REGULATION OF REPRESSOR GENES USING NUCLEIC ACID MOLECULES

Abstract: Nucleic acid molecules which inhibit the expression of repressor genes are described. Also described are methods for preparing and using such nucleic acid molecules.

Description: DESCRIPTION REGULATION OF REPRESSOR GENES USING NUCLEIC ACID MOLECULES Background of the Invention This invention relates to a novel method for the inhibition of repressor genes. Specifically, inhibition of these repressor genes allows for the increased expression of beneficially gene products. Increasing the expression of beneficial gene products may be useful as therapeutic treatments for a wide range of indications. The following is a discussion of relevant art, none of which is admitted to be prior art to the present invention. RNA synthesis in a biological system involves a number of regulatory steps. For instance in a eukaryotic cell, RNA is synthesized from DNA genes via a process termed transcription. Transcription of RNA is an exquisitely regulated process. Transcription may be positively regulated when the RNA synthesis is stimulated or negatively regulated when the RNA synthesis is inhibited. This level of RNA synthesis regulation is facilitated by the interaction of one or more protein factors that generally exert their effect on transcription by interacting with specific cis- acting elements in the gene. While, positive regulation of gene expression is far more prevalent in eukaryotic cells, negative regulation plays an important role for many genes. These protein factors involved in negative regulation ("repressors") generally bind to cis-acting elements, usually upstream to genes, and cause the down regulation of that gene's transcription into RNA ("repression"). Only when these repressors are released from their targets can unhindered gene expression take place. Repressors can also function via other mechanisms such as interacting with protein factors involved in transcription thereby blocking transcription (e.g. protein-protein interaction; modification). A number of genes have been identified in eukaryotic systems that encode repressors. A few non-limiting examples of these repressor genes include: GATA Transcription Factors: Currently 5 factors make up the human GATA family of transcription factors: hGATA-1 (also known as Eryfl, GF-1, or NF-E1) (Trainor et al, 1990, Nature 343, 92-96; Genbank Accession No. X17254); hGATA-2 (Dorfman et al, 1992, J. Biol Chem. 167, 1279-1285; Genbank Accession No. M77810); hGATA-3 (Joulin et al, 1991, EMBO J. 10, 1809-1816; Genbank Accession No. X58072); hGATA-4 (Genbank Accession No. L34357); and hGATA- 6 (Huggon et al, 1997, Biochim. Biophys. Acta 1353, 98-102; Genbank Accession No.X95701). The GATA element or binding region for the GATA protein is present ~30bp upstream of the erythropoietin (Epo) gene. Transfection of QT6 cells with hGATA-1, -2, and -3 have shown that all three factors were able to bind to the GATA element. In addition, all three factors were shown to down regulate the expression of Erythropoietin in Hep3B cells (Imagawa et al, 1996, Acta Haematol. 95, 248-256). EAR3/COUP-TF-1 : EAR3/COUP-TF-1 (Miyajima et al, 1988, Nucleic Acids Research 16, 11057-11074; Genbank Accession No.X12795) has been shown to bind to the promoter region of Erythropoietin gene and negatively regulate its expression. This transcription factor appears to compete with hepatic nuclear factor 4 (HNF-4) which is believed to positively regulate Epo expression (Galson et al, 1995, Mo/. Cell Biol. 15, 2135-2144). TR2 & TR2-11 Orphan Receptors: TR2 orphan receptor (Chang et al, 1989, Biochem. Biophys. Res. Commun. 165, 735-741 ; Genbank Accession Co. M29959) and TR2-11 orphan receptor (Chang et al., supra; Genbank Accession No. M29960) are another set of transcription factors believed to negatively regulate Epo expression. The isolated TR2 cDNA encodes for a 603 amino acid protein with a mass of 67 kDa. This protein is believed to bind to a 3' enhancer region of the Epo gene and repress the expression of Epo (Lee et al, 1996, J. Biol. Chem. 271, 10405- 10412). CCAAT Displacement Protein (CDP): CDP (Neufeld et al, 1992, Nature Genet. 1, 50-55; Genbank Accession No. M74099) is a 180-200 kDa protein that has been shown to negatively regulate a number of genes including gamma-globin, NCAM, and gp91-phox gene, neutrophil collagenase, neutrophil gelatinase, and granulocyte colony stimulating factor (G-CSF) (Khanna-Gupta et al, 1997, Blood 90, 2784- 2795). Elevated levels of G-CSF would be beneficial for treatment during myelosuppressive chemotherapy, AIDS, and chronic neutropenia. Genesis: Also known as HNF-3/Forkhead, Genesis is a member of the winged helix transcriptional regulatory family and is believed to function as a repressor gene with activity in embryonic differentiation in drosophilia (Sutton et α/.,1996, J. Biol. Chem. 271, 23126-23133). Studies in 32D cells indicate that protein products of the Genesis gene may inhibit G-CSF gene expression (Xu et al., 1997, Leukemia 12, 207-212). A human homolog of this gene may have the same effect in human cells and is likely to regulate G-CSF gene expression. Interferon regulatory Factor-2 (IRF-2): IRF-2 (Itoh et al, 1989, Nucleic Acids Research 17, 8372; Genbank Accession No. XI 5949) is a member of the interferon regulatory factors of which more than 10 members exist. IRF-2 is believed to play a role in the regulation of expression for interferon-beta, interferon-alpha, and MHC class I (Nguyen et. al, 1997, Cytokine & Growth Factor Reviews 8, 293-312). The DNA binding domain of IRF-2 is located within the N-terminus of the protein. Imagawa et al, 1997, Blood 4, 1430-1439, describes the use an antisense phosphorothioate oligodeoxynucleotide having the sequence CGGGCGCCACCTCCATGGCCGGCCGGGCGG to inhibit hGATA-2 transcription factor expression in Hep3B cells. Summary Of The Invention The invention features novel nucleic acid-based techniques (e.g., enzymatic nucleic acid molecules, antisense nucleic acids, 2-5A antisense chimeras, triplex DNA, antisense nucleic acids containing RNA cleaving chemical groups). In one aspect, the invention features use of one or more of the nucleic acid- based techniques to inhibit the expression of repressor genes. Inhibition of the repressor gene can then result in the increased expression of genes repressed by these repressor genes. By "repressor genes" is meant genes whose expression can directly or indirectly down regulate or repress or suppress the expression of other genes. By "inhibit" it is meant that the activity of repressor genes or level of mRNAs or equivalent RNAs encoding repressor genes is reduced below that observed in the absence of the nucleic acid. In one embodiment, inhibition with ribozymes preferably is below that level observed in the presence of an enzymatically attenuated nucleic acid molecule that is able to bind to the same site on the mRNA, but is unable to cleave that RNA. In another embodiment, inhibition with nucleic acid molecules, including enzymatic nucleic acid and antisense molecules, is preferably greater than that observed in the presence of for example, an oligonucleotide with scrambled sequence or with mismatches. In another embodiment, inhibition of repressor genes with the nucleic acid molecule of the instant invention is greater than in the presence of the nucleic acid molecule than in its absence. By "antisense nucleic acid" it is meant a non-enzymatic nucleic acid molecule that binds to target RNA by means of RNA-RNA or RNA-DNA or RNA- PNA (protein nucleic acid; Egholm et al, 1993 Nature 365, 566) interactions and alters the activity of the target RNA (for a review see Stein and Cheng, 1993 Science 261, 1004). By "2-5A antisense chimera" it is meant, an antisense oligonucleotide containing a 5' phosphorylated 2'-5'-linked adenylate residues. These chimeras bind to target RNA in a sequence-specific manner and activate a cellular 2-5A-dependent ribonuclease which, in turn, cleaves the target RNA (Torrence et al, 1993 Proc. Natl Acad. Sci. USA 90, 1300). By "triplex DNA" it is meant an oligonucleotide that can bind to a double- stranded DNA in a sequence-specific manner to form a triple-strand helix. Formation of such triple helix structure has been shown to inhibit transcription of the targeted gene (Duval-Valentin et al, 1992 Proc. Natl Acad. Sci. USA 89, 504). By "gene" it is meant a nucleic acid that encodes an RNA. By "enzymatic nucleic acid" it is meant a nucleic acid molecule capable of catalyzing reactions including, but not limited to, site-specific cleavage and/or ligation of other nucleic acid molecules, cleavage of peptide and amide bonds, and trans-splicing. Such a molecule with endonuclease activity may have complementarity in a substrate binding region to a specified gene target, and also has an enzymatic activity that specifically cleaves RNA or DNA in that target. That is, the nucleic acid molecule with endonuclease activity is able to intramolecularly or intermolecularly cleave RNA or DNA and thereby inactivate a target RNA or DNA molecule. This complementarity functions to allow sufficient hybridization of the enzymatic RNA molecule to the target RNA or DNA to allow the cleavage to occur. 100% complementarity is preferred, but complementarity as low as 50-75% may also be useful in this invention. The nucleic acids may be modified at the base, sugar, and/or phosphate groups. The term enzymatic nucleic acid is used interchangeably with phrases such as ribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, catalytic oligonucleotides, nucleozyme, DNAzyme, RNA enzyme, endoribonuclease, endonuclease, minizyme, leadzyme, oligozyme or DNA enzyme. All of these terminologies describe nucleic acid molecules with enzymatic activity. The specific enzymatic nucleic acid molecules described in the instant application are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target nucleic acid regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart a nucleic acid cleaving activity to the molecule (Cech et al., U.S. Patent No. 4,987,071; Cech et al, 1988, JAMA). By "enzymatic portion" or "catalytic domain" is meant that portion/region of the ribozyme essential for cleavage of a nucleic acid substrate (for example see Figure 1). By "substrate binding arm" or "substrate binding domain" is meant that portion/region 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 Figure 1 and 3. That is, these arms contain sequences within a ribozyme which are intended to bring ribozyme and target RNA together through complementary base-pairing interactions. The ribozyme of the invention may have binding arms that are contiguous or non-contiguous and may be of varying lengths. The length of the binding arm(s) are preferably greater than or equal to four nucleotides; specifically preferably 12-100 nucleotides; more preferably 14-24 nucleotides long. If two binding arms are chosen, the design is such that the length of the binding arms are symmetrical (i.e., each of the binding arms is of the same length; e.g., five and five nucleotides, six and six nucleotides or seven and seven nucleotides long) or asymmetrical (i.e., the binding arms are of different length; e.g., six and three nucleotides; three and six nucleotides long; four and five nucleotides long; four and six nucleotides long; four and seven nucleotides long; and the like). 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 the motif of a hepatitis δ virus, group I intron, group II intron or RNase P RNA (in association with an RNA guide sequence), Neurospora VS RNA, DNAzymes, NCH cleaving motifs, or G-cleavers. 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 et 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; Chowrira & McSwiggen, US. Patent No. 5,631,359; of the hepatitis δ virus motif is described by Perrotta and Been, 1992 Biochemistry 31, 16; of the RNase P 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 (Saville and Collins, 1990 Cell 61, 685-696; Saville and Collins, 1991 Proc. Natl Acad. Sci. USA 88, 8826-8830; Collins and Olive, 1993 Biochemistry 32, 2795-2799; 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; of the Group I intron by Cech et al, U.S. Patent 4,987,071 and of DNAzymes by Usman et al, International PCT Publication No. WO 95/11304; Chartrand et al, 1995, NAR 23, 4092; Breaker et al, 1995, Chem. Bio. 2, 655; Santoro et al, 1997, PNAS 94, 4262. NCH cleaving motifs are described in Ludwig & Sproat, International PCT Publication No. WO 98/58058; and G-cleavers are described in Kore et al., 1998, Nucleic Acids Research 26, 4116-4120. These specific motifs are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule (Cech et al, U.S. Patent No. 4,987,071). In preferred embodiments of the present invention, a nucleic acid molecule, e.g., an antisense molecule, a triplex DNA, or a ribozyme, is 13 to 100 nucleotides in length, e.g., in specific embodiments 35, 36, 37, or 38 nucleotides in length (e.g., for particular ribozymes). In particular embodiments, the nucleic acid molecule is 15- 100, 17-100, 20-100, 21-100, 23-100, 25-100, 27-100, 30-100, 32-100, 35-100, 40- 100, 50-100, 60-100, 70-100, or 80-100 nucleotides in length. Instead of 100 nucleotides being the upper limit on the length ranges specified above, the upper limit of the length range can be, for example, 30, 40, 50, 60, 70, or 80 nucleotides. Thus, for any of the length ranges, the length range for particular embodiments has lower limit as specified, with an upper limit as specified which is greater than the lower limit. For example, in a particular embodiment, the length range can be 35-50 nucleotides in length. All such ranges are expressly included. Also in particular embodiments, a nucleic acid molecule can have a length which is any of the lengths specified above, for example, 21 nucleotides in length. By "equivalent" RNA to repressor genes is meant to include those naturally occurring RNA molecules having homology (partial or complete) to repressor genes or encoding for proteins with similar function as repressor genes in various animals, including human, rodent, primate, rabbit and pig. The equivalent RNA sequence also includes in addition to the coding region, regions such as 5 '-untranslated region, 3 '-untranslated region, introns, intron-exon junction and the like. By "complementarity" is meant that a nucleic acid can form hydrogen bond(s) with another RNA sequence by either traditional Watson-Crick or other non- traditional types. In reference to the nucleic molecules of the present invention, the binding free energy for a nucleic acid molecule with its target or complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., ribozyme cleavage, antisense or triple helix inhibition. Determination of binding free energies for nucleic acid molecules is well-known in the art (see, e.g., Turner et al., 1987, CSH Symp. Quant. Biol. LII pp.123-133; Frier et al, 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc. 109:3783-3785. A percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson- Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Perfectly complementary" means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. In preferred embodiments of the present invention, inhibition of expression of repressor genes is related to treatment of a disease or conditions. By "related" is meant that the inhibition of repressor gene RNAs and thus reduction in the respective levels of protein activity will relieve to some extent the symptoms of the disease or condition. In another preferred embodiment, the invention features nucleic acid based techniques (e.g., enzymatic nucleic acid molecules (ribozymes), antisense nucleic acids, 2-5A antisense chimeras, triplex DNA, antisense nucleic acids containing RNA cleaving chemical groups) and methods for their use to down regulate or inhibit the expression of genes capable of repressing interferon-alpha (IFN-α). Repressors of IFN-α include, but are not limited to, IRF-2 (Lopez et al., 1997, J. Biol Chem 272, 22788-22799). In another preferred embodiment, the invention features nucleic acid techniques (e.g., enzymatic nucleic acid molecules (ribozymes), antisense nucleic acids, 2-5A antisense chimeras, triplex DNA, antisense nucleic acids containing RNA cleaving chemical groups) and methods for their use to down regulate or inhibit the expression of genes capable of repressing Granulocyte colony- stimulating factor (G-CSF). These repressor genes include, but are not limited to, CCAAT displacement protein (CDP) (Khanna-Gupta et al, 1997, Blood 90, 2784- 2795) and Genesis (Xu et al, 1998, Leukemia, 12, 207-2012). In another preferred embodiment, the invention features the use of enzymatic nucleic acids (e.g. ribozymes) that cleave the R-NAs encoded by repressor genes capable of repressing erythropoietin (Epo) expression. The list of genes capable of inhibiting Epo include, but are not limited to, TR2 Orphan Receptor (Lee et al., The Journal of Biological Chemistry, 271, 10405-10412), EAR3/COUP-TF-1 (Galson et al, 1995, Molecular and Cellular Biology, 15, 2135-2144), and GATA Transcription Factors (Imagawa et al, 1997, Blood, 89, 1430-1439). The inhibition of one or more of these repressing factors would increase cellular production of Epo which would be beneficial for applications including but not limited to: adjuvant therapy for chemotherapy and treatment during renal dialysis. In preferred embodiments, the ribozymes of the present invention have binding arms that are complementary to the target sequences in Tables III-VII (i.e., Tables III, IV, V, VI, and VII). Examples of such ribozymes are also shown in Tables III- VIII. Table m displays target sequences and ribozymes targeting GATA transcription factors (1,2,3,4,6). Table IV displays target sequences and ribozymes targeting TR2 & TR2-11 Orphan Receptors, table V displays target sequences and ribozymes for EAR3/COUP-TF-1, table VI displays target sequences and ribozymes for IRF-2, and table VII displays target sequences and ribozymes for CDP. Examples of such ribozymes consist essentially of sequences defined in these Tables. In yet another embodiment, the invention features antisense nucleic acid molecules and 2-5A chimera including sequences complementary to the target sequences shown in tables III-VII. Such nucleic acid molecules can include sequences as shown for the binding arms of the ribozymes in Tables III-VII (i.e., the left-most and right-most sequence portions in the columns headed "RZ." Similarly, triplex molecules can be provided targeted to the corresponding DNA target regions, and containing the DNA equivalent of a target sequence or a sequence complementary to the specified target (substrate) sequence. Typically, antisense molecules will be complementary to a target sequence along a single contiguous sequence of the antisense molecule. However, in certain embodiments, an antisense molecule may bind to substrate such that the substrate molecule forms a loop, and/or an antisense molecule may bind such that the antisense molecule forms a loop. Thus, the antisense molecule may be complementary to two (or even more) noncontiguous substrate sequences or two (or even more) non-contiguous sequence portions of an antisense molecule may be complementary to a target sequence or both. By "consists essentially of in connection with ribozyme sequences is meant that the active ribozyme contains an enzymatic center, or core, equivalent to those in the examples, and binding arms able to bind RNA such that cleavage at the target site occurs. Other sequences may be present which do not significantly interfere with such cleavage. Thus, a core region may, for example, include one or more loop or stem-loop structures which do not prevent enzymatic activity. "X" in the sequences in Tables III-VII can be such a loop. Thus, in one aspect, the invention features ribozymes that inhibit repressor gene expression. These chemically or enzymatically synthesized ribozyme molecules contain substrate binding domains that bind to accessible regions of their target RNAs. The ribozymes also contain domains that catalyze the cleavage of target RNA. The enzymatic nucleic acid molecules are preferably ribozymes of the hammerhead or hammerhead-like motif (Kore et al, 1998, Nucleic Acids Research 26, 4116-4120; Ludwig & Sproat, International PCT Publication No. WO 98/58058 ) or hairpin motif. Alternatively, the ribozymes are DNAzymes. Chemically synthesized ribozyme molecules also include ribozymes assembled together from various fragments of nucleic acid using a chemical or an enzymatic ligation method. Upon binding, the ribozymes cleave the target RNAs, preventing translation and protein accumulation. The expression of genes repressed by repressor genes ("repressed genes") may be elevated in the absence of or under reduced level of repressor genes. This elevated level of the repressed gene may be beneficial to the cell and target organism. In a preferred embodiment, 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 stent, with or without their incorporation in biopolymers. In another preferred embodiment, the ribozyme is administered to the site of TR2 Orphan Receptor, TR2-11 Orphan Receptor, EAR3/COUP-TF-1, and GATA transcription factors, CDP, or IRF-2 expression (e.g. liver cells, cancer cells) in an appropriate liposomal vehicle. In another aspect of the invention, ribozymes that cleave target molecules and TR2 Orphan Receptor, TR2-11 Orphan Receptor, EAR3/COUP-TF-1, and GATA transcription factors, CDP, or IRF-2 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 RNA. 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). In another aspect of the invention, ribozymes that cleave target molecules and inhibit cell proliferation are expressed from transcription units inserted into DNA, RNA, or viral vectors. Preferably, the recombinant vectors capable of expressing the ribozymes are locally delivered as described above, and transiently persist in smooth muscle cells. However, other mammalian cell vectors that direct the expression of RNA may be used for this purpose. 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. In another aspect, the nucleic acid molecule of the present invention is administered individually or in combination or in conjunction with other drugs, can be used to treat diseases or conditions. For example, to treat a disease or condition associated with cancer, the patient may be treated, or other appropriate cells may be treated, as is evident to those skilled in the art. By "comprising" is meant including, but not limited to, whatever follows the word "comprising". Thus, use of the term "comprising" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By "consisting of is meant including, and limited to, whatever follows the phrase "consisting of. Thus, the phrase "consisting of indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements. 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. Figure 1 shows the secondary structure model for seven different classes of enzymatic nucleic acid molecules. -Arrow indicates the site of cleavage. indicate the target sequence. Lines interspersed with dots are meant to indicate tertiary interactions. - is meant to indicate base-paired interaction. Group I Intron: P1-P9.0 represent various stem-loop structures (Cech et al, 1994, Nature Struc. Bio., 1, 273). RNase P (Ml RNA): EGS represents external guide sequence (Forster et al, 1990, Science, 249, 783; Pace et al, 1990, J. Biol. Chem., 265, 3587). Group II Intron: 5'SS means 5' splice site; 3'SS means 3'-splice site; IBS means intron binding site; EBS means exon binding site (Pyle et al, 1994, Biochemistry, 33, 2716). VS RNA: I- VI are meant to indicate six stem-loop structures; shaded regions are meant to indicate tertiary interaction (Collins, International PCT Publication No. WO 96/19577). HDV Ribozyme: : I-IV are meant to indicate four stem-loop structures (Been et al, US Patent No. 5,625,047). Hammerhead Ribozyme: : I-III are meant to indicate three stem-loop structures; stems I-III can be of any length and may be symmetrical or asymmetrical (Usman et al, 1996, Curr. Op. Struct. Bio., 1, 527). Hairpin Ribozyme: Helix 1, 4 and 5 can be of any length; Helix 2 is between 3 and 8 base-pairs long; Y is a pyrimidine; 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. (Burke et al, 1996, Nucleic Acids & Mol Biol, 10, 129; Chowrira et al, US Patent No. 5,631,359). Figure 2 is an example of the secondary structure of a hammerhead ribozyme targeting hGATA-2 which has the sequence contained in Seq. I.D. No. 281. Figure 3 is a schematic diagram indicating the mechanism of action by the nucleic acid molecules of the present invention. The regulation of transcription initiation can occur by one or more transcription factors working together. When more than one factor is involved the transcription factors can be present as homodimers or heterodimers. In some cases, the formation of heterodimers would result in repression of transcription, while homodimers would form inactive transcription complexes. By blocking the expression of one subunit of the heterodimer, the equilibrium would shift towards formation of more homodimers resulting in a reduced formation of active repressors and enhanced transcription. Figure 4 is a graph demonstrating increased erythropoietin synthesis in Hep3B cells following cobalt induction and administration of ribozymes targeting GATA transcription factor 2, TR2 orphan receptor and EAR3/COUP-TR1 compared to the irrelevant controls (IR1 and IR2). Figure 5 is a graph demonstrating increased erythropoietin synthesis in Hep3B cells without cobalt induction and administration of ribozymes targeting GATA transcription factor 2, TR2 orphan receptor and EAR3/COUP-TR1 compared to the irrelevant controls (IR1 and IR2). Figure 6 is a bar graph demonstrating increased Epo expression compared to irrelevant controls in Hep3B cells following continuous delivery of ribozymes targeting hGATA-2 transcription factor RNA. Figure 7 is a bar graph demonstrating increased Epo expression compared to irrelevant controls in Hep3B cells following continuous delivery of ribozymes targeting EAR3/Coup-TRl RNA. Figure 8 is a bar graph demonstrating increased Epo expression compared to irrelevant controls in Hep3B cells following pulsed delivery of ribozymes targeting hGATA-2 transcription factor RNA. Figure 9 is a bar graph demonstrating increased Epo expression compared to iπelevant controls in Hep3B cells following pulsed delivery of ribozymes targeting EAR3/Coup-TRl RNA. Eukaryotic Gene Repression For the transcription of genes, a number of transcription factors are required for gene expression and its modulation. The most prevalent type of regulator genes within eukaryotes appear to be those that function to aid RNA polymerase in the initiation of gene expression, however, many examples exist of genes under negative control. This important class of factors is known as negative regulators or repressors. These trans-acting protein factors (repressor proteins) generally modulate the rates of transcription by binding to a specific site on a gene. The binding site is typically a cis-element upstream to the target gene, often within the promoter and is in many cases less than 10 nucleotides in length. Genes under negative control are those that are generally constitutively expressed unless turned off by repressor protein(s). In certain situations, the expression of repressed genes become highly desirable. Therefore stimulating the expression of repressed genes by inhibiting the expression of repressor genes would have a beneficial effect in treating a variety of diseases. A number of proteins and/or peptides exist which would have such beneficial effects on cells or patients. Several non-limiting examples are described below. Those of ordinary skill in the art will recognize that other genes exist which an organism would benefit from their increased expression. Erythropoietin: Erythropoetin is a 30.4 kDA glycoprotein hormone which is produced in the kidney and fetal liver as a response hypoxia (Galson et al., supra). The hormone regulates erythrocyte production and functions as a survival factor for the precursors of erythrocytes in bone marrow (Maxwell & Radcliffe, 1998, Curr. Opin. in Hematol. 5, 166-170). It is believed that a hemoglobin like sensor which is present within cells producing Epo, acts as a receptor for oxygen molecules (Goldberg et al, 1988, Science 242, 1412-1415). When the level of oxygen falls below tightly regulated parameter, Erythropoietin synthesis is induced. A number of indications may be treated using Epo. For example, patients with renal disease may develop anemia which is defined as an absence of erythrocytes within blood. Treatment with recombinant Epo can significantly enhance the production of these red blood cells (Maxwell & Radcliffe, supra). By inhibiting the production of Epo repressors, the kidneys or liver and other parts of the body may be induced to synthesize erythropoietin to counter anemia. -Another application of the present invention is as an adjuvant for chemotherapy. During chemotherapy, the patient may lose a large quantity of red blood cells. By inhibiting a repressor gene for erythropoietin, the Epo protein could be expressed in elevated quantities in the kidneys or liver which would in turn stimulate the production of more erythrocytes. Granulocyte Colony Stimulating Factor (G-CSF): Granulocyte colony stimulating factor (G-CSF) is a hematopoietic growth factor that regulates the production and function of neutrophils from committed progenitor cells. It is produced in vivo by monocytes, fibroblasts and endothelial cells. Recombinant G- CSF is given clinically to decrease neutropenia associated with chemotherapy as well as treatment for congenital diseases such as severe chronic neutropenia . An alternative to exogenous addition of G-CSF would be to produce more endogenous G-CSF, thus potentially avoiding the limitations and complications associated with injection of therapeutic proteins. There are several potential molecular targets that may act as indirect or direct repressors of G-CSF production or activity. CDP or CCAAT displacement protein is a known transcription repressor that binds to a negative regulatory element to block gene expression. It has extensive homology to the Drosophila cut protein. Reports indicate that CDP binds to the Lactoferrin gene and suppresses basal promoter activity. Overexpression of CDP blocks G-CSF - induced neutrophil maturation in cultured myeloid stem cells (Blood 90, 2784-95, 1997). -Another potential target is Genesis, a transcriptional repressor which blocks granulocytic differentiation of myeloid cells (Leukemia 12, 207-212, 1998). Genesis is a member of the "winged-helix" transcription factor regulatory family. 32D myeloid cells that are over-expressing Genesis fail to mature when stimulated with G-CSF. Genesis is expressed almost exclusively in embryonic stem cells and embryonal carcinoma cells. Both CDP and Genesis appear to be involved in the regulation of development and down-regulation of these could relieve a blockage in stem cell maturation. Interferon-alpha: Interferon exhibits multiple biological effects through the induction of over 30 genes encoding proteins that have antiviral, antiproliferative, immunomodulatory and cytokine stimulation functions. Alpha interferon (IFN-A) is a critical immune system modulator. IFN-A is encoded by a large family of structurally related genes. Interferon therapy is used for cell proliferation disorders (cancer) and viral infection (HBV, HCV). Interferon-alpha differential gene expression is accomplished by a complex interaction between cis-acting DNA regulatory regions and the coπesponding trans-acting factors. One potential limitation for expression of the interferon-alpha genes is the repressor transcription factor IRF-2 (JBC 272, 22788-99, 1997). There are also other negative regulatory regions for which the trans-acting repressors have not yet been identified, but which could be additional targets inhibition by nucleic acid molecules of the present invention. Inhibiting expression of this transcriptional repressor could allow increased levels of endogenous interferon-alpha to be produced. There are also other negative regulatory regions for which the trans-acting repressors have not yet been identified, but which could be additional targets for inhibition by nucleic acid molecules of the present invention. An advantage of this approach would be the avoidance of the production of antibodies to exogenous interferon as well as the avoidance of the autoimmune complications often seen with exogenous interferon- alpha administration. Mechanism of action of Nucleic Acid Molecules of the Invention -Antisense: Antisense molecules may be modified or unmodified RNA, DNA, or mixed polymer oligonucleotides and primarily function by specifically binding to matching sequences resulting in inhibition of peptide synthesis (Wu-Pong, Nov 1994, BioPharm, 20-33). The antisense oligonucleotide binds to target RNA by Watson Crick base-pairing and blocks gene expression by preventing ribosomal translation of the bound sequences either by steric blocking or by activating RNase H enzyme. -Antisense molecules may also alter protein synthesis by interfering with RNA processing or transport from the nucleus into the cytoplasm (Mukhopadhyay & Roth, 1996, Crit. Rev. in Oncogenesis 1, 151-190). In addition, binding of single stranded DNA to RNA may result in nuclease degradation of the heteroduplex (Wu-Pong, supra; Crooke, supra). To date, the only backbone modified DNA chemistry which will act as substrates for RNase H are phosphorothioates and phosphorodithioates. Recently it has been reported that 2'- arabino and 2'-fluoro arabino- containing oligos can also activate RNase H activity. A number of antisense molecules have been described that utilize novel configurations of chemically modified nucleotides, secondary structure, and/or RNase H substrate domains (Woolf et al, International PCT Publication No. WO 98/13526; Thompson et al, USSN 60/082,404 which was filed on April 20, 1998; Hartmann et al, USSN 60/101,174 which was filed on September 21, 1998) all of these are incorporated by reference herein in their entirety. Triplex Forming Oligonucleotides (TFO): Single stranded DNA may be designed to bind to genomic DNA in a sequence specific manner. TFOs are comprised of pyrimidine-rich oligonucleotides which bind DNA helices through Hoogsteen Base-pairing (Wu-Pong, supra)T e resulting triple helix composed of the DNA sense, DNA antisense, and TFO disrupts RNA synthesis by RNA polymerase. The TFO mechanism may result in gene expression or cell death since binding may be iπeversible (Mukhopadhyay & Roth, supra) 2-5A Antisense Chimera: The 2-5A system is an interferon mediated mechanism for RNA degradation found in higher vertebrates (Mitra et al, 1996, Proc Nat Acad Sci USA 93, 6780-6785). Two types of enzymes, 2-5A synthetase and RNase L, are required for RNA cleavage. The 2-5A synthetases require double stranded RNA to form 2'-5' oligoadenylates (2-5A). 2-5A then acts as an allosteric effector for utilizing RNase L which has the ability to cleave single stranded RNA. The ability to form 2-5A structures with double stranded RNA makes this system particularly useful for inhibition of viral replication. (2 '-5') oligoadenylate structures may be covalently linked to antisense molecules to form chimeric oligonucleotides capable of RNA cleavage (Torrence, supra). These molecules putatively bind and activate a 2-5A dependent RNase, the oligonucleotide/enzyme complex then binds to a target RNA molecule which can then be cleaved by the RNase enzyme. Enzymatic Nucleic Acid: Seven basic varieties of naturally-occurring enzymatic RNAs are known presently. In addition, several in vitro selection (evolution) strategies (Orgel, 1979, Proc. R. Soc. London, B 205, 435) have been used to evolve new nucleic acid catalysts capable of catalyzing cleavage and ligation of phosphodiester linkages (Joyce, 1989, Gene, 82, 83-87; Beaudry et al, 1992, Science 257, 635-641; Joyce, 1992, Scientific American 267, 90-97; Breaker et al, 1994, TIBTECH 12, 268; Bartel et α/., 1993, Science 261 :1411-1418; Szostak, 1993, TIBS 17, 89-93; Kumar et al, 1995, FASEB J., 9, 1183; Breaker, 1996, Curr. Op. Biotech., 1, 442; Santoro et al, 1997, Proc. Natl. Acad. Sci., 94, 4262; Tang et al, 1997, RNA 3, 914; Nakamaye & Eckstein, 1994, supra; Long & Uhlenbeck, 1994, supra; Ishizaka et al., 1995, supra; Naish et al, 1997, Biochemistry 36, 6495; all of these are incorporated by reference herein). Each can catalyze a series of reactions including the hydrolysis of phosphodiester bonds in trans (and thus can cleave other RΝA molecules) under physiological conditions. Table I summarizes some of the characteristics of some of these ribozymes. In general, enzymatic nucleic acids act by first binding to a target RΝA. 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 RΝA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RΝA through complementary base-pairing, and once bound to the coπect site, acts enzymatically to cut the target R A. Strategic cleavage of such a target RΝA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RΝA target, it is released from that RΝA to search for another target and can repeatedly bind and cleave new targets. The enzymatic nature of a ribozyme has significant advantages, such as 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 RΝA. 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 RΝA, but also on the mechanism of target RΝA 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 nucleic acid molecules can be targeted to virtually any RNA transcript, and efficient cleavage achieved in vitro (Zaug et al, 324, Nature 429 1986 ; Uhlenbeck, 1987 Nature 328, 596; Kim et al., 84 Proc. Natl. Acad. Sci. USA 8788, 1987; Dreyfus, 1988, Einstein Quart. J. Bio. Med., 6, 92; Haseloff and Gerlach, 334 Nαtwre 585, 1988; Cech, 260 JAMA 3030, 1988; and Jefferies et al., 17 Nucleic Acids Research 1371, 1989; Santoro et al, 1997 supra). Because of their sequence-specificity, trα- -s-cleaving ribozymes show promise as therapeutic agents for human disease (Usman & McSwiggen, 1995 Ann. Rep. Med. Chem. 30, 285-294; Christoffersen and Man, 1995 J. Med. Chem. 38, 2023- 2037). Ribozymes can be designed to cleave specific RΝA targets within the background of cellular R A. Such a cleavage event renders the RΝA nonfunctional and abrogates protein expression from that RΝA. In this manner, synthesis of a protein associated with a disease state can be selectively inhibited. Synthesis of Nucleic acid Molecules 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 ("small refers to nucleic acid motifs no more than 100 nucleotides in length, preferably no more than 80 nucleotides in length, and most preferably no more than 50 nucleotides in length; e.g., antisense oligonucleotides, hammerhead or the hairpin ribozymes) are preferably used for exogenous delivery. The simple structure of these molecules increases the ability of the nucleic acid to invade targeted regions of RNA structure. Exemplary molecules of the instant invention were chemically synthesized, and others can similarly be synthesized. Oligodeoxyribonucleotides were synthesized using standard protocols as described in Caruthers et al, 1992, Methods in Enzymology 211,3-19, and is incorporated by reference. The method of synthesis used for normal RNA including certain enzymatic nucleic acid molecules follows the procedure 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. In a non-limiting example, small scale syntheses were conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 7.75 min coupling step for alkylsilyl protected nucleotides and a 2.5 min coupling step for 2'-O-methylated nucleotides. Table II outlines the amounts and the contact times of the reagents used in the synthesis cycle. Alternatively, syntheses at the 0.2 μmol scale can be done on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, CA) with minimal modification to the cycle. A 15-fold excess (31 μL of 0.1 M = 3.1 μmol) of phosphoramidite and a 38.7-fold excess of S-ethyl tetrazole (31 μL of 0.25 M = 7.75 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 3% 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 I2, 49 mM pyridine, 9% water in THF (PERSEPTTVE™). Burdick & Jackson Synthesis Grade acetonitrile was used directly from the reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) was made up from the solid obtained from -American International Chemical, Inc. Deprotection of the R A was performed using either a two-pot or one-pot protocol. For the two-pot protocol, the polymer-bound trityl-on oligoribonucleotide was transfeπed to a 4 mL glass screw top vial and suspended in a solution of 40% aq. methylamine (1 mL) 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:MeCΝ:H2O/3:l :l, vortexed and the supernatant was then added to the first supernatant. The combined supematants, containing the oligoribonucleotide, were dried to a white powder. The base deprotected oligoribonucleotide was resuspended in anhydrous TEA HF/NMP solution (300 μL of a solution of 1.5 mL N-methylpyrrolidinone, 750 μL TEA and 1 mL TEAβHF to provide a 1.4 M HF concentration) and heated to 65 °C. After 1.5 h, the oligomer was quenched with 1.5 M NH 4 HCO3. Alternatively, for the one-pot protocol, the polymer-bound trityl-on oligoribonucleotide was transferred to a 4 mL glass screw top vial and suspended in a solution of 33% ethanolic methylamine/DMSO:l/l (0.8 mL) at 65 °C for 15 min. The vial was brought to r.t. TEA » 3HF (0.1 mL) was added and the vial was heated at 65 °C for 15 min. The sample was cooled at -20 °C and then quenched with 1.5 M NH 4 HCO3. For purification of the trityl-on oligomers, the quenched NH 4 HCO 3 solution was loaded onto a C-18 containing cartridge that had been prewashed with acetonitrile followed by 50 mM TEAA. After washing the loaded cartridge with water, the RNA was detritylated with 0.5% TFA for 13 min. The cartridge was then washed again with water, salt exchanged with 1 M NaCl and washed with water again. The oligonucleotide was then eluted with 30% acetonitrile. Inactive hammerhead ribozymes or binding attenuated control (BAC) oligonucleotides) were synthesized by substituting a U for G5 and a U for A14 (numbering from Hertel, K. J., et al, 1992, Nucleic Acids Res., 20, 3252). The average stepwise coupling yields were >98% (Wincott et al, 1995 Nucleic Acids Res. 23, 2677-2684). Those of ordinary skill in the art will recognize that the scale of synthesis can be adapted to be larger or smaller than the example described above including but not limited to 96 well format, all that is important is the ratio of chemicals used in the reaction. Alternatively, the nucleic acid molecules of the present invention can be synthesized separately and joined together by ligation (Moore et al, 1992, Science 256, 9923; Draper et al, International PCT publication No. WO 93/23569; Shabarova et al, 1991, Nucleic Acids Research 19, 4247) Administration of Nucleic Acid Molecules Methods for the delivery of nucleic acid molecules is described in Akhtar et al, 1992, Trends Cell Bio., 2, 139; and Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995 which are both incorporated herein by reference. Sullivan et al, PCT WO 94/02595, further describes the general methods for delivery of enzymatic RNA molecules . These protocols may be utilized for the delivery of virtually any nucleic acid molecule. Nucleic acid molecules 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, nucleic acid molecules may be directly delivered ex vivo to cells or tissues with or without the aforementioned vehicles. Alternatively, the nucleic acid/vehicle combination is locally delivered by direct injection or by use of a catheter, infusion pump or stent. Other routes of delivery include, but are not limited to, intravascular, intramuscular, subcutaneous or joint injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. More detailed descriptions of nucleic acid delivery and administration are provided in Sullivan et al, supra and Draper et al, PCT WO93/23569 which have been incorporated by reference herein. The molecules of the instant invention can be used as pharmaceutical agents. Pharmaceutical agents prevent, inhibit the occwrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state in a patient. The negatively charged polynucleotides of the invention can be administered (e.g., RNA, DNA or protein) and introduced into a patient by any standard means, with or without stabilizers, buffers, and the like, to form a pharmaceutical composition. When it is desired to use a liposome delivery mechanism, standard protocols for formation of liposomes can be followed. The compositions of the present invention may also be formulated and used as tablets, capsules or elixirs for oral administration; suppositories for rectal administration; sterile solutions; suspensions for injectable administration; and the like. The present invention also includes pharmaceutically acceptable formulations of the compounds described. These formulations include salts of the above compounds, e.g., acid addition salts, for example, salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid. A pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, e.g., systemic administration, into a cell or patient, preferably a human. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, or by injection. Such forms should not prevent the composition or formulation to reach a target cell (i.e., a cell to which the negatively charged polymer is desired to be delivered to). For example, pharmacological compositions injected into the blood stream should be soluble. Other factors are known in the art, and include considerations such as toxicity and forms which prevent the composition or formulation from exerting its effect. By "systemic administration" is meant in vivo systemic absorption or accumulation of drugs in the blood stream followed by distribution throughout the entire body. Administration routes which lead to systemic absorption include, without limitations: intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular. Each of these administration routes expose the desired negatively charged polymers, e.g., nucleic acids, to an accessible diseased tissue. The rate of entry of a drug into the circulation has been shown to be a function of molecular weight or size. The use of a liposome or other drug carrier comprising the compounds of the instant invention can potentially localize the drug, for example, in certain tissue types, such as the tissues of the reticular endothelial system (RES). A liposome formulation which can facilitate the association of drug with the surface of cells, such as, lymphocytes and macrophages is also useful. This approach may provide enhanced delivery of the drug to target cells by taking advantage of the specificity of macrophage and lymphocyte immune recognition of abnormal cells, such as the cancer cells. The invention also features the use of a composition comprising surface- modified liposomes containing poly (ethylene glycol) lipids (PEG-modified, or long- circulating liposomes or stealth liposomes). These formulations offer an method for increasing the accumulation of drugs in target tissues. This class of drug carriers resists opsonization and elimination by the mononuclear phagocytic system (MPS or RES), thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated drug (Lasic et al. Chem. Rev. 1995, 95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005-1011). Such liposomes have been shown to accumulate selectively in tumors, presumably by extravasation and capture in the neovascularized target tissues (Lasic et al, Science 1995, 267, 1275-1276; Oku et α/., 1995, Biochim. Biophys. Acta, 1238, 86-90). The long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA, particularly compared to conventional cationic liposomes which are known to accumulate in tissues of the MPS (Liu et al, J. Biol. Chem. 1995, 42, 24864-24870; Choi et al, International PCT Publication No. WO 96/10391; Ansell et al, International PCT Publication No. WO 96/10390; Holland et al, International PCT Publication No. WO 96/10392; all of these are incorporated by reference herein). Long-circulating liposomes are also likely to protect drugs from nuclease degradation to a greater extent compared to cationic liposomes, based on their ability to avoid accumulation in metabolically aggressive MPS tissues such as the liver and spleen. All of these references are incorporated by reference herein. The present invention also includes compositions prepared for storage or administration which include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington 's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro edit. 1985) hereby incorporated by reference herein. For example, preservatives, stabilizers, dyes and flavoring agents may be provided. Id. at 1449. These include sodium benzoate, sorbic acid and esters of /7-hydroxybenzoic acid. In addition, antioxidants and suspending agents may be used. J A pharmaceutically effective dose is that dose required to prevent, inhibit the occuπence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state. The pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concuπent medication, and other factors which those skilled in the medical arts will recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients is administered dependent upon potency of the negatively charged polymer. The nucleic acid molecules of the present invention may also be administered to a patient in combination with other therapeutic compounds to increase the overall therapeutic effect. The use of multiple compounds to treat an indication may increase the beneficial effects while reducing the presence of side effects. Alternatively, the nucleic acid molecules of the instant invention (e.g. ribozyme and antisense molecules) can 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; Scanlon et al, 1991, Proc. Natl. Acad. Sci. USA, 88, 10591-5; Kashani-Sabet et al, 1992 Antisense Res. Dev., 2, 3-15; Dropulic et al, 1992 J. Virol, 66, 1432-41; Weerasinghe et al, 1991 J Virol, 65, 5531-4; Ojwang et al, 1992 Proc. Natl. Acad. Sci. USA 89, 10802-6; Chen et al, 1992 Nucleic Acids Res., 20, 4581-9; Sarver et al, 1990 Science 247, 1222-1225; Thompson et al, 1995 Nucleic Acids Res. 23, 2259; Good et al, 1997, Gene Therapy, 4, 45; all of the references are hereby incorporated in their totality by reference herein). 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 WO 93/23569, and Sullivan et al, PCT WO 94/02595; Ohkawa et al, 1992 Nucleic Acids Symp. Ser., 27, 15-6; Taira et al, 1991, Nucleic Acids Res., 19, 5125-30; Ventura et al, 1993 Nucleic Acids Res., 21, 3249-55; Chowrira et al, 1994 J. Biol. Chem. 269, 25856; all of the references are hereby incorporated in their totality by reference herein). In another aspect of the invention, enzymatic nucleic acid molecules that cleave target molecules are expressed from transcription units (see for example Couture et al, 1996, TIG., 12, 510) 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 RNA. The active ribozyme contains an enzymatic center or core equivalent to those in the examples, and binding arms able to bind target nucleic acid molecules such that cleavage at the target site occurs. Other sequences may be present which do not interfere with such cleavage. 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 et al, 1996, TIG., 12, 510). In one aspect the invention features, an expression vector comprising nucleic acid sequence encoding at least one of the nucleic acid molecules (ribozyme, antisense) of the instant invention is disclosed. The nucleic acid sequence encoding the nucleic acid molecule of the instant invention is operably linked in a manner which allows expression of that nucleic acid molecule. In another aspect the invention features, the expression vector comprises: a transcription initiation region (e.g., eukaryotic pol I, II or III initiation region); b) a transcription termination region (e.g., eukaryotic pol I, II or III termination region); c) a gene encoding at least one of the nucleic acid molecule of the instant invention; and wherein said gene is operably linked to said initiation region and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule. The vector may optionally include an open reading frame (ORF) for a protein operably linked on the 5' side or the 3'-side of the gene encoding the nucleic acid molecule of the invention; and/or an intron (intervening sequences). Transcription of the ribozyme or antisense sequences are driven from a promoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III). Transcripts from pol II or pol III promoters will be expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type will depend on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby. Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990 Proc. Natl. Acad. Sci. U S A, 87, 6743-7; Gao and Huang 1993 Nucleic Acids Res., 21, 2867-72; Lieber et al., 1993 Methods Enzymol, 217, 47-66; Zhou et al., 1990 Mol Cell. Biol, 10, 4529-37). Several investigators have demonstrated that ribozymes expressed from such promoters can function in mammalian cells (e.g. Kashani-Sabet et al., 1992 Antisense Res. Dev., 2, 3-15; Ojwang et al., 1992 Proc. Natl. Acad. Sci. 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, 8000-4; Thompson et al, 1995 Nucleic Acids Res. 23, 2259; Sullenger & Cech, 1993, Science, 262, 1566). More specifically, transcription units such as the ones derived from genes encoding U6 small nuclear (snRNA), transfer RNA (tRNA) and adenovirus VA RNA are useful in generating high concentrations of desired RNA molecules such as ribozymes in cells (Thompson et al, supra; Couture and Stinchcomb, 1996, supra; Noonberg et al, 1994, Nucleic Acid Res., 22, 2830; Noonberg et al, US Patent No. 5,624,803; Good et al, 1997, Gene Ther. 4, 45; Beigelman et al, International PCT Publication No. WO 96/18736; all of these publications are incorporated by reference herein. The above 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 yet another aspect the invention features an expression vector comprising nucleic acid sequence encoding at least one of the nucleic acid molecule of the invention, in a manner which allows expression of that nucleic acid molecule. The expression vector comprises in one embodiment; a) a transcription initiation region; b) a transcription termination region; c) a gene encoding at least one said nucleic acid molecule; and wherein said gene is operably linked to said initiation region and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule. In another prefeπed embodiment the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an open reading frame; d) a gene encoding at least one said nucleic acid molecule, wherein said gene is operably linked to the 3'-end of said open reading frame; and wherein said gene is operably linked to said initiation region, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule. In yet another embodiment the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) a gene encoding at least one said nucleic acid molecule; and wherein said gene is operably linked to said initiation region, said intron and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule. In another embodiment, the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) an open reading frame; e) a gene encoding at least one said nucleic acid molecule, wherein said gene is operably linked to the 3'-end of said open reading frame; and wherein said gene is operably linked to said initiation region, said intron, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule. In another aspect, the invention features a method of increasing the level of target protein in a cell comprising the step of contacting the cell with nucleic acid molecules capable of specifically inhibiting the expression of a repressor protein that represses the expression of the target protein under conditions suitable for increasing the level of target protein in the cell. In another aspect, this invention features a method of increasing the level of target protein in a cell comprising the step of isolating cells from a patient, introducing the nucleic acid molecule (synthetic or vector) capable of inhibiting the expression of a repressor of target protein, introducing the cells into same or a different patient under conditions for the increased expression of the target protein. Optimizing Ribozyme Activity Catalytic activity of the ribozymes described in the instant invention 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, 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 et al., International Publication No. WO 92/07065; Perrault et al, 1990 Nature 344, 565; Pieken et al., 1991 Science 253, 314; Usman and Cedergren, 1992 Trends in Biochem. Sci. 17, 334; Usman et al, International Publication No. WO 93/15187; and Rossi et al, International Publication No. WO 91/03162; 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 bases from stem loop structures to shorten RNA synthesis times and reduce chemical requirements are desired. (All these publications are hereby incorporated by reference herein). There are several examples in the art describing sugar, base and phosphate modifications that can be introduced into enzymatic nucleic acid molecules without significantly effecting catalysis and with significant enhancement in their nuclease stability and efficacy. 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'-fluoro, 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 35, 14090). Sugar modification of enzymatic nucleic acid molecules have been extensively described in the art (see Eckstein et al, International Publication PCT No. WO 92/07065; Peπault et al. Nature 1990, 344, 565-568; Pieken et al. Science 1991, 253, 314- 317; Usman and Cedergren, Trends in Biochem. Sci. 1992, 17, 334-339; Usman et al. International Publication PCT No. WO 93/15187; Sproat, US Patent No. 5,334,711 and Beigelman et al, 1995 J. Biol. Chem. 270, 25702; all of the references are hereby incorporated in their totality by reference herein). Such publications describe general methods and strategies to determine the location of incorporation of sugar, base and/or phosphate modifications and the like into ribozymes without inhibiting catalysis, and are incorporated by reference herein. In view of such teachings, similar modifications can be used as described herein to modify the nucleic acid catalysts of the instant invention. Nucleic acid catalysts having chemical modifications which maintain or enhance enzymatic activity are provided. Such nucleic acid is also generally more resistant to nucleases than unmodified nucleic acid. Thus, in a cell and/or in vivo the activity may not be significantly lowered. As exemplified herein such ribozymes are useful in a cell and or in vivo even if activity over all is reduced 10 fold (Burgin et al, 1996, Biochemistry, 35, 14090). Such ribozymes herein are said to "maintain" the enzymatic activity on all RNA ribozyme. Therapeutic ribozymes delivered exogenously must optimally be stable within cells until translation of the target RNA has been inhibited long enough to reduce the levels of the undesirable protein. This period of time varies between hours to days depending upon the disease state. Clearly, ribozymes must be resistant to nucleases in order to function as effective intracellular therapeutic agents. Improvements in the chemical synthesis of RNA (Wincott et al, 1995 Nucleic Acids Res. 23, 2677; incorporated by reference herein) have expanded the ability to modify ribozymes by introducing nucleotide modifications to enhance their nuclease stability as described above. 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 is increased or not significantly (less that 10 fold) decreased in vivo compared to an all RNA ribozyme. In yet another prefeπed embodiment, nucleic acid catalysts having chemical modifications which maintain or enhance enzymatic activity is provided. Such nucleic acid is also generally more resistant to nucleases than unmodified nucleic acid. Thus, in a cell and/or in vivo the activity may not be significantly lowered. As exemplified herein such ribozymes are useful in a cell and/or in vivo even if activity over all is reduced 10 fold (Burgin et al, 1996, Biochemistry, 35, 14090). Such ribozymes herein are said to "maintain" the enzymatic activity on all RNA ribozyme. Use of these molecules will lead to better treatment of the disease progression by affording the possibility of combination therapies (e.g., multiple ribozymes targeted to different genes, ribozymes coupled with known small molecule inhibitors, or intermittent treatment with combinations of ribozymes (including different ribozyme motifs) and/or other chemical or biological molecules). The treatment of patients with nucleic acid molecules may also include combinations of different types of nucleic acid molecules. Therapies may be devised which include a mixture of ribozymes (including different ribozyme motifs), antisense and/or 2-5 A chimera molecules to one or more targets to alleviate symptoms of a disease. -Animal Models In order to evaluate the therapeutic potential nucleic acid targeting repressors of Epo synthesis, two animals models of chronic anemia exist. These models are: 1) chemotherapy-induced anemia in mice and 2.) chronic renal failure-induced anemia in mice. Both of these murine models closely mimic the pathophysiology of the corresponding disease in human patients. (1) Chemotherapy-Induced -Anemia in C57/B16 Mice: The primary goal of these studies is to evaluate the effectiveness of nucleic acid therapy targeted at increasing the body's ability to produce red blood cells and thus counteract chemotherapy-induced severe anemia. Many drugs used to treat cancer patients (cytotoxic compounds) adversely effect the bone marrow and markedly reduce the number of circulating red blood cells. This is primarily due to a decrease in the hormone erythropoietin (Epo) which stimulates the production of red blood cells. Many types of chemotherapy also induce hemolytic anemia. The severe anemia that occurs with many forms of chemotherapy has a marked impact on the patients' quality of life (exercising, performing job duties, etc.) and normal daily activities are difficult to perform. Nucleic acid molecules targeting repressors of Epo are evaluated for their ability to improve severe loss of circulating red blood cells (anemia) associated with chemotherapy in C57B1/6 mice which is an indication of enhanced Epo production. Experimental Procedure: All studies are performed on pathogen-free, 20- 25g female C57B1/6 mice. Mice are housed in a pathogen-free environment and allowed food and water ad lib. For induction of anemia, all animals receive an intraperitoneal injection of 3.5 mg/kg Cisplatin (CDDP), in a 200 μL volume (Day- 0). For all blood sample collection, animals are euthanized by CO asphyxiation. Baseline blood samples are obtained via cardiac puncture for hematological and biochemical analyses prior to initiating chemotherapy (Day-0). Blood samples obtained via cardiac puncture, body weights and spleen weights from groups of 10 CDDP-treated animals are obtained beginning on Day-1 and three times weekly for 27 days. Acute renal failure with marked uremia (elevation of BUN) and anemia is apparent within 1 day post-single dose chemotherapy. Hematocrits are measured, in triplicate, using a Clay Adams microhematocrit centrifuge on a pooled whole blood sample (in EDTA) from each group of 10 animals at each termination. In addition, a complete blood cell count is obtained from whole blood. The remaining sample is spun down and plasma samples are saved at -70°C for later determination of plasma erythropoietin levels. Plasma Epo levels A determined by a commercially available ELISA (R&D Systems, Minneapolis, MN) using the manufacturer's protocol. Compound Efficacy Studies Four groups of animals are tested per drug: Group 1 receives active nucleic acid molecules (e.g. ribozyme), Group 2 receives scrambled attenuated control nucleic acid molecules as therapy and Group 3 receives vehicle as therapy. Group 4 serves as a positive therapeutic control and receives recombinant human erythropoietin (rhu-Epo; 2500 U/kg, thrice weekly). There are 10 animals per group per time point and up to three doses of nucleic acid molecules per group for groups 1 and 2. There are 13 time points (Days 0, 1, 3, 5, 8, 10, 12, 15, 17, 19, 22, 24, 26) in each study. Ten animals per group per time point per dose are euthanized and blood samples collected and tested as described above. Test agents may be delivered via an ALZET™ osmotic pump (Alza Scientific Products) subcutaneously or intravenously, subcutaneous bolus, direct i.p. injection or intravenously via the tail vein. (2) Chronic Renal Failure-Induced Anemia in C57/B16 Mice (Zhang et al, 1996, Nephron 72: 654-661): The primary goal of these studies is to evaluate the effectiveness of nucleic acid therapy targeted at increasing the body's ability to produce red blood cells and thus counteract chronic renal failure-induced severe anemia. Chronic renal failure (CRF) is a functional clinical diagnosis characterized by a progressive and iπeversible decline in the kidneys' ability to filter the blood (glomerular filtration rate; GFR). This condition is associated with a number a primary diseases including, but not limited to, glomerulonephritis, cardiovascular disease and hypertension, diabetes, kidney infections and urinary tract disease. CRF afflicts more than 370,000 patients in the U.S. alone. Most of these patients' disease will progress to end stage renal disease (ESRD) and will require renal replacement therapy (hemodialysis, peritoneal dialysis, kidney transplant) to survive. Both the loss of functional kidney tissue and the dialysis procedure cause a severe reduction in the red blood cell count of these patients. This primarily due to a decrease in the hormone erythropoietin (Epo) which stimulates the production of red blood cells. The severe chronic anemia has a marked impact on the patients' quality of life (exercising, performing job duties, etc.) and normal daily activities are difficult to perform. Experimental Procedure: All studies are performed on pathogen-free, female 20-25g C57B1/6. Mice are housed in a pathogen-free environment and allowed food and water ad lib. To establish CRF in these animals, two surgical procedures are required separated by a two week recovery period. For the first surgical procedure, animals are anesthetized with a ketamine/ xylazine cocktail (1.2 mg/kg and .14 mg/kg) and a right lateral laparotomy is performed. The entire surface of the right kidney, excluding a 2 mm rim around the hilum, is electrocoagulated using a disposable vasectomy cautery (2250°F). The kidney is returned to the renal fossa and wounds are aseptically closed with 4-0 silk suture and surgical clips Animals are allowed to recover for two weeks before the second surgical procedure is performed. For the second procedure, animals are anesthetized with a ketamine/ xylazine cocktail (1.2 mg/kg and .14 mg/kg) and a left lateral laparotomy is performed. The left kidney is removed and the wound aseptically closed with 4-0 silk suture. All animals receive penicillin G (Durapen - 30,000 U, EM) following each surgical procedure. For all blood sample collections, animals are euthanized by CO asphyxiation. Blood samples, body weights and spleen weights are obtained from groups of 8 animals each weekly beginning at week 1 post-second surgery for evaluation of disease progression up until week 14 post-Nx. A group of 8 normal animals are euthanized and blood samples obtained for control hematology and biochemistry determinations. Therefore, there are 12 euthanization time points including the control group. From reports in the literature, CRF with marked uremia (persistent elevation of BUN) and anemia will be present within 8 weeks post-Nx. At necropsy, blood samples will be obtained via cardiac puncture for clinical chemistry (BUN and creatinine) and hematology (WBC, Diff, platelet count) tests are performed by an external laboratory (IDEXX, Inc.). Compound Efficacy Studies: There are four groups of animals per drug tested: Group 1 receives active nucleic acid molecules of the invention (e.g. ribozyme), Group 2 receives scrambled attenuated nucleic acid as therapy and Group 3 receives vehicle as therapy. Group 4 serves as a positive therapeutic control and receives recombinant human erythropoietin (rhu-Epo; 250 U/kg; thrice weekly). There are 8 animals per group and up to three doses of nucleic acid per group for groups 1 and 2. Test agents may be delivered via an ALZET™ osmotic pump (Alza Scientific Products) subcutaneously or intravenously, subcutaneous bolus, direct i.p. injection or intravenously via the tail vein. (3) Chemotherapy-Induced Myelo suppression in C57/B16 Mice (Misaki et al, 1998, British Journal of Cancer 77:884-889) : The primary goal of these studies is to evaluate the effectiveness of therapy targeted at increasing the body's ability to produce white blood cells and thus counteract chemotherapy-induced neutropenia. The ability of these nucleic acid molecules to improve severe loss of circulating white blood cells (neutropenia) associated with chemotherapy in Balb/c mice is tested . A protocol modified from that of Misaki, et al. (1998) is utilized. Experimental Procedure: All studies are performed on pathogen-free, 25-30g female Balb/c mice. Mice are housed in a pathogen-free environment and allowed food and water ad lib. For induction of myelosuppression, all animals receive an intraperitoneal injection of 200 mg/kg Cyclophosphamide (CPA), in a 200 μL volume (Day-0). There are 16 time points for blood sampling. Samples are obtained to evaluate plasma G-CSF levels and CBCs. A single vehicle control group and a rhuG-CSF group is used for all ribozyme formulation testing protocols. Body and spleen weights are recorded. For all blood sample collections, animals are euthanized by CO 2 asphyxiation. Baseline blood samples are obtained via cardiac puncture for hematological analyses prior to initiating chemotherapy (Day-0). One group of ten animals is euthanized pre- CPA, 4 days post-CPA( at 6am, at 12 noon and at 6pm) and daily thereafter. Two mis of whole blood is sent to IDEXX veterinary laboratory for a complete blood cell count. The remaining samples are spun down and plasma samples are saved at -70°C for later determination of plasma G-CSF levels. Plasma G-CSF levels are determined in-house by a commercially available ELISA. Remaining plasma samples are frozen for future analyses. Compound Efficacy Studies: There are four groups of animals per drug tested: Group 1 receives active nucleic acid molecules of the invention (e.g. ribozyme), Group 2 receives scrambled attenuated nucleic acid as therapy and Group 3 receives vehicle as therapy. Group 4 serves as a positive therapeutic control and receives recombinant human rhu-G-CSF (5 μg/kg, daily). There are 10 animals per group per time point and up to three doses of ribozyme per group for groups 1 and 2. On day 0 animals receive cyclophosphamide (CPA; 200 mg/kg, IP). On day 4, nucleic acid therapy is initiated. Therapy is continued daily until Day 16. There are 16 time points (Days 0-17) in each study. Ten animals per group per time point per dose are euthanized and blood samples collected. One group of ten animals is euthanized pre- CPA, 4 days post-CPA, at 6 and at 12 hrs. after nucleic acid dosing and daily thereafter. Two mis of whole blood are sent to IDEXX veterinary laboratory for a complete blood cell count. The remaining sample is spun down and plasma samples are saved at -70°C for later determination of plasma erythropoietin levels. Plasma G-CSF levels is determined in-house by a commercially available ELISA. Remaining plasma samples are frozen for future analyses. Test agents may be delivered via an ALZET™ osmotic pump (Alza Scientific Products) subcutaneously or intravenously, subcutaneous bolus, direct i.p. injection or intravenously via the tail vein. (4) Chronic, Relapsing Experimental Autoimmune Encephalitis in Rats: Multiple sclerosis is a disorder of unknown cause that has a number of symptoms (weakness, numbness, lack of coordination, headaches) which are caused by destruction of the protective tissue (myelin) suπounding the spinal cord. Many studies have been published which support both a possible immune system problem and/or an infectious agent. It is a chronic debilitating disease with a clinical course from onset to death of approximately 35 years. There are no spontaneous animal models of MS but an autoimmune disorder which resembles MS can be induced in rodents. This is accomplished by either injection under the skin of a crude brain mixture or purified proteins obtained from the brain. The goal of this model is to evaluate the effectiveness of therapy with nucleic acid targeted against the interferon-_ repressor in improving the symptoms with this disease in an animal model. Experimental Procedure: All studies are performed on pathogen-free, male Dark Agouti (DA) rats 7-9 weeks of age obtained from Harlan, Inc. Rats are housed in a pathogen-free environment and allowed food and water ad lib for one week prior to initiation of the study. All animals are immunized with syngeneic spinal cord (SSC) in incomplete Freund's adjuvant (IF A). For the preparation of the spinal cord emulsion, cords from donor DA rats are removed and minced thoroughly. One part spinal cord to one part IFA (v/W) is used to prepare emulsion. The appropriate dose of emulsion is determined in the pilot study. 0.2 ml of homogenate (SSC and IFA) is injected into the dorsal base of the tail root on day 0. All animals receive 75 mg/kg of syngeneic spinal cord. The primary endpoint of these studies is a clinical score. The clinical scoring system is as follows: 0.0 = Normal 0.5 = Partial loss of tail tone 1.0 = Complete loss of tail tone 2.0 = Hindlimb weakness or dragging one hindlimb 3.0 = Paralysis of both hindlimbs 4.0 = Paralysis of both hindlimbs and weakness in forelimbs 5.0 = Moribund Histopathologic evidence of demyelination is a secondary endpoint. Clinical scores and body weights are determined daily for 21 days and EOD thereafter until day 90. At the termination of this study (90 days post-immunization), animals are euthanized. At necropsy, brain and spinal cord are removed, fixed in 10% buffered formalin and submitted for histopathologic analyses. The experimental method (dose of SSC) which provides the greatest reproducibility and the pathophysiology that most closely mimics the human clinical disease is then chosen for use in the compound efficacy studies. Compound Efficacy Studies: This study evaluates the efficacy of nucleic acid molecules targeted against the interferon-alpha repressor gene on severity of clinical score and on histopathological changes in the spinal cord and brain of these animals. There are two main groups of animals per drug tested: one prophylactic treatment (beginning three days post-immunization) and one therapeutic treatment (following the first paralytic episode- at approximately day 15 post-immunization). Each main group has four subgroups: Group 1 receives vehicle as therapy, Group 2 receives scrambled attenuated nucleic acid control as therapy, Group 3 receives active nucleic acid (e.g. ribozyme) and Group 4 receives recombinant human interferon-a (8 M.U., SC, per animal, EOD for 90 days). Nucleic acid molecules are administered at 30 mg/kg, EOD, SC for 90 days. There are 10 animals per subgroup and up to three doses per subgroup for dose/response studies. Test agents may be delivered via an ALZET™ osmotic pump (Alza Scientific Products) subcutaneously or intravenously, subcutaneous bolus, direct i.p. injection or intravenously via the tail vein. (6) B16 melanoma in C57/B16 Mice (Nishimura et al, 1985, Clin Exp Metastasis 3, 295-304): The primary goal of these studies is to evaluate the effectiveness of nucleic acid therapy targeted increasing the body's ability to produce interferon-alpha and thus augment the immune response and inhibit cell proliferation. Two syngeneic melanoma cell lines, B16/B16 and B16/F10, are utilized. Experimental Procedure: All studies are performed on pathogen-free, 25- 30g female C57/B16 mice. Mice are housed in a pathogen-free environment and allowed food and water ad lib. B16/B16: On Day 0, animals are injected with B16/B16 cells (5xl0 5 ), SC in 100 μl normal saline, mid-dorsal, in the scapular region. Primary tumor volume are measured using microcahpers. triplicate length and width measurements are obtained from each tumor three days per week. Tumor volumes are calculated from tumor length and width measurements according to the equation: Tumor volume = 0.5ab 2 where a=longest axis of the tumor b=shortest axis of the tumor In one set of animals (Group I), primary tumors are allowed to grow for up to 25 days. Therapeutic endpoints in this group are primary tumor volume, metastases and survival. In the second set of animals (Group II), once B16BL6 tumor growth reaches 500 mm 3 , the primary tumors are removed. Therapeutic endpoints in this group are metastases and survival. Metastatic growth in the lungs is observed at death or at day 25 (final day of experiment). Metastasis is observed in the lungs at the end of the experiment by weighing the lungs and by counting the macrometastases under 25X magnification. If no macrometastases are present, the lungs are perfusion fixed in formalin for subsequent sectioning and histological examination of micrometastases and survival time is recorded. Compound Efficacy Studies: There are four subgroups of animals for Groups I and II per drug tested: Subgroup A receives active nucleic acid molecules of the present invention, Subgroup B receives scrambled attenuated nucleic acid control as therapy, and Subgroup C receives vehicle as therapy. Subgroup D serves as a positive therapeutic control and receives recombinant human IFN-alpha A D (8 M.U., SC, per animal, EOD for 30 days). There are 15 animals per group and up to three doses of nucleic acid per group for groups A and B. Therapy begins on day-3 and is continued daily until Day 25. At necropsy, blood samples are obtained via cardiac puncture, spun down and plasma samples are saved at -70°C for future analyses. Test agents may be delivered via an ALZET™ osmotic pump (Alza Scientific Products) subcutaneously or intravenously, subcutaneous bolus, direct i.p. injection or intravenously via the tail vein. B16/F10: On Day 0, animals are injected with B16/F10 (5xl0 4 ) IN in 100 μl normal saline. Therapeutic endpoints in this group are metastases and survival. Metastatic growth in the lungs is observed at death or at day 25 (final day of experiment). Metastasis is observed in the lungs at the end of the experiment by weighing the lungs and by counting the macrometastases under 25X magnification. If no macrometastases are present the lungs are perfusion fixed in formalin for subsequent sectioning and histological examination of micrometastases and survival time is recorded. Compound Efficacy Studies: There are four groups of animals per drug tested: Group 1 receives active nucleic acid molecules (e.g. ribozymes), Group 2 receives scrambled attenuated nucleic acid control as therapy, and Group 3 receivesvehicle as therapy. Group 4 serves as a positive therapeutic control and receives recombinant human EFΝ-alpha A/D (8 M.U., SC, per animal, EOD for 30 days). There are 15 animals per group and up to three doses of nucleic acid molecules per group for groups 1 and 2. Therapy begins on day-3 and is continued daily until Day 25. At necropsy, blood samples are obtained via cardiac puncture, spun down and plasma samples are saved at -70°C for future analyses. Test agents may be delivered via an ALZET™ osmotic pump (Alza Scientific Products) subcutaneously or intravenously, subcutaneous bolus, direct i.p. injection or intravenously via the tail vein. (7) Colorectal Carcinoma (COLOΝ-26) in Balb/c Mice (Sanada et al, 1990, Acta Med Okayama 44, 217-222, Ramani et al., 1989, hit J Cancer 43, 140-146, 1989): The primary goal of these studies is to evaluate the effectiveness of nucleic acid therapy targeted increasing the body's ability to produce interferon-alpha and thus augment the immune response and inhibit cell proliferation.The study evaluates nucleic acid molecules targeting repressors of IFN-alpha on their ability to improve survival and reduce metastases in Balb/c mice with COLON-26 carcinoma. Experimental Procedure: All studies are performed on pathogen- free, 18-20g female Balb/c mice. Mice are housed in a pathogen-free environment and allowed food and water ad lib. On Day 0, animals are injected with COLON-26 cells (lxl 0 6 ) in 100 μl normal saline into the splenic capsule. On day-5 following tumor cell inoculation, the primary tumors are removed. Therapeutic endpoints are metastases and survival. Metastatic growth in the lungs and in the liver is observed at death or at day 40 (final day of experiment). Metastasis is observed in the lungs and liver at the end of the experiment by weighing the organs and by counting the macrometastases under 25X magnification. If no macrometastases are present in these tissues, the organs are perfusion fixed in formalin for subsequent sectioning and histological examination of micrometastases and survival time is recorded. Compound Efficacy Studies There are four groups of animals per drug tested: Group 1 receives active nucleic acid molecules of the invention (e.g.ribozymes), Group 2 receives scrambled attenuated nucleic acid control as therapy, and Group 3 receives vehicle as therapy. Group 4 serves as a positive therapeutic control and receives recombinant human IFN-alpha A/D (8 M.U., SC, per animal, EOD for 30 days). There are 15 animals per group and up to three doses of nucleic acid molecules per group for groups 1 and 2. Therapy begins on day-3 and is continued daily until Day 40. At necropsy, blood samples are obtained via cardiac puncture, spun down and plasma samples are saved at -70°C for future analyses. Test agents may be delivered via an ALZET™ osmotic pump (Alza Scientific Products) subcutaneously or intravenously, subcutaneous bolus, direct i.p. injection or intravenously via the tail vein. Examples The following are non-limiting examples showing the selection, isolation, synthesis and activity of enzymatic nucleic acids of the instant invention. The following examples demonstrate the selection of ribozymes that cleave TR2 Orphan Receptor, EAR3/COUP-TF-1, GATA transcription factors, IRF-2, Genesis, and CDP. The methods described herein represent a scheme by which ribozymes may be derived that cleave other RNA targets expressed from repressor genes. Those of ordinary skill in the art will recognize that other ribozymes with motifs other than hammerhead may also be devised in a similar fashion and are within the scope of the invention. Example 1 : Identification of Potential Ribozyme Cleavage Sites in GATA Transcription Factor 2 (hGATA-2) The sequences of human GATA transcription factor 2 (HUMGATA2A, Genbank Accession No. M77810 (Dorfman et al, 1997, J. Biol. Chem, 267, 1279- 1285) were screened for accessible sites using a computer folding algorithm. Regions of the RNA that did not form secondary folding structures and contained potential hammerhead cleavage sites were identified. The sequences of these cleavage sites are shown in Table III. Example 2: Selection of Ribozyme Cleavage Sites in Human GATA transcription factor To test whether the sites predicted by the computer-based RNA folding algorithm coπesponded to accessible sites in GATA transcription factor, 70 hammerhead sites were selected for analysis. Ribozyme target sites were chosen by analyzing genomic sequences of hGATA-2 (Dorfman, supra) and prioritizing the sites on the basis of folding. Hammerhead ribozymes were designed that could bind each target (see Figure 1) 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. An example of a ribozyme targeted to hGATA-2 is shown in figure 2. Example 3: Chemical Synthesis and Purification of Ribozymes for Efficient Cleavage of GATA Transcription Factor 2 RNA Ribozymes of the hammerhead and/or hammerhead like motifs 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 G5 and a U for A14 (numbering from Hertel et al, 1992 Nucleic Acids Res., 20, 3252). Hairpin ribozymes are synthesized in two parts and annealed to reconstruct the active ribozyme (Chowrira and Burke, 1992 Nucleic Acids Res., 20, 2835-2840). Ribozymes are also synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol. 180, 51). Ribozymes were modified to enhance stability by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-fluoro, 2'-O-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 ah, 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-VI. Example 4: Ribozyme Cleavage of hGATA-2 RNA Target in vitro Ribozymes targeted to the human hGATA-2 RNA are designed and synthesized as described above. These ribozymes can be tested for cleavage activity in vitro, for example using the following procedure. The target sequences and the nucleotide location within the hGATA-2 mRNA are given in Table III. Cleavage Reactions: Full-length or partially full-length, internally-labeled target RNA for ribozyme cleavage assay is prepared by in vitro transcription in the presence of [ot- 32 p] CTP, passed over a G 50 Sephadex column by spin chromatography and used as substrate RNA without further purification. Alternately, substrates are 5'-32p- e nd labeled using T4 polynucleotide kinase enzyme. Assays are performed by pre-warming a 2X concentration of purified ribozyme in ribozyme cleavage buffer (50 mM Tris-HCl, pH 7.5 at 37°C, 10 mM MgCh) 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 o cleavage buffer. As an initial screen, assays are carried out for 1 hour at 37 C using a final concentration of either 40 nM or 1 mM ribozyme, t.e., ribozyme excess. The reaction is 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 is o 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 are visualized on an autoradiograph of the gel. The percentage of cleavage is determined by PHOSPHOR IMAGER® quantitation of bands representing the intact substrate and the cleavage products. Example 5: Increased Expression of Erythropoietin by Inhibition of Repressors of Erythropoietin Transcriptional repressors of the erythropoetin gene were targeted with ribozymes in order to increase Epo levels. Ribozymes were synthesized targeting hGATA-2, TR-2, and EAR3/Coup-TFl. Ribozyme screening was performed by complexing with lipid, delivering to the appropriate cell line, and monitoring for Epo production. The ability of these ribozymes to increase Epo expression in both induced (with CoCl ) and non-induced cells was also tested. Erythropoietin (Epo) is produced in the adult kidney and fetal liver in response to hypoxia and CoCl . Two human hepatoma cell lines, Hep G2 and Hep 3B, exhibit regulated expression of Epo in response to hypoxia and CoCl 2 . Ribozymes were tested under non-induced and induced conditions to determine if Epo levels could be increased under one or both conditions. Hep3B cells were plated at 1.8 x 10 4 cells per well in a 96 well plate. Ribozymes were then transfected into cells using cationic lipids 24 hours after seeding the plates. Two concentrations of each ribozyme (100 and 400 nm) were tested using 5 or 7.5 μg/ml of cationic lipid. The sequences for the ribozymes and the irrelevant controls (IR1 & IR2) are given in table NIII. Cells were then induced to express Erythropoietin by applying cell culture media containing CoCl (50 nM). After 24 hours, 100 ml of media was removed from the plate well and added into a plate for an ELISA assay. The remaining media is aspirated off and the cells were frozen at -70°C until tested by CYQUAΝT™ assay using the manufacturer's protocol. The ELISA for quantification of erythropoietin was performed using QUAΝTIKIΝE IND™ kit sold by R&D Systems (Minneapolis, MΝ) by using the manufacturers protocol. The data indicates that a number of ribozymes were able to cause elevated expression of Epo in these cells compared to the inactive controls. Results are shown in Figures 4 and 5 for cobalt-induced and without cobalt induction respectively. Example 6: Elevated Expression of Erythropoietin Over Time Using Ribozymes Targeting Epo Repressors Hep3B cells were prepared as described in example 5. Ribozymes (RPI No. 14260 (targeting hGATA-2) & 144521 (targeting EAR3/COUP-TR1 ; table VIII) at a concentration of lOOnm were transfected into Hep3B cells using 5 μg/ml of cationic lipid. Epo expression in these cells was measured at 36 and 48 hours for continuous delivery and at 12, 24, and 36 hours for pulsed delivery using an ELISA assay from example 5. The data was compared to two irrelevant and an untreated control (Unt) . The sequences for the ribozymes and the irrelevant controls (IR-1 & IR-2) are given in table NIII. The ribozyme was either delivered continuously during the incubation period or added for just 4 hours and then replaced with fresh media (pulsed delivery). The data is shown in figures 6-9 which demonstrate that either continuous or pulsed delivery of ribozymes targeting hGATA-2 or EAR3/Coup-TRl will result in elevated expression of Epo in Hep3B cells compared to iπelevant and untreated controls. Diagnostic uses Nucleic acid molecules of this invention may be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of specific RNA in a cell. For instance, the close relationship between ribozyme activity and the structure of the target RNA allows the detection of mutations in any region of the molecule which alters the base-pairing and three-dimensional structure of the target RNA. By using multiple ribozymes described in this invention, one may map nucleotide changes which are important to RNA structure and function in vitro, as well as in cells and tissues. Cleavage of target RNAs with ribozymes may be used to inhibit gene expression and define the role (essentially) of specified gene products in the progression of disease. In this manner, other genetic targets may be defined as important mediators of the disease. 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 related conditions. 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 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 coπelated with higher risk whether RNA levels are compared qualitatively or quantitatively. Additional Uses Potential usefulness of sequence-specific enzymatic nucleic acid molecules of the instant invention might have many of the same applications for the study of RNA that DNA restriction endonucleases have for the study of DNA (Nathans et al., 1975 Ann. Rev. Biochem. 44:273). For example, the pattern of restriction fragments could be used to establish sequence relationships between two related RNAs, and large RNAs could be specifically cleaved to fragments of a size more useful for study. The ability to engineer sequence specificity of the ribozyme is ideal for cleavage of RNAs of unknown sequence. The nucleic acid molecules of the present invention may also be used for small and large scale synthesis of proteins. Nucleic acids such as enzymatic nucleic acids and antisense molecules may be administered into cells in culture to initiate in vitro synthesis of such repressed proteins as erythropoietin, G-CSF, or interferon- alpha. The method involves the steps of contacting or introducing into a cell a nucleic acid molecule (e.g. ribozyme or antisense) capable of down-regulating (inhibition) expression of a repressor protein which represses the expression of a target protein (repressed protein), such that the level of repressor protein will be decreased, resulting in the stimulation of expression of target protein in the cell. The target protein can then be purified from the cells using standard techniques known in the art. Those of ordinary skill in the art will recognize that the method could also be utilized for the increase expression of other repressed proteins in addition to the proteins mentioned above. The inhibition of expression of repressor transcription factors using nucleic acids may also be utilized in non-human organisms. Particularly since negative regulation of genes has been demonstrated in plants (Preston et al, 1998, J. Bacteriol. 180, 4532-4537). For example, plants and fungi may have repressor transcription factors which, when inhibited, would allow for the increased expression of beneficial proteins for increased crop yield, disease resistance, and increases in synthesis for desired amino acids, oils, and the like. Ladner & Bird, International Publication No. WO8806601 describe the suppression of genes to inhibit the proliferation of viruses. Applicant describes the use of nucleic acid molecules to down-regulate gene expression of repressors in bacterial, microbial, fungal, viral, and eukaryotic systems including plant, or mammalian cells. All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually. One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The methods and compositions described herein as presently representative of prefeπed embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art, which are encompassed within the spirit of the invention, are defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. Thus, such additional embodiments are within the scope of the present invention and the following claims. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising", "consisting essentially of and "consisting of may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by prefeπed embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the description and the appended claims. In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group. Thus, additional embodiments are within the scope of the invention and within the following claims TABLE I 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. • 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 [', u ]. • Complete kinetic framework established for one ribozyme ["VVN]. • Studies of ribozyme folding and substrate docking underway P rti / vm , lx ] . • Chemical modification investigation of important residues well established [Y 1 ] . • The small (4-6 nt) binding site may make this ribozyme too non-specific for targeted RNA cleavage, however, the Tetrahymena group I intron has been used to repair a "defective" β-galactosidase message by the ligation of new β-galactosidase sequences onto the defective message [ xu ], RNAse P RNA (M1 RNA) • Size: -290 to 400 nucleotides. • RNA portion of a ubiquitous ribonucleoprotein enzyme. • Cleaves tRNA precursors to form mature tRNA [ xm ] . • 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 [ xιv , xv l • Important phosphate and 2' OH contacts recently identified m l xΛ ] Group II Introns • Size: >1000 nucleotides. Trans cleavage of target RNAs recently demonstrated [ xvω xi *] Table t 51 • 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. • Only natural ribozyme with demonstrated participation in DNA cleavage [ X , XX1 ] in addition to RNA cleavage and ligation. • Major structural features largely established through phylogenetic comparisons [ xxu ] . • Important 2' OH contacts beginning to be identified [ xxm ] • Kinetic framework under development [ xxι ] Neurospora VS RNA • Size: -144 nucleotides. • Trans cleavage of hairpin target RNAs recently demonstrated [ xxv ] . • 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. • 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 [ xxv, , x * vu ] • Minimal ligation activity demonstrated (for engineering through in vitro selection) [ xxvm ] • Complete kinetic framework established for two or more ribozymes f™"]. • Chemical modification investigation of important residues well established [ *xx ] . 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 p ^^ « » « * » ] • Ligation activity (in addition to cleavage activity) makes ribozyme amenable to engineering through in vitro selection [ xxv ] • Complete kinetic framework established for one ribozyme [ x xvι ] . • Chemical modification investigation of important residues begun [ X XVU XX VU1 ] , Hepatitis Delta Virus (HDV) Ribozyme • Size: -60 nucleotides. • Trans cleavage of target RNAs demonstrated [ XXXU ]. • Binding sites and structural requirements not fully determined, although no sequences 5' of cleavage site are required. Folded ribozyme contains a pseudoknot structure [ x1 ]. • 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 [ x ] Michel, Francois; Westhof, Eric. Slippery substrates. Nat. Struct. Biol. (1994), 1(1), 5-7. " . 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! '" . 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. 1V . Herschlag, Daniel; Cech, Thomas R.. Catalysis of RNA cleavage by the Tetrahymena thermophila 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. " . 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. w . 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. "' . Li, Yi; Bevilacqua, Philip C; Mathews, David; Turner, Douglas H.. TT ermodynamic and activation parameters for binding of a pyrene-labeled substrate by the Tetrahjmiena ribozyme: docking is not diffusion-controlled and is driven by a favorable entropy change. Biochemistry (1995), 34(44), 14394-9. ,ui . 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. ix . Zamnkar, 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. " . Strobel, Scott A.; Cech, Thomas R.. Minor groove recognition of the conserved CcntdotU pair at the Tetrahymena ribozyme reaction site. Science (Washington, D. C.) (1995), 267(5198), 675-9. " . Strobel, Scott A.; Cech, Thomas R.. Exocyclic Amine of the Conserved G.cntdotU Pair at the Cleavage Site of the Tetrahymena Ribozyme Contributes to 5'-Splice Site Selection and Transition State Stabilization. Biochemistry (1996), 35(4), 1201-11. "". Sullenger, Bruce A.; Cech, Thomas R.. Ribozyme-mediated repair of defective mRNA by targeted trans-splicing. Nature (London) (1994), 371(6498), 619-22. Robertson, H.D.; Altman, S.; Smith, J.D. J. Biol. Chem., 2.4-Z, 5243-5251 (1972). " v . Forster, Anthony C; Altaian, Sidney. External guide sequences for an RNA enzyme. Science (Washington, D. C, 1883-) (1990), 249(4970), 783-6. Yuan, Y.; Hwang, E. S.; Altman, S. Targeted cleavage of mRNA by human RNase P. Proc. Natl. Acad. Sci. USA (1992) 89, 8006-10. "" . Harris, Michael E.; Pace, Norman R.. Identification of phosphates involved in catalysis by the ribozyme RNase P RNA. RNA (1995), 1(2), 210-18. * "' • Pan, Tao; Loria, Andrew; Zhong, Kun. Probing of tertiary interactions in RNA: 2'-hydroxyl- base contacts between the RNase P RNA and pre-tRNA. Proc. Natl. Acad. Sci. U. S. A. (1995), 92(26), 12510-14. ""' . Pyle, Anna Marie; Green, Justin B.. Building a Kinetic Framework for Group II Intron Ribozyme Activity: Quantitation of lnterdomain Binding and Reaction Rate. Biochemistry (1994), 33(9), 2716-25. "" . 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. " . 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. ^ . 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. " * " . Michel, Francois; Ferat, Jean Luc. Structure and activities of group π introns. Annu. Rev. Biochem. (1995), 64, 435-61. XX1 " . 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. uuv . Daniels, Danette L.; Michels, William J., Jr.; Pyle, Anna Marie. Two competing pathways for self-splicing by group II introns: a quantitative analysis of in vitro reaction rates and products. J. Mol. Biol. (1996), 256(1), 31-49. " v . 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. "" . Scott, W.G., Finch, J.T., Aaron,K. The crystal structure of an all RNA hammerhead ribozyme:Aproposed mechanism for RNA catalytic cleavage. Cell, (1995), 81, 991-1002. m " . McKay, Structure and function of the hammerhead ribozyme: an unfinished story. RNA, (1996), 2, 395-403. """ . Long, D., Uhlenbeck, O., Hertel, K. Ligation with hammerhead ribozymes. US Patent No. 5,633,133. "" . Hertel, K.J., Herschlag, D., Uhlenbeck, O. A kinetic and thermodynamic framework for the hammerhead ribozyme reaction. Biochemistry, (1994) 33, 3374-3385.Beigelman, L., et al, Chemical modifications of hammerhead ribozymes. J. Biol. Chem., (1995) 270, 25702-25708. "" • Beigelman, L., et al., Chemical modifications of hammerhead ribozymes. J. Biol. Chem., (1995) 270, 25702-25708. "" . 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. mi> . 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. XJuα " . 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. vaxv . Joseph, Simpson; Berzal-Herranz, Alfredo; Chowrira, Bharat M.; Butcher, Samuel E.. Substrate selection odes for the hairpin ribozyme determined by in vitro selection, mutation, and analysis of mismatched substrates. Genes Dev. (1993), 7(1), 130-8. " xxv . 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. axΛ . Hegg, Lisa A.; Fedor, Martha J.. Kinetics and Thermodynamics of Intermolecular Catalysis by Hairpin Ribozymes. Biochemistry (1995), 34(48), 15813-28. " aii . 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. x " xy " i . 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 internal loop B of the hairpin ribozyme: implications for secondary structure. Nucleic Acids Res. (1996), 24(4), 573-81. '"" . 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. ύ . 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. " k . 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 II: 0.2 μmol RNA Synthesis Cycle Reagents Equivalents Amounts (μL) Wait time (sec) Phosphoramidites 15 31 465 SET 38.7 31 465 Acetic anhydride 655 124 5 N-methyl-imidazole 1245 124 5 TCA 700 732 10 Iodine 20.6 244 15 Wait time does not include contact time during delivery. Table III. Hammerhead ribozymes targeting GATA Transcription factors (1, 2, 3, 4, and 6) and the complementary Sequences Table III. Hammerhead ribozymes targeting GATA transcription factors (1, 2. 3, 4, and 6) and the complementary sequences Table Id. Hammerhead ribozymes targeting GATA transcription factors (1, 2, 3, 4, and 6) and the complementary sequences Table III. Hammerhead ribozymes targeting GATA transcription factors (1, 2, 3, 4, and 6) and the complementary sequences Table EH. Hammerhead ribozymes targeting UATA transcription factors (1, 2, 3, 4, and 6) and the complementary sequences Table HI. Hammerhead ribozymes targeting GATA transcription factors (1, 2, 3, 4, and 6) and the complementary sequences Table III. Hammerhead ribozymes targeting GATA transcription factors ( I, 2, 3, 4, and 6) and the complementary sequences Table EU. Hammerhead ribozymes targeting GATA transcription factors (1, 2, 3, 4, and 6 61) a anndd t thhee ccooπm-Dplleemmeennttaarrvy s seeαquueenncceess Table HI. Hammerhead ribozymes targeting GATA transcription factors (1, 2, 3, 4, and 6) and the complementary sequences Table HI. Hammerhead ribozymes targeting GATA transcription factors (1, 2, 3, 4, and 6) and the complementary sequences Table EH. Hammerhead ribozymes targeting GATA transcription factors (1, 2, 3, 4, and 6) and the complementary sequences Table III. Hammerhead πbozymes targeting GATA transcription factors (1, 2, 3, 4, and 6) and the complementary sequences Table Hi. Hammerhead ribozymes targeting GATA transcription factors ( 1, 2, 3, 4, and 6) and the complementary sequences Table EH. Hammerhead ribozymes targeting GATA transcription factors (1, 2, 3, 4, and 6) and the complementary sequences Table EH. Hammerhead ribozymes targeting GATA transcription factors (1, 2. 3, 4, and 6) and the complementary sequences Table EH. Hammerhead ribozymes targeting GATA transcription factors (1, 2, 3, 4, and 6 6)) aanndd tthhee ccoommDplleemmeennttaarrvy s seeαquueenncceess Table IH. Hammerhead ribozymes targeting GATA transcription factors ( 1, 2, 3, 4, and 6) and the complementary sequences Table HI. Hammerhead ribozymes targeting GATA transcription factors (I, 2, 3, 4, and 6) and the complementary sequences Table III. Hammerhead ribozymes targeting GATA transcription factors ( I, 2, 3, 4, and 6 6) . a anndd t thhee ccoommDplleemmeennttaarrvy s seeαquueenncceess Table πi. Hammerhead ribozymes targeting GATA transcription factors ( 1, 2. 3, 4, and 6) and the complementary sequences Table III. Hammerhead ribozymes targeting GATA transcription factors ( 1, 2, 3, 4, and 6) and the complementary sequences Table IEE. Hammerhead ribozymes targeting GATA transcription factors ( 1, 2, 3, 4, and 6) and the complementary sequences Table III. Hammerhead ribozymes targeting GATA transcription factors ( 1, 2, 3, 4, and 6) and the complementary sequences Table πi. Hammerhead ribozymes targeting GATA transcription factors (1, 2. 3, 4, and 6) and the complementary sequences Table πi. Hammerhead ribozymes targeting GATA transcription factors ( I, 2, 3, 4, and 6) and the complementary sequences Table III- Hammerhead ribozymes targeting GATA transcription factors (1, 2, 3, 4, and 6 6)) a anndd t thhee c coommDplleemmeennttaarrvy s seeαquueenncceess Table HI. Hammerhead ribozymes targeting GATA transcription factors (1, 2, 3, 4, and 6) and the complementary sequences Table in. Hammerhead ribozymes targeting GATA transcription factors (1, 2, 3, 4, and 6) and the complementary sequences Table IH. Hammerhead ribozymes targeting GATA transcription factors (1, 2, 3, 4, and 6) and the complementary sequences Table EH. Hammerhead ribozymes targeting GATA transcription factors (1, 2, 3, 4, and 6) and the complementary sequences Table III. Hammerhead ribozymes targeting GATA transcription factors (I, 2, 3, 4, and 6) and the complementary sequences Table EH. Hammerhead ribozymes targeting GATA transcription factors ( 1, 2, 3, 4, and 6) and the complementary sequences Table HI. Hammerhead ribozymes targeting GATA transcription factors (1, 2, 3, 4, and 6) and the complementary sequences Table EH. Hammerhead ribozymes targeting GATA transcription factors (1, 2, 3, 4, and 6) and the complementary sequences Table III. Hammerhead ribozymes targeting GATA transcription factors (1, 2, 3, 4, and 6) and the complementary sequences Table EH. Hammerhead ribozymes targeting GATA transcription factors (I, 2, 3, 4, and 6) and the complementary sequences Table EH. Hammerhead ribozymes targeting GATA transcription factors (I, 2, 3, 4, and 6) and the complementary sequences Table HI. Hammerhead ribozymes targeting GATA transcription factors (I, 2, 3, 4, and 6) and the complementary sequences Table EH. Hammerhead ribozymes targeting GATA transcription factors (1, 2, 3, 4, and 6) and the complementary sequences 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. Hammerhead Ribozymes to TR2-9 and TR2-1 1 Oφhan Receptor Genes Table IV. Hammerhead Ribozymes to TR2-9 and TR2-1 1 Oφhan Receptor Genes Table IV. Hammerhead Ribozymes to TR2-9 and TR2-11 Oφhaπ Receptor Genes Table IV. Hammerhead Ribozymes to TR2-9 and TR2- 1 1 Oφhan Receptor Genes Table IV. Hammerhead Ribozymes to TR2-9 and TR2-1 1 Oφhan Receptor Genes Table IV. Hammerhead Ribozymes to TR2-9 and TR2- 11 Oφhan Receptor Genes Table IV. Hammerhead Ribozymes to TR2-9 and TR2-11 Oφhan Receptor Genes Table IV. Hammerhead Ribozymes to TR2-9 and TR2-11 Oφhan Receptor Genes Table IV. Hammerhead Ribozymes to TR2-9 and TR2-11 Oφhan Receptor Genes Table IV. Hammerhead Ribozymes to TR2-9 and TR2-11 Oφhan Receptor Genes Table IV. Hammerhead Ribozymes to TR2-9 and TR2-1 1 Oφhan Receptor Genes Table IV. Hammerhead Ribozymes to TR2-9 and TR2-11 Oφhan Receptor Genes 1&7 Table IV. Hammerhead Ribozymes to TR2-9 and TR2-1 1 Oφhan Receptor Genes Table IV. Hammerhead Ribozymes to TR2-9 and TR2-1 1 Oφhan Receptor Genes Table IV. Hammerhead Ribozymes to TR2-9 and TR2-1 1 Oφhan Receptor Genes Table IV. Hammerhead Ribozymes to TR2-9 and TR2- 11 Oφhan Receptor Genes Table IV. Hammerhead Ribozymes to TR2-9 and TR2-11 Oφhan Receptor Genes Table IV. Hammerhead Ribozymes to TR2-9 and TR2-11 Oφhan Receptor Genes 1723 H--MTR29 4156 CAUUGAAA OEAUGPG X OGAA ADUUUAAU 4935 ATEAAAAT C TITCAA-IG 1725 HUMTR29 4157 --V----A-UUGA CUGALGPG X OGRA AGADUUUA 4936 T-AAAATCT T TC-AA-IGTT 1726 HU--TR29 4158 AA-ACAUUG CUGADGPG X OGRA AP-GAUUUU 4937 AAAAICTT T CAATGT1T 1727 HUMER29 4159 CAAACAUU C--UC-AUGPG X OGRA AAAGRUUU 4938 AAA-IUTTT U AA-1GT11U 1733 H--JMER29 4160 -AI----ADUCA CUGADGAG X O AA --AC-- --UGRA 4939 ir--r3AIGT T TGAAIAIA 1734 H---JMI-R29 4161 UUAUAUUC C GAUGPG X OGRA AA-2AUUGA 4940 TCAATG-T T GAAIA--I-AA 1739 HUMIR29 4162 CX--ACAUUA (--UGAD-----PG X OGAA ADUCAAAC 4941 G1TTUAAT A '1-AA-lUlL-U 1741 HUMTR29 4163 CUCCACAU CU---ADC-AG X OGAA AU-AUUCAA 4942 T--GAAT-AT A ATC-TGC-PG 1754 H--MIR29 4164 CUCAGGUA CUCADGAG X OGAA A--AOCUCC 4943 C-GA-3--JIGT T TAO--TGPG 1755 HUM--R29 4165 CrUCRGGU ( --UGADGPG X OGRA AACPOCUC 4944 CAGGIGTT' T -M-r-TGP-GG 1756 HUMI-R29 4166 C--CC-X-3GG -GADC-AG X OGAA AAACAOCU 4945 -A---xπG-τττ A OC--TGAG3G 1768 HUMI-R29 4167 C----AGAUP-GA CUCAD--PG X CGRA -PG--D--CUC 4946 C---K3---BCCT C IU'1-AIUIU 1770 HLMI-R29 4168 QQGPGADA ( -U--AUC--PG X OGRA AGPGGCCC 4947 ( -------GCC-TCT C TATCTCCC 1772. H-JMTR29 4169 C-GGGGAGA CUGADGRG X OGRA AGPGRGGC 4948 σaiTCTUT A lUTUCCCG 1774 HJ--TR29 4170 UUCGGGGA C-UCA-X-AG X OGRA PUPGPGPG 4949 C-TCTCTAT C TCCOaGAA 1776 --CMER29 4171 -AA-U---CGGG C-UGAUGPG X CERA AGAURGPG ■ 4950 CTCI-AIUT C C--COGAA-IT 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 V. Hammerhea. jozymes to E AR3/COUP-TF- 1 113 Table V. Hammerhea-- . bozymes to EAR3/COUP-TF- 1 Table V. Hammerhe--_ xibozymes to EAR3/COUP-TF- 1 115 Table V. Hammerheav- --lbozymes to EAR3/COUP-TF-1 116 Table V. Hammerhe-- abozymes to EAR3/COUP-TF- 1 1 1 7 Table V. Hammerhe--, αbozymes to EAR3/COUP-TF-1 Table V. Hammerht -bozymes to EAR3tCOUP-TF-l 1 1 9 Res. 20: 3252). The length of stem II may be > 2 base-pairs. Table VI. Hammerh. abozymes to IRF-2 120 Table VI. Hammerheau Ribozymes to IRF-2 121 00/61729 Table VI. Hammerhe-. ,-ibozymes to IRF-2 122 Table VI. Hammerhe. ibozymes to IRF-2 123 O 00/61729 Table VI. Ham erh .ibozymes to IRF-2 124 Table VI. Hammerhe -ibozymes to ERF-2 125 Table VI. Hammerhe--- -<ibozymes to IRF-2 126 Table VI. Hammerhe- Jbozymes to IRF-2 127 Table VI. Hammerht ibozymes to IRF-2 128 Table VI. Hammer-. Aibozymes to IRF-2 1 29 5966 -----CACAG3 C-UGRDGPG X CCAA RAAUCUGA 6388 TJ---3GRDUU U (-X-UGUGGA 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 VTI. Hammerh .Ribozymes to CDP 130 Table VII. Hammerh-.- - Ribozymes to CDP 131 O 00/61729 Table VII. Hammerh .Ubozymes to CDP 1 32 Table VII. Hammerh. -ibozymes to CDP 133 Table VII. Hammerh ibozymes to CDP 134 O 00/61729 Table VII. Hammerh. --jbozymes to CDP 1 35 Table VH Hammerh dbozymes to CDP 136 O 00/61729 Table VH Hammerh- Ribozymes to CDP 1 37 Table VII. Hammerl. Ribozymes to CDP 1 38 3918 6763 -AGUL--GUGG CUCA GPG X CGAA ACCAGUUG 7303 C3ACIGGT T C-CACAAC-T 3919 6764 LrøJUGUG CUGADGRG X CGAA AAOCAGUU 7304 AACT----.1T C CACAACI-A 3927 6765 GAGACCUG C-UTGAUGAG X CGRA AGUUGDGG 7305 CCACAACT A C3G3ICTC 3933 6766 α-AUCCGA CUGADGAG X O A ACEUGUAG 7306 CTACAGG-T C TOGGA-rCC 3935 6767 α-X-XA-UCC CUGRUGRG X CERA AGR-CCUGU 7307 ACR-C--GT--T C C-EATCCOC 3940 6768 UCLT-UGCG OXADGRG X CCAA AUCCGA-G-A 7308 1ETCOGAI C QGCA-G-AGA 3954 6769 CCUCAAUG C---XAD--PG X C-GAA ACRGUUCU 7309 AGAACIGT T C3ITGAGG 3955 6770 UCCUCAAU OXGRDGRG X CXAA AAC GDUC 7310 GAA--.1GTT C A-π-GAGC-A 3958 6771 ADDUCCUC OXADGRG X CERA AUGRRCRG 7311 CTGTTUAT T GPGGAAAI 3967 6772 α-α--rαxG ox-AU AG x OAA -- -uucαr 7312 GAGGAAAT T C-AGGCC-GG 3968 6773 OX--ADGRG X CGRA AADUUOCU 7313 AG-AAA-IT C AGGCCGGG 3980 6774 OX--X---CCCU C-UGAUGPG X CCAA ACUCCOGG 7314 C--OGGGA-G-T C -AQG-GOCRG 4005 6775 CCGRGGGU CUGADGPG X OGAA A-GUCQCUG 7315 CAGO-GRCT C Aα-CTCOG 4011 6776 UO---1-3-3GOC CUGRUGRG X CGAA -PGGGUGAG 7316 σcAc-ccτ c c-c-xxαr-A 4041 6777 (-----αXUCC OXGRDGRG X CGAA PGC-UGGGC 7317 G-----CCAGCT C GGRGQ3C-E 4113 6778 OX---E-CUGA CUGRUGPG X CGRA ACUUGGGC 7318 QCOCAAGΓ C TCAGQGRG 4115 6779 CUCUCCCϋ CUGRUCAG X CGRA A-GACUUGG 7319 α---AAGICT C AGOGRGRG 4176 6780 C--GGUCCCC C--UGRDGRG X CGAA AG-3-3CQQC 7320 CXXX3-CCCT C CGQGAC-CC 4263 6781 C--3GD--X30G CUGADGRG X CGRA A-OGOGQC-G 7321 C CEOGRCCE 4307 6782 C--GOXGPGG CUGADGRG X OGAA AQOQGOGU 7322 -Aαr-O-GCT A CXTCRGOC 4311 6783 OXGRDGRG X CGAA PGGURGOG 7323 αsc-ER-ocr c -AGoααcoG 4353 6784 C-XX---X30GOG O-XGADGRG X OGAA A.O-----UCGQG 7324 000-3031 C C-----X-X-3CCAC 4407 6785 UCUQCAGC CUGRUGRG X CGRA A-C--CUGGQC 7325 σorA-G-cT c CCΪGCRGA 4420 6786 -R-O---C0GAA OXAUGAG X O-AA P-GGCUCUG 7326 CAGRGOCT T T----OGGC--CT 4421 6787 CAGQOCGA CUGRDGRG X GGAA APGGCUCU 7327 RGRGCCTΓ T TCGGCCTC 4422 6788 QGRGGOOS CUCADGPG X CCAA AAAGGCUC 7328 GAGCUTTT T 0-23C--CTCC 4423 6789 α-X-AGGCC C--0GR--XGRG X OGAA AAARG3CU 7329 -RGU.TTTT c α-x-t-πcσc 4429 6790 CS-X-XOGG CUGADGRG X OGRA -AGQOCI-AA 7330 Tl-COGOCT C C-Oα-AGQC 4455 6791 UC-UCGOGC CUGRDGRG X CGAA -AG--X-CCGG 7331 OOQGGR-Cr C QO30G-A-CA. 4494 6792 UGCUGUUC OXAUGRG X CCAA -RGUUCGCG 7332 CGOGRRCT T GAACAGC3 4504 6793 ---GaXGGRU OXGAUGRG X CGAA AD-X-UGUϋ 7333 AACR-GCAT C TCCA-OOG 4507 6794 RGGC-GGUG C-UGRDG-AG X OAA AD----ADGCU 7334 -AG--3-ICAT C (30aQ0CT 4544 6795 CCADUOGA CUGAUCAG X OCAA -RGQUUOC-U 7335 AGCAROC-T A TOGA-ATOG 4546 6796 UCCCADCC CUGRUGPG X C-GAA AUPGGUUC 7336 GA-ACC-TAT C G-RAIEQGA 4557 6797 CX--CCUCRG CUGADGRG X CGRA ACUCCCMJ 7337 A-IGQ-GP-G-T T CT--AGG-3G 4558 6798 CXX---XUCA CUGRUGRG X OGRA AftGUOOCA 7338 TO----X----AG-TT C TGRG033C 4618 6799 (---EOOGUDC CUGRDGRG X OAA AOOOOGϋC 7339 GAC-QQQGT C G-GR-0Q-33G 4656 6800 03-X3GIGCC CUGRDGRG X OCAA AGC0CAGS 7340 C--C-TGGGCT T GGCCOG0G 4713 6801 G---AD0GUG C-UGADGRG X CXAA A3333GUC 7341 (--AOOCCCT C (3CQG-TCC 4720 6802 ACX3QOQOS C-UGRDGRG X CERA AO--GUGGA 7342 TCCROSG-T C C---X-X3QCCT 4750 6803 OGQOCUUG C-UGRDGRG X CG A AD----UGG3C 7343 a-xrR-svr c CAAGOoc-E 4774 6804 C--GGCCGCA (--UGADGRG X CCAA AGUGGGUC 7344 GROOCACΓ C T-x-EGar 4801 6805 COTX3GUG C-UGRUGRG X CERA -V---JCOXA 7345 TGOGGCE-T C CRCCAAOC 4853 6806 AGU------CO-3. OXGR--GRG X CGAA AGCGGUCC 7346 C-X-A003CT T TC---E----3CT 4854 6807 -ARGUGCQC CUGRUGRG X CGAA AAGCGGUC 7347 GA--JC--UUTT T GOGCRC-TT 4862 6808 R-GGGC-QGU CUGAUGAG X CERA -R-3---X30-3--A 7348 TGCQCA-Cr T AOOGOCCT 4863 6809 (-3GGCXEG -----UCADGAG X CXAA APGUGOX 7349 a-JQCAC-TT A CXECOCIG Table VII. Hammerh Ribozymes to CDP 139 Table VII. Hammer. Ribozymes to CDP 140 Table VII. Hammerl. Ribozymes to CDP 1 41 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. O 00/61729 1 42 Table VIII. Sequences for controls & Hammerhead and Hammerhead-like Ribozymes Table VUI. Sequences for controls & Hammerhead and Hammerhead-like Ribozymes Table VE Sequences for controls & Hammerhead and Hammerhead-like Ribozymes O 00/61729 1 45 Table VUI. Sequences for controls & Hammerhead and Hammerhead-like Ribozymes Table VUI. Sequences for controls & Hammerhead and Hammerhead-like Ribozymes lower case = 2'OMe U = 2'-C-Allyl-U G,A, U= ribo G,A, U s = phosphorothioate linkages s 2=dithioate- linkages B = inverted abasic l=ribo-lnosine

Claims: Claims 1. A nucleic acid molecule capable of specifically inhibiting expression of gene encoding TR2 Oφhan Receptor. 2. The nucleic acid molecule of claims 1, wherein said nucleic acid molecule is an enzymatic nucleic acid molecule. 3. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule is an antisense nucleic acid molecule. 4. The nucleic acid molecule of claim 2, wherein the binding arms of said nucleic acid molecule comprise sequences complementary to any of sequences of Seq ID Nos 4172-4950. 5. The nucleic acid molecule of claim 3, wherein the nucleic acid molecule comprises sequences complementary to any of sequences of Seq ID Nos 4172- 4950. 6. The nucleic acid molecule of claim 2, wherein said nucleic acid molecule is in a hammerhead moti f . 7. The nucleic acid molecule of claim 2, wherein said nucleic acid molecule is in a hairpin, hepatitis Delta virus, group I intron, VS nucleic acid or RNase P nucleic acid motif. 8. The nucleic acid molecule of claims 2 or 3, wherein said nucleic acid molecule comprises between 12 and 100 bases complementary to RNA encoding the TR2 Orphan Receptor. 9. The nucleic acid molecule of claims 2 or 3, wherein said nucleic acid molecule comprises between 14 and 24 bases complementary to RNA encoding the TR2 Orphan Receptor. 10. The nucleic acid molecule of claim 6, wherein said nucleic acid molecule comprises any of sequence of Seq ID Nos 3393-4171. 11. The nucleic acid molecule of claim 2, wherein said nucleic acid molecule is an enzymatic DNA molecule. 12. The nucleic acid molecule of claim 1, wherein said TR-2 Oφhan Receptor is TR2- 11 Oφhan Receptor. 13. The nucleic acid molecule of claim 1, wherein said TR-2 Oφhan Receptor is TR2- 9 Oφhan Receptor. 14. A nucleic acid molecule capable of specifically inhibiting expression of gene encoding EAR3/ COUP-TF-1. 15. The nucleic acid molecule of any of claims 14 wherein said nucleic acid molecule is an enzymatic nucleic acid molecule. 5 16. The nucleic acid molecule of any of claims 14 wherein said nucleic acid molecule is an antisense nucleic acid molecule. 17. The nucleic acid molecule of claim 15, wherein the binding arms of said nucleic acid molecule comprise sequences complementary to any of sequences of Seq ID Nos 5248-5544. 10 18. The nucleic acid molecule of claim 16, wherein the nucleic acid molecule comprises sequences complementary to any of sequences of Seq ID Nos 5248- 5544. 19. The nucleic acid molecule of claim 15, wherein said nucleic acid molecule is in a hammerhead motif. 15 20. The nucleic acid molecule of claim 15, wherein said nucleic acid molecule is in a haiφin, hepatitis Delta virus, group I intron, VS nucleic acid or RNase P nucleic acid motif. 21. The nucleic acid molecule of claims 15 or 16, wherein said nucleic acid molecule comprises between 12 and 100 bases complementary to RNA encoding EAR3/ 20 COUP-TF-1. 22. The nucleic acid molecule of claims 15 or 16, wherein said nucleic acid molecule comprises between 14 and 24 bases complementary to RNA encoding EAR3/ COUP-TF-1. 23. The nucleic acid molecule of claim 19, wherein said nucleic acid molecule 25 comprises any of the sequences of Seq ID Nos 4951-5247. 24. The nucleic acid molecule of claim 15, wherein said nucleic acid molecule is an enzymatic DNA molecule. 25. A nucleic acid molecule with RNA cleaving activity, wherein said nucleic acid molecule cleaves RNA encoded by a GATA transcription factor gene. '30 26. The nucleic acid molecule of claim 25, wherein said GATA transcription factor gene is GATA transcription factor 2 gene. 27. The nucleic acid molecule of claim 25, wherein said GATA transcription factor gene is GATA transcription factor 3 gene. 28. The nucleic acid molecule of claim 25, wherein said GATA transcription factor gene is GATA transcription factor 4 gene. 29. The nucleic acid molecule of claim 25, wherein said GATA transcription factor gene is GATA transcription factor 6 gene. 30. The nucleic acid molecule of claim 25, wherein said nucleic acid molecule is an enzymatic nucleic acid molecule. 31. The nucleic acid molecule of claim 30, wherein the binding arms of said nucleic acid molecule comprise sequences complementary to any of sequences of Seq ID Nos 1-1696. 32. An antisense nucleic acid molecule capable of specifically inhibiting the expression of a GATA transcription factor gene, wherein said antisense nucleic acid molecule comprises sequences complementary to any of sequences of Seq ID Nos 1-1696. 33. The nucleic acid molecule of claim 30, wherein said nucleic acid molecule is in a hammerhead motif. 34. The nucleic acid molecule of claim 30, wherein said nucleic acid molecule is in a haiφin, hepatitis Delta virus, group I intron, VS nucleic acid or RNase P nucleic acid motif. 35. The nucleic acid molecule of claims 30 or 32, wherein said nucleic acid molecule comprises between 12 and 100 bases complementary to RNA encoding a GATA transcription factor. 36. The nucleic acid molecule of claims 30 or 32, wherein said nucleic acid molecule comprises between 14 and 24 bases complementary to RNA encoding GATA transcription factor. 37. The nucleic acid molecule of claim 33, wherein said nucleic acid molecule comprises any of the sequences of Seq ID Nos 1697-3392. 38. The nucleic acid molecule of claim 30, wherein said nucleic acid molecule is an enzymatic DNA molecule. 39. A nucleic acid molecule capable of specifically inhibiting expression of gene encoding IRF-2. 40. The nucleic acid molecule of any of claims 39 wherein said nucleic acid molecule is an enzymatic nucleic acid molecule. 41. The nucleic acid molecule of any of claims 39 wherein said nucleic acid molecule is an antisense nucleic acid molecule. 42. The nucleic acid molecule of claim 40, wherein the binding arms of said nucleic acid molecule comprise sequences complementary to any of sequences of Seq ID Nos 5967-6388. 43. The nucleic acid molecule of claim 41, wherein the nucleic acid molecule comprises sequences complementary to any of sequences of Seq ID Nos 5967- 6388. 44. The nucleic acid molecule of claim 40, wherein said nucleic acid molecule is in a hammerhead motif. 45. The nucleic acid molecule of claim 40, wherein said nucleic acid molecule is in a haiφin, hepatitis Delta virus, group I intron, VS nucleic acid or RNase P nucleic acid motif. 46. The nucleic acid molecule of claims 40 or 41, wherein said nucleic acid molecule comprises between 12 and 100 bases complementary to RNA encoding IRF-2. 47. The nucleic acid molecule of claims 40 or 41, wherein said nucleic acid molecule comprises between 14 and 24 bases complementary to RNA encoding IRF-2. 48. The nucleic acid molecule of claim 44, wherein said nucleic acid molecule comprises any of sequence of Seq ID Nos 5545-5966. 49. The nucleic acid molecule of claim 40, wherein said nucleic acid molecule is an enzymatic DNA molecule. 50. A nucleic acid molecule capable of specifically inhibiting expression of gene encoding CAATT Displacement Protein (CDP). 51. The nucleic acid molecule of any of claims 50 wherein said nucleic acid molecule is an enzymatic nucleic acid molecule. 52. The nucleic acid molecule of any of claims 50 wherein said nucleic acid molecule is an antisense nucleic acid molecule. 53. The nucleic acid molecule of claim 51, wherein the binding arms of said nucleic acid molecule comprise sequences complementary to any of sequences of Seq ID Nos 6929-7468. 54. The nucleic acid molecule of claim 52, wherein the nucleic acid molecule comprises sequences complementary to any of sequences of Seq ID Nos 6929- 7468. 55. The nucleic acid molecule of claim 51, wherein said nucleic acid molecule is in a hammerhead motif. 56. The nucleic acid molecule of claim 51, wherein said nucleic acid molecule is in a haiφin, hepatitis Delta virus, group I intron, VS nucleic acid or RNase P nucleic acid motif. 57. The nucleic acid molecule of claims 51 or 52, wherein said nucleic acid molecule comprises between 12 and 100 bases complementary to RNA encoding CDP. 58. The nucleic acid molecule of claims 51 or 52, wherein said nucleic acid molecule comprises between 14 and 24 bases complementary to RNA encoding CDP. 59. The nucleic acid molecule of claim 55, wherein said nucleic acid molecule comprises any of sequence of Seq ID Nos 6389-6928. 60. The nucleic acid molecule of claim 51 , wherein said nucleic acid molecule is an enzymatic DNA molecule. 61. A cell including a nucleic acid molecule of any of claims 1, 14, 25, 32, 39, or 50. 62. The cell of claim 61, wherein said cell is a mammalian cell. 63. An expression vector comprising nucleic acid sequence encoding the nucleic acid molecule of any of claims 1, 14, 25, 32, 39, or 50, in a manner which allows expression and/or delivery of that nucleic acid molecule. 64. A method for synthesis of erythropoietin protein, comprising the steps of: (a) contacting a cell with a nucleic acid molecule of any of claims 1, 14, 25 or 32 under conditions suitable for the synthesis of said erythropoietin protein; and (b) purification of the erythropoietin protein from said cell. 65. A method for synthesis of granulocyte colony-stimulating factor (G-CSF) protein, comprising the steps of: (a) contacting a cell with a nucleic acid molecule of claim 39 under conditions suitable for the synthesis of said G-CSF protein; and (b) purification of the G-CSF protein from said cell. 66. A method for synthesis of interferon alpha protein, comprising the steps of: (a) contacting a cell with a nucleic acid molecule of claim 50 under conditions suitable for the synthesis of said interferon alpha protein; and (b) purification of the interferon alpha protein from said cell. 67. A method of increasing the level of erythropoietin protein in a cell, comprising the step of contacting the cell with a nucleic acid molecule of any of claims 1, 14, 25 or 32 under conditions suitable for achieving said increase in the level of said erythropoietin protein. 68. A method of increasing the level of G-CSF protein in a cell, comprising the step of contacting a cell with a nucleic acid molecule of claim 39 under conditions suitable for achieving said increase in the level of said G-CSF protein. 69. A method of increasing the level of interferon alpha protein in a cell, comprising the step of contacting a cell with a nucleic acid molecule of claim 50 under conditions suitable for achieving said increase in the level of said interferon alpha protein.

Inventors: BLATT LAWRENCE, MCSWIGGEN JAMES, PAVCO PAMELA, ZWICK MICHAEL, BLATT, LAWRENCE, MCSWIGGEN, JAMES, PAVCO, PAMELA, ZWICK, MICHAEL
Applicants: RIBOZYME PHARM INC, RIBOZYME PHARMACEUTICALS, INC.
Application year: 2000
Main classification: C12N9
IPC codes: A61K31, A61K38, A61K48, A61P43, C12N5, C12N15, C12P21, C12R1