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  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/32—Chemical structure of the sugar
  • C12N2310/322—2'-R Modification

Definitions

• CDP CCAAT Displacement Protein
• 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 ⁇ /.,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.
• the enzymatic nucleic acid molecule is formed in a hammerhead or hairpin motif, but may also be formed in the motif of a hepatitis ⁇ 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.
• 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).
• G-CSF Granulocyte colony- stimulating factor
• 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).
• 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
• table VII displays target sequences and ribozymes for CDP. Examples of such ribozymes consist essentially of sequences defined in these Tables.
• 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.
• 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.
• genes 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).
• Erythropoietin 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.
• Epo A number of indications may be treated using Epo.
• 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).
• Epo repressors 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.
• the Epo protein could be expressed in elevated quantities in the kidneys or liver which would in turn stimulate the production of more erythrocytes.
• synthesizer using a 0.2 ⁇ mol 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.
• syntheses at the 0.2 ⁇ mol 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.
• 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.
• 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).
• stepwise coupling yields were >98% (Wincott et al, 1995 Nucleic Acids Res. 23, 2677-2684).
• 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.
• 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.
• RNA, DNA or protein e.g., RNA, DNA or protein
• 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.
• 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.
• 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 ⁇ /., 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.
• 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.
• primary tumors are allowed to grow for up to 25 days.
• Therapeutic endpoints in this group are primary tumor volume, metastases and survival.
• the second set of animals Group II
• 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.
• Subgroup A receives active nucleic acid molecules of the present invention
• Subgroup B receives scrambled attenuated nucleic acid control as therapy
• 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).
• IFN-alpha A D 8 M.U., SC, per animal, EOD for 30 days.
• Colorectal Carcinoma (COLO ⁇ -26) in Balb/c Mice (Sanada et al, 1990,
• 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.
• ribozymes with motifs other than hammerhead may also be devised in a similar fashion and are within the scope of the invention.
• ribozymes with unfavorable intramolecular interactions between the binding arms and the catalytic core were eliminated from consideration.
• varying binding arm lengths can be chosen to optimize activity.
• 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%.
• 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.
• 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.
• ribozyme cleavage buffer 50 mM Tris-HCl, pH 7.5 at 37°C, 10 mM MgCh
• Example 5 Increased Expression of Erythropoietin by Inhibition of Repressors of Erythropoietin
• Hep G2 and Hep 3B 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 ⁇ g/ml of cationic lipid.
• the sequences for the ribozymes and the irrelevant controls (IR1 & IR2) are given in table NIII.
• 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 ⁇ g/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 i ⁇ elevant 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• RNAse P RNA M1 RNA
• RNA portion of a ubiquitous ribonucleoprotein enzyme • RNA portion of a ubiquitous ribonucleoprotein enzyme.
• Reaction mechanism possible attack by M 2+ -OH to generate cleavage products with 3'- OH and 5'-phosphate.
• Reaction mechanism attack by 2' -OH 5' to the scissile bond to generate cleavage products with 2',3'-cyclic phosphate and 5'-OH ends.
• Reaction mechanism attack by 2'-OH 5' to the scissile bond to generate cleavage products with 2',3'-cyclic phosphate and 5'-OH ends.
• RNA RNA as the infectious agent.
• the length of stem II may be > 2 base-pairs.
• 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.
• 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.

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