. (Kornblith et al. , 1993) . Many detailed cytogenetic studies have been performed on malignant gliomas and these reveal commonly occurring abnormalities (Bigner & Vogelstein, 1990). For example, approximately 80% of malignant gliomas have gains of one or more copies of chromosome 7 and approximately 60% show a loss of chromosome 10. In addition, one of the most consistent genetic abnormalities is the presence of double minute chromosomes (DMs) . Double minute chromosomes refer to small portions of chromosomes which are paired but lack a centromere; they are the karyotypic manifestation of gene amplification. The presence of such DMs have been found in over 50% of glioblastomas, with some tumors possessing 50 - 100 copies of DMs per cell (Ostrowski et al . , 1994). 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The close relationship between ribozyme activity and the structure of the target RNA allows the detection of mutations in any region of the molecule which alters the base-pairing and three-dimensional structure of the target RNA. By using multiple ribozymes described in this invention, one may map nucleotide changes which are important to RNA structure and function i_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 specified gene products in the progression of disease. In this manner, other genetic targets may be defined as important mediators of the disease. These experiments will lead to better treatment of the disease progression by affording the possibility of combinational therapies (e.g., multiple ribozymes targeted to different genes, ribozymes coupled with known small molecule inhibitors, or intermittent treatment with combinations of ribozymes and/or other chemical or biological molecules) . Other in vitro uses of ribozymes of this invention are well known in the art, and include detection of the presence of mRNAs associated with EGFR related condition. Such RNA is detected by determining the presence of a cleavage product after treatment with a ribozyme using standard methodology. In a specific example, ribozymes which can cleave only wild-type or mutant forms of the target RNA are used for the assay. The first ribozyme is used to identify wild-type RNA present in the sample and the second ribozyme will be used to identify mutant RNA in the sample. As reaction controls, synthetic substrates of both wild-type and mutant RNA will be cleaved by both ribozymes to demonstrate the relative ribozyme efficiencies in the reactions and the absence of cleavage of the "non-targeted" RNA species. The cleavage products from the synthetic substrates will also serve to generate size markers for the analysis of wild-type and mutant RNAs in the sample population. Thus each analysis will require two ribozymes, two substrates and one unknown sample which will be combined into six reactions. The presence of cleavage products will be determined using an RNAse protection assay so that full-length and cleavage fragments of each RNA can be analyzed in one lane of a polyacrylamide gel. It is not absolutely required to quantify the results to gain insight into the expression of mutant RNAs and putative risk of the desired phenotypic changes in target cells. The expression of mRNA whose protein product is implicated in the development of the phenotype (i.e., EGFR) is adequate to establish risk. If probes of comparable specific activity are used for both transcripts, then a qualitative comparison of RNA levels will be adequate and will decrease the cost of the initial diagnosis. Higher mutant form to wild-type ratios will be correlated with higher risk whether RNA levels are compared qualitatively or quantitatively. Other embodiments are within the following claims. Table I Table 1: Characteristics of naturally occurring ribozymes Group I Introns • Size: -150 to >1000 nucleotides. • Requires a U in the target sequence immediately 5' of the cleavage site. • Binds 4-6 nucleotides at the 5 '-side of the cleavage site. • Reaction mechanism: attack by the 3 ' -OH of guanosine to generate cleavage products with 3 ' -OH and 5'- guanosine . • Additional protein cofactors required in some cases to help folding and maintainance of the active structure [1] • • Over 300 known members of this class. Found as an intervening sequence in Tetrahymena thermophila rRNA, fungal mitochondria, chloroplasts, phage T4, blue- green algae, and others. • Major structural features largely established through phylogenetic comparisons, mutagenesis, and biochemical studies [2,3] . • Complete kinetic framework established for one ribozyme [4,5,6,7]. • Studies of ribozyme folding and substrate docking underway [8,9,10]. • Chemical modification investigation of important residues well established [11,12]. • The small (4-6 nt) binding site may make this ribozyme too non-specific for targeted RNA cleavage, however, Table I the Tetrahymena group I intron has been used to repair a "defective" • .-galactosidase message by the ligation of new β- galactosidase sequences onto the defective message [13]. RNAse P RNA (Ml RNA) • Size: -290 to 400 nucleotides. • RNA portion of a ubiquitous ribonucleoprotein enzyme. • Cleaves tRNA precursors to form mature tRNA [14] . • Reaction mechanism: possible attack by M 2+ -OH to generate cleavage products with 3 ' -OH and 5'- phosphate . • RNAse P is found throughout the prokaryotes and eukaryotes. The RNA subunit has been sequenced from bacteria, yeast, rodents, and primates. • Recruitment of endogenous RNAse P for therapeutic applications is possible through hybridization of an External Guide Sequence (EGS) to the target RNA [15,16] • Important phosphate and 2' OH contacts recently identified [17,18] Group II Introns • Size: >1000 nucleotides. • Trans cleavage of target RNAs recently demonstrated [19,20]. • Sequence requirements not fully determined. • Reaction mechanism: 2 ' -OH of an internal adenosine generates cleavage products with 3 ' -OH and a "lariat" RNA containing a 3 '-5' and a 2 '-5' branch point. Table I • Only natural ribozyme with demonstrated participation in DNA cleavage [21,22] in addition to RNA cleavage and ligation. • Major structural features largely established through phylogenetic comparisons [23] . • Important 2' OH contacts beginning to be identified [24] • Kinetic framework under development [25] Neurospora VS RNA • Size: -144 nucleotides. • Trans cleavage of hairpin target RNAs recently demonstrated [26] . • Sequence requirements not fully determined. • Reaction mechanism: attack by 2 ' -OH 5' to the scissile bond to generate cleavage products with 2 ',3 '-cyclic phosphate and 5 ' -OH ends. • Binding sites and structural requirements not fully determined. • Only 1 known member of this class. Found in Neurospora VS RNA. Hammerhead Ribozyme (see text for references) • Size: -13 to 40 nucleotides. • Requires the target sequence UH immediately 5 'of the cleavage site. • Binds a variable number nucleotides on both sides of the cleavage site. • Reaction mechanism: attack by 2 ' -OH 5' to the scissile bond to generate cleavage products with 2 ',3 '-cyclic phosphate and 5 ' -OH ends. Table I • 14 known members of this class. Found in a number of plant pathogens (virusoids) that use RNA as the infectious agent. • Essential structural features largely defined, including 2 crystal structures [] • Minimal ligation activity demonstrated (for engineering through i_n vitro selection) [] • Complete kinetic framework established for two or more ribozymes [ ] . • Chemical modification investigation of important residues well established []. Hairpin Ribozyme • Size: -50 nucleotides. • Requires the target sequence GUC immediately 3 ' of the cleavage site. • Binds 4-6 nucleotides at the 5 '-side of the cleavage site and a variable number to the 3 '-side of the cleavage site. • Reaction mechanism: attack by 2 ' -OH 5' to the scissile bond to generate cleavage products with 2 ',3 '-cyclic phosphate and 5 ' -OH ends. • 3 known members of this class. Found in three plant pathogen (satellite RNAs of the tobacco ringspot virus, arabis mosaic virus and chicory yellow mottle virus) which uses RNA as the infectious agent. • Essential structural features largely defined [27,28,29,30] • Ligation activity (in addition to cleavage activity) makes ribozyme amenable to engineering through in vitro selection [31] • Complete kinetic framework established for one ribozyme [32] . Table I • Chemical modification investigation of important residues begun [33,34]. Hepatitis Delta Virus (HDV) Ribozyme • Size: -60 nucleotides. • Trans cleavage of target RNAs demonstrated [35] . • Binding sites and structural requirements not fully determined, although no sequences 5' of cleavage site are required. Folded ribozyme contains a pseudoknot structure [36] . • Reaction mechanism: attack by 2 ' -OH 5' to the scissile bond to generate cleavage products with 2 ' , 3 ' -cyclic phosphate and 5 ' -OH ends. • Only 2 known members of this class. Found in human HDV. • Circular form of HDV is active and shows increased nuclease stability [37] 1. Mohr, G.; Caprara, M.G.; Guo, Q. ; Lambowitz, A.M. Nature, 37fJ, 147-150 (1994) . 2. Michel, Francois; Westhof, Eric. Slippery substrates. Nat. Struct. Biol. (1994), 1(1), 5-7. 3. Lisacek, Frederique; Diaz, Yolande; Michel, Francois. Automatic identification of group I intron cores in genomic DNA sequences. J. Mol . Biol. (1994), 235(4), 1206-17. 4. Herschlag, Daniel; Cech, Thomas R.. Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 1. Kinetic description of the reaction of an RNA substrate complementary to the active site. Biochemistry (1990), 29(44), 10159-71. 5. Herschlag, Daniel; Cech, Thomas R.. Catalysis of RNA cleavage by the Tetrahymena thermophila Table I ribozyme. 2. Kinetic description of the reaction of an RNA substrate that forms a mismatch at the active site. Biochemistry (1990), 29(44), 10172-80. 6. Knitt, Deborah S.; Herschlag, Daniel. pH Dependencies of the Tetrahymena Ribozyme Reveal an Unconventional Origin of an Apparent pKa . Biochemistry (1996), 35(5), 1560-70. 7. Bevilacqua, Philip C . ; Sugimoto, Naoki; Turner, Douglas H.. A mechanistic framework for the second step of splicing catalyzed by the Tetrahymena ribozyme. Biochemistry (1996), 35(2), 648-58. 8. Li, Yi; Bevilacqua, Philip C; Mathews, David; Turner, Douglas H.. Thermodynamic and activation parameters for binding of a pyrene-labeled substrate by the Tetrahymena ribozyme: docking is not diffusion-controlled and is driven by a favorable entropy change. Biochemistry (1995), 34(44), 14394-9. 9. Banerjee, Aloke Raj; Turner, Douglas H.. The time dependence of chemical modification reveals slow steps in the folding of a group I ribozyme. Biochemistry (1995), 34(19), 6504-12. 10. Zarrinkar, Patrick P.; Williamson, James R.. The P9.1-P9.2 peripheral extension helps guide folding of the Tetrahymena ribozyme. Nucleic Acids Res. (1996), 24(5), 854-8. 11. Strobel, Scott A.; Cech, Thomas R.. Minor groove recognition of the conserved G.cntdot.U pair at the Tetrahymena ribozyme reaction site. Science (Washington, D. C.) (1995), 267(5198), 675-9. 12. Strobel, Scott A.; Cech, Thomas R.. Exocyclic Amine of the Conserved G.cntdot.U Pair at the Cleavage Site of the Tetrahymena Ribozyme Contributes to 5'- Table I Splice Site Selection and Transition State Stabilization. Biochemistry (1996), 35(4), 1201-11. 13. Sullenger, Bruce A.; Cech, Thomas R.. Ribozyme- mediated repair of defective mRNA by targeted trans-splicing. Nature (London) (1994), 371(6498), 619-22. 14. Robertson, H.D.; Altman, S . ; Smith, J.D. J. Biol. Chem., 24X, 5243-5251 (1972). 15. Forster, Anthony C; Altman, Sidney. External guide sequences for an RNA enzyme. Science (Washington, D. C, 1883-) (1990), 249(4970), 783-6. 16. Yuan, Y.; Hwang, E. S.; Altman, S. Targeted cleavage of mRNA by human RNase P. Proc. Natl. Acad. Sci. USA (1992) 89, 8006-10. 17. Harris, Michael E.; Pace, Norman R.. Identification of phosphates involved in catalysis by the ribozyme RNase P RNA. RNA (1995), 1(2), 210-18. 18. Pan, Tao; Loria, Andrew; Zhong, Kun. Probing of tertiary interactions in RNA: 2 ' -hydroxylbase contacts between the RNase P RNA and pre-tRNA. Proc. Natl. Acad. Sci. U. S. A. (1995), 92(26), 12510-14. 19. Pyle, Anna Marie; Green, Justin B.. Building a Kinetic Framework for Group II Intron Ribozyme Activity: Quantitation of Interdomain Binding and Reaction Rate. Biochemistry (1994), 33(9), 2716-25. 20. Michels, William J. Jr.; Pyle, Anna Marie. Conversion of a Group II Intron into a New Multiple-Turnover Ribozyme that Selectively Cleaves Oligonucleotides: Elucidation of Reaction Mechanism and Structure/Function Relationships. Biochemistry (1995), 34(9), 2965-77. Table I 21. Zimmerly, Steven; Guo, Huatao; Eskes, Robert; Yang, Jian; Perlman, Philip S.; Lambowitz, Alan M.. A group II intron RNA is a catalytic component of a DNA endonuclease involved in intron mobility. Cell (Cambridge, Mass.) (1995), 83(4), 529-38. 22. Griffin, Edmund A., Jr.; Qin, Zhifeng; Michels, Williams J. , Jr.; Pyle, Anna Marie. Group II intron ribozymes that cleave DNA and RNA linkages with similar efficiency, and lack contacts with substrate 2 ' -hydroxyl groups. Chem. Biol. (1995), 2(11), 761-70. 23. Michel, Francois; Ferat, Jean Luc. Structure and activities of group II introns . Annu. Rev. Biochem. (1995), 64, 435-61. 24. Abramovitz, Dana L. ; Friedman, Richard A.; Pyle, Anna Marie. Catalytic role of 2 ' -hydroxyl groups within a group II intron active site. Science (Washington, D. C.) (1996), 271(5254), 1410-13. 25. Daniels, Danette L.; Michels, William J. , Jr.; Pyle, Anna Marie. Two competing pathways for self- splicing by group 11 introns: a quantitative analysis of in vitro reaction rates and products. J. Mol. Biol. (1996), 236(1), 31-49. 26. Guo, Hans C. T.; Collins, Richard A.. Efficient trans-cleavage of a stem-loop RNA substrate by a ribozyme derived from Neurospora VS RNA. EMBO J. (1995), 14(2), 368-76. 27. Hampel, Arnold; Tritz, Richard; Hicks, Margaret; Cruz, Phillip. 'Hairpin' catalytic RNA model: evidence for helixes and sequence requirement for substrate RNA. Nucleic Acids Res. (1990), 18(2), 299-304. Table I 28. Chowrira, Bharat M. ; Berzal-Herranz, Alfredo; Burke, John M.. Novel guanosine requirement for catalysis by the hairpin ribozyme. Nature (London) (1991), 354(6351), 320-2. 29. Berzal-Herranz, Alfredo; Joseph, Simpson; Chowrira, Bharat M. ; Butcher, Samuel E.; Burke, John M.. Essential nucleotide sequences and secondary structure elements of the hairpin ribozyme. EMBO J. (1993), 12(6), 2567-73. 30. Joseph, Simpson; Berzal-Herranz, Alfredo; Chowrira, Bharat M. ; Butcher, Samuel E.. Substrate selection rules for the hairpin ribozyme determined by in vitro selection, mutation, and analysis of mismatched substrates. Genes Dev. (1993), 7(1), 130-8. 31. Berzal-Herranz, Alfredo; Joseph, Simpson; Burke, John M.. In vitro selection of active hairpin ribozymes by sequential RNA-catalyzed cleavage and ligation reactions. Genes Dev. (1992), 6(1), 129- 34. 32. Hegg, Lisa A.; Fedor, Martha J.. Kinetics and Thermodynamics of Intermolecular Catalysis by Hairpin Ribozymes. Biochemistry (1995), 34(48), 15813-28. 33. Grasby, Jane A.; Mersmann, Karin; Singh, Mohinder; Gait, Michael J.. Purine Functional Groups in Essential Residues of the Hairpin Ribozyme Required for Catalytic Cleavage of RNA. Biochemistry (1995) , 34(12), 4068-76. 34. Schmidt, Sabine; Beigelman, Leonid; Karpeisky, Alexander; Usman, Nassim; Sorensen, Ulrik S.; Gait, Michael J.. Base and sugar requirements for RNA cleavage of essential nucleoside residues in Table I internal loop B of the hairpin ribozyme: implications for secondary structure. Nucleic Acids Res. (1996), 24(4), 573-81. 35. Perrotta, Anne T . ; Been, Michael D.. Cleavage of oligoribonucleotides by a ribozyme derived from the hepatitis .delta, virus RNA sequence. Biochemistry (1992), 31(1), 16-21. 36. Perrotta, Anne T . ; Been, Michael D.. A pseudoknot- like structure required for efficient self-cleavage of hepatitis delta virus RNA. Nature (London) (1991), 350(6317), 434-6. 37. Puttaraju, M.; Perrotta, Anne T.; Been, Michael D., A circular trans-acting hepatitis delta virus ribozyme. Nucleic Acids Res. (1993), 21(18), 4253- 8. Table I I Table II: 2.5 umol RNA Synthesis Cycle Reagent Equivalents Amount Wait Time* Phosphoramidites 6.5 163 μL 2.5 S-Ethyl Tetrazole 23.8 238 μL 2.5 Acetic Anhydride 100 233 μL 5 sec N-Methyl Imidazole 186 233 μL 5 sec TCA 83.2 1.73 mL 21 sec Iodine 8.0 1.18 mL 45 sec Acetonitrile NA 6.67 mL NA * Wait time does not include contact time during delivery. Table III TABLE III : Human EGF-R Hammerhead Ribozyme and Target Sequences Table III Table III Table III Table III Table III Table III Table III Table III Table III Table III Table III Table III Table III Table III Table III Table III Table III 5482 GCAUUGUUA AGAAAGUA 811 UACUUUCU CUGAUGA X GAA AACAAUGC 1634 5490 AAGAAAGUA UUUGAUUU 812 AAAUCAAA CUGAUGA X GAA ACUUUCUU 1635 5492 GAAAGUAUU UGAUUUUU 813 AAAAAUCA CUGAUGA X GAA AUACUUUC 1636 5493 AAAGUAUUU GAUUUUUG 814 CAAAAAUC CUGAUGA X CAA AAUACUUU 1637 5497 UAUUUGAUU UUUGUCUC 815 GAGACAAA CUGAUGA X GAA AUCAAAUA 1638 5498 AUUUGAUUU UUGUCUCA 816 UGAGACAA CUGAUGA X GAA AAUCAAAU 1639 5499 UUUGAUUUU UGUCUCAA 817 UUGAGACA CUGAUGA X GAA AAAUCAAA 1640 5500 UUGAUUUUU GUCUCAAU 818 AUUGAGAC CUGAUGA X GAA AAAAUCAA 1641 5503 AUUUUUGUC UCAAUGAA 819 UUCAUUGA CUGAUGA X GAA ACAAAAAU 1642 5505 UUUUGUCUC AAUGAAAA 820 UUUUCAUU CUGAUGA X GAA AGACAAAA 1643 5515 AUGAAAAUA AAACUAUA 821 UAUAGUUU CUGAUGA X GAA AUUUUCAU 1644 5521 AUAAAACUA UAUUCAUU 822 AAUGAAUA CUGAUGA X GAA AGUUUUAU 1645 5523 AAAACUAUA UUCAUUUC 823 GAAAUGAA CUGAUGA X GAA AUAGUUUU 1646 Where "X" represents stem II region of a HH ribozyme (Hertel et al . , 1992 Nucleic Acids Res . 20 3252 ) . The length of stem II may be >2 base-pairs . Table IV TABLE IV: Human EGF-R Hairpin Ribozyme and Target Sequence Table IV Table IV Table IV Table IV

Claims: Claims 1. An enzymatic nucleic acid molecule which specifically cleaves RNA derived from an epidermal growth factor receptor (EGFR) gene. 2. The enzymatic nucleic acid molecule of claim 1, wherein said nucleic acid molecule is in a hairpin motif. 3. The enzymatic nucleic acid molecule of claim 1, wherein said nucleic acid molecule is in a hammerhead motif. 4. The enzymatic nucleic acid molecule of claim 3, wherein said nucleic acid molecule comprises a stem II region of length greater than or equal to 2 base pairs. 5. The enzymatic nucleic acid molecule of claim 3, wherein the binding arms of said nucleic acid molecule comprises sequences complementary to any of SEQ ID NOs 1- 823. 6. The enzymatic nucleic acid molecule of claim 2, wherein the binding arms of said nucleic acid molecule comprises sequences complementary to any of SEQ ID NOs 1759-1870. 7. The enzymatic nucleic acid molecule of claim 2, wherein said nucleic hairpin motif consists essentially of any ribozyme sequence shown as SEQ ID NOs 1647-1758. 8. The enzymatic nucleic acid molecule of claim 3, wherein said nucleic hammerhead motif consists essentially of any ribozyme sequence shown as SEQ ID NOs 824-1646. 9. The enzymatic nucleic acid molecule of claim 1, wherein said nucleic acid molecule is in a hepatitis delta virus, VS nucleic acid, group I intron. Group II intron, or RNase P nucleic acid motif. 10. The enzymatic nucleic acid molecule of claim 1, wherein said nucleic acid comprises between 12 and 100 bases complementary to said RNA. 11. The enzymatic nucleic acid molecule of claim 1, wherein said nucleic acid comprises between 14 and 24 bases complementary to said mRNA. 12. A mammalian cell including an enzymatic nucleic acid molecule of claim 1. 13. The cell of claim 12, wherein said cell is a human cell. 14. An expression vector comprising nucleic acid sequence encoding at least one of the enzymatic nucleic acid molecule of claim 1, in a manner which allows expression of that enzymatic nucleic acid molecule. 15. A mammalian cell including an expression vector of claim 14. 16. The cell of claim 15, wherein said cell is a human cell. 17. A method for treatment of cancer comprising the step of administering to a patient the enzymatic nucleic acid molecule of claim 1. 18. A method for treatment of a cancer comprising the step of administering to a patient the expression vector of claim 14. 19. A method for treatment of cancer comprising the steps of: a) isolating cells from a patient; b) administering to said cells the enzymatic nucleic acid molecule of claim 1 or 14 ; and c) introducing said cells back into said patient. 20. A pharmaceutical composition comprising the enzymatic nucleic acid molecule of claim 1. 21. A method of treatment of a patient having a condition associated with the level of EGFR, wherein said patient is administered the enzymatic nucleic acid molecule of claim 1. 22. A method of treatment of a patient having a condition associated with the level of EGFR, comprising contacting cells of said patient with the nucleic acid molecule of claim 1, and further comprising the use of one or more drug therapies. 23. The enzymatic nucleic acid molecule of claim 3, wherein said nucleic acid molecule comprises at least five ribose residues, and wherein said nucleic acid comprises phosphorothioate linkages at at least three of the 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. 24. The enzymatic nucleic acid of claim 22, wherein said nucleic acid comprises a 3' -3' linked inverted ribose moeity at said 3' end. 25. The enzymatic nucleic acid molecule of claim 3, wherein said nucleic acid molecule comprises at least five ribose residues, and wherein said nucleic acid molecule comprises phosphorothioate linkages at at least three of the 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 molecule, wherein said nucleic acid molecule comprises at least ten 2 ' -0- methyl modifications, and wherein said nucleic acid comprises a 3' end modification. 26. The enzymatic nucleic acid molecule of claim 3, wherein said nucleic acid molecule comprises at least five ribose residues, and wherein said nucleic acid molecule comprises phosphorothioate linkages at at least three of the 5' terminal nucleotides, and wherein said nucleic acid molecule comprises an abasic 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 molecule comprises a 3 ' -end modification. 27. The enzymatic nucleic acid molecule of claim 3, wherein said nucleic acid molecule comprises of at least five ribose residues, and wherein said nucleic acid comprises phosphorothioate linkages at at least three of the 5' terminal nucleotides, and wherein said nucleic acid molecule comprises a 6-methyl uridine substitution at position No. 4 and/or at position No. 7 of said nucleic acid molecule, wherein said nucleic acid molecule comprises at least ten 2 ' -O-methyl modifications, and wherein said nucleic acid molecule comprises a 3' end modification.

Inventors: AKHTAR SAGHIR, FELL PATRICIA, MCSWIGGEN JAMES A, AKHTAR, SAGHIR, FELL, PATRICIA, MCSWIGGEN, JAMES, A.
Applicants: RIBOZYME PHARM INC, UNIV ASTON, ASTON UNIVERSITY, RIBOZYME PHARMACEUTICALS, INC.
Application year: 1998
Main classification: C12N9
IPC codes: A61K31, A61K38, A61P35, C12N5, C12N9, C12N15