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WO2002011674A2 - Procede et reactif permettant d'inhiber le canal chlorure active par le calcium 1 (clca-1) - Google Patents

Procede et reactif permettant d'inhiber le canal chlorure active par le calcium 1 (clca-1) Download PDF

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WO2002011674A2
WO2002011674A2 PCT/US2001/024970 US0124970W WO0211674A2 WO 2002011674 A2 WO2002011674 A2 WO 2002011674A2 US 0124970 W US0124970 W US 0124970W WO 0211674 A2 WO0211674 A2 WO 0211674A2
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gccguuaggc cgaa
cugaugag gccguuaggc
nucleic acid
acid molecule
rna
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PCT/US2001/024970
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WO2002011674A3 (fr
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James Mcswiggen
James Thompson
Timothy Mckenzie
David Ayers
David E. Szymkowski
Andrew Grupe
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Ribozyme Pharmaceuticals, Incorporated
Syntex (U.S.A.) Llc
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Priority to AU2001283222A priority Critical patent/AU2001283222A1/en
Publication of WO2002011674A2 publication Critical patent/WO2002011674A2/fr
Publication of WO2002011674A3 publication Critical patent/WO2002011674A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • C12N15/1138—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 against receptors or cell surface proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N2310/121—Hammerhead

Definitions

  • the present invention concerns compounds, compositions, and methods for the study, diagnosis, and treatment of conditions and diseases related to the expression of CLCA (Ch Channel Ca 2+ - Activated) genes.
  • CLCA proteins are emerging as a new class of channel proteins that mediate Ca 2+ -activated CI" conductance in a variety of tissues.
  • Members of the CLCA family have been cloned, isolated, and partially characterized from human, bovine, and murine species. These proteins demonstrate a high degree of homology in their size, sequence, and predicted structure yet can vary considerably in tissue distribution.
  • Bovine CLCAl (bCLCAl or CaCC) was the first reported CLCA homolog.
  • the bCLCAl protein which was isolated from and is exclusively detected in trachial epithelial cells, functions as a Ca 2+ -activated CI" channel (Ran > and Benos, 1992, J Biol.
  • Lu-ECAM-1 bovine lung-endothelial cell adhesion molecule- 1
  • Lu-ECAM-1 can mediate the binding of lung-metastatic mouse B16F10 melanoma cells to endothelial. cells (Zhu et al, 1992, J. Clin. Invest, 89, 1718-1724), however, due to sequence similarity to bCLCAl, the role of Lu-ECAM-1 as a chloride channel has been suggested (Elble et al, supra).
  • the mouse homolog, mCLCAl appears to have an expression pattern similar to the cystic fibrosis transmembrane conductance regulator (CFTR), with expression seen in various secretory epithelial cells, ' squamous epithelia, and in some lymphocytes (Gruber et al, 1998, Histochem. Cell Biol, 110, 43-49).
  • the three human CLCA homologs (hCLCAl, hCLCA2, and hCLCA3) thus far cloned, isolated, and partially characterized, all retain sequence homology, similar cDNA length, and are all located on the short arm of chromosome 1 (lp22- p31).
  • Human CLCA proteins show a restricted pattern of expression in differing secretory tissues.
  • Human CLCAl was the first reported calcium activated chloride channel in humans.
  • the 31,902-bp hCLCAl gene is located on chromosome lp22- p31, contains 14 introns, and is preceded by a canonic promoter region that contains an LI transposable element. Expression of hCLCAl is predominant in intestinal basal crypt epithelia and goblet cells.
  • hCLCAl A protein processing model has been proposed for hCLCAl in which the primary translation product (125-kDa) is cleaved to a 90- kDa and a group of 37- to 41 -kDa proteins, the latter apparently representing different glycosylation products of the same polypeptide (Gruber et al, 1998, Genomics, 54, 200-214); Transient expression of hCLCAl cDNA in HEK 293 cells is associated with an increase in whole-cell Ca 2+ -activated CI" conductance that is susceptible to inhibition with anion channel blocking compounds. Cell attached patch recordings of transfected cells in this study revealed single channels with a slope conductance of 13.4 pS (Gruber et al, supra).
  • hCLCA2 homolog is processed in a similar manner as is hCLCAl, resulting in the formation of a heterodimer consisting of a 90-kDa amino terminal and an approximately 35 -kDa carboxy terminal subunit with anchorage to the plasma membrane via four or five transmembrane domains.
  • Expression of hCLCA2 is somewhat less restricted than that of hCLCAl, being expressed from human lung, trachea, and breast tissue (Gruber et al, 1999, Am. J. Physiol, 276, C1261-C1270).
  • Human CLCA2 is expressed in normal breast epithelium but not in breast tumors of different stages of progression, suggesting that hCLCA2 may act as a tumor suppressor in breast cancer (Gruber et al, 1999, Cancer Res., 59, 5488-5491).
  • Human CLCA3 is a truncated, secreted member of the CLCA family which is expressed in numerous tissues including lung, trachea, spleen, thymus, and breast tissue.
  • hCLCA3 mRNA encodes a 37-kDa glycoprotein that corresponds to the N-terminal extracellular domain of its homologs.
  • the 37-kDa glycoprotein is secreted (Gruber and Pauli, 1999, Biochem. Biophys. Acta, 1444, 418-423).
  • the invention features novel 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 modulate the expression of CLCA (CI" Channel
  • the invention features the use of one or more of the nucleic acid-based techniques independently or in combination to inhibit the expression of the genes encoding hCLCAl, hCLCA2, hCLCA3, and hCLCA4.
  • the invention features the use of nucleic acid-based techniques to specifically inhibit the expression of CLCAl (GenBank accession Nos. NM_001285, AF039400, AF039401, AF127036), CLCA2 (GenBank accession No. NM_006536), CLCA3 (GenBank accession No. NM_004921), and CLCA4 (GenBank accession No. NM_012128) genes.
  • the invention features the inhibition of CLCAl gene using the nucleic acid-based techniques of the instant invention.
  • the invention features the use of an enzymatic nucleic acid molecule, preferably in the hammerhead, NCH (hiozyme), G- cleaver, amberzyme, zinzyme and/or DNAzyme motif, to inhibit the expression of CLCA genes.
  • inhibitor it is meant that the activity of CLCAl or level of RNAs or equivalent RNAs encoding one or more protein subunits of CLCAl is reduced below that observed in the-absence of the nucleic acid molecules of the invention.
  • inhibition with enzymatic nucleic acid molecules preferably is below that level observed in the presence of an enzymatically inactive or attenuated molecule that is able to bind to the same site on the target RNA, but is unable to cleave that RNA.
  • inhibition with antisense oligonucleotides is preferably below that level observed in the presence of, for example, an oligonucleotide with scrambled sequence or with mismatches.
  • inhibition of CLCAl 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, or the presence of a control, irrelevant, or non-inhibitory oligonucleotide.
  • enzymatic nucleic acid molecule it is meant a nucleic acid molecule which has complementarity in a substrate binding region to a specified gene target, and also has an enzymatic activity which is active to specifically cleave target RNA. That is, the enzymatic nucleic acid molecule is able to intermolecularly cleave RNA and thereby inactivate a target RNA molecule. These complementary regions allow sufficient hybridization of the enzymatic nucleic acid molecule to the target RNA and thus permit cleavage. One hundred percent 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.
  • enzymatic nucleic acid is used interchangeably with phrases such as ribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, aptazyme or aptamer-binding ribozyme, regulatable ribozyme, 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.
  • enzymatic nucleic acid molecules described in the instant application are not meant to be limiting 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 have 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).
  • nucleic acid molecule as used herein is meant a molecule having nucleotides.
  • the nucleic acid can be single, double, or multiple stranded and may comprise modified or unmodified nucleotides or non-nucleotides or various mixtures and combinations thereof.
  • enzymatic portion or “catalytic domain” is meant that portion/region of the enzymatic nucleic acid molecule essential for cleavage of a nucleic acid substrate (for example, see Figures 1-4).
  • 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. Examples of such arms are shown generally in Figures 1-4. 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 and of sufficient length to stably interact with the target RNA; specifically 12-100 nucleotides; more specifically 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).
  • NCH or "Inozyme” motif is meant, an enzymatic nucleic acid molecule comprising a motif as described in Ludwig et al, USSN No. 09/406,643, filed September 27, 1999, entitled “COMPOSITIONS HAVING RNA CLEANING ACTIVITY", and International PCT publication Nos. WO 98/58058 and WO 98/58057, all incorporated by reference herein in their entirety including the drawings.
  • G-cleaver an enzymatic nucleic acid molecule comprising a motif as described in Eckstein et al, mtemational PCT publication No. WO 99/16871, incorporated by reference herein in its entirety including the drawings.
  • zinzyme motif is meant, a class ⁇ enzymatic nucleic acid molecule comprising a motif as described in Beigelman et al, International PCT publication
  • Zinzymes represent a non-limiting example of an enzymatic nucleic acid molecule that does not require a ribonucleotide (2' -OH) group within its own nucleic
  • Amberzyme motif is meant, a class I enzymatic nucleic acid molecule comprising a motif as described in Beigelman et al, International PCT publication No. WO 99/55857, incorporated by reference herein in its entirety including the drawings.
  • Amberzymes represent a non-limiting example of an enzymatic nucleic acid molecule that does not require a ribonucleotide (2'-OH) group within its own nucleic acid sequence for activity.
  • DNAzyme' is meant, an enzymatic nucleic acid molecule that does not require the presence of a ribonucleotide (2' -OH) group within the DNAzyme molecule for its activity.
  • the enzymatic nucleic acid molecule may have an attached linker(s) or other attached or associated groups, moieties, or chains containing one or more nucleotides with 2'-OH groups.
  • DNAzyme can be synthesized chemically or expressed endogenously in vivo, by means of a single stranded DNA vector or equivalent thereof.
  • sufficient length is meant an oligonucleotide of greater than or equal to 3 nucleotides that is of a length great enough to provide the intended function under the expected condition.
  • sufficient length means that the binding arm sequence is long enough to provide stable binding to a target site under the expected binding conditions. Preferably, the binding arms are not so long as to prevent useful turnover.
  • stably interact is meant, interaction of the oligonucleotides with target nucleic acid (e.g., by forming hydrogen bonds with complementary nucleotides in the target under physiological conditions).
  • RNA to CLCAl is meant to include those naturally occurring RNA molecules having homology (partial or complete) to CLCAl proteins or encoding for proteins with similar function as CLCAl in various organisms, including human, rodent, primate, rabbit, pig, protozoans, fungi, plants, and other microorganisms and parasites.
  • 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.
  • nucleotide sequence of two or more nucleic acid molecules is partially or completely identical.
  • antisense nucleic acid 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 and Woolf et al, US patent No. 5,849,902).
  • antisense molecules will be complementary to a target sequence along a single contiguous sequence of the antisense molecule.
  • 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.
  • 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.
  • antisense DNA can be used to target RNA by means of DNA-RNA interactions, thereby activating RNase H, which digests the target RNA in the duplex.
  • Antisense DNA can be synthesized chemically or expressed via the use of a single stranded DNA expression vector or equivalent thereof.
  • 2-5A antisense chimera an antisense oligonucleotide containing a 5'-phosphorylated 2'-5'-linked adenylate residue. 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.
  • 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).
  • RNA By “gene” it is meant a nucleic acid that encodes an RNA.
  • nucleic acid can form hydrogen bond(s) with another RNA sequence by either traditional Watson-Crick or other non-traditional types.
  • 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, CSHSymp. Quant. Biol. UL pp.123-133; Frier et al, 1986, Proc. Nat. Acad.
  • 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.
  • enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA.
  • the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA.
  • the ribozyme is a highly specific inhibitor of gene expression, with the specificity of inhibition depending not only on the base-pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage. Single mismatches, or base-substitutions, near the site of cleavage can completely eliminate catalytic activity of a ribozyme.
  • the enzymatic nucleic acid molecule that cleave the specified sites in CLCAl - specific RNAs represent a novel therapeutic approach to treat Chronic Obstructive
  • COPDs Pulmonary Diseases
  • chronic bronchitis chronic bronchitis
  • asthma chronic bronchitis
  • cystic fibrosis chronic fibrosis
  • obstructive bowel syndrome and other indications that may respond to the level of
  • the enzymatic nucleic acid molecule is formed in a hammerhead or hairpin motif, but may also be formed in the motif of a hepatitis delta vims, group I intron, group ⁇ intron or RNase P RNA (in association with an RNA guide sequence), Neurospora VS RNA, DNAzymes, NCH cleaving motifs, or G-cleavers.
  • hammerhead motifs are described by Dreyfus, supra, Rossi et al, 1992, AIDS Research and Human Retroviruses 8, 183;
  • hairpin motifs are described by Hampel et ah, EP0360257, Hampel and Tritz, 1989 Biochemistry 28, 4929, Feldstein et al, 1989, Gene 82, 53, Haseloff and Gerlach, 1989, Gene, 82, 43, Hampel et al, 1990 Nucleic Acids Res. 18, 299; Chowrira & McSwiggen, US. Patent No. 5,631,359.
  • the hepatitis delta vims motif is described by Perrotta and Been, 1992 Biochemistry 31, 16.
  • the RNase P motif is described 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.
  • WO 98/58058 and G-cleavers are described in Kore et al., 1998, Nucleic Acids Research 26, 4116-4120 and Eckstein et al, International PCT Publication No. WO 99/16871. Additional motifs such as the Aptazyme (Breaker et al, WO 98/43993), Amberzyme (Class I motif; Figure 3; Beigelman et al, International PCT publication No. WO 99/55857) and Zinzyme (Beigelman et al, International PCT publication No. WO 99/55857), all these references are incorporated by reference herein in their totalities, including drawings and can also be used in the present invention.
  • a nucleic acid molecule e.g., an antisense molecule, a triplex DNA, or a ribozyme
  • a nucleic acid molecule 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 or. antisense).
  • 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.
  • the upper limit of the length range can be, for example, 30, 40, 50, 60, 70, or 80 nucleotides.
  • the length range for particular embodiments has lower limit as specified, with an upper limit as specified which is greater than the lower limit.
  • the length range can be 35-50 nucleotides in length. All such ranges are expressly included.
  • a nucleic acid molecule can have a length which is any of the lengths specified above, for example, 21 nucleotides in length.
  • the invention provides a method for producing a class of nucleic acid-based gene inhibiting agents which exhibit a high degree of specificity for the RNA of a desired target.
  • the enzymatic nucleic acid molecule is preferably targeted to a highly conserved sequence region of target RNAs encoding CLCA proteins (for example, CLCAl, CLCA2, CLCA3 and/or CLCA4) such that specific treatment of a disease or condition can be provided with either one or several nucleic acid molecules of the invention.
  • Such nucleic acid molecules can be delivered exogenously to specific tissue or cellular targets as required.
  • the nucleic acid molecules e.g., ribozymes and antisense
  • the invention features the use of nucleic acid-based inhibitors of the invention to specifically target genes that share homology with the CLCAl gene.
  • cell is used in its usual biological sense, and does not refer to an entire multicellular organism, e.g., specifically does not refer to a human.
  • the cell may be present in a non-human multicellular organism, e.g., birds, plants and mammals such as cows, sheep, apes, monkeys, swine, dogs, and cats.
  • CLCA proteins is meant, a protein or a mutant protein derivative thereof, comprising a calcium activated chloride channel protein.
  • highly conserved sequence region a nucleotide sequence of one or more regions in a target gene does not vary significantly from one generation to the other or from one biological system to the other.
  • the nucleic acid-based inhibitors of CLCAl expression are useful for the prevention and/or treatment of diseases and conditions including Chronic Obstructive Pulmonary Disease (COPD), chronic bronchitis, asthma, cystic fibrosis, obstructive bowel syndrome, and any other diseases or conditions that are related to or will respond to the levels of CLCAl in a cell or tissue, alone or in combination with other therapies.
  • COPD Chronic Obstructive Pulmonary Disease
  • COPD Chronic Obstructive Pulmonary Disease
  • chronic bronchitis asthma
  • cystic fibrosis obstructive bowel syndrome
  • any other diseases or conditions that are related to or will respond to the levels of CLCAl in a cell or tissue, alone or in combination with other therapies.
  • the nucleic acid-based inhibitors of the invention are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells or tissues.
  • 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.
  • the enzymatic nucleic acid inhibitors comprise sequences, which are complementary to the substrate sequences in Tables III to IX. Examples of such enzymatic nucleic acid molecules also are shown in Tables III to IX. Examples of such enzymatic nucleic acid molecules consist essentially of sequences defined in these Tables.
  • the invention features antisense nucleic acid molecules and 2-5A chimera including sequences complementary to the substrate sequences shown in Tables III to IX.
  • nucleic acid molecules can include sequences as shown for the binding arms of the enzymatic nucleic acid molecules in Tables III to VIII and sequences shown as GeneBlocTM sequences in Table IX.
  • 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.
  • antisense molecules will be complementary to a target sequence along a single contiguous sequence of the antisense molecule.
  • 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.
  • the antisense molecule may be complementary to two (or even more) non- contiguous substrate sequences or two (or even more) non-contiguous sequence portions of an antisense molecule may be complementary to a target sequence or both.
  • the active nucleic acid molecule of the invention for example, an enzymatic nucleic acid molecule, 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.
  • a core region can, for example, include one or more loop, stem-loop structure, or linker which does not prevent enzymatic activity.
  • the underlined regions in the sequences in Tables III, IV and VIII can be such a loop, stem-loop, nucleotide linker, and/or non- nucleotide linker and can be represented generally as sequence "X".
  • a core sequence for a hammerhead enzymatic nucleic acid can comprise a conserved sequence, such as 5'-CUGAUGAG-3' and 5'-CGAA-3' connected by "X", where X is 5'-GCCGUUAGGC-3' (SEQ ID NO 5450), or any other Stem ⁇ region known in the art.
  • ribozymes or antisense molecules that interact with target RNA molecules and inhibit CLCAl (specifically CLCAl gene) activity are expressed from transcription units inserted into DNA or RNA vectors.
  • the recombinant vectors are preferably DNA plasmids or viral vectors.
  • Ribozyme or antisense expressing viral vectors could be constracted based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus.
  • the recombinant vectors capable of expressing the ribozymes or antisense are delivered as described above, and persist in target cells.
  • viral vectors may be used that provide for transient expression of ribozymes or antisense. Such vectors can be repeatedly administered as necessary. Once expressed, the ribozymes or antisense bind to the target RNA and inhibit its function or expression.
  • ribozyme or antisense expressing vectors can 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.
  • Antisense DNA can be expressed endogenously via the use of a single stranded DNA intracellular expression vector.
  • RNA is meant a molecule comprising at least one ribonucleotide residue.
  • ribonucleotide is meant a nucleotide with a hydroxyl group at the 2' position of a ⁇ -D-ribo-furanose moiety.
  • vectors any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.
  • 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 the nucleic acid molecules of the invention can be administered.
  • a patient is a mammal or mammalian cells. More preferably, a patient is a human or human cells.
  • nucleic acid molecules of the instant invention can be used to treat diseases or conditions discussed above.
  • the patient may be treated, or other appropriate cells may be treated, as is evident to those skilled in the art, individually or in combination with one or. more drugs under conditions suitable for the treatment.
  • the described molecules can be used in combination with other known treatments to treat conditions or diseases discussed above.
  • the described molecules could be used in combination with one or more known therapeutic agents to treat Chronic Obstructive Pulmonary Diseases (COPDs), chronic bronchitis, asthma, cystic fibrosis, obstructive bowel syndrome, and/or other disease states or conditions which respond to the modulation of CLCAl expression.
  • COPDs Chronic Obstructive Pulmonary Diseases
  • chronic bronchitis chronic bronchitis
  • asthma cystic fibrosis
  • obstructive bowel syndrome obstructive bowel syndrome
  • the invention features nucleic acid-based inhibitors (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 (e.g., CLCAl) capable of progression and/or maintenance of Chronic Obstructive Pulmonary Diseases (COPDs), chronic bronchitis, asthma, cystic fibrosis, obstructive bowel syndrome, and/or other disease states or conditions which respond to the modulation of CLCAl expression.
  • genes e.g., CLCAl
  • COPDs Chronic Obstructive Pulmonary Diseases
  • COPDs Chronic Obstructive Pulmonary Diseases
  • Figure 1 shows examples of chemically stabilized ribozyme motifs.
  • HH Rz represents hammerhead ribozyme motif (Usman et al, 1996, Curr. Op. Struct. Bio., 1, 527);
  • NCH Rz represents the NCH ribozyme motif (Ludwig & Sproat, Intemational PCT Publication No. WO 98/58058);
  • G-CIeaver represents G-cleaver ribozyme motif (Kore et al, 1998, Nucleic Acids Research 26, 4116-4120).
  • N or n represent independently a nucleotide which may be same or different and have complementarity to each other; ri, represents ribo-Inosine nucleotide; arrow indicates the site of cleavage within the target.
  • Position 4 of the HH Rz and the NCH Rz is shown as having 2'-C-allyl modification, but those skilled in the art will recognize that this position can be modified with other modifications well known in the art, so long as such modifications do not significantly inhibit the activity of the ribozyme.
  • Figure 2 shows an example of the Amberzyme ribozyme motif that is chemically stabilized (see, for example, Beigelman et al, Intemational PCT publication No. WO 99/55857, incorporated by reference herein; also referred to as
  • the Amberzyme motif is a class of enzymatic nucleic molecules that do not require the presence of a ribonucleotide (2'-OH) group for its activity.
  • Figure 3 shows an example of the Zinzyme A ribozyme motif that is chemically stabilized (Beigelman et al, International PCT publication No. WO
  • the Zinzyme motif is a class of enzymatic nucleic molecules that do not require the presence of a ribonucleotide (2' -OH) group for its activity.
  • Figure 4 shows an example of a DNAzyme motif described by Santoro et al., 1991, PNAS, 94, 4262.
  • Figures 5A and 5B are diagrammatic schemes representative of the process used for Target Discovery in the instant invention.
  • the process for Target Discovery is described in Jarvis et al, Intemational PCT publication No. WO 98/50530, incorporated by reference herein in its entirety including the Figures.
  • Antisense molecules may be modified or unmodified RNA, DNA, or mixed polymer oligonucleotides which 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 can also alter protein synthesis by interfering with RNA processing or transport from the nucleus into the cytoplasm (Mukhopadhyay & Roth, 1996, Crit. Rev. in Oncogenesis 7, 151-190).
  • binding of single stranded DNA to RNA may result in nuclease degradation of the heteroduplex (Wu-Pong, supra; Crooke, supra).
  • the only backbone modified DNA chemistry which will act as substrates for RNase H are phosphorothioates, phosphorodithioates, and borontrifluoridates.
  • 2'-arabino and 2'-fluoro arabino- containing oligos can also activate RNase H activity.
  • antisense molecules have been described that utilize novel configurations of chemically modified nucleotides, secondary stracture, and/or RNase H substrate domains (Woolf et al, International PCT Publication No. WO 98/13526; Thompson et al, Intemational PCT Publication No. WO 99/54459; 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.
  • antisense deoxyoligoribonucleotides can be used to target RNA by means of DNA-RNA interactions, thereby activating RNase H, which digests the target RNA in the duplex.
  • Antisense DNA can be expressed endogenously in vivo via the use of a single stranded DNA intracellular expression vector or equivalents and variations thereof.
  • TFO Triplex Forming Oligonucleotides
  • 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). The 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 irreversible (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 (2-5 A).
  • 2-5 A 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 2-5 A 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 presently known.
  • several in vitro selection (evolution) strategies 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 al., 1993, Science 261:1411-1418; Szostak, 1993, IWS 17, 89-93; Kumar et al, 1995, FASEB J., 9, 1183; Breaker, 1996, Curr.
  • Nucleic acid molecules of this invention will block to some extent CLCAl protein expression and can be used to treat disease or diagnose disease associated with the levels of CLCAl.
  • 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.
  • a single ribozyme molecule is able to cleave many molecules of target RNA.
  • the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base-pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage. Single mismatches, or base-substitutions, near the site of cleavage can be chosen to completely eliminate catalytic activity of a ribozyme.
  • Nucleic acid molecules having an endonuclease enzymatic activity are able to repeatedly cleave other separate RNA molecules in a nucleotide base sequence- specific manner. Such enzymatic nucleic acid molecules can be targeted to virtually any RNA transcript, and achieve efficient cleavage 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.
  • Ribozymes can be designed to cleave specific RNA targets within the background of cellular RNA. Such a cleavage event renders the RNA nonfunctional and abrogates protein expression from that RNA. In this manner, synthesis of a protein associated with a disease state can be selectively inhibited (Warashina et al, 1999, Chemistry and Biology, 6, 237-250).
  • the nucleic acid molecules of the instant invention are also referred to as GeneBloc reagents, which are essentially nucleic acid molecules (e.g.; ribozymes, antisense) capable of down-regulating gene expression.
  • GeneBlocs are modified oligonucleotides including ribozymes and modified antisense oligonucleotides that bind— to and target specific mRNA molecules. Because GeneBlocs can be designed to target any specific mRNA, their potential applications are quite broad. Traditional antisense approaches have often relied heavily on the use of phosphorothioate modifications to enhance stability in biological samples, leading to a myriad of specificity problems stemming from nonspecific protein binding and general cytotoxicity (Stein, 1995, Nature Medicine, 1, 1119).
  • GeneBlocs contain a number of modifications that confer nuclease resistance while making minimal use of phosphorothioate linkages, which reduces toxicity, increases binding affinity and minimizes non-specific effects compared with traditional antisense oligonucleotides. Similar reagents have recently been utilized successfully in various cell culture systems (Vassar, et al, 1999, Science, 286, 735) and in vivo (Jarvis et al., manuscript in preparation). In addition, novel cationic lipids can be utilized to enhance cellular uptake in the presence of serum.
  • Targets for useful ribozymes and antisense nucleic acids can be determined as disclosed in Draper et al, WO 93/23569; Sullivan et al, WO 93/23057; Thompson et al, WO 94/02595; Draper et al, WO 95/04818; McSwiggen et al, US Patent No. 5,525,468. All of these publications are hereby incorporated by reference herein in their totality. Other examples include the following PCT applications, which concern inactivation of expression of disease-related genes: WO 95/23225, WO 95/13380, WO 94/02595, all of which are incorporated by reference herein.
  • Ribozymes and antisense to such targets are designed as described in those applications and synthesized to be tested in vitro and in vivo, as also described.
  • the sequences of human CLCAl RNAs were screened for optimal enzymatic nucleic acid and antisense target sites using a computer-folding algorithm. Antisense, hammerhead, DNAzyme, NCH, amberzyme, zinzyme, or G-Cleaver ribozyme binding/cleavage sites were identified.
  • Antisense, hammerhead, DNAzyme, NCH, amberzyme, zinzyme or G-Cleaver ribozyme binding/cleavage sites were identified.
  • the nucleic acid molecules are individually analyzed by computer folding (Jaeger et al, 1989 Proc. Natl Acad. Sci. USA, 86, 7706) to assess whether the sequences fold into the appropriate secondary stracture. Those nucleic acid molecules with unfavorable intramolecular interactions such as between the binding arms and the catalytic core are eliminated from consideration. Varying binding arm lengths can be chosen to optimize activity.
  • Antisense, hammerhead, DNAzyme, NCH, amberzyme, zinzyme or G-Cleaver ribozyme binding/cleavage sites were identified and were designed to anneal to various sites in the RNA target.
  • the binding arms are complementary to the target site sequences described above.
  • the nucleic acid molecules were chemically synthesized. The method of synthesis used follows the procedure for normal DNA/RNA synthesis as described below and in Usman et al, 1987 J. Am. Chem. Soc, 109, 7845; Scaringe et al, 1990 Nucleic Acids Res., 18, 5433; Wincott et al, 1995 Nucleic Acids Res. 23, 2677-2684; and Carafhers et al., 1992, Methods in Enzymology 211,3-19.
  • nucleic acids greater than 100 nucleotides in length is difficult using automated methods, and the therapeutic cost of such molecules is prohibitive.
  • 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 NCH 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 are chemically synthesized, and others can similarly be synthesized.
  • Oligonucleotides are synthesized using protocols known in the art as described in Carathers et al, 1992, Methods in Enzymology 211, 3-19, Thompson et al, Intemational PCT Publication No. WO 99/54459, Wincott et al, 1995, Nucleic Acids Res. 23, 2677-2684, Wincott et al, 1997, Methods Mol. Bio., 74, 59, Brennan et al, 1998, Biotechnol Bioeng., 61, 33-45, and Brennan, US patent No. 6,001,311. All of these references are incorporated herein by reference.
  • oligonucleotides makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end.
  • small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 ⁇ mol scale protocol with a 2.5 min coupling step for 2'-O-methylated nucleotides and a 45 sec coupling step for 2'- deoxy 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 performed on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, CA) with minimal modification to the cycle.
  • synthesizer include; detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); and oxidation solution is 16.9 mM 12, 49 mM pyridine, 9% water in THF (PERSEPTIVETM). Burdick & Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc.
  • Beaucage reagent (3H-l,2-Benzodithiol-3-one 1,1 -dioxide, 0.05 M in acetonitrile) is used.
  • Deprotection of the antisense oligonucleotides is performed as follows: the polymer-bound trityl-on oligoribonucleotide is transferred 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 is removed from the polymer support. The support is washed three times with 1.0 mL of EtOH:MeC ⁇ :H2O/3:l:l, vortexed and the supernatant is then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, are dried to a white powder.
  • 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; Wincott et al, 1995, Nucleic Acids Res. 23, 2677-2684 and Wincott et al, 1997, Methods Mol. Bio., 74, 59, and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end.
  • common nucleic acid protecting and coupling groups such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end.
  • small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 ⁇ mol scale protocol with a 7.5 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.
  • Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, are typically 97.5- 99%.
  • synthesizer include; detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); oxidation solution is 16.9 mM ⁇ 2, 49 mM pyridine, 9% water in THF (PERSEPTIVETM). Burdick & Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American Intemational Chemical, Inc.
  • Beaucage reagent (3H-l,2-Benzodifhiol-3-one l,l-dioxide0.05 M in acetonitrile) is used.
  • Deprotection of the R ⁇ A is performed using either a two-pot or one-pot protocol.
  • the polymer-bound trityl-on oligoribonucleotide is transferred 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 is removed from the polymer support. The support is washed three times with 1.0 mL of EtOH:MeC ⁇ :H2O/3:l:l, vortexed and the supernatant is then added to the first supernatant.
  • the combined supernatants, containing the oligoribonucleotide, are dried to a white powder.
  • the base deprotected oligoribonucleotide is resuspended in anhydrous TEA HF/NMP solution (300 ⁇ L of a solution of 1.5 mL N- methylpyrrolidinone, 750 ⁇ L TEA and 1 mL TEA-3HF to provide a 1.4 M HF concentration) and heated to 65 °C. After 1.5 h, the oligomer is quenched with 1.5 M N ⁇ 4HCO3.
  • the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 33% ethanolic methylamine/DMSO: 1/1 (0.8 mL) at 65 °C for 15 min.
  • the vial is brought to r.t. TEA « 3HF (0.1 mL) is added and the vial is heated at 65 °C for 15 min.
  • the sample is cooled at -20 °C and then quenched with 1.5 M NH4HCO3.
  • the quenched NH4HCO3 solution is 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 is detritylated with 0.5% TFA for 13 min. The cartridge is then washed again with water, salt exchanged with 1 M NaCl and washed with water again. The oligonucleotide is then eluted with 30% acetonitrile.
  • Inactive hammerhead ribozymes or binding attenuated control (BAC) oligonucleotides are synthesized by substituting a U for G5 and a U for A14
  • nucleotide substitutions can be introduced in other enzymatic nucleic acid molecules to inactivate the molecule and such molecules can serve as a negative control.
  • the average stepwise coupling yields are typically >98% (Wincott et al, 1995
  • nucleic acid molecules of the present invention can be synthesized separately and joined together post- synthetically, for example by ligation (Moore et al, 1992, Science 256, 9923; Draper et al, Intemational PCT publication No. WO 93/23569; Shabarova et al, 1991, Nucleic Acids Research 19, 4247; Bellon et al, 1997, Nucleosides & Nucleotides, 16, 951; Bellon et al, 1997, Bioconjugate Chem. 8, 204).
  • nucleic acid molecules of the present invention are modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2'- amino, 2'-C-allyl, 2'-flouro, 2'-O-mefhyl, 2'-H (for a review see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al, 1994, Nucleic Acids Symp. Ser. 31, 163).
  • Ribozymes are purified by gel electrophoresis using general methods or are purified by high pressure liquid chromatography (HPLC; See Wincott et al, supra, the totality of which is hereby inco ⁇ orated herein by reference) and are resuspended in water.
  • the ribozyme and antisense constract sequences listed in Tables III to IX may be formed of ribonucleotides or other nucleotides or non- nucleotides. Such ribozymes with enzymatic activity are equivalent to the ribozymes described specifically in the Tables. Optimizing Activity of the nucleic acid molecule of the invention.
  • oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-flouro, -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).
  • nuclease resistant groups for example, 2'-amino, 2'-C-allyl, 2'-flouro, -O- methyl, 2'-H, nucleotide base modifications
  • Nucleic acid molecules having chemical modifications which maintain or enhance 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.
  • Therapeutic nucleic acid molecules 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.
  • nucleic acid molecules must be resistant to nucleases in order to function as effective intracellular therapeutic agents. Improvements in the chemical synthesis of RNA and DNA (Wincott et al, 1995 Nucleic Acids Res.
  • nucleic acid-based molecules of the invention will lead to better treatment of the disease progression by affording the possibility of combination therapies (e.g., multiple antisense or enzymatic nucleic acid molecules targeted to different genes, nucleic acid molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations of molecules (including different motifs) and/or other chemical or biological molecules).
  • combination therapies e.g., multiple antisense or enzymatic nucleic acid molecules targeted to different genes, nucleic acid molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations of molecules (including different 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.
  • Therapeutic nucleic acid molecules e.g., enzymatic nucleic acid molecules and antisense nucleic acid molecules
  • 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.
  • nucleic acid molecules must be resistant to nucleases in order to function as effective intracellular therapeutic agents. Improvements in the chemical synthesis of nucleic acid molecules described in the instant invention and in the art have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability as described above.
  • 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.
  • the product of these properties is increased or not significantly (less than 10- fold) decreased in vivo compared to an all RNA ribozyme or all DNA enzyme.
  • 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.
  • the activity may not be significantly lowered.
  • 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 of an all RNA ribozyme.
  • nucleic acid molecules comprise a 5' and/or a 3'- cap structure.
  • cap stracture is meant chemical modifications, which have been inco ⁇ orated at either terminus of the oligonucleotide (see, for example, Wincott et al, WO 97/26270, inco ⁇ orated by reference herein). These terminal modifications protect the nucleic acid molecule from exonuclease degradation, and may help in delivery and/or localization within a cell.
  • the cap may be present at the 5 '-terminus (5 '-cap) or at the 3 '-terminus (3 '-cap) or may be present on both termini.
  • the 5 '-cap is selected from the group comprising inverted abasic residue (moiety), 4',5'-methylene nucleotide; l-(beta-D-erythrofuranosyl) nucleotide, 4'-thio nucleotide, , carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L- nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; t/zreo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; acyclic 3,4- dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3'-3 '-inverted nucleotide moiety; 3 '-3 '-inverted abasic moiety; 3'-2'
  • the 3 '-cap is selected from a group comprising, 4',5'-mefhylene nucleotide; l-(beta-D-erythrofuranosyl) nucleotide; 4'- thio nucleotide, carbocyclic nucleotide; 5'-amino-alkyl phosphate; l,3-diamino-2- propyl phosphate, 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2- aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide;
  • non-nucleotide any group or compound which can be inco ⁇ orated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity.
  • the group or compound is abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine.
  • alkyl refers to a saturated aliphatic hydrocarbon, including straight-chain, branched-chain, and cyclic alkyl groups.
  • the alkyl group has 1 to 12 carbons. More preferably it is a lower alkyl of from 1 to 7 carbons, more preferably 1 to 4 carbons.
  • the term also includes alkenyl groups which are unsaturated hydrocarbon groups containing at least one carbon-carbon double bond, including straight-chain, branched-chain, and cyclic groups.
  • the alkenyl group has 1 to 12 carbons. More preferably it is a lower alkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons.
  • alkyl also includes alkynyl groups which have an unsaturated hydrocarbon group containing at least one carbon-carbon triple bond, including straight-chain, branched-chain, and cyclic groups.
  • the alkynyl group has 1 to 12 carbons. More preferably it is a lower alkynyl of from 1 to 7 carbons, more preferably 1 to 4 carbons.
  • alkyl groups may also include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups.
  • An "aryl” group refers to an aromatic group which has at least one ring having a conjugated ⁇ electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which may be optionally substituted.
  • the preferred substituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups.
  • alkylaryl refers to an alkyl group (as described above) covalently joined to an aryl group (as described above).
  • Carbocyclic aryl groups are groups wherein the ring atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted.
  • Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms.
  • Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted.
  • An "amide” refers to an -C(O)-NH-R,. where R is either alkyl, aryl, alkylaryl or hydrogen.
  • An “ester” refers to an -C(O)-OR', where R is either alkyl, aryl, alkylaryl or hydrogen.
  • nucleotide as used herein is as recognized in the art to include natural bases (standard), and modified bases well known in the art. Such bases are generally located at the 1' position of a nucleotide sugar moiety. Nucleotides generally comprise a base, sugar and a phosphate group. The nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and other; see for example, Usman and McSwiggen, supra; Eckstein et al, International PCT Publication No.
  • base modifications that can be introduced into nucleic acid molecules include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3 -methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6- alkylpyrimidines (e.g.
  • modified bases in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at 1' position or their equivalents; such bases may be used at any position, for example, within the catalytic core of an enzymatic nucleic acid molecule and/or in the substrate-binding regions of the nucleic acid molecule.
  • the invention features modified ribozymes with phosphate backbone modifications comprising one or more phosphorothioate, phosphorodithioate, methylphosphonate, mo ⁇ holino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl, substitutions.
  • abasic sugar moieties lacking a base or having other chemical groups in place of a base at the 1' position, (for more details, see Wincott et al, Intemational PCT publication No. WO 97/26270).
  • unmodified nucleoside is meant one of the bases adenine, cytosine, guanine, thymine, uracil joined to the 1' carbon of ⁇ -D-ribo-furanose.
  • modified nucleoside any nucleotide base which contains a modification in the chemical structure of an unmodified nucleotide base, sugar and/or phosphate.
  • amino 2'-NH 2 or 2'-O- NH , which may be modified or unmodified.
  • modified groups are described, for example, in Eckstein et al,
  • 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 2-5A chimera molecules to one or more targets to alleviate symptoms of a disease.
  • nucleic acid molecules Methods for the delivery of nucleic acid molecules are described in Akhtar et al, 1992, Trends Cell Bio., 2, 139; and Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995 which are both inco ⁇ orated 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 inco ⁇ oration into other vehicle ' s " such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres.
  • nucleic acid molecules may be directly delivered ex vivo to cells or tissues with or without the aforementioned vehicles.
  • the nucleic acid/vehicle combination is locally delivered by direct injection or by use of a catheter, infusion pump or stent.
  • nucleic acid delivery and administration are provided in Sullivan et al, supra, Draper et al, PCT WO93/23569, Beigelman et al, PCT WO99/05094, and Klimuk et al, PCT WO99/04819 all of which have been inco ⁇ orated by reference herein.
  • nucleic acid molecules of the instant invention used to treat pulmonary diseases and disorders, may be administered directly to the lungs via pulmonary delivery.
  • the molecules of the instant invention can be used as pharmaceutical agents.
  • Pharmaceutical agents prevent, inhibit the occurrence, 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.
  • 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 other compositions known in the art.
  • the present invention also includes pharmaceutically acceptable formulations of the compounds described.
  • formulations include salts of the above compounds, e.g., acid addition salts, including 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 from reaching 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.
  • systemic administration is meant in vivo systemic abso ⁇ tion or accumulation of drags in the blood stream followed by distribution throughout the entire body.
  • Administration routes that lead to systemic abso ⁇ tion include, without limitations: intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular.
  • Each of these administration routes exposes 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 drag, for example, in certain tissue types, such as the tissues of the reticular endothelial system (RES).
  • RES reticular endothelial system
  • a liposome formulation that 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 cancer cells.
  • compositions or formulation that allows for the effective distribution of the nucleic acid molecules of the instant invention in the physical location most suitable for their desired activity.
  • agents suitable for formulation with the nucleic acid molecules of the instant invention include: P-glycoprotein inhibitors (such as Pluronic P85) which can enhance entry of drags into the CNS (Jolliet-Riant and Tillement, 1999, Fundam. Clin. Pharmacol, 13, 16-26); biodegradable polymers, such as poly (DL-lactide-coglycolide) microspheres for sustained release delivery after intracerebral implantation (Emerich, DF et al, 1999, Cell Transplant, 8, 47-58) Alkermes, Inc.
  • nanoparticles such as those made of polybutylcyanoacrylate, which can deliver drags across the blood brain barrier and can alter neuronal uptake mechanisms (Prog Neuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999).
  • Other non-limiting examples of delivery strategies for the nucleic acid molecules of the instant invention include material described in Boado et al, 1998, J. Pharm. Sci., 87, 1308-1315; Tyler et al, 1999, FEBS Lett, 421, 280-284; Pardridge et al, 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv. Drug Delivery Rev., 15, 73-107; Aldrian-Herrada et al, 1998, Nucleic Acids Res., 26, 4910-4916; and Tyler et al, 1999, PNAS USA., 96, 7053-7058.
  • the invention also features the use of the composition comprising surface- modified liposomes containing poly (ethylene glycol) lipids (PEG-modified, or long- circulating liposomes or stealth liposomes).
  • PEG-modified, or long- circulating liposomes or stealth liposomes These formulations offer a method for increasing the accumulation of drags in target tissues.
  • This class of drag 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 drag (Lasic et al. Chem. Rev. 1995, 95, 2601-2627; Ishiwata et al, Chem. Pharm. Bull 1995, 43, 1005-1011). All inco ⁇ orated by reference herein.
  • 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 al, 1995, Biochim. Biophys. Acta, 1238, 86- 90). All inco ⁇ orated by reference herein.
  • 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, Intemational PCT Publication No. WO 96/10392; all of which are inco ⁇ orated by reference herein).
  • Long-circulating liposomes are also likely to protect drags 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.
  • the invention features the use of methods to deliver the nucleic acid molecules of the instant invention to hematopoietic cells, including monocytes and lymphocytes. These methods are described in detail by Hartmann et al, 1998, J. Phamacol Exp. Ther., 285(2), 920-928; Kronenwett et al, 1998, Blood, 91(3), 852- 862; Filion and Phillips, 1997, Biochim. Biophys. Acta., 1329(2), 345-356; Ma and Wei, 1996, Leuk. Res., 20(11/12), 925-930; and Bongartz et al, 1994, Nucleic Acids Research, 22(22), 4681-8.
  • Such methods include the use of free oligonucleotide, cationic lipid formulations, liposome formulations including pH sensitive liposomes and immunoliposomes, and bioconjugates including oligonucleotides conjugated to fusogenic peptides, for the transfection of hematopoietic cells with oligonucleotides.
  • 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 inco ⁇ orated by reference herein.
  • preservatives, stabilizers, dyes and flavoring agents may be provided. These include sodium benzoate, sorbic acid and esters of j9-hydroxybenzoic acid.
  • antioxidants and suspending agents may be used.
  • a pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, 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, concurrent 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.
  • Oxygen therapy, bronchodilators, corticosteroids, antibacterials, vaccinations, acetylcysteine, mucokinetic agents, and DNase (Pulmozyme) are non-limiting examples of compounds and/or methods that can be combined with or used in conjunction with the nucleic acid molecules (e.g. ribozymes and antisense molecules) of the instant invention.
  • ribozymes and antisense molecules e.g. ribozymes and antisense molecules
  • nucleic acid molecules of the instant invention 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.
  • 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.
  • 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 ⁇ /., 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 these references are hereby inco ⁇ orated in their totalities by reference herein).
  • a ribozyme Draper et al, PCT WO 93/23569, and Sullivan et al, PCT 94/02595; Ohkawa et al, 1992, Nucleic Acids Symp. Ser., 27, 15-6
  • RNA molecules of the present invention are preferably 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 alphaviras.
  • the recombinant vectors capable of expressing the nucleic acid molecules are delivered as described above, and persist in target cells.
  • viral vectors may be used that provide for transient expression of nucleic acid molecules. Such vectors might be repeatedly administered as necessary.
  • nucleic acid molecule binds to the target mRNA.
  • Delivery of nucleic acid molecule expressing vectors could be systemic, such as by intravenous or intra-muscular 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).
  • the invention features an expression vector comprising a nucleic acid sequence encoding at least one of the nucleic acid molecules disclosed in the instant invention.
  • 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.
  • the invention features an expression vector comprising: a) a transcription initiation region (e.g., eukaryotic pol I, II or in initiation region); b) a transcription termination region (e.g., eukaryotic pol I, ⁇ or HI termination region); c) a nucleic acid sequence encoding at least one of the nucleic acid catalyst of the instant invention; and wherein said sequence 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 sequence encoding the nucleic acid catalyst of the invention; and/or an intron (intervening sequences).
  • ORF open reading frame
  • RNA polymerase I eukaryotic RNA polymerase I
  • poly TL RNA polymerase II
  • RNA polymerase HI RNA polymerase HI
  • Transcripts from pol H or pol HI promoters will be expressed at high levels in all cells; the levels of a given pol H 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.
  • nucleic acid molecules such as 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. Nat Acad. Sci. USA, 89, 10802-6; Chen etal, 1992, Nucleic Acids Res., 20, 4581-9; Yu et al, 1993, Proc. Natl. Acad. Sci. USA, 90, 6340-4; L'Huillier et al, 1992, EMBO J., 11, 4411-8; Lisziewicz et al, 1993, Proc. Natl. Acad.
  • 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.
  • ribozyme transcription units can be inco ⁇ orated 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 alphaviras vectors) (for a review, see Couture and Stinchcomb, 1996, supra).
  • plasmid DNA vectors such as adenovirus or adeno-associated virus vectors
  • viral RNA vectors such as retroviral or alphaviras vectors
  • the invention features an expression vector comprising a nucleic acid sequence encoding at least one of the nucleic acid molecules 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 nucleic acid sequence encoding at least one said nucleic acid molecule; and wherein said sequence 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 expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an open reading frame; d) a nucleic acid sequence encoding at least one said nucleic acid molecule, wherein said sequence is operably linked to the 3 '-end of said open reading frame; and wherein said sequence 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.
  • the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) a nucleic acid sequence encoding at least one said nucleic acid molecule; and wherein said sequence 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.
  • the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) an open reading frame; e) a nucleic acid sequence encoding at least one said nucleic acid molecule, wherein said sequence is operably linked to the 3'-end of said open reading frame; and wherein said sequence 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.
  • the reporter system consists of a plasmid construct, termed pMUC5AC- EGFP, bearing a gene coding for Green Fluorescent Protein (GFP).
  • GFP Green Fluorescent Protein
  • the promoter region of the GFP gene is replaced by a portion of the Mucin 5AC promoter sufficient to direct efficient transcription of the GFP gene.
  • the plasmid also contains the neomycin drug resistance gene.
  • NCI-H292 (ATCC CRL-1848), is derived from a human lung mucoepidermoid carcinoma.
  • the cells retain mucoepidermoid characteristics in culture and endogenously express mucin 5AC and mucin 2.
  • the ⁇ MUC5AC-EGFP plasmid was transfected into NCI-H292 using a cationic lipid formulation. Following transfection, the cells were subjected to limiting dilution cloning under selection by 600 ⁇ g/mL Geneticin.
  • a clonal cell line highly responsive to mucin promoter induction designated
  • H292/MUC5AC/EGFP Clone8 (H292 Clone 8) was selected as the reporter line for subsequent studies.
  • the process for Target Discovery is described in Jarvis et al, International PCT publication No. WO 98/50530, inco ⁇ orated by reference herein in its entirety including the Figures.
  • a ribozyme library was constracted with oligonucletides containing ribozymes with two randomized regions comprising six-nucleotide binding "arms" (Stem I and Stem HI of a ribozyme-substrate complex). Oligo sequence 5' and 3' of the ribozyme contains restriction endonuclease cleavage sites for cloning. The 3' trailing sequence forms a stem-loop for priming DNA polymerase extension to form a double stranded molecule.
  • the double-stranded ribozyme library was cloned into the U6+27 transcription unit located in the 5' LTR region of a retroviral vector containing the human nerve growth factor receptor (hNGFr) reporter gene. Positioning the U6+27/ribozyme transcription unit in the 5' LTR results in a duplication of the transcription unit when the vector integrates into the host cell genome. As a result, the ribozyme is transcribed by RNA polymerase HI from U6+27 and by RNA polymerase H activity directed by the 5' LTR. The ribozyme library was packaged into retroviral particles that were used to infect and transduce H292 Clone 8 cells.
  • hNGFr human nerve growth factor receptor
  • FIG. 5A and 5B describe the generalized scheme used in the ribozyme library constraction and target discovery.
  • randomized region is meant a region of completely random sequence and/or partially random sequence.
  • completely random sequence is meant a sequence wherein theoretically there is equal representation of A, T, G and C nucleotides or modified derivatives thereof, at each position in the sequence.
  • partially random sequence is meant a sequence wherein there is an unequal representation of A, T, G and C nucleotides or modified derivatives thereof, at each position in the sequence.
  • a partially random sequence can therefore have one or more positions of complete randomness and one or more positions with defined nucleotides.
  • Sorting of ribozyme library-containing cells was performed to enrich for cells that produce less GFP after treatment with PCM and PMA.
  • Lower GFP production may be due to ribozyme action upon genes involved in the activation of the mucin promoter.
  • ribozymes may directly target the mucin/GFP transcript resulting in reduced GFP expression.
  • Cells were seeded at a density of 1 x 10 per 150 cm style cell culture flasks. After 72 hours the standard cell culture medium was replaced with medium without fetal bovine serum. After 24 hours of seram deprivation the cells were treated with serum-containing medium supplemented with PCM (to 40%) and PMA (to 50 nM) to induced GFP production via the mucin promoter. After 20 to 22 hours, cells were monitored for GFP level on a FACStar Plus cell sorter.
  • Sorting was performed if 90% of ribozyme library cells from an unsorted control sample were induced to produce GFP above background levels. Two cell fractions were collected in each round of sorting.
  • the Ml gate collected cells in luminescence channels 1 to 4.5; those cells with the lowest GFP signal (5% of the induced population).
  • the M2 sort gate collected cells in luminescence channels 4.5 to 20; cells with low GFP signal (10% of the induced population).
  • the Ml and M2 fractions together represented the 15% of the induced population responding least to the GFP induction treatment.
  • the Ml and M2 fractions wee cultured separately and representative portions of each were cryopreserved after each round of sorting.
  • the Ml fraction Prior to the third round of sorting the Ml fraction showed a three-fold enrichment of GFP negative cells. Prior to the fourth round of sorting both the Ml and M2 fractions were significantly enriched in cells unresponsive to the GFP induction treatment.
  • the Ml fraction was selected to generate a database of ribozymes present in the sorted cells.
  • Genomic DNA was obtained from sorted ribozyme library cells by standard methods. Nested polymerase chain reaction (PCR) primers (Sequence ID Nos. 5468 and 5469) that hybridized to the retroviral vector 5' and 3' of the ribozyme were used to recover and amplify the ribozyme sequences from the Clone 8 library cell DNA. The PCR product was ligated into a bacterial cloning vector. Two methods were developed to use the recovered ribozyme library, in plasmid form, to generate a database of ribozyme binding arm sequences. In the first approach the library was cloned into E. coli. DNA was prepared by plasmid isolation from bacterial colonies or by direct colony.
  • PCR polymerase chain reaction
  • PCR and _ribozyme..arm sequence. was determined. Over 450 sequences have been obtained by this method.
  • a second method used the ribozyme library to transfect H292 Clone 8 cells. Clonal lines of stably transfected cells were established and induced with PCM and PMA. Those lines which failed to respond to GFP induction were probed by PCR for single ribozyme integration events. Over 300 sequences were obtained in this manner. The unique ribozyme sequences obtained by both methods were added to a Target Sequence Tag (TST) database.
  • TST Target Sequence Tag
  • Ribozyme binding arm sequences were compared to public and private gene data banks. Gene matches were compiled according to perfect and imperfect matches. Potential gene targets were categorized by the number of different ribozyme sequences matching each gene. Multiple ribozyme matches have been found for 180 genes. Genes with more than one perfect ribozyme match were given close attention. A total of 34 genes have been verified to date to have multiple perfect ribozyme matches. Of those at least 17 have protein products of known function.
  • hCLCAl as a candidate for target validation was based on bioinformatics and on emerging data in murine models of mucous hypersecretion in the trachea and lung.
  • Two ribozymes (Seq. ID Nos. 2332 and 2273) recovered from cells that no longer respond to mucin promoter/GFP induction match perfectly to hCLCAl.
  • a third has a single mismatch.
  • Evidence from two murine models indicates a correlation between mucous hypersecretion in the lung and strong upregulation of gob-5 (GenBank ABO17156), a murine homologue of hCLCAl.
  • GeneBIoc reagents were designed (Table IX) to the hCLCAl cDNA sequence (GenBank AF039400). GeneBIoc reagents are complexed with a cationic lipid formulation prior to administration to H292/MUC5AC/GFP Clone 8 cells. Concentrations of the GeneBIoc reagents administered range from 30 nM to 120 nM at cationic lipid concentrations of 4-6 ⁇ g/mL. Cells are treated with GeneBIoc reagents for 72 to 96 hours.
  • the sequence of human CLCAl is screened for accessible sites using a computer-folding algorithm. Regions of the RNA are identified that do not form secondary folding structures. These regions contain potential ribozyme and/or antisense binding/cleavage sites. The sequences of these binding/cleavage sites are shown in Tables III-IX.
  • Ribozyme target sites are chosen by analyzing sequences of Human CLCAl (GenBank accession numbers: NM_001285 and AF039400) and prioritizing the sites on the basis of folding. Ribozymes are designed that could bind each target and are 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 are eliminated from consideration. As noted below, varying . binding arm lengths can be chosen to optimize activity. Generally, at least 5 bases on each arm are able to bind to, or otherwise interact with, the target RNA.
  • Example 11 Chemical Synthesis and Purification of Ribozymes and Antisense for Efficient Cleavage and/or blocking of CLCAl RNA
  • Ribozymes and antisense constructs are designed to anneal to various sites in the RNA message.
  • the binding arms of the ribozymes are complementary to the target site sequences described above, while the antisense constructs are fully complimentary to the target site sequences described above.
  • the ribozymes and antisense constructs were chemically synthesized. The method of synthesis used followed the procedure for normal RNA synthesis as described above and in Usman et al, (1987 J. Am. Chem.
  • Ribozymes and antisense constructs are purified by gel electrophoresis using general methods or are purified by high pressure liquid chromatography (HPLC; see Wincott et al, supra; the totality of which is hereby inco ⁇ orated herein by reference) and are resuspended in water.
  • HPLC high pressure liquid chromatography
  • the sequences of the chemically synthesized ribozymes and antisense constructs used in this study are shown below in Table III-IX.
  • Particular conditions and disease states that can be associated with CLCAl expression modulation include but are not limited to Chronic Obstructive Pulmonary Disease (COPD), chronic bronchitis, asthma, cystic fibrosis, obstructive bowel syndrome, and any other diseases or conditions that are related to or will respond to the levels of CLCAl in a cell or tissue, alone or in combination with other therapies.
  • COPD Chronic Obstructive Pulmonary Disease
  • chronic bronchitis chronic bronchitis
  • asthma chronic bronchitis
  • cystic fibrosis obstructive bowel syndrome
  • any other diseases or conditions that are related to or will respond to the levels of CLCAl in a cell or tissue alone or in combination with other therapies.
  • 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.
  • Oxygen therapy, bronchodilators, corticosteroids, antibacterials, vaccinations, acetylcysteine, mucokinetic agents, and DNase (Pulmozyme) are non-limiting examples of methods and/or treatments that can be used in combination with nucleic acid molecules of the invention.
  • Those skilled in the art will recognize that other drug compounds and therapies can be similarly and readily combined with the nucleic acid molecules of the instant invention (e.g. ribozymes and antisense molecules) and are, therefore, within the scope of the instant invention.
  • Example 8 The cell culture system described in Example 8 can be used to evaluate nucleic acid molecules of the invention for efficacy in CLCAl and mucin modulation. Animal Models
  • COPD chronic pulmonary disease
  • cystic fibrosis animal models relevant to disease states such as COPD and cystic fibrosis. These models can be used to determine efficacy of the nucleic acid molecules of the instant invention targeting such disease states or conditions.
  • Animal models for chronic pulmonary disease (COPD) are described by Shapiro, 2000, Am. J. Respir. Cell Mol Biol, 22(1), 4-7; Hogg, 1998, Ika Daigaku Zasshi, 56(3), 429-432; and Garssen et al, 1997, Inhalation Toxicol, 9(6), 581-599.
  • cystic fibrosis are described by Kent et al, 1997, J. Clin.
  • the 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 CLCAl RNA in a cell.
  • the close relationship between ribozyme activity and the structure of the target RNA allows the detection of mutations in any region of the molecule which alters the base-pairing and three- dimensional stracture 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.
  • ribozymes of this invention include detection of the presence of mRNAs associated with CLCAl -related condition. Such RNA is detected by determining the presence of a cleavage product after treatment with a ribozyme using standard methodology.
  • 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.
  • each analysis can require two ribozymes, two substrates and one unknown sample, which will be combined into six reactions.
  • the presence of cleavage products will be determined using an RNAse protection assay so that full-length and cleavage fragments of each RNA can be analyzed in one lane of a polyacrylamide gel. It is not absolutely required to quantify the results to gain insight into the expression of mutant RNAs and putative risk of the desired phenotypic changes in target cells.
  • the expression of mRNA whose protein product is implicated in the development of the phenotype i.e., CLCAl
  • a qualitative comparison of RNA levels will be adequate and will decrease the cost of the initial diagnosis. Higher mutant form to wild-type ratios will be correlated with higher risk whether RNA levels are compared qualitatively or quantitatively.
  • 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).
  • 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 enzymatic nucleic acid molecule is ideal for cleavage of RNAs of unknown sequence.
  • Applicant describes the use of nucleic acid molecules to down-regulate gene expression of target genes in bacterial, microbial, fungal, viral, and eukaryotic systems including plant, or mammalian cells.
  • Reaction mechanism attack by the 3' -OH of guanosine to generate cleavage products with 3'-OH and 5'-guanosine.
  • 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" D -galactosidase message by the ligation of new D- galactosidase sequences onto the defective message [ xu ].
  • RNAse P RNA Ml RNA
  • RNA portion of a ubiquitous ribonucleoprotein enzyme • RNA portion of a ubiquitous ribonucleoprotein enzyme.
  • Reaction mechanism possible attack by M -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.
  • 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.
  • 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.
  • 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.
  • Ligation activity (in addition to cleavage activity) makes ribozyme amenable to engineering through in vitro selection [ x xv ]
  • HDV Hepatitis Delta Virus
  • 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.
  • Circular form of HDV is active and shows increased nuclease stability [ xli ]
  • RNA is a catalytic component of a DNA endonuclease involved in intron mobility.
  • Cell Cell (Cambridge, Mass.) (1995), 83(4), 529-38.
  • xxi 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. x ⁇ . Michel, Francois; Ferat, Jean Luc. Structure and activities of group II introns. Annu. Rev.
  • Substrate selection rules for the hairpin ribozyme determined by in vitro selection, mutation, and analysis of mismatched substrates. Genes Dev. (1993), 7(1), 130-8. xx x v 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-
  • Wait time does not include contact time during delivery.
  • AAAUAUUU A CAAGUACG 25 CGUACUUG CUGAUGAG GCCGUUAGGC CGAA AAAUAUUU 2214
  • AAACACAU C AGGUCAGG 42 CCUGACCU CUGAUGAG GCCGUUAGGC CGAA AUGUGUUU 2231
  • AAACCACU U CCGAUAAG 47 CUUAUCGG CUGAUGAG GCCGUUAGGC CGAA AGUGGUTJU 2236
  • AAAAGAUU A CCUGCAGC 251 GCUGCAGG CUGAUGAG GCCGUUAGGC CGAA AAUCUUUU 2440
  • AAAUCAUU C AUCCUUUU 531 AAAAGGAU CUGAUGAG GCCGUUAGGC CGAA AAUGAUUU 2720
  • AAAAUUUU C UAAAAUGU 545 ACAUUUUA CUGAUGAG GCCGUUAGGC CGAA AAAAUUUU 2734
  • NM_001285 Homo sapiens chloride channel, calcium activated, 1 (CLCAl) mRNA, 3311 bp

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Abstract

L'invention concerne des molécules d'acide nucléique, notamment des molécules d'acide nucléique antisens et enzymatiques telles que les ribozymes en tête de marteau, les ADNzymes et les GeneBlocs, qui modulent l'expression des canaux chlorure activés par le calcium (CLCA1, CLCA2, CLCA3, et CLCA4).
PCT/US2001/024970 2000-08-09 2001-08-09 Procede et reactif permettant d'inhiber le canal chlorure active par le calcium 1 (clca-1) WO2002011674A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2001283222A AU2001283222A1 (en) 2000-08-09 2001-08-09 Method and reagent for the inhibition of calcium activated chloride channel-1 (clca-1)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US22438300P 2000-08-09 2000-08-09
US60/224,383 2000-08-09

Publications (2)

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WO2002011674A2 true WO2002011674A2 (fr) 2002-02-14
WO2002011674A3 WO2002011674A3 (fr) 2003-09-25

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PCT/US2001/024970 WO2002011674A2 (fr) 2000-08-09 2001-08-09 Procede et reactif permettant d'inhiber le canal chlorure active par le calcium 1 (clca-1)

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AU (1) AU2001283222A1 (fr)
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1234878A4 (fr) * 1999-11-24 2003-06-11 Takeda Chemical Industries Ltd Utilisation d'un gene associe a une maladie
JP2007505162A (ja) * 2003-05-28 2007-03-08 ワイス 可溶性ｃｌｃａ−１およびｃｌｃａ−１に対するアンタゴニスト
EP3263115A1 (fr) * 2016-06-30 2018-01-03 Technische Hochschule Mittelhessen Invention concernant un agent actif contre le pseudomonas aeruginosa
US12378559B2 (en) 2018-11-02 2025-08-05 University Of Essex Enterprises Limited Enzymatic nucleic acid molecules

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5861288A (en) * 1993-10-18 1999-01-19 Ribozyme Pharmaceuticals, Inc. Catalytic DNA
US6103890A (en) * 1994-05-18 2000-08-15 Ribozyme Pharmaceuticals, Inc. Enzymatic nucleic acids that cleave C-fos
DE69637256T2 (de) * 1996-01-16 2008-06-19 Sirna Therapeutics, Inc., Boulder Synthese von Methoxynukleoside und enzymatische Nukleisäure Moleküle
AU751480B2 (en) * 1998-04-29 2002-08-15 Ribozyme Pharmaceuticals, Inc. Nucleoside triphosphates and their incorporation into ribozymes

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1234878A4 (fr) * 1999-11-24 2003-06-11 Takeda Chemical Industries Ltd Utilisation d'un gene associe a une maladie
JP2007505162A (ja) * 2003-05-28 2007-03-08 ワイス 可溶性ｃｌｃａ−１およびｃｌｃａ−１に対するアンタゴニスト
EP3263115A1 (fr) * 2016-06-30 2018-01-03 Technische Hochschule Mittelhessen Invention concernant un agent actif contre le pseudomonas aeruginosa
US12378559B2 (en) 2018-11-02 2025-08-05 University Of Essex Enterprises Limited Enzymatic nucleic acid molecules

Also Published As

Publication number Publication date
WO2002011674A3 (fr) 2003-09-25
AU2001283222A1 (en) 2002-02-18

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