WO2001057206A2 - Procede et reactif destines a inhiber la checkpoint kinase-1 (chk 1) - Google Patents

Procede et reactif destines a inhiber la checkpoint kinase-1 (chk 1) Download PDF

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WO2001057206A2
WO2001057206A2 PCT/US2001/003504 US0103504W WO0157206A2 WO 2001057206 A2 WO2001057206 A2 WO 2001057206A2 US 0103504 W US0103504 W US 0103504W WO 0157206 A2 WO0157206 A2 WO 0157206A2
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nucleic acid
acid molecule
ucaaggacaucguccggg
ggaggaaacucc
rna
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WO2001057206A3 (fr
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Thale Jarvis
James Mcswiggen
Robert N. Booher
Patricia S. Holman
Ali R. Fattaey
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Ribozyme Pharmaceuticals, Inc.
Onyx Pharmaceuticals
Warner-Lambert
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Publication of WO2001057206A3 publication Critical patent/WO2001057206A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7115—Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/712—Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • 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
    • C12N15/1137—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 enzymes
    • 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
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    • 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
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
    • C12N2310/121—Hammerhead
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
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    • C12N2310/31—Chemical structure of the backbone
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/332—Abasic residue

Definitions

  • the present invention concerns compounds, compositions, and methods for the study, diagnosis, and treatment of conditions and diseases related to the expression of kinases which phosphorylate Cdc25 S216, such as Chkl (checkpoint kinase 1) enzyme.
  • Mammalian cells treated with agents that inhibit DNA replication or cause DNA damage undergo cell cycle arrest due to the presence of multiple checkpoint response mechanisms. Cancer cells frequently lack the p53-induced Gl DNA damage checkpoint response and instead arrest in G2 due to a checkpoint pathway directed towards preventing Cdc2 kinase activation. Inhibition of Cdc2 kinase activity is mediated by Weel-like kinases, which phosphorylate key residues within the ATP-binding pocket of Cdc2 (accession No. X05360).
  • Cdc25 function the phosphatase that removes the Cdc2 inhibitory phosphorylations
  • S216 a mechanism involving the binding of 14-3-3 proteins to a phosphorylated serine residue (S216) in Cdc25.
  • Multiple kinases including Chkl (accession No. AFO16582), Chk2 (Cdsl) (accession No. NM_007194), and C-TAK1 (accession No. AL050393), can phosphorylate Cdc25 S216 (accession No. M34065) in-vitro. These kinases may function in the DNA replication and/or DNA damage checkpoint response in vivo.
  • 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 kinases which phosphorylate Cdc25 S216, such as Chkl (checkpoint kinase 1) enzyme, Chk2 (Cdsl) and C-TAK1.
  • kinases which phosphorylate Cdc25 S216, such as Chkl (checkpoint kinase 1) enzyme, Chk2 (Cdsl) and C-TAK1.
  • 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 Chkl. Specifically, the invention features the use of nucleic acid-based techniques to specifically inhibit the expression of Chkl gene.
  • the invention features the use of an enzymatic nucleic acid molecule, preferably in the hammerhead, NCH, G-cleaver, amberzyme, zinzyme and/or DNAzyme motif, to inhibit the expression of Chkl gene.
  • inhibit it is meant that the activity of Chkl or level of RNAs or equivalent RNAs encoding one or more protein subunits of Chkl is reduced below that observed in the absence of the nucleic acid molecules of the invention.
  • inhibition with enzymatic nucleic acid molecule 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 Chkl 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.
  • 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.
  • 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 limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target nucleic acid regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart a nucleic acid cleaving and/or ligation activity to the molecule (Cech et al., U.S. Patent No. 4,987,071; Cech et al., 1988, 260 JAMA 3030).
  • 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 1-5).
  • substrate binding arm or “substrate binding domain” is meant that portion/region of a enzymatic nucleic acid which is able to interact, for example via complementarity (i.e., able to base-pair with), with a portion of its substrate.
  • complementarity i.e., able to base-pair with
  • such complementarity is 100%, but can be less if desired.
  • as few as 10 bases out of 14 can be base-paired (see for example Werner and Uhlenbeck, 1995, Nucleic Acids Research, 23, 2092-2096; Hammann et al, 1999, Antisense and Nucleic Acid Drug Dev., 9, 25-31). Examples of such arms are shown generally in Figures 1-5.
  • these arms contain sequences within a enzymatic nucleic acid which are intended to bring enzymatic nucleic acid and target RNA together through complementary base-pairing interactions.
  • the enzymatic nucleic acid 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; preferably 12-100 nucleotides; more preferably 14- 24 nucleotides long (see for example Werner and Uhlenbeck, supra; Hamman et al, supra; Ha pel et al, EP0360257; Berzal-Herrance et al, 1993, EMBO J., 12, 2567-73).
  • 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, or 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).
  • Inozyme or "NCH” motif is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described as NCH Rz in Figure 2. Inozymes possess endonuclease activity to cleave RNA substrates having a cleavage triplet NCH/, where N is a nucleotide, C is cytidine and H is adenosine, uridine or cytidine, and / represents the cleavage site. H is used interchangeably with X.
  • Inozymes can also possess endonuclease activity to cleave RNA substrates having a cleavage triplet NCN/, where N is a nucleotide, C is cytidine, and / represents the cleavage site.
  • "I” in Figure 2 represents an Inosine nucleotide, preferably a ribo-Inosine or xylo-Inosine nucleoside.
  • G-cleaver motif is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described as G-cleaver in Figure 2.
  • G-cleavers possess endonuclease activity to cleave RNA substrates having a cleavage triplet NYN/, where N is a nucleotide, Y is uridine or cytidine and / represents the cleavage site.
  • G-cleavers may be chemically modified as is generally shown in Figure 2.
  • amberzyme motif an enzymatic nucleic acid molecule comprising a motif as is generally described in Figure 3.
  • Amberzymes possess endonuclease activity to cleave RNA substrates having a cleavage triplet NG/N, where N is a nucleotide, G is guanosine, and / represents the cleavage site.
  • Amberzymes may be chemically modified to increase nuclease stability through substitutions as are generally shown in Figure 3.
  • differing nucleoside and/or non-nucleoside linkers can be used to substitute the 5'-gaa-3' loops shown in the figure.
  • 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.
  • Zinzyme motif is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described in Figure 4.
  • Zinzymes possess endonuclease activity to cleave RNA substrates having a cleavage triplet including but not limited to YG/Y, where Y is uridine or cytidine, and G is guanosine and / represents the cleavage site.
  • Zinzymes may be chemically modified to increase nuclease stability through substitutions as are generally shown in Figure 4, including substituting 2'-O-methyl guanosine nucleotides for guanosine nucleotides.
  • 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 acid sequence for activity.
  • DNAzyme' is meant, an enzymatic nucleic acid molecule that does not require the presence of a 2'-OH group 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.
  • DNAzymes can be synthesized chemically or expressed endogenously in vivo, by means of a single stranded DNA vector or equivalent thereof. An example of a DNAzyme is shown in Figure 5 and is generally reviewed in Usman et al., International PCT Publication No.
  • 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) that is sufficient to the intended purpose (e.g., cleavage of target RNA by an enzyme).
  • RNA to Chkl is meant to include those naturally occurring RNA molecules having homology (partial or complete) to Chkl proteins or encoding for proteins with similar function as Chkl 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.
  • antisense nucleic acid By “homology” is meant the nucleotide sequence of two or more nucleic acid molecules is partially or completely identical.
  • antisense nucleic acid it is meant a non-enzymatic nucleic acid molecule that binds to target RNA by means of RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al, 1993 Nature 365, 566) interactions and alters the activity of the target RNA (for a review, see Stein and Cheng, 1993 Science 261, 1004 and Woolf et al, US patent No. 5,849,902).
  • antisense molecules are 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.
  • 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.
  • the antisense oligonucleotides can comprise one or more RNAse H activating region, which is capable of activating RNAse H cleavage of a target RNA.
  • Antisense DNA can be synthesized chemically or expressed via the use of a single stranded DNA expression vector or equivalent thereof.
  • RNase H activating region is meant a region (generally greater than or equal to 4-25 nucleotides in length, preferably from 5-11 nucleotides in length) of a nucleic acid molecule capable of binding to a target RNA to form a non-covalent complex that is recognized by cellular RNase H enzyme (see for example Arrow et al, US 5,849,902; Arrow et al, US 5,989,912).
  • the RNase H enzyme binds to the nucleic acid molecule-target RNA complex and cleaves the target RNA sequence.
  • the RNase H activating region comprises, for example, phosphodiester, phosphorothioate (preferably at least four of the nucleotides are phosphorothiote substitutions; more specifically, 4-11 of the nucleotides are phosphorothiote substitutions); phosphorodithioate, 5'-thiophosphate, or methylphosphonate backbone chemistry or a combination thereof.
  • the RNase H activating region can also comprise a variety of sugar chemistries.
  • the RNase H activating region can comprise deoxyribose, arabino, fluoroarabino or a combination thereof, nucleotide sugar chemistry.
  • 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. Natl. Acad. Sci. USA 90, 1300; Silverman et al, 2000, Methods Enzymol, 313, 522-533; Player and Torrence, 1998, Pharmacol. Ther., 78, 55-113).
  • triplex forming oligonucleotides 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; Fox, 2000, Curr. Med. Chem., 7, 17-37; Praseuth et. al, 2000, Biochim. Biophys. Acta, 1489, 181-206).
  • RNA RNA sequences including but not limited to structural genes encoding a polypeptide.
  • “Complementarity” refers to the ability of a nucleic acid to 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., enzymatic nucleic acid cleavage, antisense or triple helix inhibition. Determination of binding free energies for nucleic acid molecules is well known in the art (see, e.g., Turner et al., 1987, CSH Symp. Quant. Biol. LII pp.123-133; Frier et al, 1986, Proc.
  • 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.
  • RNA is meant a molecule comprising at least one ribonucleotide residue.
  • ribonucleotide or “2'-OH” is meant a nucleotide with a hydroxyl group at the 2' position of a ⁇ -D-ribo-furanose moiety.
  • decoy RNA is meant a RNA molecule that mimics the natural binding domain for a ligand. The decoy RNA therefore competes with natural binding target for the binding of a specific ligand.
  • TAR HIV trans-activation response
  • RNA can act as a "decoy” and efficiently binds HIV tat protein, thereby preventing it from binding to TAR sequences encoded in the HIV RNA (Sullenger et al., 1990, Cell, 63, 601-608). This is but a specific example and those in the art will recognize that other embodiments can be readily generated using techniques generally known in the art.
  • 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. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA.
  • 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.
  • 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 Chkl -specific RNAs represent a novel therapeutic approach to treat a variety of pathologic indications, including cancer.
  • 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 virus, group I intron, group II intron or RNase P RNA (in association with an RNA guide sequence), Neurospora VS RNA, DNAzymes, NCH cleaving motifs, or G- cleavers.
  • 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 al, 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; and Chowrira & McSwiggen, US. Patent No. 5,631,359.
  • the hepatitis delta virus 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 Altaian, 1990, Science 249, 783; and Li and Airman, 1996, Nucleic Acids Res. 24, 835.
  • the 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; and 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 include 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 of the instant invention can be between 13 and 100 nucleotides in length.
  • Exemplary enzymatic nucleic acid molecules of the invention are shown in Tables III-XIII.
  • enzymatic nucleic acid molecules of the invention are preferably between 15 and 50 nucleotides in length, more preferably between 25 and 40 nucleotides in length, e.g., 34, 36, or 38 nucleotides in length (for example see Jarvis et al, 1996, J. Biol. Chem., 271, 29107-29112).
  • Exemplary DNAzymes of the invention are preferably between 15 and 40 nucleotides in length, more preferably between 25 and 35 nucleotides in length, e.g., 29, 30, 31, or 32 nucleotides in length (see for example Santoro et al, 1998, Biochemistry, 37, 13330-13342; Chartrand et al, 1995, Nucleic Acids Research, 23, 4092-4096).
  • Exemplary antisense molecules of the invention are preferably between 15 and 75 nucleotides in length, more preferably between 20 and 35 nucleotides in "length, e.g., 25, 26, 27, or 28 nucleotides in length (see for example Woolf et al, 1992, EN4S., 89, 7305-7309; Milner et al, 1997, Nature Biotechnology, 15, 537-541).
  • Exemplary triplex forming oligonucleotide molecules of the invention are preferably between 10 and 40 nucleotides in length, more preferably between 12 and 25 nucleotides in length, e.g., 18, 19, 20, or 21 nucleotides in length (see for example Maher et al, 1990, Biochemistry, 29, 8820-8826; Srrobel and Dervan, 1990, Science, 249, 73-75).
  • Those skilled in the art will recognize that all that is required is for the nucleic acid molecule are of length and conformation sufficient and suitable for the nucleic acid molecule to catalyze a reaction contemplated herein.
  • the length of the nucleic acid molecules of the instant invention are not limiting within the general limits stated.
  • a nucleic acid molecule that down regulates the replication of Chkl comprises between 12 and 100 bases complementary to a RNA molecule of Chkl. Even more preferably, a nucleic acid molecule that down regulates the replication of Chkl comprises between 14 and 24 bases complementary to a RNA molecule of Chkl .
  • 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 kinases which phosphorylate Cdc25 S216, such as Chkl proteins (specifically Chkl gene) 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 Chkl 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 an organism which may be a human but is preferably a non-human multicellular organism, e.g., birds, plants and mammals such as cows, sheep, apes, monkeys, swine, dogs, and cats.
  • the cell may be prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., mammalian or plant cell).
  • Chkl proteins is meant, a protein or a mutant protein derivative thereof, comprising phosphorylation activity, preferably to serine residue (S216), or its equivalent, in Cdc25 phosphatase.
  • 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 Chkl expression are useful for the prevention and/or treatment of diseases and conditions such as cancer, including cancer of the colon, rectum, lung, breast, prostate and any other diseases or conditions that are related to or will respond to the levels of Chkl in a cell or tissue, alone or in combination with other therapies.
  • Chkl inhibition may be used as a therapeutic target for abrogating the G2 DNA damage checkpoint arrest; a situation that may selectively sensitize p53-deficient tumor cells to radiation or chemotherapy treatment.
  • Chkl expression specifically Chkl gene
  • reduction in the level of the respective protein will relieve, to some extent, the symptoms of the disease or condition.
  • 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 VIII. Examples of such enzymatic nucleic acid molecules also are shown in Tables III to VIII. 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 may, 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 and IV 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 a sequence X, where X is 5'-GCCGUUAGGC-3' (SEQ ID NO 3173) or any other stem II region known in the art or a nucleotide and/or non-nucleotide linker.
  • nucleic acid molecules of the instant invention such as Inozyme, G-cleaver, amberzyme, zinzyme, DNAzyme, antisense, 2-5A antisense, triplex forming nucleic acid, and decoy nucleic acids
  • other sequences or non-nucleotide linkers may be present that do not interfere with the function of the nucleic acid molecule.
  • Sequence X may be a linker of > 2 nucleotides in length, preferably 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 26, 30, where the nucleotides may preferably be internally base-paired to form a stem of preferably > 2 base pairs.
  • sequence X may be a non-nucleotide linker.
  • the nucleotide linker X can be a nucleic acid aptamer, such as an ATP aptamer, HIV Rev aptamer (RRE), HIV Tat aptamer (TAR) and others (for a review see Gold et al, 1995, Annu. Rev.
  • nucleic acid aptamer as used herein is meant to indicate a nucleic acid sequence capable of interacting with a ligand.
  • the ligand can be any natural or a synthetic molecule, including but not limited to a resin, metabolites, nucleosides, nucleotides, drugs, toxins, transition state analogs, peptides, lipids, proteins, amino acids, nucleic acid molecules, hormones, carbohydrates, receptors, cells, viruses, bacteria and others.
  • non-nucleotide linker X is as defined herein.
  • non-nucleotide include either abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, or polyhydrocarbon compounds. Specific examples include those described by Seela and Kaiser, Nucleic Acids Res. 1990, 75:6353 and Nucleic Acids Res. 1987, 75:3113; Cload and Schepartz, J. Am. Chem. Soc. 1991, 773:6324; Richardson and Schepartz, J Am. Chem. Soc. 1991, 773:5109; Ma et al., Nucleic Acids Res.
  • non-nucleotide further means any group or compound which can be incorporated 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 can be abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine.
  • the invention features an enzymatic nucleic acid molecule having one or more non-nucleotide moieties, and having enzymatic activity to cleave an RNA or DNA molecule.
  • ribozymes or antisense molecules that cleave target RNA molecules and inhibit Chkl (specifically Chkl 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 constructed 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. Delivery of 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.
  • 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.
  • enhanced enzymatic activity is meant to include activity measured in cells and/or in vivo where the activity is a reflection of both the catalytic activity and the stability of the nucleic acid molecules of the invention.
  • the product of these properties can beincreased in vivo compared to an all RNA enzymatic nucleic acid or all DNA enzyme.
  • the activity or stability of the nucleic acid molecule can bedecreased (i.e., less than tenfold), but the overall activity of the nucleic acid molecule is enhanced, in vivo.
  • the nucleic acid molecules of the instant invention individually, or in combination or in conjunction with other drugs, can be used to treat diseases or conditions discussed above. For example, to treat a disease or condition associated with the levels of Chkl, 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 cancer, including but not limited to cancer of the colon, rectum, lung, breast and prostate.
  • 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., Chkl) capable of progression and/or maintenance of cancer.
  • 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
  • methods for their use to down regulate or inhibit the expression of genes e.g., Chkl
  • the invention provides mammalian cells containing one or more nucleic acid molecules and/or expression vectors of this invention.
  • the one or more nucleic acid molecules may independently be targeted to the same or different sites.
  • Figure 1 shows the secondary structure model for seven different classes of enzymatic nucleic acid molecules. Arrow indicates the site of cleavage. indicate the target sequence. Lines interspersed with dots are meant to indicate tertiary interactions. - is meant to indicate base-paired interaction.
  • Group I Intron: P1-P9.0 represent various stem-loop structures (Cech et al, 1994, Nature Struc. Bio., 1, 273).
  • Group II Intron 5'SS means 5' splice site; 3'SS means 3'-splice site; IBS means intron binding site; EBS means exon binding site (Pyle et al, 1994, Biochemistry, 33, 2716).
  • VS RNA I- VI are meant to indicate six stem-loop structures; shaded regions are meant to indicate tertiary interaction (Collins, International PCT Publication No. WO 96/19577).
  • Hammerhead Ribozyme I-III are meant to indicate three stem-loop structures; stems I-III can be of any length and may be symmetrical or asymmetrical (Usman et al, 1996, Curr. Op. Struct. Bio., 1, 527).
  • Helix 2 and helix 5 may be covalently linked by one or more bases (i.e., r is > 1 base). Helix 1, 4 or 5 may also be extended by 2 or more base pairs (e.g., 4 - 20 base pairs) to stabilize the ribozyme structure, and preferably is a protein binding site.
  • each N and N' independently is any normal or modified base and each dash represents a potential base-pairing interaction. These nucleotides may be modified at the sugar, base or phosphate. Complete base-pairing is not required in the helices, but is preferred.
  • Helix 1 and 4 can be of any size (i.e., o and p is each independently from 0 to any number, e.g., 20) as long as some base-pairing is maintained.
  • Essential bases are shown as specific bases in the structure, but those in the art will recognize that one or more may be modified chemically (abasic, base, sugar and/or phosphate modifications) or replaced with another base without significant effect.
  • Helix 4 can be formed from two separate molecules, i.e., without a connecting loop.
  • the connecting loop when present may be a ribonucleotide with or without modifications to its base, sugar or phosphate, "q" > is 2 bases.
  • the connecting loop can also be replaced with a non-nucleotide linker molecule.
  • H refers to bases A, U, or C.
  • Y refers to pyrimidine bases.
  • Figure 2 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, International PCT Publication No. WO 98/58058);
  • G-Cleaver 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.
  • FIG 3 shows an example of the Amberzyme ribozyme motif that is chemically stabilized (see, for example, Beigelman et al, International PCT publication No. WO 99/55857, incorporated by reference herein; also referred to as Class I Motif).
  • 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.
  • FIG 4 shows an example of the Zinzyme A ribozyme motif that is chemically stabilized (Beigelman et al, International PCT publication No. WO 99/55857, incorporated by reference herein; also referred to as Class A or Class II Motif).
  • 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 5 shows an example of a DNAzyme motif described by Santoro et al, 1997, PNAS, 94, 4262.
  • Figure 6 shows a bar graph of a nucleic acid inhibitor (50 to 200 nM GeneBlocTM screen against Chkl RNA in HeLa cells using 1.25 ⁇ g/ml GSV lipid with 24 hour sustained delivery in a 96-well format. Relative amounts of target RNA were measured normalized to actin using real-time PCR monitoring of amplification compared to mismatch nucleic acid and untreated controls. The sequences of GeneBlocTM reagents used in this experiment are shown in Table IX.
  • Figure 7 shows a bar graph of a lipid optimization study utilizing lead nucleic acid inhibitors (GeneBlocsTM) targeting Chkl RNA in HeLa cells; 96-well plate format, 5000 cells/well, GSV lipid. Six different lipid concentrations are shown in conjunction with two different concentrations of the nucleic acid inhibitors.
  • GeneBlocsTM lead nucleic acid inhibitors
  • Figure 8 shows a bar graph displaying a time-course inhibition study of a lead nucleic acid inhibitor (GeneBlocTM) targeting Chkl RNA compared to a scrambled nucleic acid control, both at 5 and 100 nM concentrations; 96-well plate format, 5000 cells/well, 1.0 ⁇ g/ml GSV lipid.
  • Figure 9 shows a bar graph representing inhibition of Chkl RNA via primary lead (GeneBlocTM) inhibition as described in Figure 6, however utilizing a 6-well plate format with a cell density of 150,000 cells per well.
  • GeneBlocTM lead nucleic acid inhibitor
  • Figure 10 shows a bar graph representing inhibition of Chkl RNA via primary lead (GeneBlocTM) inhibition in conjunction with +/- etoposide and nocodazole treatment; 50 nM GeneBlocTM, 1.25 ⁇ g/ml GSV lipid, HeLa cells, 6-well plate format, 100,000 cells/well.
  • GeneBlocTM primary lead
  • Figure 11 shows a bar graph of a lipid optimization study utilizing a lead nucleic acid inhibitor (GeneBlocTM) targeting Chkl RNA in DLD-1 cells; 96-well plate format, 15,000 cells/well, GSV lipid. Four different lipid concentrations are shown in conjunction with two different concentrations of the nucleic acid inhibitor.
  • GeneBlocTM lead nucleic acid inhibitor
  • Figure 12 shows a bar graph of a lipid optimization study utilizing a lead nucleic acid inhibitor (GeneBlocTM) targeting Chkl RNA in MCF-7 cells; 96-well plate format, 10,000 cells/well, GSV lipid. Four different lipid concentrations are shown in conjunction with two different concentrations of the nucleic acid inhibitor.
  • GeneBlocTM lead nucleic acid inhibitor
  • Figure 13 shows a dose curve of primary and secondary nucleic acid inhibitor (GeneBlocTM) leads targeting Chkl RNA in HeLa cells using 1.25 ⁇ g/ml GSV lipid, 24 hr time- point, 96-well plate format with a density of 5000 cells/well.
  • GeneBlocTM primary and secondary nucleic acid inhibitor
  • Antisense molecules can 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.
  • a number of antisense molecules have been described that utilize novel configurations of chemically modified nucleotides, secondary structure, and/or RNase H substrate domains (Woolf et al, International PCT Publication No. WO 98/13526; Thompson et al, International 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 via the use of a single stranded DNA intracellular expression vector or equivalents and variations thereof.
  • TFO Triplex Forming Oligonucleotides
  • Single stranded DNA can 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 can 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 (2-5A).
  • 2-5A then acts as an allosteric effector for utilizing RNase L which has the ability to cleave single stranded RNA.
  • the ability to form 2-5A structures with double stranded RNA makes this system particularly useful for inhibition of viral replication.
  • (2'-5') oligoadenylate structures can be covalently linked to antisense molecules to form chimeric oligonucleotides capable of RNA cleavage (Torrence, supra). These molecules putatively bind and activate a 2-5A dependent RNase, the oligonucleotide/enzyme complex then binds to a target RNA molecule which can then be cleaved by the RNase enzyme.
  • Enzymatic Nucleic Acid Seven basic varieties of naturally occurring enzymatic RNAs are 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 ⁇ .,1993, Science 261:1411-1418; Szostak, 1993, TIBS 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 Chkl protein expression and can be used to treat disease or diagnose disease associated with the levels of Chkl.
  • 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.
  • tr ⁇ s-cleaving ribozymes can be used as therapeutic agents for human disease (Usman & McSwiggen, 1995 Ann. Rep. Med. Chem. 30, 285-294; Christoffersen and Marr, 1995 J. Med. Chem. 38, 2023-2037). Ribozymes can be designed to cleave specific RNA targets within the background of cellular RNA. Such a cleavage event renders the RNA non-functional and abrogates protein expression from that RNA. In this manner, synthesis of a protein associated with a disease state can be selectively inhibited (Warashina et ⁇ ., 1999, Chemistry and Biology, 6, 237-250).
  • the nucleic acid molecules of the instant invention are also referred to as GeneBlocTM 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 non-specific 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 Chkl 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 structure. Those nucleic acid molecules with unfavorable intramolecular interactions such as between the binding arms and the catalytic core are eliminated from consideration. Varying binding ami 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 Caruthers 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 Caruthers et al, 1992, Methods in Enzymology 211, 3-19, Thompson et al, International PCT Publication No. WO 99/54459, Wincott et al, 1995, Nucleic Acids Res. 23, 2677-2684, Wincott et al, 1997, Methods Mol. Bio., IA, 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.
  • 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); and oxidation solution is 16.9 mM 12, 49 mM pyridine, 9% water in THF
  • 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 12, 49 mM pyridine, 9% water in THF (PERSEPTTVETM). 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. Alternately, for the introduction of phosphorothioate linkages, Beaucage reagent (3H-l,2-Benzodithiol-3-one 1,1- dioxide ⁇ .05 M in acetonitrile) is used.
  • RNA deprotection of the RNA 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.
  • the supernatant is removed from the polymer support.
  • the support is washed three times with 1.0 mL of EtOH:MeCN: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 NH4HCO3.
  • 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 ⁇ HF
  • 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 (B AC) oligonucleotides are synthesized by substituting a U for G5 and a U for A14 (numbering from Hertel, K. J., et al,
  • nucleic Acids Res_. 20, 3252.
  • 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 Acids Res. 23, 2677-2684).
  • the scale of synthesis can be adapted to be larger or smaller than the examples described above including but not limited to 96-well format, all that is important is the ratio of chemicals used in the reaction.
  • 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, International 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-methyl, 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 elecrrophoresis using general methods or are purified by high pressure liquid chromatography (HPLC; See Wincott et al, supra, the totality of which is hereby incorporated herein by reference) and are re-suspended in water.
  • the ribozyme and antisense construct 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.
  • 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, 2'-O-methyl, 2'-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al, 1994, Nucleic Acids Symp. Ser. 31, 163; Burgin et al, 1996, Biochemistry , 35, 14090).
  • nuclease resistant groups for example, 2'- amino, 2'-C-allyl, 2'-flouro, 2'-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 present 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 can also include combinations of different types of nucleic acid molecules.
  • 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. This period of time varies between hours to days depending upon the disease state.
  • these 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.
  • 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 structure is meant chemical modifications, which have been incorporated at either terminus of the oligonucleotide (see, for example, Wincott et al, WO 97/26270, incorporated 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'- inverted nucle
  • the 3 '-cap is selected from a group comprising, 4',5'- methylene 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; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; t/Veo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; 3,4- dihydroxy
  • non-nucleotide any group or compound which can be incorporated 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. Preferably, the alkynyl group has 1 to 12 carbons.
  • alkynyl of from 1 to 7 carbons, more preferably 1 to 4 carbons.
  • the alkynyl group may be substituted or unsubstituted.
  • 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 is meant a heterocyclic nitrogenous base in N-glycosidic linkage with a phosphorylated sugar.
  • Nucleotides are 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. WO 92/07065; Usman et al., International PCT Publication No. WO 93/15187; Uhlman & Peyman, supra all are hereby incorporated by reference herein).
  • modified nucleic acid bases known in the art as summarized by Limbach et al., 1994, Nucleic Acids Res. 22, 2183.
  • nucleic acids Some of the non-limiting examples of chemically modified and other natural nucleic acid bases that can be introduced into nucleic acids 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.
  • 6-methyluridine 6-methyluridine
  • propyne quesosine, 2-thiouridine, 4-thiouridine, wybutosine, wybutoxosine, 4-acetylcytidine, 5- (carboxyhydroxymethyl)uridine, 5 '-carboxymethylaminomethyl-2-thiouridine, 5- carboxymethylaminomethyluridine, beta-D-galactosylqueosine, 1-methyladenosine, 1- methylinosine, 2,2-dimethylguanosine, 3-methylcytidine, 2-methyladenosine, 2- methylguanosine, N6-methyladenosine, 7-methylguanosine, 5-methoxyaminomethyl-2- thiouridine, 5-methylaminomethyluridine, 5-methylcarbonylmethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine, -D-mannosylqueos
  • 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.
  • nucleoside is meant a heterocyclic nitrogenous base in N-glycosidic linkage with a sugar.
  • Nucleosides are 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 nucleoside sugar moiety.
  • Nucleosides generally comprise a base and sugar group.
  • the nucleosides can be unmodified or modified at the sugar, and/or base moiety, (also referred to interchangeably as nucleoside analogs, modified nucleosides, non-natural nucleosides, non-standard nucleosides and other; see for example, Usman and McSwiggen, supra; Eckstein et al., International PCT Publication No.
  • nucleic acids Some of the non-limiting examples of chemically modified and other natural nucleic acid bases that can be introduced into nucleic acids 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.
  • 6-methyluridine 6-methyluridine
  • propyne quesosine, 2-thiouridine, 4-thiouridine, wybutosine, wybutoxosine, 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 5'- carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, -D- galactosylqueosine, 1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine, 3- methylcytidine, 2-methyladenosine, 2-methylguanosine, N6-methyladenosine, 7- methylguanosine, 5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5- methylcarbonylmethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N6- isopentenyladenosine, beta-D-mannosylqueosine
  • modified bases in this aspect is meant nucleoside 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, morpholino, 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, International 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 beta-D-ribo-furanose.
  • modified nucleoside is meant 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 2 , which may be modified or unmodified.
  • modified groups are described, for example, in Eckstein et al, U.S. Patent 5,672,695 and Matulic-Adamic et al, W ⁇ 98/28317, respectively, which are both incorporated by reference herein in their entireties.
  • nucleic acid e.g., antisense and ribozyme
  • modifications to nucleic acid can be made to enhance the utility of these molecules. Such modifications will enhance shelf-life, half-life in vitro, stability, and ease of introduction of such oligonucleotides to the target site, e.g., to enhance penetration of cellular membranes, and confer the ability to recognize and bind to targeted cells.
  • 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 incorporated herein by reference.
  • Sullivan et al, PCT WO 94/02595 further describes the general methods for delivery of enzymatic RNA molecules. These protocols may be utilized for the delivery of virtually any nucleic acid molecule.
  • Nucleic acid molecules may be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres.
  • 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.
  • routes of delivery include, but are not limited to, intravascular, intramuscular, subcutaneous or joint injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. More detailed descriptions of nucleic acid delivery and administration are provided in Sullivan et al. , supra, Draper et al, PCT WO93/23569, Beigelman et al, PCT WO99/05094, and Klimuk et al, PCT WO99/04819 all of which have been incorporated by reference herein.
  • 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. Other factors are known in the art, and include considerations such as toxicity and forms which prevent the composition or formulation from exerting its effect.
  • systemic administration in vivo systemic absorption or accumulation of drugs in the blood stream followed by distribution throughout the entire body.
  • Administration routes that lead to systemic absorption 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 drug, 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 drugs 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.
  • P- glycoprotein inhibitors such as Pluronic P85
  • 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 drugs 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 drugs in target tissues.
  • This class of drug carriers resists opsonization and elimination by the mononuclear phagocytic system (MPS or RES), thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated drug (Lasic et al. Chem. Rev. 1995, 95, 2601- 2627; Ishiwata et al, Chem. Pharm. Bull. 1995, 43, 1005-1011). All incorporated 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 t " ., 1995, Biochim. Biophys. Ada, 1238, 86-90). All incorporated 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, International PCT Publication No. WO 96/10392; all of which are incorporated by reference herein).
  • Long-circulating liposomes are also likely to protect drugs from nuclease degradation to a greater extent compared to cationic liposomes, based on their ability to avoid accumulation in metabolically aggressive MPS tissues such as the liver and spleen.
  • compositions prepared for storage or administration which include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent.
  • Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro edit. 1985) hereby incorporated by reference herein.
  • preservatives, stabilizers, dyes and flavoring agents may be provided. These include sodium benzoate, sorbic acid and esters of j3-hydroxybenzoic acid.
  • antioxidants and suspending agents may be used.
  • a phannaceutically 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.
  • nucleic acid molecules of the instant invention can be expressed within cells from eukaryotic promoters (e.g., Izant and Weintraub, 1985, Science, 229, 345; McGa ⁇ y 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.
  • eukaryotic promoters e.g., Izant and Weintraub, 1985, Science, 229, 345; McGa ⁇ y 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.
  • nucleic acid can be expressed in eukaryotic cells from the appropriate DNA/RNA vector.
  • the activity of such nucleic acids can be augmented by their release from the primary transcript by a ribozyme (Draper et al, PCT WO 93/23569, and Sullivan et al, PCT WO 94/02595; Ohkawa et al, 1992, Nucleic Acids Symp. Ser., 27, 15-6; Taira et ⁇ /., 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 incorporated in their totalities by reference herein).
  • 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 alphavirus.
  • 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 operable 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 III initiation region); b) a transcription termination region (e.g., eukaryotic pol I, II or III 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 RNA polymerase I
  • polymerase II RNA polymerase II
  • poly III RNA polymerase III
  • Transcripts from pol II or pol III promoters will be expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type will depend on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby.
  • Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci.
  • 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. Natl. Acad. Sci. U S A, 89, 10802-6; Chen et al, 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 , 11, 4411-8; Lisziewicz et al, 1993, Proc. Natl.
  • 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 incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated virus vectors), or viral RNA vectors (such as retroviral or alphavirus vectors) (for a review, see Couture and Stinchcomb, 1996, supra).
  • plasmid DNA vectors such as adenovirus or adeno-associated virus vectors
  • viral RNA vectors such as retroviral or alphavirus 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.
  • Control of the cell cycle is one of the most highly orchestrated events in the cell. There is a great deal of interest in discovering the function of genes involved in mitotic checkpoint abrogation, since inhibition of these genes or activities of these gene products could sensitize cells to DNA damaging agents. In these studies, the cell cycle regulatory role of Chkl (GeneBank Accession # AF016582 is investigated).
  • Chkl also known as p56chkl, is a Wee 1-like protein kinase, which phosphorylates and inactivates Cdc25.
  • Cdc25 is a phosphatase that acts directly on Cdc2. Chkl is required for the DNA damage checkpoint, whereas the rad gene products are required for both S-M and DNA damage checkpoints.
  • Chkl has recently been cloned from mammalian cells.
  • the Chkl protein is modified in response to DNA damage, and has been shown to bind and phosphorylate Cdc25A, Cdc25B and Cdc25C.
  • the phosphorylation of Cdc25C prevents activation of the Cdc2/CyclinB complex and blocks entry into mitosis, thereby validating the inhibition of Chkl as a target for nucleic acid based therapeutics.
  • Chkl kinase plays an essential role in both the DNA replication and DNA damage checkpoint responses.
  • the sequence of human Chkl 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 Chkl (Genbank accession number: AF016582) 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.
  • 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 also synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol. 180, 51). 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 incorporated herein by reference) and are resuspended in water. The sequences of the chemically synthesized ribozymes and antisense constructs used in this study are shown below in Table III-IX.
  • Ribozymes targeted to the human Chkl RNA are designed and synthesized as described above. These ribozymes can be tested for cleavage activity in vitro, for example, using the following procedure.
  • the target sequences and the nucleotide location within the Chkl RNA are given in Tables III-IX.
  • Full-length or partially full-length, internally-labeled target RNA for ribozyme cleavage assay is prepared by in vitro transcription in the presence of [a-32p] CTP, passed over a G 50 Sephadex® column by spin chromatography and used as substrate RNA without further purification.
  • substrates are 5'- 32 P-end labeled using T4 polynucleotide kinase enzyme.
  • Assays are performed by pre-warming a 2X concentration of purified ribozyme in ribozyme cleavage buffer (50 mM Tris-HCl, pH 7.5 at 37°C, 10 mM MgCl2) and the cleavage reaction was initiated by adding the 2X ribozyme mix to an equal volume of substrate RNA (maximum of 1-5 nM) that was also pre-warmed in cleavage buffer. As an initial screen, assays are carried out for 1 hour at 37 C using a final concentration of either 40 nM or 1 mM ribozyme, i.e., ribozyme excess.
  • the reaction is quenched by the addition of an equal volume of 95% formamide, 20 mM EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol after which the sample is heated to 95°C for 2 minutes, quick chilled and loaded onto a denaturing polyacrylamide gel.
  • Substrate RNA and the specific RNA cleavage products generated by ribozyme cleavage are visualized on an autoradiograph of the gel. The percentage of cleavage is determined by Phosphor Imager® quantitation of bands representing the intact substrate and the cleavage products.
  • Antisense nucleic acid molecules targeted to the human Chkl RNA are designed and synthesized as described above. These nucleic acid molecules can be tested for cleavage activity in vivo, for example, using the following procedure.
  • the target sequences and the nucleotide location within the Chkl RNA are given in Tables III-IX.
  • surrogate mitotic markers include decreased phosphorylation of cdc-2 at Thrl4 and Tyrl5, phosphorylation of Myt-1, and phosphorylation of PP1. This study set out to determine whether inhibiting expression of the Chkl gene would allow the G2/M checkpoint to be bypassed after DNA damage, as well as determining if the presence of p53 influences the DNA-damage checkpoint response.
  • RNA inhibition was measured after delivery of these reagents by GSV lipid (Glenn Research) to HeLa cells. Relative amounts of target RNA were measured versus actin using realtime PCR monitoring of amplification (ABI 7700 Taqman®). The results are shown in Figure 6. The comparison is made to a mixture of 5 oligonucleotide sequences made to unrelated targets (GB-3) or to a randomized oligonucleotide control with the same overall length and chemistry, but randomly substituted at each position (GBC3.2). Primary and secondary lead reagents were chosen for the target and optimization performed.
  • the optimal GSV lipid concentration was chosen after screening for RNA inhibition with oligonucleotides at 5 and 50 nM ( Figure 7). After optimal lipid concentration was chosen, a RNA time-course of inhibition was performed with the lead nucleic acid molecule (GeneBlocTM) ( Figure 8). In addition, a cell- plating format was tested for RNA inhibition. The use of a 96-well (5000 cells/well) versus six- well (150,000 cells/well) plating density made no difference in the extent of RNA inhibition ( Figure 9). The phenotypic assays require treatment with etoposide and nocodazole as described above, and RNA inhibition in this assay was also determined ( Figure 10). The various treatments had essentially no effect on RNA levels.
  • Chkl expression modulation includes but are not limited to cancers of the colon, rectum, lung, breast and prostate
  • the present body of knowledge in Chkl research indicates the need for methods to assay Chkl activity and for compounds that can regulate Chkl expression for research, diagnostic, and therapeutic use.
  • Radiation and chemotherapeutic treatments are non-limiting examples of 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.
  • nucleic acid molecules e.g. ribozymes and antisense molecules
  • Those skilled in the art will recognize that other drug compounds and therapies can be similarly be readily combined with the nucleic acid molecules of the instant invention (e.g. ribozymes and antisense molecules) are hence within the scope of the instant invention. Diagnostic uses
  • the nucleic acid molecules of this invention can be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of Chkl 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 structure of the target RNA.
  • ribozymes described in this invention one can 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 can 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 Chkl -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 is used to identify mutant RNA in the sample.
  • synthetic substrates of both wild-type and mutant RNA is 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 also serve to generate size markers for the analysis of wild-type and mutant RNAs in the sample population.
  • each analysis requires two ribozymes, two substrates and one unknown sample, which is combined into six reactions.
  • the presence of cleavage products is 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., Chkl
  • 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 are correlated with higher risk whether RNA levels are compared qualitatively or quantitatively. Additional Uses
  • 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 can 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 has described 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" beta-galactosidase message by the ligation of new beta- galactosidase sequences onto the defective message [ xii ].
  • RNAse P RNA Ml RNA
  • Size -290 to 400 nucleotides.
  • RNA portion of a ubiquitous ribonucleoprotein enzyme • RNA portion of a ubiquitous ribonucleoprotein enzyme.
  • 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.
  • RNA RNA as the infectious agent.
  • Ligation activity (in addition to cleavage activity) makes ribozyme amenable to engineering through in vitro selection [ xxxv ]
  • 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 ]
  • a group II intron RNA is a catalytic component of a DNA endonuclease involved in intron mobility.
  • Cell (Cambridge, Mass.) (1995), 83(4), 529-38.
  • 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.
  • XXX1 Hampel, Arnold; Tritz, Richard; Hicks, Margaret; Cruz, Phillip. 'Hairpin' catalytic RNA model: evidence for helixes and sequence requirement for substrate RNA. Nucleic Acids Res. (1990), 18(2), 299- 304. xxx ⁇ . Chowrira, Bharat M.; Berzal-Herranz, Alfredo; Burke, John M.. Novel guanosine requirement for catalysis by the hairpin ribozyme. Nature (London) (1991), 354(6351), 320-2.
  • AAAAUU A AGAAAGAG 21 CUCUUUCU CUGAUGAG GCCGUUAGGC CGAA AAUAUUUU 1443
  • AAGAUUAU C CUGUCCUG 278 CAGGACAG CUGAUGAG GCCGUUAGGC CGAA AUAAUCUU 1700
  • AF016582 Homo sapiens checkpoint kinase Chkl (CHK1) mRNA; 1821 bp)
  • Underlined region can be any X sequence or linker as previously defined herein.
  • CAGUCCGC C GAGGUGCU 361 AGCACCUC CUGAUGAG GCCGUUAGGC CGAA ICGGACUG 1783
  • GAAGAAGC A GUCGCAGU 380 ACUGCGAC CUGAUGAG GCCGUUAGGC CGAA ICUUCUUC 1802
  • GACUGUCC A GAAAAUAU 385 AUAUUUUC CUGAUGAG GCCGUUAGGC CGAA IGACAGUC 1807
  • AUAGAGCC A GACAUAGG 401 CCUAUGUC CUGAUGAG GCCGUUAGGC CGAA IGCUCUAU 1823
  • AF016582 Homo sapiens checkpoint kinase Chkl (CHK1) mRNA; 1821 bp)
  • Underlined region can be any X sequence or linker as previously defined herein.
  • I Inosine 5
  • Table V Human Chkl G-Cleaver Ribozyme and Substrate Sequence
  • AF016582 Homo sapiens checkpoint kinase Chkl (CHK1) mRNA; 1821 bp)
  • AF016582 Homo sapiens checkpoint kinase Chkl (CHK1) mRNA; 1821 bp)
  • AF016582 Homo sapiens checkpoint kinase Chkl (CHK1) mRNA; 1821 bp)
  • AAAAAAUC G AUUCUGCU 1295 AGCAGAAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GAUUUUUU 3045

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Abstract

La présente 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 enzymes d'ADN et les antisens qui modulent l'expression du gène Chk-1.
PCT/US2001/003504 2000-02-03 2001-02-02 Procede et reactif destines a inhiber la checkpoint kinase-1 (chk 1) WO2001057206A2 (fr)

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WO2002038143A2 (fr) * 2000-11-07 2002-05-16 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Amelioration de l'efficacite et de la securite d'une therapie genotoxique par modulation de la proteine kinase mapk p38
WO2002038143A3 (fr) * 2000-11-07 2003-12-31 Us Gov Health & Human Serv Amelioration de l'efficacite et de la securite d'une therapie genotoxique par modulation de la proteine kinase mapk p38
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EP2330105A1 (fr) 2005-03-29 2011-06-08 ICOS Corporation Dérivés d'uree d'heteroaryle utilises pour inhiber CHK1
US7872118B2 (en) 2006-09-08 2011-01-18 Opko Ophthalmics, Llc siRNA and methods of manufacture
US8470792B2 (en) 2008-12-04 2013-06-25 Opko Pharmaceuticals, Llc. Compositions and methods for selective inhibition of VEGF
WO2013160642A3 (fr) * 2012-04-23 2014-06-05 Ucl Business Plc Traitement des plaies

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