EP1212416A2 - Nucleic acid based modulators of gene expression - Google Patents

Nucleic acid based modulators of gene expression

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Publication number
EP1212416A2
EP1212416A2 EP00963298A EP00963298A EP1212416A2 EP 1212416 A2 EP1212416 A2 EP 1212416A2 EP 00963298 A EP00963298 A EP 00963298A EP 00963298 A EP00963298 A EP 00963298A EP 1212416 A2 EP1212416 A2 EP 1212416A2
Authority
EP
European Patent Office
Prior art keywords
nucleic acid
acid molecule
enzymatic nucleic
rna
enzymatic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00963298A
Other languages
German (de)
English (en)
French (fr)
Inventor
James Mcswiggen
Nassim Usman
Lawrence Blatt
Leonid Beigelman
Alex Burgin
Alexander Karpeisky
Jasenka Matulic-Adamic
David Sweedler
Kenneth Draper
Bharat Chowrira
Dan Stinchcomb
Amber Beaudry
Shawn Zinnen
Janos Lugwig
Brian S. Sproat
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sirna Therapeutics Inc
Original Assignee
Ribozyme Pharmaceuticals Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US09/474,432 external-priority patent/US6528640B1/en
Priority claimed from US09/476,387 external-priority patent/US6617438B1/en
Application filed by Ribozyme Pharmaceuticals Inc filed Critical Ribozyme Pharmaceuticals Inc
Publication of EP1212416A2 publication Critical patent/EP1212416A2/en
Withdrawn legal-status Critical Current

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    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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Definitions

  • This invention relates to reagents useful as inhibitors of gene expression relating to diseases such as cancers, diabetes, obesity, Alzheimer's disease, cardiac diseases, age- related diseases, and/or hepatitis B infections and related conditions.
  • 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 (for example, Cook et al., U.S.
  • Patent 5,359,051 and methods for their use to modulate the expression of molecular targets impacting the development and progression of cancers, diabetes, obesity, Alzheimer's disease, cardiac diseases, age-related diseases, and or hepatitis B infections and related conditions
  • 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 (for exaple, Cook et al., U.S.
  • 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 (for exaple, Cook et al., U.S.
  • Patent 5,359,051)] and methods for their use for inhibiting the expression of disease related genes e.g., Protein-Tyrosine-Phosphatase- lb (PTP-IB, Genbank accession No. NM_002827), Methionine Aminopeptidase (MetAP- 2, Genbank accession No. U29607), beta-Secretase (BACE, Genbank accession No. AF190725), Presenilin-1 (ps-1, Genbank accession No. L76517), Presenilin-2 (ps-2, Genbank accession No. L43964), Human Epidermal Growth Factor Receptor-2 (HER2/c- erb2/neu, Genbank accession No.
  • disease related genes e.g., Protein-Tyrosine-Phosphatase- lb (PTP-IB, Genbank accession No. NM_002827), Methionine Aminopeptidase (MetAP- 2, Genbank
  • ribozymes can be used in a method for treatment of diseases caused by the expression of these genes in man and other animals, including other primates.
  • the invention features novel nucleic acid-based techniques such as enzymatic nucleic acid molecules and antisense molecules and methods for their use to down regulate or inhibit the expression of genes encoding Protein-Tyrosine-Phosphatase-lb (PTP-IB), Methionine Aminopeptidase (MetAP -2), beta-Secretase (BACE), Presenilin-1 (ps-1), Presenilin-2 (ps-2), Human Epidermal Growth Factor Receptor-2 (HER2/c-erb2/neu), Phospholamban (PLN), Telomerase (hTERT) PKC alpha, and Hepatitis B (HBV) proteins.
  • PTP-IB Protein-Tyrosine-Phosphatase-lb
  • MetAP-2 Methionine Aminopeptidase
  • BACE beta-Secretase
  • Presenilin-1 ps-1
  • Presenilin-2 Presenilin-2
  • nucleic acid molecules capable of cleaving RNAs encoded by these genes and their use to reduce levels of PTP-IB, MetAP-2, BACE, ps-1 , ps-2, HER2, PLN, TERT, and/or HBV proteins in various tissues to treat the diseases discussed herein.
  • Such nucleic acid molecules are also useful for diagnostic uses.
  • the invention features the use of one or more ofthe nucleic acid-based techniques independently or in combination to inhibit the expression of the genes encoding PTP-IB, MetAP-2, BACE, ps-1 , ps-2, HER2, PLN, TERT, and/or HBV.
  • the invention features the use of nucleic acid-based techniques to - specifically inhibit the expression of PTP-IB, MetAP-2, BACE, ps-1, ps-2, HER2, PLN, TERT, PKC alpha, and/or HBV genes.
  • the invention features the use of an enzymatic nucleic acid molecule, preferably in the hammerhead, NCH (Inozyme), G-cleaver, amberzyme, zinzyme, and/or DNAzyme motif, to inhibit the expression of PTP-IB, MetAP-2, BACE, ps-1, ps-2, HER2, PLN, TERT, PKC alpha and/or HBV RNA.
  • NCH Inozyme
  • nucleic acid molecules are able to inhibit expression of PTP-IB, MetAP-2, BACE, ps-1, ps-2, HER2, PLN, TERT, PKC alpha, and/or HBV genes.
  • inhibit it is meant that the activity of target genes or level of mRNAs or equivalent RNAs encoding target genes is reduced below that observed in the absence of the nucleic acid molecules of the instant invention (e.g., enzymatic nucleic acid molecules), antisense nucleic acids, 2-5A antisense chimeras, triplex DNA, antisense nucleic acids containing RNA cleaving chemical groups).
  • inhibition with an enzymatic nucleic acid molecule preferably is below that level observed in the presence of an enzymatically attenuated nucleic acid molecule that is able to bind to the same site on the mRNA, but is unable to cleave that RNA.
  • inhibition with nucleic acid molecules is preferably greater than that observed in the presence of, for example, an oligonucleotide with scrambled sequence or with mismatches.
  • inhibition of target genes with the nucleic acid molecule ofthe instant invention is greater than in the presence ofthe nucleic acid molecule than in its absence.
  • the activity of telomerase enzyme or the level of RNA encoding one or more portein subunits ofthe telomerase enzyme is inhibited if it is at least 10% less, 20% less, 50% less, 75% less or even not active or present at all, in the presence of a nucleic acid of the invention relative to the level in the absence of such a nucleic acid.
  • 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 ofthe enzymatic nucleic acid molecule to the target RNA and thus permit cleavage. One hundred percent complementarity is preferred, but complementarity as low as 50-75% may also be useful in this invention.
  • the nucleic acids may be modified at the base, sugar, and/or phosphate groups.
  • enzymatic nucleic acid is used interchangeably with phrases such as ribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, aptazyme or aptamer-binding ribozyme, regulatable ribozyme, catalytic oligonucleotides, nucleozyme, DNAzyme, RNA enzyme, endoribonuclease, endonuclease, minizyme, leadzyme, oligozyme or DNA enzyme. All of these terminologies describe nucleic acid molecules with enzymatic activity.
  • 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.
  • An example of a nucleic acid molecule according to the invention is a gene which encodes for a macromolecule such as a protein.
  • enzymatic portion or “catalytic domain” is meant that portion/region ofthe 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 ribozyme which is complementary to (i.e., able to base-pair with) a portion of its substrate. Generally, such complementarity is 100%, but can be less if desired. For example, as few as 10 bases out of 14 may be base-paired. Such arms are shown generally in Figures 1-5. That is, these arms contain sequences within a ribozyme which are intended to bring ribozyme and target RNA together through complementary base-pairing interactions.
  • the ribozyme ofthe invention may have binding arms that are contiguous or non-contiguous and may be of varying lengths.
  • the length ofthe binding arm(s) are preferably greater than or equal to four nucleotides and of sufficient length to stably interact with the target RNA; specifically 12-100 nucleotides; more specifically 14- 24 nucleotides long. If two binding arms are chosen, the design is such that the length of the binding arms are symmetrical (i.e., each ofthe binding arms is ofthe same length; e.g., five and five nucleotides, six and six nucleotides or seven and seven nucleotides long) or asymmetrical (i.e., the binding arms are of different length; e.g., six and three nucleotides; three and six nucleotides long; four and five nucleotides long; four and six nucleotides long; four and seven nucleotides long; and the like).
  • Binding arms can be complementary to the specified substrate, to a portion ofthe indicated substrate, to the indicated substrate sequence and additional adjacent sequence, or a portion ofthe indicated sequence and additional adjacent sequence.
  • NCH or "Inozyme” motif is meant, an enzymatic nucleic acid molecule comprising a motif as described in Ludwig et al, USSN No. 09/406,643, filed September 27, 1999, entitled “COMPOSITIONS HAVING RNA CLEAVING ACTIVITY", and International PCT publication Nos. WO 98/58058 and WO 98/58057, all incorporated by reference herein in their entirety, including the drawings.
  • G-cleaver an enzymatic nucleic acid molecule comprising a motif as described in Eckstein et al, International PCT publication No. WO 99/16871, inco ⁇ orated by reference herein in its entirety, including the drawings.
  • zinzyme a class II enzymatic nucleic acid molecule comprising a motif as described herein and in Beigelman et al, International PCT publication No. WO 99/55857, inco ⁇ orated by reference herein in its entirety, including the drawings.
  • amberzyme motif is meant, a class I enzymatic nucleic acid molecule comprising a motif as described herein and in Beigelman et al, International PCT publication No. WO 99/55857, inco ⁇ orated by reference herein in its entirety, including the drawings.
  • 'DNAzyme' is meant, an enzymatic nucleic acid molecule lacking a ribonucleotide (2' -OH) group.
  • 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.
  • a DNAzyme can be synthesized chemically or can be expressed by means of a single stranded DNA vector or equivalent thereof.
  • sufficient length 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 ofthe oligonucleotides with target nucleic acid (e.g., by forming hydrogen bonds with complementary nucleotides in the target under physiological conditions).
  • RNA to PTP-IB, MetAP-2, BACE, ps-1, ps-2, HER2, PLN, TERT, and/or HBV is meant to include those naturally occurring RNA molecules having homology (partial or complete) to PTP-IB, MetAP-2, BACE, ps-1 , ps-2, HER2, PLN, TERT, and/or HBV proteins or encoding for proteins with similar function as PTP-IB, MetAP-2, BACE, ps-1, ps-2, HER2, PLN, TERT, and/or HBV in various organisms, including human, rodent, primate, rabbit, pig, protozoans, fungi, plants, and other microorganisms and parasites.
  • 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 in HBV.
  • regions such as 5 '-untranslated region, 3 '-untranslated region, introns, intron-exon junction and the like in HBV.
  • homoology is meant the nucleotide sequence of two or more nucleic acid molecules is partially or completely identical.
  • antisense nucleic acid a non-enzymatic nucleic acid molecule that binds to target RNA by means of RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al, 1993 Nature 365, 566) interactions and alters the activity ofthe target RNA (for a review, see Stein and Cheng, 1993 Science 261, 1004 and Woolf et al, US patent No. 5,849,902).
  • antisense molecules will be complementary to a target sequence along a single contiguous sequence ofthe 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.
  • Antisense DNA can be synthesized chemically or can be expressed via the use of a single stranded DNA expression vector or the equivalent thereof.
  • 2-5A antisense chimera an antisense oligonucleotide containing a 5'-phosphorylated 2'-5'-linked adenylate residue. These chimeras bind to target RNA in a sequence-specific manner and activate a cellular 2-5 A-dependent ribonuclease which, in turn, cleaves the target RNA (Torrence et al, 1993 Proc. Natl. Acad. Sci. USA 90, 1300).
  • trim DNA it is meant an oligonucleotide that can bind to a double-stranded DNA in a sequence-specific manner to form a triple-strand helix.
  • nucleic acid that encodes a RNA.
  • complementarity is meant that a nucleic acid can form hydrogen bond(s) with another RNA sequence by either traditional Watson-Crick or other non-traditional types.
  • the binding free energy for a nucleic acid molecule with its target or complementary sequence is sufficient to allow the relevant function ofthe nucleic acid to proceed, e.g., ribozyme cleavage, antisense or triple helix inhibition.
  • a percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule which can form hydrogen bonds (e.g.,
  • Watson-Crick base pairing with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary).
  • Perfectly complementary means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.
  • enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion ofthe molecule that acts to cleave the target RNA.
  • the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA.
  • the ribozyme is a highly specific inhibitor of gene expression, with the specificity of inhibition depending not only on the base-pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage. Single mismatches, or base-substitutions, near the site of cleavage can completely eliminate catalytic activity of a ribozyme.
  • the enzymatic nucleic acid molecule that cleave the specified sites in PTP-IB, MetAP-2, BACE, ps-1, ps-2, HER2, PLN, TERT, and/or HBV-specific RNAs represent a novel therapeutic approach to treat a variety of pathologic indications, including, HBV infection, hepatitis, hepatocellular carcinoma, tumorigenesis, cirrhosis, liver failure, cancers including breast, ovarian, prostate, and esophogeal cancer, tumorigenesis, retinopathy, arthritis, psoriasis, female reproduction, restinosis, certain infectious diseases, transplant rejection and autoimmune disease such as multiple sclerosis, lupus, and AIDS, age related diseases such as macular degeneration and skin ulceration, Alzheimer's disease, dementia, diabetes, obesity and any other condition related to the level of PTP-IB, MetAP-2, BACE, ps-1, ps-2, HER2, PLN, TERT, and or
  • the enzymatic nucleic acid molecule is formed in a hammerhead or hai ⁇ in motif, but may also be formed in the motif of a hepatitis delta virus, group I intron, group U 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.
  • hai ⁇ in motifs are described by Hampel et al, EP0360257, Hampel and Tritz, 1989 Biochemistry 28, 4929, Feldstein et al, 1989, Gene 82, 53, Haseloff and Geriach, 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 Airman, 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 inco ⁇ orated by reference herein in their totalities, including drawings and can also be used in the present invention.
  • a nucleic acid molecule e.g., an antisense molecule, a triplex DNA, or a ribozyme
  • the nucleic acid molecule is 15-100, 17-100, 20-100, 21-100, 23-100, 25-100, 27-100, 30-100, 32-100, 35-100, 40-100, 50-100, 60-100, 70-100, or 80-100 nucleotides in length.
  • the upper limit ofthe length range can be, for example, 30, 40, 50, 60, 70, or 80 nucleotides.
  • the length range for particular embodiments has lower limit as specified, with an upper limit as specified which is greater than the lower limit.
  • the length range can be 35-50 nucleotides in length. All such ranges are expressly included.
  • a nucleic acid molecule can have a length which is any ofthe lengths specified above, for example, 21 nucleotides in length.
  • the invention provides a method for producing a class of nucleic acid-based gene inhibiting agents which exhibit a high degree of specificity for the RNA of a desired target.
  • the enzymatic nucleic acid molecule is preferably targeted to a highly conserved sequence region of target RNAs encoding PTP-IB, MetAP- 2, BACE, ps-1, ps-2, HER2, PLN, TERT, and/or HBV proteins (specifically PTP-IB, MetAP-2, BACE, ps-1, ps-2, HER2, PLN, TERT, and/or HBV RNA) such that specific treatment of a disease or condition can be provided with either one or several nucleic acid molecules ofthe invention.
  • nucleic acid molecules can be delivered exogenously to specific tissue or cellular targets as required.
  • the nucleic acid molecules e.g., ribozymes and antisense
  • 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).
  • PTP-IB MetAP-2, BACE, ps-1, ps-2, HER2, PLN, TERT, and/or HBV proteins
  • PTP-IB MetAP-2, BACE, ps-1, ps-2, HER2, PLN, TERT, and/or HBV proteins
  • highly conserved sequence region is meant 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 enzymatic nucleic acid-based inhibitors of PTP-IB, MetAP-2, BACE, ps-1, ps- 2, HER2, PLN, TERT, and/or HBV expression are useful for the prevention ofthe diseases and conditions including HBV infection, hepatitis, hepatocellular carcinoma, tumorigenesis, cirrhosis, liver failure, cancers including breast, ovarian, prostate, and esophogeal cancer, tumorigenesis, retinopathy, arthritis, psoriasis, female reproduction, restinosis, certain infectious diseases, transplant rejection and autoimmune disease such as multiple sclerosis, lupus, and AIDS, age related diseases such as macular degeneration and skin ulceration, Alzheimer's disease, dementia, diabetes, obesity and any other condition related to the level of PTP-IB, MetAP-2, BACE, ps-1, ps-2, HER2, PLN, TERT, and/or HBV in a cell or tissue, and any other diseases or conditions that are
  • RNA levels by “related” is meant that the reduction of PTP-IB, MetAP-2, BACE, ps-1, ps-2, HER2, PLN, TERT, and/or HBV expression (specifically PTP-IB, MetAP-2, BACE, ps-1, ps-2, HER2, PLN, TERT, and/or HBV genes) RNA levels and thus reduction in the level ofthe respective protein will relieve, to some extent, the symptoms ofthe disease or condition.
  • nucleic acid-based inhibitors ofthe 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 inco ⁇ oration in biopolymers.
  • the enzymatic nucleic acid inhibitors comprise sequences, which are complementary to the subsfrate sequences in Tables 3-31, 33, 34, 36-43, 56, 58, 59, 62, 63.
  • Examples of such enzymatic nucleic acid molecules also are shown in Tables 3-29, 31, 33, 34, 37-43, 56, 58, 59, 62, 63. Examples of such enzymatic nucleic acid molecules consist essentially of sequences defined in these tables.
  • the invention features antisense nucleic acid molecules including sequences complementary to the substrate sequences shown in Tables 3-31, 33, 34, 36, 37-43, 56, 58, 59, 62, 63.
  • Such nucleic acid molecules can include sequences as shown for the binding arms ofthe enzymatic nucleic acid molecules in Tables 3-29, 31, 33, 34, 37-43, 56, 58, 59, 62, 63.
  • 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 (subsfrate) sequence.
  • antisense molecules will be complementary to a target sequence along a single contiguous sequence ofthe antisense molecule.
  • an antisense molecule may bind to subsfrate such that the subsfrate molecule forms a loop, and/or an antisense molecule may bind such that the antisense molecule forms a loop.
  • the antisense molecule may be complementary to two (or even more) noncontiguous substrate sequences or two (or even more) non-contiguous sequence portions of an antisense molecule may be complementary to a target sequence or both.
  • 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.
  • “consists essentially of is meant that the active nucleic acid molecule ofthe 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 mRNA such that cleavage at the target site occurs.
  • a core region may, for example, include one or more loop or stem-loop structures, which do not prevent enzymatic activity.
  • "X" in the sequences in Tables 3, 4, 9, 10, 13, 14, 18, 19, 24, 25, 33, 34, 37, 38, 63 can be such a loop.
  • ribozymes or antisense molecules that interact with target RNA molecules and inhibit PTP-IB, MetAP-2, BACE, ps-1, ps-2, HER2, PLN, TERT, and/or HBV (specifically PTP-IB, MetAP-2, BACE, ps-1, ps-2, HER2, PLN, TERT, and/or HBV RNA) 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 might be repeatedly administered as necessary. Once expressed, the ribozymes or antisense bind to the target RNA and inhibit its function or expression.
  • ribozyme or antisense expressing vectors could be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex -planted from the patient followed by reinfroduction into the patient, or by any other means that would allow for introduction into the desired target cell.
  • Antisense DNA can be expressed via the use of a single stranded DNA intracellular expression vector.
  • RNA is meant a molecule comprising at least one ribonucleotide residue.
  • ribonucleotide is meant a nucleotide with a hydroxyl group at the 2' position of a ⁇ -D- ribo-furanose moiety.
  • vectors is meant 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 ofthe invention can be administered.
  • a patient is a mammal or mammalian cells. More preferably, a patient is a human or human cells.
  • nucleic acid molecules ofthe instant invention can be used to treat diseases or conditions discussed above.
  • the patient may be treated, or other appropriate cells may be treated, as is evident to those skilled in the art, individually or in combination with one or more drugs under conditions suitable for the treatment.
  • the described molecules can be used in combination with other known treatments to treat conditions or diseases discussed above.
  • the described molecules could be used in combination with one or more known therapeutic agents to treat HBV infection, hepatitis, hepatocellular carcinoma, tumorigenesis, cirrhosis, liver failure, cancers including breast, ovarian, prostate, and esophogeal cancer, tumorigenesis, retinopathy, arthritis, psoriasis, female reproduction, restinosis, certain infectious diseases, transplant rejection and autoimmune disease such as multiple sclerosis, lupus, and AIDS, age related diseases such as macular degeneration and skin ulceration, Alzheimer's disease, dementia, diabetes, and/or obesity.
  • the invention features nucleic acid-based inhibitors (e.g., enzymatic nucleic acid molecules (ribozymes), antisense nucleic acids, triplex DNA, antisense nucleic acids containing RNA cleaving chemical groups) and methods for their use to down regulate or inhibit the expression of RNA (e.g., PTP-IB, MetAP-2, BACE, ps-1, ps-2, HER2, PLN, TERT, and or HBV) capable of progression and/or maintenance of HBV infection, hepatitis, hepatocellular carcinoma, tumorigenesis, cirrhosis, liver failure, cancers including breast, ovarian, prostate, and esophogeal cancer, tumorigenesis, retinopathy, arthritis, psoriasis, female reproduction, restinosis, certain infectious diseases, fransplant rejection and autoimmune disease such as multiple sclerosis, lupus, and ADDS, age related diseases such as macular degeneration and skin ulceration,
  • RNA
  • the invention features nucleic acid-based techniques (e.g., enzymatic nucleic acid molecules (ribozymes), antisense nucleic acids, triplex DNA, antisense nucleic acids containing RNA cleaving chemical groups) and methods for their use to down regulate or inhibit the expression of PTP-IB, MetAP-2, BACE, ps- 1 , ps-2, HER2, PLN, TERT, and or HBV RNA expression.
  • nucleic acid-based techniques e.g., enzymatic nucleic acid molecules (ribozymes), antisense nucleic acids, triplex DNA, antisense nucleic acids containing RNA cleaving chemical groups
  • 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).
  • HDV Ribozyme I-IV are meant to indicate four stem-loop structures (Been et al, US Patent No. 5,625,047).
  • Hammerhead Ribozyme I- HI are meant to indicate three stem- loop structures; stems I-HI 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., - 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.
  • Helix 1 and 4 can be of any size (i.e., o and p is each independently from 0 to any number, e.g., 20) as long as some base-pairing is maintained.
  • Essential bases are shown as specific bases in the structure, but those in the art will recognize that one or more may be modified chemically (abasic, base, sugar and/or phosphate modifications) or replaced with another base without significant effect.
  • Helix 4 can be formed from two separate molecules, i.e., without a connecting loop. The connecting loop when present may be a ribonucleotide with or without modifications to its base, sugar or phosphate, "q" > is 2 bases.
  • the connecting loop can also be replaced with a non-nucleotide linker molecule.
  • H refers to bases A, U, or C.
  • Y refers to pyrimidine bases. " " refers to a covalent bond.
  • Figure 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 (described herein and in Ludwig & Sproat, International PCT Publication No. WO 98/58058);
  • G-Cleaver represents G- cleaver ribozyme motif (Kore et ⁇ /., 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; rl, represents ribo-Inosine nucleotide; arrow indicates the site of cleavage within the target.
  • Position 4 ofthe 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 ofthe ribozyme.
  • FIG 3 shows an example ofthe Amberzyme ribozyme motif that is chemically stabilized (see, for example, Beigelman et al, International PCT publication No. WO 99/55857; also referred to as Class I Motif).
  • the Amberzyme motif is a class of enzymatic nucleic acid molecules that do not require the presence of a ribonucleotide (2'-OH) group for activity.
  • FIG 4 shows an example ofthe Zinzyme A ribozyme motif that is chemically stabilized (see, for example, International PCT publication No. WO 99/55857; also referred to as Class A Motif).
  • the Zinzyme motif is a class of enzymatic nucleic acid molecules that do not require the presence of a ribonucleotide (2' -OH) group for activity.
  • Figure 5 shows an example of a DNAzyme motif described by Santoro et al., 1997, PNAS, 94, 4262.
  • FIG. 6 is a diagrammatic representation ofthe hammerhead ribozyme motif known in the art and the NCH motif.
  • Stem JJ can be 2 base-pair long, preferably, 2, 3, 4, 5, 6, 7, 8, and 10 base-pairs long.
  • Each N and N' is independently any base or non- nucleotide as used herein;
  • X is adenosine, cytidine or uridine;
  • Stem I-HI are meant to indicate three stem-loop structures; stems I-HI can be of any length and may be symmetrical or asymmetrical (Usman et al, 1996, Curr. Op. Struct.
  • Loop II may be present or absent. If Loop II is present it is greater than or equal to three nucleotides, preferably four nucleotides.
  • the Loop II sequence is preferably 5'-GAAA-3' or 5'- GUUA-3'.
  • Figure 7 shows examples of chemically stabilized ribozyme motifs.
  • HH Rz represents hammerhead ribozyme motif (Usman et al, 1996, Curr. Op. Struct. Bio., 1, 527);
  • NCH-Inosine Rz represents the NCH ribozyme motif with riboinosine at 15.1 position;
  • NCH-Xylo Rz represents the NCH ribozyme with xylo inosine at 15.1 position.
  • N or n represent independently a nucleotide which may be same or different and may have complementarity to each other; ri, represents ribo-Inosine nucleotide; xl represent xylo- inosine; arrow indicates the site of cleavage within the target. Position 4 ofthe HH Rz and the NCH Rzs 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 ofthe ribozyme.
  • Figure 8 is a graphical representation of data showing inhibition of cell proliferation mediated by NCH and HH ribozymes targeted against HER2/new/ErbB2 gene.
  • Figure 9 is a schematic diagram ofthe process for the synthesis of beta-D- xylofuranosyl hypoxantine 3 '-phosphoramidite.
  • Figure 10 displays a schematic representation of NTP synthesis using nucleoside substrates.
  • Figure 11 shows a scheme for an in vitro selection method.
  • a pool of nucleic acid molecules is generated with a random core region and one or more region(s) with a defined sequence. These nucleic acid molecules are bound to a column containing immobilized oligonucleotide with a defined sequence, where the defined sequence is complementary to region(s) of defined sequence of nucleic acid molecules in the pool.
  • Those nucleic acid molecules capable of cleaving the immobilized oligonucleotide (target) in the column are isolated and converted to complementary DNA (cDNA), followed by transcription using NTPs to form a new nucleic acid pool.
  • cDNA complementary DNA
  • Figure 12 shows a scheme for a two column in vitro selection method.
  • a pool of nucleic acid molecules is generated with a random core and two flanking regions (region A and region B) with defined sequences.
  • the pool is passed through a column which has immobilized oligonucleotides with regions A' and B' that are complementary to regions A and B ofthe nucleic acid molecules in the pool, respectively.
  • the column is subjected to conditions sufficient to facilitate cleavage ofthe immobilized oligonucleotide target.
  • the molecules in the pool that cleave the target (active molecules) have A' region ofthe target bound to their A region, whereas the B region is free.
  • the column is washed to isolate the active molecules with the bound A' region ofthe target.
  • This pool of active molecules may also contain some molecules that are not active to cleave the target (inactive molecules) but have dissociated from the column.
  • the pool is passed through a second column (column 2) which contains immobilized oligonucleotides with the A' sequence but not the B' sequence.
  • the inactive molecules will bind to column 2 but the active molecules will not bind to column 2 because their A region is occupied by the A' region ofthe target oligonucleotide from column 1.
  • Column 2 is washed to isolate the active molecules for further processing as described in the scheme shown in Figure 11.
  • Figure 13 is a diagram of a novel 48 nucleotide enzymatic nucleic acid motif which was identified using in vitro methods described in the instant invention.
  • the molecule shown is only exemplary.
  • the 5' and 3' terminal nucleotides (referring to the nucleotides ofthe subsfrate binding arms rather than merely the single terminal nucleotide on the 5' and 3' ends) can be varied so long as those portions can base-pair with target substrate sequence.
  • the guanosine (G) shown at the cleavage site ofthe subsfrate can be changed to other nucleotides so long as the change does not eliminate the ability of enzymatic nucleic acid molecules to cleave the target sequence. Substitutions in the nucleic acid molecule and/or in the subsfrate sequence can be readily tested, for example, as described herein.
  • Figure 14 is a schematic diagram of HCV luciferase assay used to demonstrate efficacy of class I enzymatic nucleic acid molecule motif.
  • Figure 15 is a graph indicating the dose curve of an enzymatic nucleic acid molecule targeting site 146 on HCV RNA.
  • Figure 16 is a bar graph showing enzymatic nucleic acid molecules targeting 4 sites within the HCV RNA are able to reduce RNA levels in cells.
  • Figure 17 shows secondary structures and cleavage rates for characterized Class ⁇ enzymatic nucleic acid motifs.
  • Figure 18 is a diagram of a novel 35 nucleotide enzymatic nucleic acid motif which was identified using in vitro methods described in the instant invention.
  • the molecule shown is only exemplary.
  • the 5' and 3' terminal nucleotides (referring to the nucleotides ofthe subsfrate binding arms rather than merely the single terminal nucleotide on the 5' and 3' ends) can be varied so long as those portions can base-pair with target substrate sequence.
  • the guanosine (G) shown at the cleavage site ofthe subsfrate can be changed to other nucleotides so long as the change does not eliminate the ability of enzymatic nucleic acid molecules to cleave the target sequence. Substitutions in the nucleic acid molecule and/or in the substrate sequence can be readily tested, for example, as described herein.
  • Figure 19 is a bar graph showing subsfrate specificities for Class ⁇ (zinzyme) ribozymes.
  • Figure 20 is a bar graph showing Class II enzymatic nucleic acid molecules targeting 10 representative sites within the HER2 RNA in a cellular proliferation screen.
  • Figure 21 is a synthetic scheme outlining the synthesis of 5-[3- aminopropynyl(propyl)]uridine 5'-triphosphates and 4-imidazoleaceticacid conjugates.
  • Figure 22 is a synthetic scheme outlining the synthesis of 5-[3-(N-4- imidazoleacetyl)aminopropynyl(propyl)]uridine 5 '-triphosphates.
  • Figure 23 is a synthetic scheme outlining the synthesis of carboxylate tethered uridine 5'-triphosphoates.
  • Figure 24 is a synthetic scheme outlining the synthesis of 5-(3-aminoalkyl) and 5- [3(N-succinyl)aminopropyl] functionalized cytidines.
  • Figure 25 is a diagram of a class I ribozyme stem truncation and loop replacement analysis.
  • Figure 26 is a diagram of class I ribozymes with truncated stem(s) and/or non- nucleotide linkers used in loop structures.
  • Figure 27 is a diagram of "no-ribo" class LI ribozymes.
  • Figure 28 is a graph showing cleavage reactions with class II ribozymes under differing divalent metal concentrations.
  • Figure 29 is a diagram of differing class ⁇ ribozymes with varying ribo content and their relative rates of catalysis.
  • Figure 30 is a graph showing class II ribozyme (zinzyme) mediated reduction of
  • FIG. 31 is a graph showing class II ribozyme (zinzyme) mediated dose response anti-prolferation assay in SKBR3 breast carcinoma cells.
  • zinzyme RPI 18656
  • a corresponding scrambled attenuated control complexed with 2.0 ⁇ g/ml of lipid Active zinzymes and scrambled attenuated controls were compared to untreated cells after 24 hours post treatment.
  • Figure 32 is a graph which shows the dose dependent reduction of HER2 RNA in SKOV-3 cells treated with RPI 19293 from 0 to 100 nM with 5.0 ⁇ g/ml of cationic lipid.
  • Figure 33 is a graph which shows the dose dependent reduction of HER2 RNA and inhibition of cellular proliferation in SKBR-3 cells freated with RPI 19293 from 0 to 400 nM with 5.0 ⁇ g/ml of cationic lipid.
  • Figure 34 shows a non-limiting example ofthe replacement of a 2'-O-methyl 5'- CA-3' with a ribo G in the class LI (zinzyme) motif.
  • the representative motif shown for the pu ⁇ ose ofthe figure is a "seven-ribo" zinzyme motif, however, the interchangeability of a G and a CA in the position shown in Figure 25 of the class II (zinzyme) motif extends to any combination of 2-O-methyl and ribo residues.
  • a 2'-O-methyl G can replace the 2'-O-methyl 5'-CA-3' and vise versa.
  • RPI 19727 no ribo
  • RPI 19728 one ribo
  • RPI 19723 two ribo
  • RPI 19729 three ribo
  • RPI 19730 four ribo
  • 19731 five ribo
  • RPI 19292 seven ribo
  • Figure 36 summarizes the results of functional group modification studies in which various nucleoside analogs were tested for activity in the NCH ribozyme motif.
  • K re ⁇ values describe the cleavage values of a given substituent at position 15.1 relative the Inosine at position 15.1 (1-15.1).
  • Figure 37 summarizes reported functional group modification studies performed at the A 15.1 residue in the A-15.1 »U-16.1 context of NUH cleaving ribozymes.
  • K re ⁇ values describe the cleavage values of a given substituent at position 15.1 relative the adenosine at position 15.1 (A-15.1).
  • Antisense molecules may be modified or unmodified RNA, DNA, or mixed polymer oligonucleotides and primarily function by specifically binding to matching sequences resulting in inhibition of peptide synthesis (Wu-Pong, Nov 1994, BioPharm, 20-33).
  • the antisense oligonucleotide binds to target RNA by Watson Crick base-pairing and blocks gene expression by preventing ribosomal translation ofthe bound sequences either by steric blocking or by activating RNase H enzyme.
  • Antisense molecules may also alter protein synthesis by interfering with RNA processing or transport from the nucleus into the cytoplasm (Mukhopadhyay & Roth, 1996, Crit. Rev. in Oncogenesis 7, 151 - 190).
  • binding of single stranded DNA to RNA may result in nuclease degradation ofthe heteroduplex (Wu-Pong, supra; Crooke, supra).
  • the only backbone modified DNA chemistry which will act as substrates for RNase H are phosphorothioates, phosphorodithioates, and borontrifluoridates.
  • 2'-arabino and 2'-fluoro arabino- containing oligos can also activate RNase H activity.
  • antisense molecules have been described that utilize novel configurations of chemically modified nucleotides, secondary 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, International PCT Publication No. WO 00/17346) all of these are inco ⁇ orated by reference herein in their entirety.
  • Antisense DNA can be used to target RNA by means of DNA-RNA interactions, thereby activating RNase H, which digests the target RNA in the duplex.
  • Antisense DNA can be chemically synthesized or can be expressed via the use of a single stranded DNA intracellular expression vector or the equivalent thereof.
  • TFO Triplex Forming Oligonucleotides
  • the TFO mechanism may result in gene expression or cell death since binding may be irreversible (Mukhopadhyay & Roth, supra) 2'-5' Oligoadenylates:
  • the 2-5 A system is an interferon-mediated mechanism for RNA degradation found in higher vertebrates (Mifra et al, 1996, Proc Nat Acad Sci USA 93, 6780-6785).
  • Two types of enzymes, 2-5 A synthetase and RNase L are required for RNA cleavage.
  • the 2-5 A synthetases require double stranded RNA to form 2'-5' oligoadenylates (2-5 A).
  • 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 2-5 A structures with double stranded RNA makes this system particularly useful for inhibition of viral replication.
  • (2'-5') oligoadenylate structures may be covalently linked to antisense molecules to form chimeric oligonucleotides capable of RNA cleavage (Torrence, supra). These molecules putatively bind and activate a 2-5 A dependent RNase, the oligonucleotide/enzyme complex then binds to a target RNA molecule which can then be cleaved by the RNase enzyme.
  • the covalent attachment of 2'-5' oligoadenylate structures is not limited to antisense applications, and can be further elaborated to include attachment to nucleic acid molecules ofthe instant invention.
  • Enzymatic Nucleic Acid Seven basic varieties of naturally-occurring enzymatic RNAs are presently known.
  • several in vitro selection (evolution) strategies (Orgel, 1979, Proc. R. Soc. London, B 205, 435) have been used to evolve new nucleic acid catalysts capable of catalyzing cleavage and ligation of phosphodi ester linkages (Joyce, 1989, Gene, 82, 83-87; Beaudry et al, 1992, Science 257, 635-641 ; Joyce, 1992, Scientific American 267, 90-97; Breaker et al, 1994, TIBTECH 12, 268; Bartel et al, 1993, Science 261:1411-1418; Szostak, 1993, TIBS 17, 89-93; Kumar et al, 1995, FASEB J., 9, 1183; Breaker, 1996, Curr.
  • enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of an enzymatic nucleic acid which is held in close proximity to an enzymatic portion ofthe molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.
  • Nucleic acid molecules of this invention will block to some extent PTP-IB, MetAP- 2, BACE, ps-1, ps-2, HER2, PLN, TERT, and/or HBV protein expression and can be used to treat disease or diagnose disease associated with the levels of PTP-IB, MetAP-2, BACE, ps-1, ps-2, HER2, PLN, TERT, and/or HBV.
  • the enzymatic nature of a ribozyme has significant advantages, such as the concentration of ribozyme necessary to affect a therapeutic treatment is low. This advantage reflects the ability ofthe ribozyme to act enzymatically.
  • ribozyme is able to cleave many molecules of target RNA.
  • the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base-pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage. Single mismatches, or base-substitutions, near the site of cleavage can be chosen to completely eliminate catalytic activity of a ribozyme.
  • Nucleic acid molecules having an endonuclease enzymatic activity are able to repeatedly cleave other separate RNA molecules in a nucleotide base sequence-specific manner. Such enzymatic nucleic acid molecules can be targeted to virtually any RNA transcript, and achieve efficient cleavage in vitro (Zaug et al, 324, Nature, 429 1986 ; Uhlenbeck, 1987 Nature, 328, 596; Kim et al., 84 Proc. Natl. Acad. Sci. USA, 8788, 1987; Dreyfus, 1988, Einstein Quart. J. Bio.
  • Ribozymes can be designed to cleave specific RNA targets within the background of cellular RNA. Such a cleavage event renders the RNA 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 al, 1999, Chemistry and Biology, 6, 237-250.
  • the nucleic acid molecules ofthe 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.
  • 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, and all hereby inco ⁇ orated in their entireties by reference herein.
  • 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 inco ⁇ orated 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 sequence of human PTP-IB, MetAP-2, BACE, ps-1, ps-2, HER2, PLN, TERT, and/or HBV RNAs for example, GenBank accession Nos. (PTP-IB,.
  • NM_002827 (MetAP-2, U29607), (BACE, AF190725), (ps-1, L76517), (ps-2, L43964), (HER2/c-erb2/neu, X03363), (PLN, NM_002667), (TERT, NM_003219) and (HBV, AF100308.1, HBV strain 2-18; additionally, other HBV strains can be screened by one skilled in the art, see Table 35 for other possible strains) were screened for optimal enzymatic nucleic acid and antisense target sites using a computer-folding algorithm.
  • Antisense, hammerhead, DNAzyme, NCH (Inozyme), amberzyme, zinzyme or G-Cleaver ribozyme binding/cleavage sites were identified. These sites are shown in Tables 3-29, 31, 33, 34, 37-43, 56, 58, 59, 62, 63 (all sequences are 5' to 3' in the tables; X can be any base-paired sequence, the actual sequence is not relevant here).
  • the nucleotide base position is noted in the Tables as that site to be cleaved by the designated type of enzymatic nucleic acid molecule.
  • Table 36 shows substrate positions selected from Renbo et al, 1987, Sci. Sin., 30, 507, used in Draper, US patent No.
  • Cleaver ribozyme binding/cleavage sites were identified, as discussed above.
  • the nucleic acid molecules were 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 were eliminated from consideration. Varying binding arm lengths can be chosen to optimize activity.
  • Antisense, hammerhead, DNAzyme, NCH, amberzyme, zinzyme or G-Cleaver ribozyme binding/cleavage sites were identified and were designed to anneal to various sites in the RNA target.
  • the binding arms are complementary to the target site sequences described above.
  • the nucleic acid molecules were chemically synthesized. The method of synthesis used follows the procedure for normal DNA/RNA synthesis as described below and in Usman et al, 1987 J. Am. Chem. Soc, 109, 7845; Scaringe et al, 1990 Nucleic Acids Res., 18, 5433; Wincott et al, 1995 Nucleic Acids Res. 23, 2677-2684; and 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 ofthe nucleic acid to invade targeted regions of RNA structure.
  • Exemplary molecules ofthe 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, Intemational PCT Publication No. WO 99/54459, Wincott et al, 1995, Nucleic Acids Res. 23, 2677-2684, Wincott et al, 1997, Methods Mol. Bio., 74, 59, Brennan et al, 1998, Biotechnol Bioeng., 61, 33-45, and Brennan, US patent No.
  • oligonucleotides make 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 ofthe reagents used in the synthesis cycle.
  • syntheses at the 0.2 ⁇ mol scale can be performed on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, CA) with minimal modification to the cycle.
  • synthesizer include the following: 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 I2, 49 mM pyridine, 9% water in THF (PERSEPTIVETM). Burdick & Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle. S-Ethyltefrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc.
  • Beaucage reagent (3H- l,2-Benzodithiol-3-one 1,1 -dioxide, 0.05 M in acetonitrile) is used.
  • Deprotection ofthe 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 supematant is removed from the polymer support.
  • the support is washed three times with 1.0 mL of EtOH:MeCN:H2O/3 : 1 : 1 , 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; and Wincott et al, 1995, Nucleic Acids Res. 23, 2677-2684 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 ofthe 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 ofthe trityl fractions, are typically 97.5-99%.
  • synthesizer include the following: 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 I2, 49 mM pyridine, 9% water in THF (PERSEPTIVETM). Burdick & Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc.
  • Beaucage reagent (3H- l,2-Benzodithiol-3-one l,l-dioxide0.05 M in acetonitrile) is used.
  • Deprotection ofthe 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. 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:MeCN:H2O/3 : 1 : 1 , vortexed and the supernatant is then added to the first supematant. 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 NH 4 HCO 3 .
  • the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 33% ethanolic methylamine/DMSO: 1/1 (0.8 mL) at 65 °C for 15 min.
  • the vial is brought to r.t. TEA*3HF (0.1 mL) is added and the vial is heated at 65 °C for 15 min.
  • the sample is cooled at -20 °C and then quenched with 1.5 M NH 4 HCO3.
  • 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 confrol (B AC) oligonucleotides are synthesized by substituting a U for G5 and a U for A14 (numbering from Hertel, K. J., et al, 1992, Nucleic Acids Res.. 20, 3252). Similarly, one or more nucleotide substitutions can be introduced in other enzymatic nucleic acid molecules to inactivate the molecule and such molecules can serve as a negative confrol.
  • 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 example 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 ofthe 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 ofthe 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 elecfrophoresis using general methods or are purified by high pressure liquid chromatography (HPLC; see Wincott et al, supra, the totality of which is hereby inco ⁇ orated herein by reference) and are re-suspended in water.
  • the sequences ofthe ribozymes and antisense constructs that are chemically synthesized, useful in this study, are shown in Tables 3-31, 33, 34, 37-43, 56, 58, 59, 62, 63. Those in the art will recognize that these sequences are representative only of many more such sequences where the enzymatic portion ofthe ribozyme (all but the binding arms) is altered to affect activity.
  • the ribozyme and antisense construct sequences listed in Tables 3-31, 33, 34, 37-43, 56, 58, 59, 62, 63 maybe formed of ribonucleotides or other nucleotides or non-nucleotides. Such ribozymes with enzymatic activity are equivalent to the ribozymes described specifically in the Tables.
  • Enzymatic nucleic acid molecules are modified to enhance stability and/or enhance catalytic activity by modification with nuclease resistant groups, for example, 2'-amino, 2"-C-allyl, 2'-fluoro, 2'-O-methyl, 2'-O-allyl, 2'-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992 TIBS 17, 34; Usman et al, 1994 Nucleic Acids Symp. Ser. 31, 163; Burgin et al, 1996 Biochemistry 35, 14090). Sugar modification of enzymatic nucleic acid molecules have been extensively described in the art (see Eckstein et al, International Publication PCT No.
  • Such publications describe general methods and strategies to determine the location of inco ⁇ oration of sugar, base and/or phosphate modifications and the like into enzymatic nucleic acid molecules without inhibiting catalysis, and are inco ⁇ orated by reference herein.
  • the 2'-position ofthe sugar in a nucleotide present in the nucleic acid molecules ofthe instant invention which tolerates substitution is selected from the group comprising -H, -OH, -COOH, -CONH , - CONHR 1 , -CONR'R 2 , -N ⁇ -NHR'.
  • the substituents for sugar 2' position preferably are independently halogen, cyano, amino, carboxy, ester, ether, carboxamide, hydroxy, or mercapto.
  • R 1 and R 2 can be substituted or unsubstituted alkyl, alkenyl, or alkynyl groups, where the substituents are independently halogen, cyano, amino, carboxy, ester, ether, carboxamide, hydroxy, or mercapto.
  • similar modifications can be used as described herein to modify the nucleic acid molecules ofthe instant invention.
  • modified bases in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at 1' position or their equivalents; such bases may be used within the catalytic core ofthe enzyme and/or in the substrate-binding regions.
  • the nucleic acid bases can be hypoxanthin-9-yl, or a functional equivalent thereof, in position 15 'of the ribozyme; the base at other positions may be guanin-9-yl, hypoxanthin-9-yl or 7-deazaguanin-9-yl in positions 5, 8 and 12 in the ribozyme; adenin-9- yl, 2,6-diaminopurin-9-yl, purin-9-yl or 7-deaza adenin-9-yl in positions 6, 9, 13 and 14; uracil-1-yl, uracil-5-yl, thymin-1-yl or 5-propynyluracil-l-yl in position 4; cytosin-1-yl, 5- methylcytosin-1-yl or 5-propynylcytosin-l-yl in position 3; and adenin-9-yl, cytosin-1-yl, guanin-9-yl, uracil
  • the base at position 15.1 is preferably hypoxanthin-9-yl or an analog where no hydrogen bond can form between any group at the 2 position ofthe base and the 2-oxo group of C 16 ' 1 .
  • B is not guanin-9-yl in position 15.1.
  • the invention features modified ribozymes having a base substitution selected from pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyluracil, dihydrouracil, naphthyl, 6-methyl-uracil and aminophenyl.
  • a base substitution selected from pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyluracil, dihydrouracil, naphthyl, 6-methyl-uracil and aminophenyl.
  • chemical modification of oligonucleotide intemucleotide linkages with phosphorothioate, phosphorothioate, and/or 5'-methylphosphonate linkages improves stability, too many of these modifications may cause some toxicity. Therefore, when designing nucleic acid molecules, the amount of these intemucleotide linkages should be minimized.
  • nucleic acid molecules having chemical modifications which maintain or enhance activity are provided. Such nucleic acid molecules are 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 ofthe target RNA has been inhibited long enough to reduce the levels ofthe 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 infracellular therapeutic agents.
  • nucleic acid-based molecules ofthe invention will lead to better treatment ofthe disease progression by affording the possibility of combination therapies (e.g., multiple antisense or enzymatic nucleic acid molecules targeted to different genes, nucleic acid molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations of molecules (including different motifs) and/or other chemical or biological molecules).
  • combination therapies e.g., multiple antisense or enzymatic nucleic acid molecules targeted to different genes, nucleic acid molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations of molecules (including different motifs) and/or other chemical or biological molecules).
  • the treatment of patients with nucleic acid molecules may also include combinations of different types of nucleic acid molecules.
  • Therapeutic nucleic acid molecules e.g., enzymatic nucleic acid molecules and antisense nucleic acid molecules
  • delivered exogenously must optimally be stable within cells until translation ofthe target RNA has been inhibited long enough to reduce the levels ofthe undesirable protein.
  • nucleic acid molecules must be resistant to nucleases in order to function as effective intracellular therapeutic agents. Improvements in the chemical synthesis of nucleic acid molecules described in the instant invention and in the art have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability as described above.
  • enhanced enzymatic activity is meant to include activity measured in cells and/or in vivo where the activity is a reflection of both catalytic activity and ribozyme stability.
  • the product of these properties is increased or not significantly (less than 10- fold) decreased in vivo compared to an all RNA ribozyme or all DNA enzyme.
  • nucleic acid catalysts having chemical modifications which maintain or enhance enzymatic activity are provided.
  • Such nucleic acid catalysts are 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.
  • 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.
  • the nucleic acid molecules comprise a 5' and/or a 3'- cap structure.
  • cap structure is meant chemical modifications, which have been inco ⁇ orated at either terminus ofthe oligonucleotide (see, for example, Wincott et al, WO 97/26270, inco ⁇ orated by reference herein). These terminal modifications protect the nucleic acid molecule from exonuclease degradation, and may help in delivery and/or localization within a cell.
  • the cap may be present at the 5'-terminus (5'-cap) or at the 3'-terminal (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; 1- (beta-D-erythrofuranosyl) nucleotide, 4'-thio nucleotide; carbocyclic nucleotide; 1,5- anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; t ⁇ reo-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 nucleo
  • 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/zreo-pentofuranosyl nucleotide; acyclic 3
  • 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 pi 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 ofthe ring atoms are carbon atoms.
  • Suitable heteroatoms include oxygen, sulfur, and nifrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted.
  • An "amide” refers to an -C(O)-NH-R, where R is either alkyl, aryl, alkylaryl or hydrogen.
  • An “ester” refers to an -C(O)-OR', where R is either alkyl, aryl, alkylaryl or hydrogen.
  • nucleotide as used herein is as recognized in the art to include natural bases (standard), and modified bases well known in the art. Such bases are generally located at the 1' position of a nucleotide sugar moiety. Nucleotides generally comprise a base, sugar and a phosphate group. The nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and other; see, for example, Usman and McSwiggen, supra; Eckstein et al, International PCT Publication No.
  • base modifications that can be introduced into nucleic acid molecules include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3- methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, and others (Burgin et al, 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra).
  • 5-alkylcytidines e.g., 5-methylcytidine
  • 5-alkyluridines e.g., ribothymidine
  • 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 ofthe nucleic acid molecule.
  • modified nucleotides include dideoxynucleotides which have pharmaceutical utility well known in the art, as well as utility in basic molecular biology methods such as sequencing.
  • the invention features modified ribozymes with phosphate backbone modifications comprising one or more phosphorothioate, phosphorodithioate, methylphosphonate, mo ⁇ holino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl, substitutions.
  • abasic sugar moieties lacking a base or having other chemical groups in place of a base at the 1' position, (for more details, see Wincott et al, International PCT publication No. WO 97/26270).
  • unmodified nucleoside or “unmodified nucleotide” is meant one ofthe bases adenine, cytosine, guanine, thymine, uracil joined to the 1' carbon of ⁇ -D-ribo-furanose.
  • modified nucleoside or “modified nucleotide” 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, which are both inco ⁇ orated by reference 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 2-5 A chimera molecules to one or more targets to alleviate symptoms of a disease.
  • Nucleic acid molecules may be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by inco ⁇ oration into other 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 oral (tablet or pill form) and/or intrathecal delivery (Gold, 1997, Neuroscience, 76, 1153-1158).
  • routes of delivery include, but are not limited to, infravascular, intramuscular, subcutaneous or joint injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intrathecal delivery.
  • nucleic acid delivery and administration 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 are inco ⁇ orated by reference herein.
  • the molecules ofthe instant invention can be used as pharmaceutical agents.
  • Pharmaceutical agents prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all ofthe symptoms) of a disease state in a patient.
  • the negatively charged polynucleotides ofthe 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 ofthe present invention may also be formulated and used as tablets, capsules or elixirs for oral administration; suppositories for rectal administration; sterile solutions; suspensions for injectable administration; and the other compositions known in the art.
  • the present invention also includes pharmaceutically acceptable formulations ofthe compounds described.
  • 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 abso ⁇ tion or accumulation of drugs in the blood stream followed by distribution throughout the entire body.
  • Administration routes which lead to systemic abso ⁇ tion include, without limitations: intravenous, subcutaneous, intraperitoneal, inhalation, oral, infrapulmonary and intramuscular. Each of these administration routes expose the desired negatively charged polymers, e.g., nucleic acids, to an accessible diseased tissue.
  • the rate of entry of a drug into the circulation has been shown to be a function of molecular weight or size.
  • the use of a liposome or other drug carrier comprising the compounds ofthe instant invention can potentially localize the drug, for example, in certain tissue types, such as the tissues ofthe reticular endothelial system (RES).
  • RES reticular endothelial system
  • a liposome formulation which can facilitate the association of drug with the surface of cells, such as, lymphocytes and macrophages is also useful. This approach may provide enhanced delivery ofthe drug to target cells by taking advantage ofthe specificity of macrophage and lymphocyte immune recognition of abnormal cells, such as cancer cells.
  • compositions or formulation that allows for the effective distribution ofthe nucleic acid molecules ofthe instant invention in the physical location most suitable for their desired activity.
  • agents suitable for formulation with the nucleic acid molecules ofthe 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.
  • 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 ofthe 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 ofthe 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).
  • liposomes have been shown to accumulate selectively in tumors, presumably by extravasation and capture in the neovascularized target tissues (Lasic et al, Science 1995, 267, 1275-1276; Oku et al., 995, Biochim. Biophys. Acta, 1238, 86-90).
  • the long- circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA, particularly compared to conventional cationic liposomes which are known to accumulate in tissues ofthe MPS (Liu et al, J. Biol. Chem. 1995, 42, 24864-24870; Choi et al, International PCT Publication No.
  • compositions prepared for storage or administration which include a pharmaceutically effective amount ofthe 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.
  • a pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all ofthe 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 freated, the physical characteristics ofthe specific mammal under consideration, concurrent medication, and other factors which those skilled in the medical arts will recognize.
  • an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients is administered dependent upon potency ofthe negatively charged polymer.
  • the nucleic acid molecules ofthe 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 freat an indication may increase the beneficial effects while reducing the presence of side effects.
  • nucleic acid molecules ofthe instant invention can be expressed within cells from eukaryotic promoters (e.g., Izant and Weintraub, 1985, Science, 229, 345; McGarry and Lindquist, 1986, Proc. Natl. Acad. Sci., USA 83, 399; Scanlon et al, 1991, Proc. Natl. Acad. Sci. USA, 88, 10591-5; Kashani-Sabet et al, 1992, Antisense Res. Dev., 2, 3-15; Dropulic et al, 1992, J. Virol, 66, 1432-41; Weerasinghe et al, 1991, J.
  • eukaryotic promoters e.g., Izant and Weintraub, 1985, Science, 229, 345; McGarry and Lindquist, 1986, Proc. Natl. Acad. Sci., USA 83, 399; Scanlon et al, 1991,
  • nucleic acids can be augmented by their release from the primary transcript by a ribozyme (Draper et al, PCT WO 93/23569, and Sullivan et al, PCT WO 94/02595; Ohkawa et al, 1992, Nucleic Acids Symp. Ser., 27, 15-6; Taira et ⁇ /., 1991, Nucleic Acids Res., 19, 5125- 30; Ventura et al, 1993, Nucleic Acids Res., 21, 3249-55; Chowrira et al, 1994, J. Biol. Chem., 269, 25856; all of these references are hereby inco ⁇ orated in their totality by reference herein).
  • a ribozyme Draper et al, PCT WO 93/23569, and Sullivan et al, PCT 94/02595; Ohkawa et al, 1992, Nucleic Acids Symp. Ser., 27, 15-6
  • RNA molecules ofthe 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.
  • the invention features an expression vector comprising a nucleic acid sequence encoding at least one ofthe nucleic acid molecules ofthe instant invention is disclosed.
  • the nucleic acid sequence encoding the nucleic acid molecule ofthe instant invention is operably linked in a manner which allows expression of that nucleic acid molecule.
  • the invention features an expression vector comprising: a) a transcription initiation region (e.g., eukaryotic pol I, II or HI initiation region); b) a transcription termination region (e.g., eukaryotic pol I, LI or LU termination region); c) a nucleic acid sequence encoding at least one ofthe nucleic acid catalyst ofthe 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 ofthe sequence encoding the nucleic acid catalyst ofthe invention; and/or an intron (intervening sequences).
  • ORF open reading frame
  • RNA polymerase I RNA polymerase I
  • polymerase II RNA polymerase II
  • pol HI RNA polymerase HI
  • Transcripts from pol ⁇ or pol LU promoters will be expressed at high levels in all cells; the levels of a given pol LI promoter in a given cell type will depend on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby.
  • Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci. USA, 87, 6743-7; Gao and Huang 1993, Nucleic Acids Res., 21, 2867-72;
  • 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. USA, 89,
  • transcription units such as the ones derived from genes encoding U6 small nuclear
  • RNA molecules such as ribozymes in cells
  • snRNA transfer RNA
  • tRNA transfer RNA
  • adenovirus VA RNA RNA molecules
  • desired RNA molecules such as ribozymes in cells
  • the above ribozyme transcription units can be inco ⁇ orated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated virus vectors), or viral RNA vectors (such as retroviral or alphavirus vectors) (for a review see Couture and Stinchcomb, 1996, supra).
  • the invention features an expression vector comprising nucleic acid sequence encoding at least one ofthe nucleic acid molecules ofthe 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 infron; 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 infron; 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 infron, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.
  • Example 1 Telomerase
  • telomere The ribonucleoprotein enzyme telomerase consists of an RNA template subunit and one or more protein subunits including telomerase reverse transcriptase (TERT), which function together to direct the synthesis of telomeres.
  • Telomeres exist as non-nucleosome DNA/protein complexes at the physical ends of eukaryotic chromosomes. These capping structures maintain chromosome stability and replicative potential (Zakian, V. A., 1995, Science, 270, 1601-1607). Telomere structure is characterized by tandem repeats of conserved DNA sequences rich in G-C base pairs.
  • telomere elements include a terminal 3 '-overhang in the G-rich strand and non-histone structural proteins that are complexed with telomeric DNA in the nucleus.
  • Observed shortening of telomeres coincides with the onset of cellular senescence in most somatic cell lines lacking significant levels of telomerase. This finding has had a profound impact on our views concerning the mechanisms of aging, age related disease, and cancer.
  • Conventional DNA polymerases are unable to fully replicate the ends of linear chromosomes (Watson, J. D., 1972, Nature, 239, 197-201).
  • telomere This inability stems from the 3' G-rich overhang that is a product of ribonuclease cleavage ofthe RNA primer used in DNA replication.
  • the overhang prevents DNA polymerase replication since the recessed C-rich parent strand cannot be used as a template.
  • Telomerase overcomes this limitation by extending the 3' end ofthe chromosome using deoxyribonucleotides as substrates and a sequence within the telomerase RNA subunit as a template. (Lingner, J., 1995, Science, 269, 1533-1534). As such, telomerase is considered a reverse transcriptase that is responsible for telomere maintenance.
  • telomerase was first discovered by in Tetrahymena thermophila in 1985 (Greider, C. W., 1995, Cell, 43, 405-413). The RNA subunits and their respective genes were later discovered and characterized in protozoa, budding yeast, and mammals. Genetic studies of these genes confirmed the role of telomerase RNA (TR) in determining telomere sequence by mutating genes which encode the telomeric RNA (Yu, G. L., 1990, Nature, 344, 126-132), (Singer, M. S., 1994, Science, 266, 404-409), (Blasco, M. A., 1995, Science, 269, 1267-1270).
  • TR telomerase RNA
  • telomerase activity parallels TR expression in protozoa, yeast and mice.
  • hTR human telomerase RNA
  • Many human tissues express hTR but are devoid of telomerase activity (Feng, J., 1995, Science, 269, 1236-1241).
  • Knockout mice in which the mTR gene has been deleted from germline cells, have been shown to be viable for at least six generations. Cells from later generations of these mice showed chromosomal abnormalities consistent with telomere degradation, indicating that mTR is necessary for telomere length maintenance, but is not required for embryonic development, oncogenic transformation, or tumor formation in mice (Blasco, M. A., 1997, Cell, 91, 25-34).
  • telomere catalytic subunit of telomerase (pi 23) was isolated from Euplotes aediculatus along with another subunit (p43) and a 66-kD RNA subunit (Linger, J., 1996, Proc. Natl. Acad. Sci., 93, 10712-10717). Subsequent studies revealed telomerase catalytic subunit homologs from fission yeast (Est2p) and human genes (TRT1). The human homolog, TRT1 encoding hTERT, expressed mRNA with a strong correlation to telomerase activity in human cells (Nakamura, T. M., 1997, Science, 277, 955-959).
  • telomere activity Reconstitution of telomerase activity with in vitro transcribed and translated hTERT and hTR, either co-synthesized or simply mixed, demonstrated that hTERT and hTR represent the minimal components of telomerase. Furthermore, transient expression of hTERT in normal diploid human cells restored telomerase activity, demonstrating that hTERT is the only component necessary to restore telomerase activity in normal human cells (Weinrich, S. L., 1997, Nature Genetics, 17, 498-502). The introduction of telomerase into normal human cells using hTERT expression via fransfection has resulted in the extension of life span in these cells.
  • telomere loss in the absence of telomerase is the "mitotic clock” that controls the replicative potential of a cell prior to senescence (Bodnar, A. G., 1998, Science, 279, 349-352).
  • telomere maintenance is essential for the formation of human tumor cells (Hahn, W. C, 1999, Nature, 400, 464-468).
  • TRAP telomeric repeat amplification protocol
  • a method based on Kim is as follows. Briefly, for the telomerase assay, 2 ⁇ g of protein exfract is used. The exfract is assayed in 50 ⁇ l of reaction mixture containing 0.1 ⁇ g TS substrate primer (5 ' -AATCCGTCGAGCAGAGTT-3', end-labeled using alpha- 32 P- ATP and T4 polynucleotide kinase), 0.1 ⁇ g ACX return primer(5'-GCGCGG[CTTACC] 3 CTAACC-3'), 0.1 ⁇ g NT internal control primer (5*-ATCGCTTCTCGGCCTTTT-3'), 0.01 micromol TSNT internal confrol template (5'- AATCCGTCGAGCAGAGTTAAAAGGCCGAGAACGAT-3), 50 ⁇ M each deoxynucleoside triphosphate, 2 U of Taq DNA polymerase, and 2 ⁇ l CHAPS protein exfract, all in IX TRAP buffer (20 mM Tris (pH 8.3),
  • reaction products are separated on a denaturing 8% polyacrylamide gel, followed by drying ofthe gel and autoradiography.
  • the internal control to control for possible Taq polymerase inhibition
  • Comparison of radioactive signal integrated e.g., by pho ⁇ horimager analysis
  • telomerase-extended bands with the radioactive signal from a reaction performed with a known amount of quantification standard template (termed R8; 5'-
  • AATCCGTCGAGCAGAGTTAG [GGTTAG] 7 -3) allows expression of telomerase activity as an absolute value.
  • TPG values of 0-10,000 are possible, with the linear range being from approximately 1 to 1000 TPG.
  • the range of 1 to 1000 TPG encompasses the minimum and maximum levels of telomerase activity in most tumor samples tested, while non-tumor cells most often have no telomerase activity (TPG approximately zero). Telomerase activity may also be assayed as follows.
  • Samples to be assayed for telomerase activity are prepared by extraction into CHAPS lysis buffer (lOmM Tris pH 7.5, ImM MgCl 2 , ImM EGTA, 0.1 mM PMSF, 5mM -mercaptoethanol, ImM DTT, 0.5% 3-[(3-cholamidopropyl)-dimethyl-amino]-l- propanesulfonate (CHAPS), 10% glycerol and 40 U/ml RNAse inhibitor (Promega, Madison, WI, U.S.A.). Cells are suspended in CHAPS lysis buffer and incubated on ice for 30 minutes, which allows lysis of 90-100%) of cells.
  • CHAPS lysis buffer LOmM Tris pH 7.5, ImM MgCl 2 , ImM EGTA, 0.1 mM PMSF, 5mM -mercaptoethanol, ImM DTT, 0.5% 3-[(3-cholamidopropyl)-dimethyl-amino
  • Lysate is then transferred to polyallomer centrifuge tubes and spun at 100,000 x g for 1 hour at 4 degrees C.
  • the supernatant is the protein exfract, and concentration ranges of 4-10 ⁇ g/ ⁇ l are suitable for telomerase assay.
  • Extracts may be concentrated if necessary using a Microcon Microfilter 30 (Amicron, Beverly, MA U.S.A.) according to the manufactureris instructions. Exfracts may be stored frozen at -80 degrees C until assayed.
  • telomere activity has been analyzed in rats via cell proliferation studies with MNU (N-methyl-N-nitosurea) induced mammary carcinomas in response to treatment with 4-(hydroxyphenyl)retinamide (4-HPR), a known inhibitor of mammary carcinogenesis in animal models and premenopausal women (Bednarek, A., 1999, Carcinogenesis, 20, 879-883). Additional studies have focused on the up-regulation of telomerase in transformed cell lines from animal and human model systems (Zhang, P. B., 1998, Leuk. Res., 22, 509-516), (Chadeneau, C, 1995, Oncogene, 11, 893-898), (Greenberg, R., 1999, Oncogene, 18, 1219-1226).
  • telomere expression As related to various other cancers are described including cervical cancer (Nakano, K., 1998, Am. J. Pathol, 153, 857-864), endometrial cancer (Kyo, S., 1999, Int. J. Cancer, 80, 60-63), meningeal carcinoma (Kleinschmidt-DeMasters, B. K., 1998, J. Neurol.
  • telomere expression modulation includes but are not limited to:
  • telomere activity Treatment with telomerase inhibitors may provide effective cancer therapy with minimal side effects in normal somatic cells that lack telomerase activity.
  • the therapeutic potential exists for the treatment of a wide variety of cancer types.
  • telomerase inhibition in vascular smooth muscle cells may inhibit restinosis by limiting proliferation of these cells.
  • telomerase inhibition in infectious cell types that express telomerase activity may provide selective anti-infectious agent activity. Such treatment may prove especially effective in protozoan-based infection such as Giardia and Lesh Meniesis.
  • telomerase inhibition in endothelial cell types may demonstrate selective immunnosuppressant activity. Activation of telomerase in transplant cells could benefit grafting success through increased proliferative potential.
  • Autoimmune disease Telomerase modulation in various immune cells may prove beneficial in treating diseases such as multiple sclerosis, lupus, and AIDS.
  • Age related disease Activation of telomerase expression in cells at or nearing senescence as a result of advanced age or premature aging could benefit conditions such as macular degeneration, skin ulceration, and rheumatoid arthritis.
  • the present body of knowledge in telomerase research indicates the need for methods to assay telomerase activity and for compounds that can regulate telomerase expression for research, diagnostic, trait alteration, animal health and therapeutic use.
  • Gemcytabine and cyclophosphamide are non-limiting examples of chemotherapeutic agents that can be combined with or used in conjunction with the nucleic acid molecules (e.g. ribozymes and antisense molecules) ofthe instant invention.
  • chemotherapeutic agents that can be combined with or used in conjunction with the nucleic acid molecules (e.g. ribozymes and antisense molecules) ofthe instant invention.
  • other drugs such as anti-cancer compounds and therapies can be similarly be readily combined with the nucleic acid molecules ofthe instant invention (e.g. ribozymes and antisense molecules) and are hence within the scope ofthe instant invention.
  • Such compounds and therapies are well known in the art (see for example Cancer: Principles and Pranctice of Oncology, Volumes 1 and 2, eds Devita, V.T., Hellman, S., and Rosenberg, S.A., J.B.
  • the nucleic acids ofthe invention are prepared in one of two ways.
  • the agents are physically combined in a preparation of nucleic acid and chemotherapeutic agent, such as a mixture of a nucleic acid ofthe invention encapsulated in liposomes and ifosfamide in a solution for intravenous administration, wherein both agents are present in a therapeutically effective concentration (e.g., ifosfamide in solution to deliver 1000-1250 mg/m 2 /day and liposome-associated nucleic acid ofthe invention in the same solution to deliver 0.1-100 mg/kg/day).
  • the agents are administered separately but simultaneously in their respective effective doses (e.g., 1000-1250 mg/m 2 /d ifosfamide and 0.1 to 100 mg/kg/day nucleic acid ofthe invention).
  • Gaeta et al US patents No. 5,760,062; 5,767,278; 5,770,613 have described small molecule inhibitors of human telomerase RNA (hTR) subunit.
  • hTR human telomerase RNA
  • telomerase RNA subunit may not be very beneficial, because as demonstrated by Feng et al, (Feng, J., 1995, Science, 269, 1236-1241), telomerase activity in humans does not correlate well to hTR concentration.
  • telomere activity Four human telomerase subunit proteins are described called pi 40, pi 05, p48 and p43.
  • hybridization probes and primers are described as inhibitors of telomerase gene function.
  • Antibody based inhibitors of telomerase protein subunits are described.
  • telomerase regulation would involve the regulation of human telomerase by modulating the expression ofthe protein subunits ofthe enzyme, preferably the reverse transcriptase (hTERT) subunit.
  • hTERT and hTR represent the minimal components of telomerase. Since hTR expression does not correlate well with telomerase activity in human cells and since many human cells express hTR without telomerase activity, targeting hTERT may prove more beneficial than targeting hTR.
  • hTERT is the only component necessary to restore telomerase activity in normal human cells.
  • hTERT is a rate limiting determinant of enzymatic activity of human telomerase (Kyo, S., 1999, Int. J. Cancer, 80, 60-63). Additional protein subunits that have been isolated most likely serve only a structural role in telomerase activity, but may be important in enhancing the activity ofthe telomerase enzyme. As such, hTERT is one ofthe better targets for the ectopic regulation of telomerase activity.
  • Cech et al, International PCT publication No. WO 98/14593 describe compositions and methods related to hTERT for diagnosis, prognosis and treatment of human diseases, for altering proliferative capacity in cells and organisms, and for screening compounds and treatments with potential use as human therapeutics.
  • Cech et al, International PCT publication No. WO 98/14592 describe nucleic acid and amino acid sequences encoding various telomerase protein subunits and motifs of Euplotes aediculatus, and related sequences from Schizosaccharomyces, Saccharomyces sequences, and human telomerase.
  • polypeptides comprising telomeric subunits and functional polypeptides and ribonucleoproteins that contain these subunits are described as well.
  • Cech et al International PCT Publication No. WO 98/14592, mentions in general terms the the possibility of using antisense and ribozymes to down regulate the expression of human telomerase reverse transcriptase enzyme.
  • RNA sequence of human TERT was screened for accessible sites using a computer folding algorithm. Regions ofthe RNA that did not form secondary folding structures and contained potential ribozyme and/or antisense binding/cleavage sites were identified. The sequences of these cleavage sites are shown in Tables 13-17.
  • Ribozyme target sites were chosen by analyzing sequences of Human TERT (Nakamura et al, 1997 Science 277, 955-959; Genbank sequence accession number: NM 003219) and prioritizing the sites on the basis of folding. Ribozymes were designed that could bind each target and were individually analyzed by computer folding (Christoffersen et al, 1994 J. Mol. Struc. Theochem, 311, 273; Jaeger et al, 1989, Proc. Natl. Acad.
  • ribozyme sequences fold into the appropriate secondary structure.
  • Those ribozymes with unfavorable intramolecular interactions between the binding arms and the catalytic core were eliminated from consideration.
  • 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 for Efficient Cleavage of TERT RNA Ribozymes were designed to anneal to various sites in the RNA message. The binding arms are complementary to the target site sequences described above. The ribozymes were chemically synthesized. The method of synthesis used followed the procedure for normal RNA synthesis as described above and in Usman et al., (1987 J. Am. Chem.
  • Ribozymes were also synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol. 180, 51). Ribozymes were purified by gel electrophoresis using general methods or were purified by high pressure liquid chromatography (HPLC; See Wincott et al., supra; the totality of which is hereby inco ⁇ orated herein by reference) and were resuspended in water. The sequences of the chemically synthesized ribozymes used in this study are shown below in Table 13-17.
  • Ribozyme Cleavage of TERT RNA Target in vitro Ribozymes targeted to the human TERT 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
  • TERT RNA are given in Tables 13-17.
  • substrates are 5'-32p- en( i labeled using T4 polynucleotide kinase enzyme.
  • Assays are performed by pre- warming 15 ⁇ l of a 2X concentration of purified ribozyme in ribozyme cleavage buffer (50 mM Tris-HCI, pH 7.5 at 37°C, 10 mM MgC ) and the cleavage reaction was initiated by adding the 2X ribozyme mix to an equal volume (15 ⁇ l) of subsfrate RNA (maximum of 1-5 nM; 5 x 10 5 to 1 x 10 7 cpm) that was also pre- warmed in cleavage buffer.
  • ribozyme cleavage buffer 50 mM Tris-HCI, pH 7.5 at 37°C, 10 mM MgC
  • assays o 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 (30 ⁇ l) of 95% formamide, 20 mM EDTA, 0.05% bromophenol blue and 0.05% o xylene cyanol after which the sample is heated to 95 C for 2 minutes, quick chilled and loaded onto a denaturing polyacrylamide gel.
  • Subsfrate 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 subsfrate and the cleavage products.
  • Protein tyrosine phosphorylation and dephosphorylation are important mechanisms in the regulation of signal transduction pathways that control the processes of cell growth, proliferation, and differentiation (Fantl, W. J., 1993, Annu. Rev. Biochem., 62, 453-481).
  • Cooperative enzyme classes regulate protein tyrosine phosphorylation and dephosphorylation events. These broad classes of enzymes consist ofthe protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs).
  • PTKs and PTPs can exist as both receptor-type transmembrane proteins and as cytoplasmic protein enzymes.
  • Receptor tyrosine kinases propagate signal transduction events via extracellular receptor-ligand interactions that result in the activation ofthe tyrosine kinase portion ofthe PTK in the cytoplasmic domain.
  • Receptor-like transmembrane PTPs function through extracellular ligand binding that modulates dephosphorylation of infracellular phosphotyrosine proteins via cytoplasmic phosphatase domains. Cytoplasmic PTKs and PTPs exert enzymatic activity without receptor-mediated ligand interactions, however, phosphorylation can regulate the activity of these enzymes.
  • Protein tyrosine phosphatase IB was the first PTP to be isolated in homogeneous form (Tonks, N. K., 1988, J. Biol. Chem., 263, 6722-6730), characterized (Tonks, N. K., 1988, J. Biol. Chem., 263, 6731-6737), and sequenced (Charbonneau, H., 1989, Biochemistry, 86, 5252-5256).
  • Cytoplasmic and receptor-like PTPs both share a catalytic domain characterized by eleven conserved amino acids containing cysteine and arginine residues that are critical for phosphatase activity (Streuli, M., 1990, EMBO, 9, 2399-2407).
  • a cysteine residue at position 215 is responsible for the covalent attachment of phosphate to the enzyme (Guan, K, 1991, J. Biol. Chem., 266, 17026-17030).
  • the crystal structure of human PTP IB defined the phosphate binding site ofthe enzyme as a glycine rich cleft at the surface of the molecule with cysteine 215 positioned at the base of this cleft.
  • cysteine 215 and the shape ofthe cleft provide specificity of PTPase activity for tyrosine residues but not for serine or threonine residues (Barford, D., 1994, Science, 263, 1397-1404).
  • Receptor tyrosine kinase and protein tyrosine phosphatase localization plays a key role in the regulation of phosphotyrosine mediated signal fransduction.
  • PTP-IB activity and specificity against a panel of receptor tyrosine kinases demonstrated clear differences between substrates, suggesting that cellular comparrmentalization is a determinant in defining the activity and function ofthe enzyme (Lammers, R.,1993, J. Biol. Chem., 268, 22456-22462).
  • Experiments have indicated that PTP-IB is localized predominantly in the endoplasmic reticulum via its 35 amino acid carboxyterminal sequence.
  • PTP-IB is also tightly associated with microsomal membranes with its catalytic phosphatase domain oriented towards the cytoplasm (Frangioni, J. V., 1992, Cell, 68, 545-560).
  • PTP-IB has been identified as a negative regulator ofthe insulin response. PTP-IB is widely expressed in insulin sensitive tissues (Goldstein, B. J., 1993, Receptor, 3, 1-15). Isolated PTP-IB dephosphorylates the insulin receptor in vitro (Tonks, N. K., 1988, J. Biol. Chem., 263, 6731-6737). PTP-IB dephosphorylation of multiple phosphotyrosine residues ofthe insulin receptor proceeds sequentially and with specificity for the three tyrosine residues that are critical for receptor autoactivation (Ramachandran, C, 1992, Biochemistry, 31, 4232-4238).
  • PTP-IB In addition to insulin receptor dephosphorylation, PTP-IB also dephosphorylates the insulin related subfrate 1 (IRS-1), a principal substrate ofthe insulin receptor (Lammers, R., 1993, J. Biol. Chem., 268, 22456-22462). Microinjection of PTP IB into Xenopus oocytes results in the inhibition of insulin stimulated tyrosine phosphorylation of endogenous proteins, including the ⁇ -subunit ofthe insulin and insulin-like growth factor receptor proteins. The resulting 3 to 5 fold increase over endogenous PTPase activity also blocks the activation of an S6 peptide kinase (Cicirelli, M. F., 1990, Proc, Natl. Acad.
  • Increased PTP-IB expression correlates with insulin resistance in hyperglycemic cultured fibroblasts.
  • desensitized insulin receptor function was observed via impaired insulin-induced autophosphorylation ofthe receptor.
  • Treatment with insulin sensitivity normalizing thiazolidine derivatives resulted in the amelioration ofthe hyperglycemic insulin resistance via a normalization in PTP-IB expression (Maegawa, H., 1995, J. Biol. Chem., 270, 7724-7730).
  • a murine model of insulin resistance with a knockout of the hefrerotrimeric GTP-binding protein subunit Gi ⁇ 2 provides a type 2 diabetis phenotype that correlates with the increased expression of PTP-IB (Moxam, C.
  • PTP-IB interacts directly with the activated insulin receptor ⁇ -subunit.
  • An inactive homolog of PTP-IB was used to precipitate the activated insulin receptor in both purified receptor preparations and whole-cell lysates. Phosphorylation ofthe insulin receptor's triple tyrosine residues in the kinase domain is necessary for PTP-IB interaction.
  • insulin stimulates tyrosine phosphorylation of PTP-IB (Seely, B. L., 1996, Diabetes, 45, 1379-1385).
  • Knockout mice lacking the PTP-IB gene have been used to study the specific role of PTP-IB relating to insulin action in vivo.
  • the resulting PTP-IB deficient mice were healthy and, in the fed state, had lower blood glucose and circulating insulin levels that were half that of their PTP-1B +/+ expressing littermates.
  • These PTP-IB deficient mice demonstrated enhanced insulin sensitivity in glucose and insulin tolerance tests.
  • the PTP-IB deficient mice showed increased phosphorylation ofthe insulin receptor after insulin administration.
  • Type 1 diabetes may be treated by modulation of PTP-IB expression.
  • Type 2 diabetes correlates to desensitized insulin receptor function (White et al, 1994).
  • Disruption ofthe PTP-IB dephosphorylation ofthe insulin receptor in vivo manifests in insulin sensitivity and increased insulin receptor autophosphorylation (Elchebly et al, 1999).
  • Insulin dependant diabetes, type 1 may respond to PTP-IB modulation through increased insulin sensitivity.
  • Troglitazone is a non-limiting example of a pharmaceutical agent that can be combined with or used in conjunction with the nucleic acid molecules (e.g. ribozymes and antisense molecules) ofthe instant invention.
  • nucleic acid molecules e.g. ribozymes and antisense molecules
  • other drugs such as anti-diabetes and anti-obesity compounds and therapies can be similarly be readily combined with the nucleic acid molecules ofthe instant invention (e.g. ribozymes and antisense molecules) are hence within the scope ofthe instant invention.
  • Methods have been developed to assay PTP-IB activity.
  • Tonks et al International PCT publication No. WO 97/US13016, describe subsfrate-frapping protein PTPase mutants for identification of tyrosine-phosphorylated protein substrates and their clinical uses.
  • the human genome is thought to contain up to 100 PTPases, each varying slightly in chemistry but vastly in function.
  • Compounds designed to inhibit PTP-IB activity specifically by covalent binding to or modification of PTP-IB have the potential for multiple side effects.
  • Conventional drug substances that will potently suppress PTP-IB activity with few or no side effects from interaction with other PTPs are difficult to envision.
  • a more attractive approach to PTP-IB modulation would involve the specific regulation of PTP-IB expression with oligonucleotides.
  • RNA sequence of human PTP-IB was screened for accessible sites using a computer folding algorithm. Regions ofthe RNA that did not form secondary folding structures and contained potential ribozyme and/or antisense binding/cleavage sites were identified. The sequences of these cleavage sites are shown in Tables 3-8.
  • Ribozyme target sites were chosen by analyzing sequences of Human PTP-IB (Genbank accession number M33689) and prioritizing the sites on the basis of folding. Ribozymes were designed that could bind each target and were individually analyzed by computer folding (Christoffersen et al, 1994 J. Mol. Struc. Theochem, 311, 273; Jaeger et al, 1989, Proc. Natl. Acad. Sci.
  • Ribozymes were designed to anneal to various sites in the RNA message.
  • the binding arms are complementary to the target site sequences described above.
  • the ribozymes were chemically synthesized. The method of synthesis used followed the procedure for normal RNA synthesis as described above and in Usman et al., (1987 J. Am. Chem. Soc, 109, 7845), Scaringe et al., (1990 Nucleic Acids Res., 18, 5433) and Wincott et al., supra, and made use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end. The average stepwise coupling yields were >98%. Ribozymes were also synthesized from DNA templates using bacteriophage T7
  • RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol. 180, 51). Ribozymes were purified by gel electrophoresis using general methods or were purified by high pressure liquid chromatography (HPLC; see Wincott et al., supra; the totality of which is hereby inco ⁇ orated herein by reference) and were resuspended in water. The sequences of the chemically synthesized ribozymes used in this study are shown below in Tables 3-8.
  • Ribozymes targeted to the human PTP-IB 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 PTP-IB RNA are given in Tables 3-8.
  • Full-length or partially full-length, internally-labeled target RNA for ribozyme cleavage assay is prepared by in vitro transcription in the presence of [ ⁇ - 32 p] CTP, passed over a G 50 Sephadex column by spin chromatography and used as subsfrate RNA without further purification.
  • substrates are 5'-32p-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-HCI, 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 o
  • Methionyl aminopeptidases are metalloproteases that are known to possess post- franslational enzymatic activity by hydrolytically cleaving amino-terminal methionine residues from nascent peptide substrates in a non-processive manner (Kendall, R. L., 1992, J. Biol. Chem., 267, 20667-20673). This family of enzymes is divided into two classes (type 1 and type 2) based on differences in sequence, although the overall structure ofthe two classes are similar (Liu, S., 1998, Science, 282, 1324-1327). Methionine aminopeptidase expression appears to be involved in the confrol of cellular proliferation.
  • NMT N-myristoyl transferase
  • VEGF vascular endothelial growth factor
  • Fumagillin, a sesquite ⁇ ene diepoxide metabolite ofthe fungus Aspergillus fumigatus, and a related compound TNP-470 are strong inhibitors of growth in cultured endothelial cells.
  • the antiproliferative and angiostatic activity of fumagillin was originally discovered by the serendipitous contamination of Aspergillus fumigatus in an endothelial cell culture dish in which cells closest to the fungal colony displayed growth inhibition.
  • Synthetic analogs of fumagillin were later synthesized resulting in the discovery of TNP- 470, which is 50 times more potent of an inhibitor than fumagillin and is less toxic in mice (Ingber, D., 1990, Nature, 348, 555-557).
  • TNP-470 inhibits the signaling pathway of retinoblastoma gene product phosphorylation, cyclin dependent kinases cdk2 and cdk4 activation, and cyclins E and A expression (Abe, J., 1994, Cancer Res., 54, 3407-3412). TNP-470 has also been shown to potently inhibit endothelial cell proliferation induced by the growth factors VEGF and bFGF (Toi, M., 1994, Oncology Reports, 1, 423-426). The bifunctional protein MetAP-2 has been identified as the molecular target for fumagillin and related compounds that demonstrate antiproliferative activity in endothelial cells.
  • MetAP-2 is the molecular target for these fumagillin-related compounds (Griffith, E. C, 1997, Chemistry & Biology, 4, 461-471). MetAP-2 expression correlates with cellular growth. Non-dividing cells in culture have no detectable levels ofthe 67-kDa MetAP-2 protein by immunoassay. MetAP-2 has been shown to affect franslational initiation by association with eukaryotic initiation factor 2 ⁇ (eIF-2 ) (Ray, M. K., 1992, Proc. Natl. Acad. Sci., 89, 539-543).
  • eIF-2 eukaryotic initiation factor 2 ⁇
  • MetAP-2 The binding of MetAP-2 with eLF-2 ⁇ inhibits the heme-regulated inhibitor kinase (HRI) phosphorylation of eLF-2 ⁇ in vitro in reticulocyte lysates (Datta, B., 1988, Proc. Natl. Acad. Sci., 85, 3324- 3328). MetAP-2/eIF-2 ⁇ binding results in the partial reversal of protein synthesis inhibition by double stranded RNA dependent kinase mediated phosphorylation in vivo (Wu, S., 1996, Biochemistry, 35, 8275-8280). Griffith et al.
  • HRI heme-regulated inhibitor kinase
  • angiogenesis related degenerative and disease states that can be associated with MetAP expression modulation include but are not limited to: Cancer: Solid tumors are unable to grow or metastasize without the formation of new blood vessels (Hanahan, D., 1996, Cell, 86, 353-364). Inhibition of angiogenesis via MetAP modulation can potentially be used to freat a wide variety of cancers.
  • Diabetic retinopathy and age related macular degeneration Ocular neovascularization is observed in diabetic retinopathy, which is mediated by up-regulation of VEGF (Adamis, A. P., 1994, Amer. J. Ophthal, 118, 445-450).
  • VEGF vascular endothelial growth factor
  • the requirement of protein kinase Src in hypoxia induced VEGF expression indicates that MetAP modulation of aminopeptidase activity can potentially be used to freat conditions involving ocular neovascularization.
  • Arthritis The ingrowth of a vascular pannus in arthritis may be mediated by the overexpression of angiogenic factors from infiltrating inflammatory cells, macrophages, and immune cells (Peacock, D. J., 1992, J. exp. Med., 175, 1135-1138).
  • Angiogenesis inhibition through MetAP modulation can potentially be used to freat arthritis.
  • Psoriasis Angiogenesis has been implicated in psoriasis due to overexpression of the angiogenic polypeptide interleukin-8 and decreased expression ofthe angiogenesis inhibitor thrombospondin (Nickoloff, B. J., 1994, Amer. J. Pathol. 44, 820-828).
  • Angiogenesis inhibition through MetAP modulation can potentially be used to treat psoriasis.
  • Angiogenesis in the female reproductive system has been implicated in several disorders ofthe reproductive tract (Reynolds, L. P., 1992, FASEB, 6, 886-892). Modulation of angiogenesis through confrol of MetAP may have various applications in the area of female reproduction and fertility. Various methods have been developed to assay MetAP activity.
  • Quantitative methods have been developed to assay the efficacy of antiangiogenic therapies.
  • angiogenic peptides (bFGF) in human serum as a prognostic test for breast cancer.
  • Nguyen et al, 1994, J. Narn. Cancer Inst., 86, 356-361 describe the quantitation of angiogenic peptides (bFGF) in the urine of patients with a wide spectrum of cancers.
  • Li et al, 1994, The Lancet, 344, 82-86 describe the quantitation of angiogenic peptides (bFGF) in the cerebrospinal fluid of children with brain tumors.
  • This work also describes determining the extent of neovascularization in histological sections by utilizing microvessel count.
  • the present body of knowledge in angiogenesis research indicates the need for compounds that can modulate MetAP activity for research, diagnostic, trait alteration, animal health and therapeutic use.
  • Chang et al, US patent No. 5,888,796 describe a clone of a nucleotide sequence encoding a protein having two functions comprising methionine aminopeptidase activity and anti eIF-2 phosphorylation activity.
  • a rat corneal model has been developed to study ribozyme inhibition of VEGF receptor-mediated angiogenesis (Pavco, P. A., 1999, Nucleic Acids Research, 27, 2569- 2577).
  • MetAP-2 inhibition could be used to study ribozyme based inhibition of MetAP-2 induced angiogenesis in vivo.
  • the sequence of human MetAP-2 was screened for accessible sites using a computer- folding algorithm. Regions ofthe RNA that did not form secondary folding structures and contained potential ribozyme and/or antisense binding/cleavage sites were identified. The sequences of these cleavage sites are shown in Tables 9-12.
  • Ribozyme target sites were chosen by analyzing sequences of Human MetAP-2 (Genbank accession number HSU29607) and prioritizing the sites on the basis of folding. Ribozymes were designed that could bind each target and were individually analyzed by computer folding (Christoffersen et al, 1994 J. Mol. Struc. Theochem, 311, 273; Jaeger et al, 1989, Proc.
  • ribozyme sequences fold into the appropriate secondary structure.
  • Those ribozymes with unfavorable intramolecular interactions between the binding arms and the catalytic core were eliminated from consideration.
  • 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 were designed to anneal to various sites in the RNA message.
  • the binding arms are complementary to the target site sequences described above.
  • the ribozymes were chemically synthesized. The method of synthesis used followed the procedure for normal RNA synthesis as described above and in Usman et al., (1987 J. Am. Chem. Soc, 109, 7845), Scaringe et al., (1990 Nucleic Acids Res., 18, 5433) and Wincott et al., supra, and made use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end. The average stepwise coupling yields were >98%.
  • Ribozymes were also synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol. 180, 51). Ribozymes were purified by gel electrophoresis using general methods or were purified by high pressure liquid chromatography (HPLC; see Wincott et al, supra; the totality of which is hereby inco ⁇ orated herein by reference) and were resuspended in water. The sequences of the chemically synthesized ribozymes used in this study are shown below in Table 9-12.
  • Ribozyme Cleavage of MetAP-2 RNA Target in vitro Ribozymes targeted to the human MetAP-2 RNA are designed and synthesized as described above. These ribozymes can be tested for cleavage activity in vitro, for example, using the following procedure. The target sequences and the nucleotide location within the MetAP-2 RNA are given in Tables 9-12.
  • 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- 32 p] CTP, passed over a G 50 Sephadex column by spin chromatography and used as subsfrate RNA without further purification.
  • substrates are 5'-32p-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-HCI, pH 7.5 at 37°C, 10 mM MgC ) 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 o
  • Alzheimer's disease is a progressive, degenerative disease ofthe brain which affects approximately 4 million people in the United States alone. An estimated 14 million Americans will have Alzheimer's disease by the middle ofthe next century if no cure or definitive prevention ofthe disease is found. Nearly one out often people over age 65 and nearly half of those over 85 have Alzheimer's disease. Alzheimer's disease is not confined to the elderly, a small percentage of people in their 30' s and 40 's are afflicted with early onset AD. Alzheimer's disease is the most common form of dementia, and amounts to the third most expensive disease in the US following heart disease and cancer. An estimated 100 billion dollars are spent annually on Alzheimer's disease (National Alzheimer's Association, 1999).
  • Alzheimer's disease is characterized by the progressive formation of insoluble plaques and vascular deposits in the brain consisting ofthe 4 kD amyloid ⁇ peptide (A ⁇ ). These plaques are characterized by dysfrophic neurites that show profound synaptic loss, neurofibrillary tangle formation, and gliosis.
  • a ⁇ arises from the proteolytic cleavage of the large type I transmembrane protein, ⁇ -amyloid precursor protein (APP) (Kang et al, 1987, Nature, 325, 733). Processing of APP to generate A ⁇ requires two sites of cleavage by a ⁇ -secretase and a ⁇ -secretase.
  • APP ⁇ -amyloid precursor protein
  • ⁇ -secretase cleavage of APP results in the cytoplasmic release of a 100 kD soluble amino-terminal fragment, APPs ⁇ , leaving behind a 12 kD transmembrane carboxy-terminal fragment, C99.
  • APP can be cleaved by a ⁇ - secretase to generate cytoplasmic APPs ⁇ and transmembrane C83 fragments.
  • Early onset familial Alzheimer's disease is characterized by mutant APP protein with a Met to Leu substitution at position PI, characterized as the "Swedish" familial mutation (Mullan et al, 1992, Nature Genet., 1, 345). This APP mutation is characterized by a dramatic enhancement in ⁇ -secretase cleavage (Citron et al, 1992, Nature, 360, 672).
  • ⁇ -secretase and ⁇ -secretase constituents involved in the release of ⁇ -amyloid protein is of primary importance in the development of treatment strategies for Alzheimer's disease. Characterization of ⁇ -secretase is also important in this regard since ⁇ -secretase cleavage may compete with ⁇ -secretase cleavage resulting in non- pathogenic vs. pathogenic protein production. Involvement ofthe two metalloproteases, ADAM 10, and TACE has been demonstrated in ⁇ -cleavage of AAP (Buxbaum et al, 1999, J. Biol. Chem., 273, 27765, and Lammich et al, 1999, Proc. Natl. Acad. Sci.
  • anti-inflammatory drugs may be associated with a reduced risk of Alzheimer's as well.
  • Calcium channel blockers such as Nimodipine® are considered to have a potential benefit in treating Alzheimer's disease due to protection of nerve cells from calcium overload, thereby prolonging nerve cell survival.
  • Nootropic compounds such as acetyl-L-carnitine (Alcar®) and insulin, have been proposed to have some benefit in treating Alzheimer's due to enhancement of cognitive and memory function based on cellular metabolism.
  • Alzheimer's patients there exists an unmet need in the comprehensive treatment and prevention of this disease. As such, there exists the need for therapeutics effective in reversing the physiological changes associated with Alzheimer's disease, specifically, therapeutics that can eliminate and/or reverse the deposition of amyloid ⁇ peptide.
  • therapeutics effective in reversing the physiological changes associated with Alzheimer's disease, specifically, therapeutics that can eliminate and/or reverse the deposition of amyloid ⁇ peptide.
  • the use of compounds to modulate the expression of proteases that are instrumental in the release of amyloid ⁇ peptide, namely ⁇ -secretase (BACE), and ⁇ -secretase (presenilin), is of therapeutic significance.
  • Specific antisense nucleic acid molecules targeting BACE mRNA were used for inhibition studies of endogenous BACE expression in 101 cells and APPsw
  • RNA sequence of human BACE was screened for accessible sites using a computer- folding algorithm. Regions ofthe RNA that did not form secondary folding structures and contained potential ribozyme and/or antisense binding/cleavage sites were identified. The sequences of these cleavage sites are shown in Tables 18-23.
  • Ribozyme target sites were chosen by analyzing sequences of Human BACE (Genbank sequence accession number: AF 190725) and prioritizing the sites on the basis of folding. Ribozymes were designed that could bind each target and were individually analyzed by computer folding (Christoffersen et al, 1994 J. Mol. Struc. Theochem, 311, 273; Jaeger et al, 1989, Proc. Natl. Acad. Sci. USA, 86, 7706) to assess whether the ribozyme sequences fold into the appropriate secondary structure. Those ribozymes with unfavorable intramolecular interactions between the binding arms and the catalytic core were eliminated from consideration. As noted below, varying binding arm lengths can be chosen to optimize activity. Generally, at least 5 bases on each arm are able to bind to, or otherwise interact with, the target RNA.
  • Ribozymes and antisense constructs were designed to anneal to various sites in the RNA message.
  • the binding arms ofthe 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 were also synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymo 180, 51). Ribozymes and antisense constructs were purified by gel elecfrophoresis using general methods or were purified by high pressure liquid chromatography (HPLC; See Wincott et al., supra; the totality of which is hereby inco ⁇ orated herein by reference) and were resuspended in water. The sequences ofthe chemically synthesized ribozymes and antisense constructs used in this study are shown below in Table 18-23.
  • Ribozymes targeted to the human BACE 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 BACE RNA are given in Tables 18-23.
  • 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- 32 p] CTP, passed over a G 50 Sephadex column by spin chromatography and used as substrate RNA without further purification.
  • substrates are 5'-32p-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-HCI, pH 7.5 at 37°C, 10 mM MgCl 2 ) and the cleavage reaction was initiated by adding the 2X ribozyme mix to an equal volume of substrate RNA (maximum of 1-5 nM) that was also pre-warmed in cleavage buffer. As an initial screen, assays are carried out for 1 hour at o 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 ofthe gel. The percentage of cleavage is determined by Phosphor Imager® quantitation of bands representing the intact subsfrate and the cleavage products.
  • Heart failure related disease represents a major public health issue due to an overall increase in prevalence and incidence in aging populations with a greater proportion of survivors of acute myocardial infarction (AMI) (Kannel et al, 1994, Br. Heart. J., 72 (suppl), 3).
  • AMD acute myocardial infarction
  • Heart failure related disease represents the most common reason for hospitalization of elderly patients in the US. The resulting life expectancy of these patients is less than that of many common cancers, with five year survival rates for men and women at only 25% and 38% respectively, and with one year mortality rates for severe heart failure at 50% (Ho et al, 1993, Circulation, 88, 107).
  • Heart disease is characterized by a progressive decrease in cardiac output resulting from insufficient pumping activity ofthe diseased heart.
  • the resulting venous back- pressure results in peripheral and pulmonary dysfunctional congestion.
  • the heart responds to a variety of mechanical, hemodynamic, hormonal, and pathological stimuli by increasing muscle mass in response to an increased demand for cardiac output.
  • the resulting transformation of heart tissue can arise as a result of genetic, physiologic, and environmental factors, and represents an early indication of clinical heart disease and an important risk factor for subsequent heart failure (Hunter and Chien, 1999, New England J. of Medicine, 99, 313-322).
  • Coronary heart disease is a predominant factor in the development ofthe cardiac disease state, along with prior AMI, hypertension, diabetes mellitus, and valvular heart disease.
  • Diagnosis of cardiac disease includes determination of coronary heart disease associated left ventricular systolic dysfunction (LVSD) and/or left ventricular diastolic dysfunction (LVDD) by echocaardiographic imaging (Cleland, 1997, Dis Management Health Outcomes, 1, 169). Promising diagnosis may also rely on assaying atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) concentrations. ANP and BNP levels are indicative ofthe level of ventricular dysfunction (Davidson et al, 1996, Am. J. Cardiol., 77, 828). Current treatment strategies for cardiac disease associated failure are varied.
  • Diuretics are often used to reduce pulmonary edema and dyspnea in patients with fluid overload, and are usually used in conjunction with angiotensin converting enzyme (ACE) inhibitors for vasodilation.
  • ACE angiotensin converting enzyme
  • Digoxin is another popular choice for treating cardiac disease as an ionofropic agent, however, doubts remain concerning the long-term efficacy and safety of Digoxin (Harnish, 1999, Drug & Market Development, 10, 114- 119).
  • Carvedilol a beta-blocker, has been introduced to complement the above treatments in order to slow down the progression of cardiac disease.
  • Antiarrhythmic agents can be used in order to reduce the risk of sudden death in patients suffering from cardiac disease.
  • heart transplants have been effective in the treatment of patients with advanced stages of cardiac disease, however, the limited supply of donor hearts greatly limits the scope of this treatment to the broad population (Hamish, 1999, Drug & Market Development, 10, 114- 119).
  • Endothelin 1 and angiotensin ⁇ receptor antagonists and antagonists of ras, p38, and c-jun ⁇ -terminal kinase (J ⁇ K) for inhibition of pathologic hypertrophy.
  • Insulin like growth factor I and growth hormone receptor stimulation for promotion of physiologic hypertrophy.
  • beta- 1 -adrenergic receptor blockers for inhibition of neurohumoral over stimulation.
  • Phospholamban and Sarcolipin small molecule inhibitors for relief of sarcoplasmic reticulum calcium ATPase inhibition to provide enhancement of myocardial contractile and relaxation responses.
  • Enhancement of angiogenic growth factors (VEGF, FGF-5) for relief of energy deprivation in cardiac tissues.
  • ⁇ euregulin for the inhibition of apoptosis of myocytes.
  • Inhibitors of apoptosis such as Caspase inhibitors for the inhibition of apoptosis of myocytes.
  • Inhibitors of cytokines such as T ⁇ F-alpha for the inhibition of apoptosis of myocytes are nonlimiting examples of disorders and disease states that can be associated with the above classes of molecular targets.
  • the release and uptake of cytosolic Ca 2+ by the sarcoplasmic reticulum plays an integral role in each cycle of cardiac confraction and excitation (Minamisawa et al, 1999, Cell, 99, 313-322).
  • SERCA2a activity is regulated by phospholamban, a p52 muscle specific sarcoplasmic reticulum phosphoprotein (Koss et al, 1996, Circ. Res., 79, 1059-1063, and Simmerman et al, 1998, Physiol. Rev., 78, 921-947). In its active, unphosphorylated state, phospholamban is a potent inhibitor of SERCA2a activity.
  • Phosphorylation of phospholamban at serine 16 by cyclic AMP-dependent protein kinase (PKA) or calmodulin kinase results in the inhibition of phospholamban interaction with SERCA2a.
  • PKA cyclic AMP-dependent protein kinase
  • calmodulin kinase results in the inhibition of phospholamban interaction with SERCA2a.
  • This phosphorylation event is predominantly responsible for the proportional increase in the rate of Ca 2+ uptake into the sarcoplasmic reticulum and resultant ventricular relaxation (Tada et al, 1982, Mol. Cell Biochem., 46, 73-95, and Luo et al, 1998, J. Biol. Chem., 273, 4734-4739).
  • nucleic acid molecules ofthe instant invention permits highly specific regulation ofthe molecular targets of interest, including phospholamban (PLN) (GenBank accession No. NM_002667), sarcolipin (SLN) (GenBank accession No. NM_003063), angiotensin II receptor (GenBank accession No. U20860), endothelin 1 receptor (GenBank accession No. NM 001957), K-ras (GenBank accession No. NM 004985), p38 (GenBank accession No.
  • c-jun N-terminal kinase (GenBank accession No. NM_002750, L31951, NM_002753), growth hormone receptor (GenBank accession No. NM_000163), insulin- like growth factor I receptor (GenBank accession No. NM_000875), beta- 1 -adrenergic receptor (GenBank accession No. NM_000024), ⁇ l-adrenergic receptor kinase (GenBank accession No. NM_001619, NM_005160), VEGF receptor (GenBank accession No. U43368, M27281 X15997), fibroblast growth factor 5 (GenBank accession No.
  • NM_004464 cardiofrophin I (GenBank accession No. NM_001330), neuregulin (GenBank accession No. AF009227), TNF-alpha (GenBank accession No. X02910 X02159), PI3 kinase (GenBank accession No. NM_006218, NM_006219, U86453, NM_002649, M61906), and AKT kinase (GenBank accession No. NM_005163, M77198).
  • Minamisawa et al, 1999, Cell, 99, 313-322 describe a phospholamban knockout mouse model which affords protection from induced dilated cardiomyopathy.
  • Dillmann et al, 1999, Am. J. Cardiol, 83, 89H-91H describe a fransgenic rat model for the study of altered expression of calcium regulatory proteins, including phospholamban, and their effect on myocyte contractile response.
  • Cardiol, 30, 1877-1888 describe a rat pressure-overload model to investigate alterations in gene expression of phospholamban, atrial natriuretic peptide (ANP), sarcoplasmic endoplasmic reticular calcium ATPase 2 (SERCA2), collagen LLI ⁇ l, and calsequestrin (CSQ).
  • ABP atrial natriuretic peptide
  • SERCA2 sarcoplasmic endoplasmic reticular calcium ATPase 2
  • CSQ calsequestrin
  • Jones et al, 1998, J. Clin. Invest., 101, 1385-1393 describe a mouse model for investigating the regulation of calcium signaling in transgenic mouse cardiac myocytes overexpressing calsequestrin. In this study, the upregulation and downregulation of calcium uptake and release proteins were determined, including phospholamban. Lorenz et al, 1997, Am J.
  • Physiol, 273, 6 describe a mouse model for the study of regulatory effects of phospholamban on cardiac function in intact mice. This study makes use of animal models with altered levels of phospholamban to permit in vivo evaluation ofthe physiological role of phospholamban.
  • Arai et al, 1996, Saishin Igaku, 51, 1095-1104 presents a review article of gene targeted animal models expressing cardiovascular abnormalities. The study of phospholamban and other protein expression modification effects in mice is presented. Wankerl et al, 1995, J. Mol. Med., 73, 487-496, presents a review article describing the study of calcium transport proteins in the nonfailing and failing heart.
  • Endpoints may be, but are not limited to, left ventricular pressure, left ventricular pressure as a function of time (LVdP/dt), and mean arterial blood pressure. Endpoints will be evaluated under basal and stimulated (cardiac load) conditions.
  • Particular degenerative and disease states that can be associated with phospholamban expression modulation include but are not limited to congestive heart failure, heart failure, dilated cardiomyopathy and pressure overload hypertrophy: Digoxin, Bendrofluazide, Dofetilide, and Carvedilol are non-limiting examples of pharmaceutical agents that can be combined with or used in conjunction with the nucleic acid molecules (e.g. ribozymes and antisense molecules) ofthe instant invention.
  • ribozymes and antisense molecules are non-limiting examples of pharmaceutical agents that can be combined with or used in conjunction with the nucleic acid molecules (e.g. ribozymes and antisense molecules) ofthe instant invention.
  • drugs such as diuretic and antihypertensive compounds and therapies can be similarly be readily combined with the nucleic acid molecules ofthe instant invention (e.g. ribozymes and antisense molecules) are hence within the scope ofthe instant invention.
  • RNA sequence of human phospholamban was screened for accessible sites using a computer folding algorithm. Regions ofthe RNA that did not form secondary folding structures and contained potential ribozyme and/or antisense binding/cleavage sites were identified. The sequences of these cleavage sites are shown in Tables 24-30.
  • Ribozyme target sites were chosen by analyzing sequences of Human phospholamban (Genbank sequence accession number: NM 002667) and prioritizing the sites on the basis of folding. Ribozymes were designed that could bind each target and were individually analyzed by computer folding (Christoffersen et al, 1994 J. Mol. Struc. Theochem, 311, 273; Jaeger et al, 1989, Proc. Natl. Acad. Sci. USA, 86, 7706) to assess whether the ribozyme sequences fold into the appropriate secondary structure.
  • 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 were designed to anneal to various sites in the RNA message.
  • the binding arms ofthe 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 were also synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol. 180, 51). Ribozymes and antisense constructs were purified by gel elecfrophoresis using general methods or were purified by high pressure liquid chromatography (HPLC; see Wincott et al., supra; the totality of which is hereby inco ⁇ orated herein by reference) and were resuspended in water. The sequences ofthe chemically synthesized ribozymes and antisense constructs used in this study are shown below in Table 24-30.
  • Ribozymes targeted to the human phospholamban 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 phospholamban RNA are given in Tables 24-30.
  • 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- 32 p] CTP, passed over a G 50 Sephadex column by spin chromatography and used as substrate RNA without further purification.
  • substrates are 5'-32p_end labeled using T4 polynucleotide kinase enzyme.
  • Assays are performed by pre-warming a 2X concenfration of purified ribozyme in ribozyme cleavage buffer (50 mM Tris-HCI, pH 7.5 at 37°C, 10 mM MgC ) 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
  • CD1 mice were injected with a single bolus (30 mg/kg) of a BrdU-labeled antisense oligonucleotide or a similar molar amount of BrdU (as a control). At various time points (30 min, 2h and 6 h), mice were sacrificed and major tissues isolated and fixed. Distribution of antisense oligonucleotides was determined by probing with an anti-BrdU antibody and immunohistochemical staining. Tissue slices were probed with an anti-BrdU antibody followed by a reporter enzyme-conjugated second antibody and finally an enzyme subsfrate. Visualization ofthe colored product by microscopy indicated nuclear staining, demonstrating effective distribution of antisense oligonucleotide in cardiac tissue.
  • Rhesus monkeys were dosed with BrdU-labeled ribozyme by intravenous bolus injection at 0.1 , 1.0, and 10 mg/kg once daily over five days. Saline injection was used in control animals. Animals were sacrificed and major tissues isolated and fixed. Tissue samples were probed with an anti-BrdU antibody followed by a reporter enzyme- conjugated second antibody and finally an enzyme subsfrate. Significant quantities of chemically modified ribozyme are detected in cardiac tissue following this dosing regimen.
  • HBV hepatitis B virus
  • infected blood or other body fluids especially saliva and semen
  • saliva and semen a virus that causes sexual activity, or sharing of needles contaminated by infected blood.
  • Individuals may be "carriers" and transmit the infection to others without ever having experienced symptoms ofthe disease.
  • Persons at highest risk are those with multiple sex partners, those with a history of sexually transmitted diseases, parenteral drug users, infants bom to infected mothers, "close” contacts or sexual partners of infected persons, and healthcare personnel or other service employees who have contact with blood. Transmission is also possible via tattooing, ear or body piercing,
  • Hepatitis B has never been documented as being a food-borne disease. The average incubation period is 60 to 90 days, with a
  • the determinants of severity include: (1) The size ofthe dose to which the person was exposed; (2) the person's age with younger patients experiencing a milder form ofthe disease; (3) the status ofthe immune system with those who are immunosuppressed experiencing milder cases; and (4) the presence or absence of co-infection with the Delta virus (hepatitis D), with more severe cases resulting from co-infection.
  • hepatitis D Delta virus
  • the acute phase ofthe disease may be accompanied by severe depression, meningitis, Guillain-Barre syndrome, myelitis, encephalitis, agranulocytosis, and/or thrombocytopenia.
  • Hepatitis B is generally self-limiting and will resolve in approximately 6 months. Asymptomatic cases can be detected by serologic testing, since the presence ofthe virus
  • HBsAg This antigen is the first and most useful diagnostic marker for active infections. However, if HBsAg remains positive for 20 weeks or longer, the person is likely to remain positive indefinitely and is now a carrier. While only 10% of persons over age 6 who contract HBV become carriers, 90% of infants infected during the first year of life do so.
  • Hepatitis B virus infects over 300 million people worldwide (Imperial, 1999, Gastroenterol. Hepatol, 14 (suppl), Sl-5). In the United States approximately 1.25 million individuals are chronic carriers of HBV as evidenced by the fact that they have measurable hepatitis B virus surface antigen HBsAg in their blood. The risk of becoming a chronic HBsAg carrier is dependent upon the mode of acquisition of infection as well as the age ofthe individual at the time of infection. For those individuals with high levels of viral replication, chronic active hepatitis with progression to cirrhosis, liver failure and hepatocellular carcinoma (HCC) is common, and liver transplantation is the only treatment option for patients with end-stage liver disease from HBV.
  • HCC hepatocellular carcinoma
  • Hepatitis B virus is a double-stranded circular DNA virus. It is a member ofthe Hepadnaviridae family. The virus consists of a central core that contains a core antigen (HBcAg) surrounded by an envelope containing a surface protein surface antigen (HBsAg)
  • HBcAg core antigen
  • HBsAg surface protein surface antigen
  • HBV uses a reverse transcriptase to transcribe a positive-sense full length RNA version of its genome back into DNA. This reverse transcriptase also contains DNA polymerase activity and thus begins replicating the newly synthesized minus-sense DNA strand. However, it appears that the core protein encapsidates the reverse-transcriptase/polymerase before it completes replication.
  • the virus From the free-floating form, the virus must first attach itself specifically to a host cell membrane. Viral attachment is one ofthe crucial steps which determines host and tissue specificity. However, currently there are no in vitro cell-lines that can be infected by HBV. There are some cells lines, such as HepG2, which can support viral replication only upon transient or stable transfection using HBV DNA.
  • the complete closed circular DNA genome of HBV remains in the nucleus and gives rise to four transcripts. These transcripts initiate at unique sites but share the same 3'-ends.
  • the 3.5-kb pregenomic RNA serves as a template for reverse transcription and also encodes the nucleocapsid protein and polymerase.
  • a subclass of this transcript with a 5 '-end extension codes for the precore protein that, after processing, is secreted as HBV e antigen.
  • the 2.4-kb RNA encompasses the pre-Sl open reading frame (ORF) that encodes the large surface protein.
  • the 2.1-kb RNA encompasses the pre-S2 and S ORFs that encode the middle and small surface proteins, respectively.
  • the smallest transcript ( ⁇ 0.8- kb) codes for the X protein, a transcriptional activator.
  • Multiplication ofthe HBV genome begins within the nucleus of an infected cell.
  • RNA polymerase LI transcribes the circular HBV DNA into greater-than-full length mRNA. Since the mRNA is longer than the actual complete circular DNA, redundant ends are formed. Once produced, the pregenomic RNA exits the nucleus and enters the cytoplasm.
  • RNA encapsidation is believed to occur as soon as binding occurs.
  • the HBV polymerase also appears to require associated core protein in order to function.
  • the HBV polymerase initiates reverse transcription from the 5' epsilon stem-loop three to four base pairs at which point the polymerase and attached nascent DNA are transferred to the 3' copy ofthe DR1 region. Once there, the (-)DNA is extended by the HBV polymerase while the RNA template is degraded by the HBV polymerase RNAse H activity.
  • RNAse H activity When the HBV polymerase reaches the 5' end, a small stretch of RNA is left undigested by the RNAse H activity. This segment of RNA is comprised of a small sequence just upstream and including the DR1 region. The RNA oligomer is then translocated and annealed to the DR2 region at the 5' end ofthe (-)DNA. It is used as a primer for the (+)DNA synthesis which is also generated by the HBV polymerase. It appears that the reverse transcription as well as plus strand synthesis may occur in the completed core particle.
  • the pregenomic RNA is required as a template for DNA synthesis, this RNA is an excellent target for ribozyme cleavage.
  • Nucleoside analogues that have been documented to inhibit HBV replication target the reverse transcriptase activity needed to convert the pregenomic RNA into DNA. Ribozyme cleavage ofthe pregenomic RNA template would be expected to result in a similar inhibition of HBV replication. Further, targeting the 3 '-end ofthe pregenomic RNA that is common to all HBV transcripts could result in reduction of all HBV gene products and an additional level of inhibition of HBV replication.
  • HBV does not infect cells in culture.
  • fransfection of HBV DNA (either as a head-to-tail dimer or as an "overlength" genome of >100%) into HuH7 or Hep G2 hepatocytes results in viral gene expression and production of HBV virions released into the media.
  • HBV replication competent DNA would be co-fransfected with ribozymes in cell culture.
  • Such an approach has been used to report infracellular ribozyme activity against HBV (zu Putlitz, et al, 1999, J. Virol, 73, 5381- 5387, and Kim et al, 1999, Biochem. Biophys. Res. Commun., 257, 759-765).
  • stable hepatocyte cell lines have been generated that express HBV. In these cells only ribozyme would need to be delivered; however, a delivery screen would need to be performed. In addition, stable hepatocyte cell lines have been generated that express HBV. Infracellular HBV gene expression can be assayed by a Taqman® assay for HBV RNA or by ELISA for HBV protein. Exfracellular virus can be assayed by PCR for DNA or ELISA for protein. Antibodies are commercially available for HBV surface antigen and core protein. A secreted alkaline phosphatase expression plasmid can be used to normalize for differences in transfection efficiency and sample recovery.
  • HBV replication There are several small animal models to study HBV replication. One is the fransplantation of HBV-infected liver tissue into irradiated mice. Viremia (as evidenced
  • HBV DNA is detectable in both liver and serum (Morrey et al, 1999, Antiviral Res., 42, 97-108).
  • An additional model is to establish subcutaneous tumors in nude mice with Hep G2 cells transfected with HBV. Tumors develop in about 2 weeks after inoculation and express HBV surface and core antigens. HBV DNA and surface antigen is also detected in the circulation of tumor-bearing mice (Yao et al, 1996, J. Viral Hepat., 3, 19-22).
  • Woodchuck hepatitis virus (WHV) is closely related to HBV in its virus structure
  • HCC chronic hepatitis and hepatocellular carcinoma
  • nucleoside analogue 3T3 was observed to cause dose dependent reduction in virus (50% reduction after two daily treatments at the highest dose) (Hurwitz et al, 1998. Antimicrob. Agents Chemother., 42, 2804-2809).
  • Interferon alpha is the most common therapy for HBV; however, recently Lamivudine (3TC) has been approved by the FDA.
  • Interferon alpha is one treatment for chronic hepatitis B. The standard duration of IFN-alpha therapy is 16 weeks, however, the optimal treatment length is still poorly defined.
  • a complete response HBV
  • Influenza-like symptoms include, fatigue, fever; myalgia, malaise, appetite loss, tachycardia, rigors, headache and arthralgias.
  • the influenza-like symptoms are usually short-lived and tend to abate after the first four weeks of dosing (Dusheiko et al, 1994, Journal of Viral Hepatitis, 1, 3-5).
  • Neuropsychiatric side effects include irritability, apathy, mood changes, insomnia, cognitive changes, and depression.
  • Lamivudine (3TC) is a nucleoside analogue, which is a very potent and specific inhibitor of HBV DNA synthesis. Lamivudine has recently been approved for the treatment of chronic Hepatitis B. Unlike treatment with interferon, treatment with 3TC does not eliminate the HBV from the patient. Rather, viral replication is controlled and chronic administration results in improvements in liver histology in over 50%> of patients. Phase m studies with 3TC, showed that treatment for one year was associated with reduced liver inflammation and a delay in scarring ofthe liver. In addition, patients treated with Lamivudine (lOOmg per day) had a 98 percent reduction in hepatitis B DNA and a significantly higher rate of seroconversion, suggesting disease improvements after completion of therapy.
  • HBV infection hepatitis
  • cancer hepatitis
  • tumorigenesis cirrhosis
  • Lamivudine (3TC), L-FMAU, adefovir dipivoxil, type 1 Interferon, therapeutic 5 vaccines, steriods, and 2 '-5' Oligoadenylates are non- limiting examples of pharmaceutical agents that can be combined with or used in conjunction with the nucleic acid molecules (e.g. ribozymes and antisense molecules) ofthe instant invention.
  • nucleic acid molecules e.g. ribozymes and antisense molecules
  • Oligoadenylates are non- limiting examples of pharmaceutical agents that can be combined with or used in conjunction with the nucleic acid molecules (e.g. ribozymes and antisense molecules) ofthe instant invention.
  • Those skilled in the art will recognize that other drugs such as diuretic and antihypertensive compounds or other therapies can similarly and readily be combined with the nucleic acid molecules ofthe
  • Draper US patent No. 6,017,756, describes the use of ribozymes for the inhibition of Hepatitis B Virus.
  • the methods of this invention can be used to treat human hepatitis B virus infections, which include productive virus infection, latent or persistent virus infection, and HBV-induced hepatocyte transformation.
  • the utility can be extended to other species of HBV which infect non-human animals where such infections are of veterinary importance.
  • Preferred target sites are genes required for viral replication, a non-limiting example includes genes for protein synthesis, such as the 5' most 1500 nucleotides ofthe HBV pregenomic mRNAs.
  • genes for protein synthesis such as the 5' most 1500 nucleotides ofthe HBV pregenomic mRNAs.
  • This region confrols the franslational expression ofthe core protein (C), X protein (X) and DNA polymerase (P) genes and plays a role in the replication ofthe viral DNA by serving as a template for reverse transcriptase. Disruption of this region in the RNA results in deficient protein synthesis as well as incomplete DNA synthesis (and inhibition of transcription from the defective genomes).
  • Target sequences 5' ofthe encapsidation site can result in the inclusion ofthe disrupted 3' RNA within the core virion structure and targeting sequences 3' ofthe encapsidation site can result in the reduction in protein expression from both the 3' and 5' fragments.
  • Targets outside ofthe 5' most 1500 nucleotides ofthe pregenomic mRNA also make suitable targets of enzymatic nucleic acid mediated inhibition of HBV replication.
  • targets include the mRNA regions that encode the viral S gene.
  • Targets in the minor mRNAs can also be used, especially when folding or accessibility assays in these other RNAs reveal additional target sequences that are unavailable in the pregenomic mRNA species.
  • a desirable target in the pregenomic RNA is a proposed bipartite stem-loop structure in the 3 '-end ofthe pregenomic RNA which is believed to be critical for viral replication (Kidd and Kidd-Ljunggren, 1996. Nuc. Acid Res. 24:3295-3302).
  • the 5'end ofthe HBV pregenomic RNA carries a cw-acting encapsidation signal, which has inverted repeat sequences that are thought to form a bipartite stem-loop structure. Due to a terminal redundancy in the pregenomic RNA, the putative stem-loop also occurs at the 3'-end. While it is the 5' copy which functions in polymerase binding and encapsidation, reverse franscription actually begins from the 3' stem-loop. To start reverse transcription, a 4 nt primer which is covalently attached to the polymerase is made, using a bulge in the 5' encapsidation signal as template.
  • This primer is then shifted, by an unknown mechanism, to the DR1 primer binding site in the 3' stem-loop structure, and reverse franscription proceeds from that point.
  • the 3' stem-loop, and especially the DR1 primer binding site, appear to be highly effective targets for ribozyme intervention.
  • Sequences ofthe pregenomic RNA are shared by the mRNAs for surface, core, polymerase, and X proteins. Due to the overlapping nature ofthe HBV transcripts, all share a common 3'-end. Ribozyme targeting this common 3'-end will thus cleave the pregenomic RNA as well as all ofthe mRNAs for surface, core, polymerase and X proteins.
  • the invention features a method for the analysis of HBV proteins. This method is useful in determining the efficacy of HBV inhibitors. Specifically, the instant invention features an assay for the analysis of HBsAg proteins and secreted alkaline phosphatase (SEAP) confrol proteins to determine the efficacy of agents used to modulate HBV expression.
  • SEAP alkaline phosphatase
  • the method consists of coating a micro-titer plate with an antibody such as anti- HBsAg Mab (for example, Biostride B88-95-31ad,ay) at 0.1 to 10 ⁇ g/ml in a buffer (for example, carbonate buffer, such as Na 2 CO 3 15 mM, NaHCO 3 35 mM, pH 9.5) at 4°C overnight.
  • a buffer for example, carbonate buffer, such as Na 2 CO 3 15 mM, NaHCO 3 35 mM, pH 9.5
  • the microtiter wells are then washed with PBST or the equivalent thereof, (for example, PBS, 0.05% Tween 20) and blocked for 0.1-24 hr at 37° C with PBST, 1% BSA or the equivalent thereof. Following washing as above, the wells are dried (for example, at 37° C for 30 min).
  • Biotinylated goat anti-HBsAg or an equivalent antibody is diluted (for example at 1:1000) in PBST and incubated in the wells (for example, 1 hr. at 37° C). The wells are washed with PBST (for example, 4x).
  • a conjugate, for example, Streptavidin/ Alkaline Phosphatase Conjugate, Pierce 21324 is diluted to 10-10,000 ng/ml in PBST, and incubated in the wells (for example, 1 hr. at 37° C).
  • a substrate for example, p-nitrophenyl phosphate substrate, Pierce 37620
  • a substrate for example, p-nitrophenyl phosphate substrate, Pierce 37620
  • the optical density is then determined (for example, at 405 nm).
  • SEAP levels are then assayed, for example, using the Great EscAPe® Detection Kit (Clontech K2041-1), as per the manufacturers instructions. In the above example, incubation times and reagent
  • Example 6 5 concentrations may be varied to achieve optimum results, a non-limiting example is described in Example 6.
  • the sequence of human HBV was screened for accessible sites using a computer- folding algorithm. Regions ofthe RNA that did not form secondary folding structures and ! 5 contained potential ribozyme and/or antisense binding/cleavage sites were identified. The sequences of these cleavage sites are shown in Tables 36-43.
  • Ribozyme target sites were chosen by analyzing sequences of Human HBV
  • Ribozymes were designed that could bind each target and were individually analyzed by computer folding (Christoffersen et al, 1994 J. Mol. Struc. Theochem, 311, 273; Jaeger et al, 1989, Proc. Natl. Acad. Sci. USA, 86, 7706) to assess whether the ribozyme sequences fold into the appropriate secondary structure. Those ribozymes with unfavorable
  • 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 were designed to anneal to various sites in the RNA message.
  • the binding arms ofthe ribozymes are complementary to the target site sequences described above, while the antisense constructs are fully complementary 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 were also synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol. 180, 51). Ribozymes and antisense constructs were purified by gel electrophoresis using general methods or were purified by high pressure liquid chromatography (HPLC; see Wincott et al., supra; the totality of which is hereby inco ⁇ orated herein by reference) and were resuspended in water. The sequences ofthe chemically synthesized ribozymes used in this study are shown below in Table 43.
  • Ribozymes targeted to the human HBV 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 !5 HBV RNA are given in Tables 36-43.
  • Full-length or partially full-length, internally-labeled target RNA for ribozyme cleavage assay is prepared by in vitro franscription in the presence of [ ⁇ - 32 p] CTP, passed over a G 50 Sephadex® column by spin chromatography and used as substrate RNA without further purification.
  • substrates are 5'-32p-end labeled 0 using T4 polynucleotide kinase enzyme.
  • Assays are performed by pre-warming a 2X concentration of purified ribozyme in ribozyme cleavage buffer (50 mM Tris-HCI, 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 o
  • the human hepatocellular carcinoma cell line Hep G2 was grown in Dulbecco's modified Eagle media supplemented with 10% fetal calf serum, 2 mM glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 25 mM Hepes, 100 units penicillin, and 100 ⁇ g/ml streptomycin.
  • To generate a replication competent cDNA prior to fransfection the HBV genomic sequences are excised from the bacterial plasmid sequence contained in the psHBV-1 vector (Those skilled in the art understand that other methods may be used to generate a replication competent cDNA). This was done with an EcoRI and Hind IU restriction digest. Following completion ofthe digest, a ligation was performed under dilute conditions (20 ⁇ g/ml) to favor intermolecular ligation. The total ligation mixture was then concentrated using Qiagen spin columns.
  • SEAP Secreted alkaline phosphatase
  • the pSEAP2-TK control vector was constructed by ligating a Bgl II-Hind HI fragment ofthe pRL-TK vector (Promega), containing the he ⁇ es simplex virus thymidine kinase promoter region, into Bgl HIHind HI digested pSEAP2- Basic (Clontech). Hep G2 cells were plated (3 x IO 4 cells/well) in 96-well microtiter plates and incubated overnight.
  • a lipid/DNA/ribozyme complex was formed containing (at final concentrations) cationic lipid (15 ⁇ g/ml), prepared psHBV-1 (4.5 ⁇ g/ml), pSEAP2-TK (0.5 ⁇ g/ml), and ribozyme (100 ⁇ M). Following a 15 min. incubation at 37° C, the complexes were added to the plated Hep G2 cells. Media was removed from the cells 96 hr. post-fransfection for HBsAg and SEAP analysis.
  • HBV ribozymes were co-transfected with HBV genomic DNA into Hep G2 cells, and the subsequent levels of secreted HBV surface antigen (HBsAg) were analyzed by ELISA.
  • HBV surface antigen HBV surface antigen
  • SEAP secreted alkaline phosphatase
  • Immulon 4 (Dynax) microtiter wells were coated overnight at 4° C with anti-HBsAg Mab (Biostride B88-95-31ad,ay) at 1 ⁇ g/ml in Carbonate Buffer (Na 2 CO 3 15 mM, NaHCO 3 35 mM, pH 9.5). The wells were then washed 4x with PBST (PBS, 0.05%
  • p-nitrophenyl phosphate substrate (Pierce 37620) was added to the wells, which were then incubated for 1 hr. at 37° C. The optical density at 405 nm was then determined. SEAP levels were assayed using the Great EscAPe® Detection Kit (Clontech K2041-1), as per the manufacturers instructions.
  • Example 7 X-gene Reporter Assay The effect of ribozyme treatment on the level of fransactivation of a SV40 promoter driven firefly luciferase gene by the HBV X-protein was analyzed in transfected Hep G2 cells. As a confrol for variability in transfection efficiency, a Renilla luciferase reporter driven by the TK promoter, which is not transactivated by the X protein, was used. Hep G2 cells were plated (3 x IO 4 cells/well) in 96-well microtiter plates and incubated overnight.
  • a lipid/DNA ribozyme complex was formed containing (at final concentrations) cationic lipid (2.4 ⁇ g/ml), the X-gene vector pSBDR(2.5 ⁇ g/ml), the firefly reporter pSV40HCVluc (0.5 ⁇ g/ml), the Renilla luciferase control vector pRL-TK (0.5 ⁇ g/ml), and ribozyme (100 ⁇ M). Following a 15 min. incubation at 37° C, the complexes were added to the plated Hep G2 cells. Levels of firefly and Renilla luciferase were analyzed 48 hr. post transfection, using Promega' s Dual-Luciferase Assay System.
  • the HBV X protein is a transactivator of a number of viral and cellular genes. Ribozymes which target the X region were tested for their ability to cause a reduction in X protein fransactivation of a firefly luciferase gene driven by the S V40 promoter in transfected Hep G2 cells. As a control for fransfection variability, a vector containing the Renilla luciferase gene driven by the TK promotor, which is not activated by the X protein, was included in the co-transfections.
  • the efficacy ofthe HBV ribozymes was determined by comparing the ratio of firefly luciferase: Renilla luciferase to that of a scrambled attenuated confrol (SAC) ribozyme. Eleven ribozymes (RPI18365, RPI18367, RPI18368, RPI18371, RPI18372, RPI18373, RPI18405, RPI18406, RPI18411, RPI18418, RPI18423) were identified which cause a reduction in the level of fransactivation of a reporter gene by the X protein, as compared to the corresponding SAC ribozyme.
  • a fransgenic mouse strain (founder strain 1.3.32 with a C57B1/6 background) that expresses HBV RNA and forms HBV viremia (Morrey et al, 1999, Antiviral Res., 42, 97- 108; Guidotti et al, 1995, J. Virology, 69, 10, 6158-6169) was utilized to study the in vivo activity of ribozymes ofthe instant invention. This model is predictive in screening for anti-HBV agents. Ribozyme or the equivalent volume of saline was administered via a continuous s.c infusion using Alzet® mini-osmotic pumps for 14 days.
  • Alzet® pumps were filled with test material(s) in a sterile fashion according to the manufacturer's instructions. Prior to in vivo implantation, pumps were incubated at 37°C overnight (> 18 hours) to prime the flow modulators. On the day of surgery, animals were lightly anesthetized with a ketamine/xylazine cocktail (94 mg/kg and 6 mg/kg, respectively; 0.3 ml, IP). Baseline blood samples (200 ⁇ l) were obtained from each animal via a retro- orbital bleed. A 2 cm area near the base ofthe tail was shaved and cleansed with betadine surgical scrub and sequentially with 70%> alcohol.
  • a 1 cm incision in the skin was made with a #15 scalpel blade or a blunt pair of scissors near the base ofthe tail. Forceps were used to open a pocket rosfrally (i.e., towards the head) by spreading apart the subcutaneous connective tissue. The pump was inserted with the delivery portal pointing away from the incision. Wounds were closed with sterile 9-mm stainless steel clips or with sterile 4-0 suture. Animals were then allowed to recover from anesthesia on a warm heating pad before being returned to their cage. Wounds were checked daily. Clips or sutures were replaced as needed. Incisions typically healed completely within 7 days post-op.
  • Table 44 is a summary ofthe group designation and dosage levels used in the HBV fransgenic mouse study.
  • animals treated with a ribozyme targeting site 273 (RPI.18341) of the HBV RNA showed a significant reduction in serum HBV DNA concenfration, compared to the saline treated animals as measured by a quantitative PCR assay.
  • the saline treated animals had a 69% increase in serum HBV DNA concentrations over this 2-week period while treatment with the 273 ribozyme (RPI.18341) resulted in a 60% decrease in serum HBV DNA concentrations.
  • HER2 (also known as neu, erbB2 and c-erbB2) is an oncogene that encodes a 185- kDa transmembrane tyrosine kinase receptor.
  • HER2 is a member ofthe epidermal growth factor receptor (EGFR) family and shares partial homology with other family members. In normal adult tissues HER2 expression is low. However, HER2 is overexpressed in at least 25-30% of breast (McGuire & Greene, 1989) and ovarian cancers (Berchuck, et al, 1990).
  • HER2 expression is high in aggressive human breast and ovarian cancers, but low in normal adult tissues, it is an attractive target for ribozyme-mediated therapy (Thompson et al, supra).
  • the greatest HER2 specific effects have been observed in cancer cell lines that express high levels of HER2 protein (as measured by ELISA).
  • Phenotypic endpoints include
  • HER2 protein expression 5 inhibition of cell proliferation, apoptosis assays and reduction of HER2 protein expression. Because overexpression of HER2 is directly associated with increased proliferation of breast and ovarian tumor cells, a proliferation endpoint for cell culture assays will be our primary screen. There are several methods by which this endpoint can be measured. Following treatment of cells with ribozymes, cells are allowed to grow (typically 5 days)
  • the inco ⁇ oration of [ 3 H] thymidine into cellular DNA and/or the cell density can be measured.
  • the assay of cell density is very straightforward and can be done in a 96-well format using commercially available fluorescent nucleic acid stains (such as Syto 13 or CyQuant).
  • the assay using CyQuant is in place at RPI and is currently being employed to screen -100 ribozymes targeting HER2 (details below).
  • HER2 protein expression can be evaluated using a HER2-specific ELISA.
  • the SKBR-3 cell line were be used for the initial screen because it has the higher level of HER2 protein, and thus should be most susceptible to a HER2-specific ribozyme.
  • follow- up studies can be carried out in T47D cells to confirm leads as necessary.
  • Ribozyme screens were be performed using an automated, high throughput 96-well cell proliferation assay. Cell proliferation were measured over a 5-day freatment period using the nucleic acid stain CyQuant for determining cell density.
  • the growth of cells treated with ribozyme/lipid complexes were compared to both untreated cells and to cells treated with Scrambled-arm attenuated core Confrols (SAC; or A; Figure 8). SACs can no longer bind to the target site due to the scrambled arm sequence and have nucleotide changes in the core that greatly diminish ribozyme cleavage. These SACs are used to determine non-specific inhibition of cell growth caused by ribozyme chemistry (i. e.
  • Electrode ribozymes are chosen from the primary screen based on their ability to inhibit cell proliferation in a specific manner. Dose response assays are carried out on these leads and a subset was advanced into a secondary screen using the level of HER2 protein as an endpoint.
  • a Taqman assay for measuring the ribozyme-mediated decrease in HER2 RNA has also been established.
  • This assay is based on PCR technology and can measure in real time the production of HER2 mRNA relative to a standard cellular mRNA such as GAPDH.
  • This RNA assay is used to establish proof that lead ribozymes are working through an RNA cleavage mechanism and result in a decrease in the level of HER2 mRNA, thus leading to a decrease in cell surface HER2 protein receptors and a subsequent decrease in tumor cell proliferation.
  • HER2 sensitive mouse tumor xenografts are those derived from human breast carcinoma cells that express high levels of HER2 protein.
  • nude mice bearing BT-474 xenografts were sensitive to the anti-HER2 humanized monoclonal antibody Herceptin, resulting in an 80%) inhibition of tumor growth at a 1 mg kg dose (ip, 2 X week for 4-5 weeks). Tumor eradication was observed in 3 of 8 mice freated in this manner (Baselga et al, 1998).
  • T47D Three human breast tumor cell lines (T47D, SKBR-3 and BT-474) were characterized to establish their growth curves in mice. These three cell lines have been implanted into the mammary papillae of both nude and SCLD mice and primary tumor volumes are measured 3 times per week. Growth characteristics of these tumor lines using a Matrigel implantation format will also be established. In addition, the use of two other breast cell lines that have been engineered to express high levels of HER2 are also being used. The tumor cell line(s) and implantation method that supports the most consistent and reliable tumor growth is used in animal studies testing the lead HER2 ribozyme(s).
  • Ribozyme are administered by daily subcutaneous injection or by continuous subcutaneous infusion from Alzet mini osmotic pumps beginning 3 days after tumor implantation and continuing for the duration ofthe study. Group sizes of at least 10 animals are employed. Efficacy is determined by statistical comparison of tumor volume of ribozyme-treated animals to a control group of animals freated with saline alone. Because the growth of these tumors is generally slow (45-60 days), an initial endpoint will be the time in days it takes to establish an easily measurable primary tumor (i.e. 50-100 mm 3 ) in the presence or absence of ribozyme treatment.
  • Stage I breast cancer the cancer is no larger than 2 centimeters and has not spread outside ofthe breast.
  • Stage LI the patient's tumor is 2-5 centimeters but cancer may have spread to the axillary lymph nodes.
  • Stage LU metastasis to the lymph nodes is typical, and tumors are 5 centimeters. Additional tissue involvement (skin, chest wall, ribs, muscles etc.) may also be noted.
  • the therapy regimen employed depends not only on the stage ofthe cancer at its time of removal, but other variables such the type of cancer (ductal or lobular), whether lymph nodes were involved and removed, age and general health ofthe patient and if other organs are involved.
  • Common chemotherapies include various combinations cytotoxic drugs to kill the cancer cells. These drugs include paclitaxel (Taxol), docetaxel, cisplatin, methotrexate, cyclophosphamide, doxorubin, fluorouracil etc.
  • Significant toxicities are associated with these cytotoxic therapies.
  • Well-characterized toxicities include nausea and vomiting, myelosuppression, alopecia and mucosity.
  • Serious cardiac problems are also associated with certain ofthe combinations, e.g. doxorubin and paclitaxel, but are less common.
  • SERMs selective esfrogen receptor modulators
  • Tamoxifen is one such compound.
  • the primary toxic effect associated with the use of tamoxifen is a 2 to 7-fold increase in the rate of endometrial cancer. Blood clots in the legs and lung and the possibility of stroke are additional side effects.
  • tamoxifen has been determined to reduce breast cancer incidence by 49% in high-risk patients and an extensive, somewhat controversial, clinical study is underway to expand the prophylactic use of tamoxifen.
  • Another SERM, raloxifene was also shown to reduce the incidence of breast cancer in a large clinical trial where it was being used to freat osteoporosis.
  • removal ofthe ovaries and/or drugs to keep the ovaries from working are being tested.
  • Bone marrow fransplantation is being studied in clinical trials for breast cancers that have become resistant to traditional chemotherapies or where >3 lymph nodes are involved. Marrow is removed from the patient prior to high-dose chemotherapy to protect it from being destroyed, and then replaced after the chemotherapy.
  • Another type of "fransplant” involves the exogenous freatment of peripheral blood stem cells with drugs to kill cancer cells prior to replacing the freated cells in the bloodstream.
  • One biological freatment a humanized monoclonal anti-HER2 antibody, Herceptin
  • Herceptin binds with high affinity to the exfracellular domain of HER2 and thus blocks its signaling action. Herceptin can be used alone or in combination with chemotherapeutics (i.e. paclitaxel, docetaxel, cisplatin, etc.) (Pegram, et al, 1998). In Phase LTJ studies, Herceptin significantly improved the response rate to chemotherapy as well as improving the time to progression (Ross & Fletcher, 1998).
  • Herceptin The most common side effects attributed to Herceptin are fever and chills, pain, asthenia, nausea, vomiting, increased cough, diarrhea, headache, dyspnea, infection, rhinitis, and insomnia.
  • Herceptin in combination with chemotherapy can lead to cardiotoxicity (Sparano, 1999), leukopenia, anemia, diarrhea, abdominal pain and infection.
  • HER2 levels can be detected in at least 30% of breast cancers
  • breast cancer patients can be pre-screened for elevated HER2 prior to admission to initial clinical trials testing an anti-HER2 ribozyme.
  • Initial HER2 levels can be determined (by ELISA) from tumor biopsies or resected tumor samples. During clinical trials, it may be possible to monitor circulating HER2 protein by ELISA (Ross and Fletcher, 1998). Evaluation of serial blood/serum samples over the course ofthe anti-HER2 ribozyme freatment period could be useful in determining early indications of efficacy. In fact, the clinical course of Stage TV breast cancer was correlated
  • CA27.29 levels are higher than CA15.3 in breast cancer patients; the reverse is
  • CA27.29 was found to better discriminate primary cancer from healthy subjects.
  • a statistically significant and direct relationship was shown between CA27.29 and large vs small tumors and node postive vs node negative disease (Gion, et al, 1999).
  • both cancer antigens were found to be suitable for the detection of possible metastases during follow-up (Rodriguez
  • CA27.29 and CA15.3 for monitoring metastatic breast cancer patients have been filed (reviewed in Beveridge, 1999). Fully automated methods for measurement of either of these markers are commercially available.
  • Ribozyme for Breast Cancer In Methods in Molecular Medicine, Vol. 11, Therapeutic Applications of Ribozmes, Human Press, Inc., Totowa, NJ. aughn, J.P., Iglehart, J.D., Demirdji, S., Davis, P., Babiss, L.E., Caruthers, M.H., Marks, J.R. (1995) Antisense DNA downregulation ofthe ERBB2 oncogene measured by a flow cytometric assay. Proc Natl Acad Sci USA 92: 8338-8342.
  • Applicant has designed, synthesized and tested several NCH ribozymes and HH ribozymes targeted against HER2 RNA (see for example Tables 31 and 34) in cell proliferation assays.
  • the model proliferation assay used in the study can require a cell plating density of 2000 cells/well in 96-well plates and at least 2 cell doublings over a 5-day freatment period.
  • the FFPS fluoro-imaging processing system
  • This method allows for cell density measurements after nucleic acids are stained with CyQuant dye, and has the advantage of accurately measuring cell densities over a very wide range 1,000-100,000 cells/well in 96- well format.
  • Ribozymes (50-200 nM) were delivered in the presence of cationic lipid at 2.0 ⁇ g/mL and inhibition of proliferation was determined on day 5 post-treatment. Two full ribozyme screens were completed and 4 lead HH and 11 lead NCH ribozymes were chosen for further testing. Ofthe 15 lead Rzs chosen from primary screens, 4 NCH and 1 HH Rzs continued to inhibit cell proliferation in subsequent experiments. NCH Rzs against sites, 2001 (RPI No. 17236), 2783 (RPI No. 17249), 2939 (RPI No. 17251) or 3998 (RPI No. 17262) caused inhibition of proliferation ranging from 25-60% as compared to a scrambled confrol Rz (LA; RPI No.
  • NCH Rz Rz (RPI No. 17251) against site 2939 of HER2 RNA.
  • FIG 8. An example of results from cell culture assay is shown in Figure 8. Referring to Figure 8, NCH ribozymes and a HH ribozyme targeted against HER2 RNA, are shown to cause significant inhibition of proliferation of cells. This shows that ribozymes, for instance the NCH ribozymes are capable of inhibiting HER2 gene expression in mammalian cells.
  • Example 8 Activity of Class LI (Zinzyme) nucleic acid catalysts to inhibit HER2 gene expression
  • Applicant has designed, synthesized and tested several class ⁇ (zinzyme) ribozymes targeted against HER2 RNA (see, for example, Tables 58, 59, and 62) in cell proliferation RNA reduction assays.
  • the model proliferation assay used in the study requires a cell-plating density of 2000-10000 cells/well in 96-well plates and at least 2 cell doublings over a 5-day treatment period.
  • Cells used in proliferation studies were either human breast or ovarian cancer cells (SKBR-3 and SKOV-3 cells respectively).
  • the FLPS (fluoro-imaging processing system) method well known in the art was used. This method allows for cell density measurements after nucleic acids are stained with CyQuant® dye, and has the advantage of accurately measuring cell densities over a very wide range 1,000-100,000 cells/well in 96-well format.
  • Ribozymes (50-200 nM) were delivered in the presence of cationic lipid at 2.0-5.0 ⁇ g/mL and inhibition of proliferation was determined on day 5 post-treatment. Two full ribozyme screens were completed resulting in the selection of 14 ribozymes.
  • FIG. 20 An example of results from a cell culture assay is shown in Figure 20.
  • Class II ribozymes targeted against HER2 RNA are shown to cause significant inhibition of proliferation of cells. This shows that ribozymes, for instance the Class II (zinzyme) ribozymes are capable of inhibiting HER2 gene expression in mammalian cells.
  • Real time RT-PCR (TaqMan® assay) was performed on purified RNA samples using separate primer/probe sets specific for either target HER2 RNA or control actin RNA (to normalize for differences due to cell plating or sample recovery). Results are shown as the average of triplicate determinations of HER2 to actin RNA levels post- treatment.
  • Figure 30 shows class LI ribozyme (zinzyme) mediated reduction in HER2 RNA targeting site 972 vs a scrambled attenuated confrol.
  • Dose response assays Active ribozyme was mixed with binding arm-attenuated confrol (BAC) ribozyme to a final oligonucleotide concenfration of either 100, 200 or 400 nM and delivered to cells in the presence of cationic lipid at 5.0 ⁇ g/mL. Mixing active and BAC in this manner maintains the lipid to ribozyme charge ratio throughout the dose response curve.
  • HER2 RNA reduction was measured 24 hours post-treatment and inhibition of proliferation was determined on day 5 post-treatment.
  • the dose response antiproliferation results are summarized in Figure 31 and the dose-dependent reduction of HER2 RNA results are summarized in Figure 32.
  • Figure 33 shows a combined dose response plot of both anti- proliferation and RNA reduction data for a class LI ribozyme targeting site 972 of HER2 RNA (RPI 19293).
  • Example 9 Compositions having RNA cleaving activity
  • Hammerhead ribozymes are an example of catalytic RNA molecules which are able to recognize and cleave a given specific RNA subsfrate (Hutchins et ⁇ /.,1986, Nucleic Acids Res. 14:3627; Keese and Symons, in Viroids and viroid-like pathogens (J.J. Semanchik, publ., CRC-Press, Boca Raton, Florida, 1987, pages 1-47).
  • the catalytic center of hammerhead ribozymes is flanked by three stems and can be formed by adjacent sequence regions ofthe RNA or also by regions, which are separated from one another by many nucleotides.
  • Figure 6 shows a diagram of such a catalytically active hammerhead structure.
  • the stems have been denoted I, IL and ILI.
  • the nucleotides are numbered according to the standard nomenclature for hammerhead ribozymes (Hertel et al, 1992, Nucleic Acids Res. 20:3252).
  • bases are denoted by a number, which relates their position relative to the 5' side ofthe cleavage site.
  • each base that is involved in a stem or loop region has an additional designation (which is denoted by a decimal point and then another number) that defines the position of that base within the stem or loop.
  • a designation of A 15 ⁇ would indicate that this base is involved in a paired region and that it is the first nucleotide in that stem going away from the core region.
  • This accepted convention for describing hammerhead-derived ribozymes allows for the nucleotides involved in the core ofthe enzyme to always have the same number relative to all ofthe other nucleotides.
  • the size ofthe stems involved in substrate binding or core formation can be any size and of any sequence, and the position of A 9 , for example, will remain the same relative to all ofthe other core nucleotides.
  • Nucleotides designated, for example, N ⁇ 12 or N 9 ⁇ represent an inserted nucleotide where the position ofthe caret ( ⁇ ) relative to the number denotes whether the insertion is before or after the indicated nucleotide.
  • N ⁇ represents a nucleotide inserted before nucleotide position 12
  • N 9 ⁇ represents a nucleotide inserted after nucleotide position 9.
  • this special hammerhead structure exhibits a very effective self-catalytic cleavage despite the more open central stem.
  • hammerhead ribozymes Possible uses include, for example, generation of RNA restriction enzymes and the specific inactivation ofthe expression of genes in, for example, animal, human or plant cells and prokaryotes, yeasts and plasmodia.
  • a particular biomedical interest is based on the fact that many diseases, including many forms of tumors, are related to the overexpression of specific genes. Inactivating such genes by cleaving the associated mRNA represents a possible way to confrol and eventually treat such diseases.
  • Ribozymes have potential as such anti-infective agents since RNA molecules vital to the survival ofthe organism can be selectively destroyed.
  • N can e my nuc ieotide
  • U is uridine
  • H is either adenosine, cytidine, or uridine
  • NUH is sometimes designated as NUX.
  • Efficient catalytic molecules with reduced or altered requirements in the cleavage region are highly desirable because their isolation would greatly increase the number of available target sequences that molecules of this type could cleave. For example, it would be desirable to have a ribozyme variant that could efficiently cleave substrates containing triplets other than N U H since this would increase the number of potential target cleavage sites.
  • oligonucleotides which contain a block of deoxyribonucleotides in the middle region ofthe molecule have potential as pharmaceutical agents for the specific inactivation ofthe expression of genes (Giles et al, 1992, Nucleic Acids Res. 20:763-770). These oligonucleotides can form a hybrid DNA- RNA duplex in which the DNA bound RNA strand is degraded by RNase H. Such oligonucleotides are considered to promote cleavage ofthe RNA and so cannot be characterized as having an RNA-cleaving activity nor as cleaving an RNA molecule (the RNase H is cleaving). A significant disadvantage of these oligonucleotides for in vivo applications is their low specificity, since hybrid formation, and thus cleavage, can also take place at undesired positions on the RNA molecules.
  • 5,334,711 describe such chemically modified active substances based on synthetic catalytic oligonucleotide structures with a length of 35 to 40 nucleotides which are suitable for cleaving a nucleic acid target sequence and contain modified nucleotides that contain an optionally substituted alkyl, alkenyl or alkynyl group with 1 - 10 carbon atoms at the 2'-O atom ofthe ribose. These oligonucleotides contain modified nucleotide building blocks and form a structure resembling a hammerhead structure. These oligonucleotides are able to cleave specific RNA subsfrates.
  • Sullivan et al US Patent No. 5,616,490 describe enzymatic RNA molecules targeted against protein kinase C (PKC) RNA.
  • PLC protein kinase C
  • Sioud International PCT publication No. WO 99/63066 describe hammerhead ribozymes targeted against specific sites within protein kinase C alpha (PKC alpha), VEGF, and TNF alpha RNA.
  • Jarvis et al International PCT publication No. WO 98/505030, describe the synthesis of xylo-ribonucleosides and oligonucleotides comprising xylo modifications.
  • This invention relates to novel enzymatic nucleic acid molecules having an RNA- cleavage activity, as well as their use for cleaving RNA substrates in vitro and in vivo.
  • the compositions contain an active center, the subunits of which are selected from nucleotides and/or nucleotide analogues, as well as flanking regions contributing to the formation of a specific hybridization with an RNA subsfrate.
  • compositions form, in combination with an RNA substrate, a structure resembling a hammerhead structure.
  • the active center ofthe disclosed compositions is characterized by the presence of I 15 ' which allows cleavage of RNA substrates having C 16 -1 . It is therefore an object of the present invention to provide compositions that cleave RNA, and in particular to provide RNA- cleaving oligomers which at the same time have a high stability, activity, and specificity.
  • This invention relates to novel nucleic acid molecules with catalytic activity, which are particularly useful for cleavage of RNA or DNA or combination thereof.
  • the nucleic acid catalysts ofthe instant invention are distinct from other nucleic acid catalysts known in the art.
  • nucleic acid catalysts ofthe instant invention are capable of catalyzing an intermolecular or intramolecular endonuclease reaction. It is another object ofthe present invention to provide compositions that cleave RNA subsfrates having a cleavage site triplet other than N 16,2 U 16 1 H 17 (NUH; Figure 6), where N is a nucleotide, U is uridine and H is adenosine, uridine or cytidine. H is used interchangably with X.
  • the enzymatic nucleic acid molecule ofthe instant invention has an endonuclease activity to cleave RNA substrates having a cleavage triplet N i6 . 2 .
  • the invention features an enzymatic nucleic acid molecule ofthe instant invention has an endonuclease activity to cleave RNA substrates having a cleavage triplet N 162 C 16 1 N 17 (NCN; Figure 6), where N is a nucleotide, C is cytidine.
  • the invention features an enzymatic nucleic acid molecule having formula 1 :
  • N represents independently a nucleotide or a non-nucleotide linker, which may be same or different;
  • D and E are independently oligonucleotides of length sufficient to stably interact (e.g., by forming hydrogen bonds with complementary nucleotides in the target) with a target nucleic acid molecule (the target can be an RNA, DNA or mixed polymers), preferably, the length of D and E are independently between 3-20 nucleotides long, specifically, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, and 20;
  • o and n are integers independently greater than or equal to 1 and preferably less than about 100, specifically 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 50, wherein if (N) 0 and (N)n are nucleotides, (N)o and (N)n are optional
  • A, U, I, C and G represent adenosine, uridine, inosine, cytidine and guanosine nucleotides, respectively.
  • the N in 5'-CUGANGA-3' region of formula 1 is preferably U.
  • the nucleotides in the formula 1 are unmodified or modified at the sugar, base, and/or phosphate as known in the art.
  • the invention features an enzymatic nucleic acid molecule having formula 2:
  • N represents independently a nucleotide or a non-nucleotide linker, which may be same or different;
  • D and E are independently oligonucleotides of length sufficient to stably interact (e.g., by forming hydrogen bonds with complementary nucleotides in the target) with a target nucleic acid molecule (the target can be an RNA, DNA or mixed polymers), preferably, the length of D and E are independently between 3-20 nucleotides long, specifically, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, and 20;
  • o and n are integers independently greater than or equal to 0 and preferably less than about 100, specifically 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 50, wherein if (N) 0 and (N) n are nucleotides, (N)o and (N)n are optionally able to interact by hydrogen bond interaction; • indicates base- paired interaction;
  • L is a linker which may be present or absent (i.e
  • A, U, I, C and G represent adenosine, uridine, inosine, cytidine and guanosine nucleotides, respectively.
  • the N in 5'-CUGANGA-3' region of formula 2 is preferably U.
  • the nucleotides in the formula 2 are unmodified or modified at the sugar, base, and/or phosphate as known in the art.
  • the I (inosine) in formula 1 and 2 is preferably a riboinosine or a xylo-inosine.
  • the nucleotide linker (L) is 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. Biochem., 64, 763; and Szostak & Ellington, 1993, in The RNA World, ed. Gesteland and Atkins, pp 511, CSH Laboratory Press).
  • RRE HIV Rev aptamer
  • TAR HIV Tat aptamer
  • a "nucleic acid aptamer” as used herein is meant to indicate 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.
  • L has the sequence 5 '-GAAA-3 ' or 5 '-GUUA-3 ' .
  • non-nucleotide linker (L) is as defined herein.
  • non-nucleotide includes 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, 775:5109; Ma et al., Nucleic Acids Res.
  • Non-nucleotide linkers can be any molecule, which is not an oligomeric sequence, that can be covalently coupled to an oligomeric sequence.
  • Preferred non-nucleotide linkers are oligomeric molecules formed of non-nucleotide subunits. Examples of such non-nucleotide linkers are described by Letsinger and Wu, (J. Am. Chem. Soc. 117:7323-7328 (1995)), Benseler et al, (J. Am. Chem. Soc. 115:8483-8484 (1993)) and Fu et al. , (J. Am. Chem. Soc.
  • non-nucleotide linkers or subunits for non-nucleotide linkers, include substituted or unsubstituted Ci-Cio straight chain or branched alkyl, substituted or unsubstituted C 2 -C 10 straight chain or branched alkenyl, substituted or unsubstituted C 2 - C 10 straight chain or branched alkynyl, substituted or unsubstituted C do straight chain or branched alkoxy, substituted or unsubstituted C 2 -C 10 straight chain or branched alkenyloxy, and substituted or unsubstituted C 2 -C ⁇ 0 straight chain or branched alkynyloxy.
  • the substituents for these prefe ⁇ ed non-nucleotide linkers can be halogen, cyano, amino, carboxy, ester, ether, carboxamide, hydroxy, or mercapto.
  • 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.
  • non-nucleotide any group or compound which can be inco ⁇ orated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity.
  • the group or compound is abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine.
  • abasic or “abasic nucleotide” as used herein encompass sugar moieties lacking a base or having other chemical groups in place of nucleotide base at the 1' position.
  • the invention features modified ribozymes with phosphate backbone modifications comprising one or more phosphorothioate, phosphorodithioate, methylphosphonate, mo ⁇ holino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl, substitutions.
  • an inverted deoxy abasic moiety is utilized at the 3' end ofthe enzymatic nucleic acid molecule.
  • pyrimidines nucleotides comprising modified or unmodified derivatives of a six membered pyrimidine ring.
  • An example of a pyrimidine is modified or unmodified uridine.
  • the nucleosides ofthe instant invention include, 2'-O- methyl-2,6-diaminopurine riboside; 2'-deoxy-2'amino-2,6-diaminopurine riboside; 2'-(N- alanyl) amino-2'-deoxy-uridine; 2'-(N-phenylalanyl)amino-2'-deoxy-uridine; 2'-deoxy -2'- (N-beta-alanyl) amino ; 2'-deoxy-2'-(lysiyl) amino uridine; 2'-C-allyl uridine; 2'-O-amino- uridine; 2'-O-methylthiomethyl adenosine; 2'-O-methylthiomethyl cytidine ; 2'-O- methylthiomethyl guanosine; 2'-O-methylthiomethyl-uridine; 2'-Deoxy-2'-(N- alanyl) amino-2'-de
  • oligonucleotide as used herein is meant a molecule having two or more nucleotides.
  • the polynucleotide can be single, double or multiple stranded and may have modified or unmodified nucleotides or non-nucleotides or various mixtures and combinations thereof.
  • the enzymatic nucleic acid molecule of formula 1 or 2 include at least three ribonucleotide residues, preferably 4, 5, 6, 7, 8, 9, and 10 ribonucleotide residues.
  • the enzymatic nucleic acid ofthe instant invention includes one or more stretches of R ⁇ A, which provide the enzymatic activity ofthe molecule, linked to the non-nucleotide moiety.
  • R ⁇ A The necessary R ⁇ A components are known in the art (see for e.g., Usman et al, supra).
  • the invention features enzymatic nucleic acid molecules that inhibit gene expression and/or cell proliferation in vitro or in vivo (e.g. in patients).
  • These chemically or enzymatically synthesized nucleic acid molecules contain substrate binding domains that bind to accessible regions of specific target nucleic acid molecules.
  • the nucleic acid molecules also contain domains that catalyze the cleavage of target.
  • the enzymatic nucleic acid molecules cleave the target molecules, preventing for example, translation and protein accumulation. In the absence ofthe expression ofthe target gene, cell proliferation, for example, is inhibited.
  • catalytic activity ofthe molecules described in the instant invention can be optimized as described by Draper et al., supra. The details will not be repeated here, but include altering the length ofthe ribozyme binding arms, or chemically synthesizing ribozymes with modifications (base, sugar and/or phosphate) that prevent their degradation by serum ribonucleases and/or enhance their enzymatic activity (see e.g., Eckstein et al, International Publication No. WO 92/07065; Pe ⁇ ault et al, 1990 Nature 344, 565; Pieken et al., 1991 Science 253, 314; Usman and Cedergren, 1992 Trends in Biochem. Sci.
  • nucleic acid catalyst as used herein is meant a nucleic acid molecule (e.g., the molecule of formulae 1 and 2) capable of catalyzing (altering the velocity and/or rate of) a variety of reactions including the ability to repeatedly cleave other separate nucleic acid molecules (endonuclease activity) in a nucleotide base sequence-specific manner.
  • a molecule with endonuclease activity may have complementarity in a subsfrate binding region to a specified gene target, and also has an enzymatic activity that specifically cleaves RNA or DNA in that target.
  • the nucleic acid molecule with endonuclease activity is able to inframolecularly or intermolecularly cleave RNA or DNA and thereby inactivate a target RNA or DNA molecule.
  • This complementarity functions to allow sufficient hybridization ofthe enzymatic RNA molecule to the target RNA or DNA to allow the cleavage to occur. 100%> complementarity is prefe ⁇ ed, but complementarity as low as 50-75%) may also be useful in this invention.
  • the nucleic acids may be modified at the base, sugar, and/or phosphate groups.
  • enzymatic nucleic acid as used herein is used interchangeably with phrases such as ribozymes, catalytic RNA, enzymatic RNA, catalytic oligonucleotides, nucleozyme, RNA enzyme, endoribonuclease, endonuclease, minizyme, oligozyme, finderon or nucleic acid catalyst. All of these terminologies describe nucleic acid molecules ofthe instant invention 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 ofthe target nucleic acid regions, and that it have nucleotide sequences within or su ⁇ ounding that substrate binding site which impart a nucleic acid cleaving activity to the molecule (Cech et al., U.S. Patent No. 4,987,071; Cech et al, 1988, 260 JAMA 3030).
  • the enzymatic nucleic acid molecule of Formula 1 or 2 may independently comprise a cap structure which may independently be present or absent.
  • chimeric nucleic acid molecule or “mixed polymer” is meant that, the molecule maybe comprised of both modified or unmodified nucleotides.
  • the 3 '-cap is selected from a group comprising,
  • nucleotide 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 ⁇ re ⁇ -pentofuranosyl nucleotide; acyclic
  • 3',4'-seco nucleotide 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5'-5'- inverted nucleotide moiety; 5'-5'-inverted abasic moiety; 5'-phosphoramidate; 5'- phosphorothioate; 1,4-butanediol phosphate; 5'-amino; bridging and/or non-bridging 5'- phosphoramidate, phosphorothioate and/or phosphorodithioate, bridging or non bridging methylphosphonate and 5'-mercapto moieties (for more details, see Beaucage and Iyer, 1993, Tetrahedron 49, 1925; inco ⁇ orated by reference herein).
  • non- nucleotide any group or compound which can be inco ⁇ orated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity.
  • the group or compound is abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine.
  • abasic or “abasic nucleotide” as used herein encompass sugar moieties lacking a base or having other chemical groups in place of a base at the 1' position.
  • the invention features l-(beta-D-xylofuranosyl)- xypoxanthine phosphoramidite and a process for the synthesis thereof and inco ⁇ oration into oligonucleotides, such as enzymatic nucleic acid molecule.
  • the invention features enzymatic nucleic acid molecules targeted against HER2 RNA, specifically, ribozymes in the hammerhead and NCH motifs.
  • the invention features enzymatic nucleic acid molecules targeted against PKC alpha RNA, specifically, ribozymes in the hammerhead and NCH motifs.
  • Targets, for example PKC alpha RNA, for useful ribozymes and antisense nucleic acids can be determined, for example, as described in Draper et al, WO 95/04818; McSwiggen et al, U.S. Patent Nos.
  • 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 subsfrate binding site (e.g., D and E of Formula 1 above) which is complementary to one or more ofthe target nucleic acid regions, and that it have nucleotide sequences within or su ⁇ ounding that substrate binding site which impart a nucleic acid cleaving activity to the molecule.
  • a specific subsfrate binding site e.g., D and E of Formula 1 above
  • compositions preferably synthetic oligomers, which cleave a nucleic acid target sequence containing the triplet N 16 ' 2 C 16 1 H 17 . It is prefe ⁇ ed that H 17 is not guanosine, however, under certain circumstances, NCG triplet containing RNA can be cleaved by the ribozymes ofthe instant invention.
  • N C X triplets effectively doubles the number of targets available for cleavage by compositions ofthe type disclosed.
  • Example 10 Synthesis of l-(beta-D-xylofuranosyl)-xypoxanthine phosphoramidite
  • Inosine (1) was 5'-O-monomethoxyfritylated and 2'-O- silylated under standard conditions to afford 2 (Charubala, R; Pfleiderer, W. Heterocycles 1990, 30, 1141).
  • NCH ribozymes with xylo-inosine at position 15.1 were designed ( Figure 7) to cleave RNA containing GCA, ACA, UCA or the CCA triplet. These ribozymes were synthesized and purified as described herein and tested using standard RNA cleavage reaction conditions (see Table 31, for example, and see below).
  • ribozymes 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. Soc, 109, 7845), Scaringe et al., (1990 Nucleic Acids Res., 18, 5433) and
  • Ribozymes were purified by gel elecfrophoresis using general methods or were purified by high pressure liquid chromatography (HPLC; See Wincott et al., supra; the totality of which is hereby inco ⁇ orated herein by reference) and were resuspended in water.
  • HPLC high pressure liquid chromatography
  • [alpha- 32 p] CTP passed over a G 50 Sephadex column by spin chromatography and used as substrate RNA without further purification.
  • substrates were 5'-32p-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-HCI, pH 7.5 at 37°C, 10 mM MgCt ⁇ ) 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 o
  • Example 12 Activity of NCH Ribozyme variants
  • the nucleic acid molecules ofthe instant invention allow for the ability to cleave a new set of 12 NCH triplets. Determination of single turnover rate constants at pH 6 of these ribozymes in the all ribo form show that with NCA type triplets, the cleavage rate is higher than at NUA sites. NCC and NUC site rates are similar, and NCU sites are slightly lower than NUU sites.
  • nucleoside analogs were inco ⁇ orated at position 15.1 ofthe ribozyme. Cleavage activity was tested with the complementary Fl* labeled substrates at pH 7.4 in the presence of 10 mM Mg ++ under conditions of ribozyme excess (i.e. single turnover conditions).
  • the modified oligonucleotides were synthesized by standard oligonucleotide synthesis procedures. Xanthosine was protected using O-2 ,0-4pivaloyloxymethyl groups; N,N-dimethylguanosine with 6-O-( 2-nitrophenyl-)ethyl and 6-thio-inosine with S- cyanoethyl protecting groups.
  • Figure 36 The cleavage activity ofthe ribozymes containing the 15.1 analogs is summarized in Figure 36.
  • Figure 37 summarizes reported functional group modification studies performed at the A 15.1 residue in the A-15.1 » U- 16.1 context of NUH cleaving ribozymes. Modifications at the purine 15.1 Nl and/or C6 positions ( Figure 36 A, B, C)
  • Xanthosine 15.1 contains the same functional groups as inosine at the Nl and C6 sites but contains an additional hydrogen-bond donor site at position N3 along with a C2 carbonyl group. The complete lack ofactivity seen with this construct reinforces the importance ofthe purine N3 acceptor functionality in transition state formation. Similarly, 3-deaza-adenosine ( Figure 37, F) containing ribozymes were also inactive. The C2 carbonyl ofthe 15.1 purine shows no significant negative interference in iso-guanosine containing 15.1 ribozymes. Activity of modified core variants
  • Example 13 Activity of NCH Ribozyme to inhibit HER2 gene expression
  • Applicant has designed, synthesized and tested several NCH ribozymes and HH ribozymes targeted against HER2 RNA (see, for example, Tables 31 and 34) in cell proliferation assays.
  • the model proliferation assay used in the study can require a cell plating density of 2000 cells/well in 96-well plates and at least 2 cell doublings over a
  • FIPS fluoro- imaging processing system
  • Ribozymes (50-200 nM) were delivered in the presence of cationic lipid at 2.0 ⁇ g/mL and inhibition of proliferation was determined on day 5 post- treatment. Two full ribozyme screens were completed and 4 lead HH and 11 lead NCH ribozymes were chosen for further testing. Ofthe 15 lead Rzs chosen from primary screens, 4 NCH and 1 HHRzs continued to inhibit cell proliferation in subsequent experiments. NCH Rzs against sites, 2001 (RPI No. 17236), 2783 (RPI No. 17249), 2939 (RPI No. 17251) or 3998 (RPI No. 17262) caused inhibition of proliferation ranging from 25-60%) as compared to a scrambled confrol Rz (LA; RPI No. 17263).
  • NCH Rz Rz (RPI No. 17251) against site 2939 of ⁇ ER2 RNA.
  • FIG 3 An example of results from cell culture assay is shown in Figure 3.
  • NCH ribozymes and a HH ribozyme targeted against HER2 RNA are shown to cause significant inhibition of proliferation of cells. This shows that ribozymes, for instance, the NCH ribozymes are capable of inhibiting HER2 gene expression in mammalian cells.
  • the Protein Kinase C family contains twelve cu ⁇ ently known isozymes divided into three classes: the classic, Ca ⁇ dependent (PKC ⁇ , ⁇ l, ⁇ H, ⁇ ), the novel, non-Ca** dependent (PKC ⁇ , ⁇ , ⁇ , ⁇ , ⁇ ) and the atypical (PKC ⁇ , i/ ⁇ ); all of which are serine/threonine kinases.
  • PKC ⁇ , ⁇ l, ⁇ H, ⁇ novel, non-Ca** dependent
  • PIC ⁇ , i/ ⁇ atypical serine/threonine kinases.
  • These isozymes show distinct and overlapping tissue, cellular, and subcellular distribution. They aid in the regulation of cell growth and differentiation through their response to second messenger products of lipid metabolism (Blobe, et al, 1996, Cancer Surveys, 27, 213-248).
  • DAG diacylglyceral
  • LP3 inositol-friphosphate
  • lysophospholipids free fatty acids
  • phosphatidate phosphatidate
  • PKC's phosphorylation of c-Raf which phosphorylates MEK, which phosphorylates MAP, which phosphorylates transcription factors such as Jun and thereby activates a mitogenic program in the nucleus.
  • MEK phosphorylates kinase
  • MAP phosphorylates transcription factors
  • Jun thereby activates a mitogenic program in the nucleus.
  • PKC's There are numerous substrates for the various PKC's, one which for PKC ⁇ ultimately stimulates franscription factors that activate P- glycoprotein (P-gp) causing the multi-drug resistant phenotype (MDR) (Blobe, et al, 1994, Cancer and Metastasis Reviews, 13, 411-431).
  • MDR multi-drug resistant phenotype
  • PKC's have been implicated in tumor promotion since the discovery that these molecules can serve as receptors for tumor-promoting phorbol esters.
  • An increase in PKC overexpression in numerous tumor cell lines and tumor tissues has also been demonsfrated.
  • PKC overexpression has been shown to be associated with increased invasion and metastasis in mouse Lewis lung carcinoma, mouse B16 melanoma (Lee et al, 1997, Molecular Carcinogenesis, 18, 44-53), mouse mammary adenocarcinoma, mouse fibrosarcoma, human lung carcinoma (Wang and Liu, 1998, Acta Pharmacologica Sinica, 19, 265-268), human bladder carcinoma, human pancreatic cancer (Denham et al., 1998, Surgery, 124, 218-223), and human gastric cancer (Dean et al, 1996, Cancer Research, 56, 3499-3507).
  • PKC ⁇ can stimulate adhesion molecule expression and can directly act on these membrane bound species as subsfrates, thereby modulating cellular adhesion to the extracellular matrix and increasing metastic potential.
  • human surgical specimens have demonsfrated elevated PKC in breast tumors, thyroid carcinomas and melanomas (Becker et al, 1990, Oncogene, 5, 1133- 1139).
  • PKC expression co ⁇ elates with resistance to doxorubicin and high P-gp levels in human renal carcinoma and non-small cell lung carcinoma, inhibitors of PKC partially reverse the MDR phenotype and decrease phosphorylation of P-gp (Caponigro et al, 1997, Anti-Cancer Drugs, 8, 26-33).
  • BC1-XL is overexpressed in glioma cells and is an apoptosis inhibitor.
  • the ribozyme mediated inhibition of cell proliferation presumably results from apoptosis induction of transformed glioma cells through suppression of PKC ⁇ and Bcl-x L (Leirdal and Sioud, 1999, British J. of Cancer, 80, 1558- 1564).
  • Animal Models
  • mice xenograft models using human tumor cell lines have been developed using cell lines which express high levels of PKC ⁇ protein.
  • McGraw et ⁇ /, 1997 ', Anti-Cancer Drug Design, 12, 315-326 describe mouse xenograft models using human breast (MDA MB-321), prostate (Du-145), colon (Colo 205, WiDr), lung (NCI H69, H209, J460, H520, A549), bladder (T-24), and melanoma (SK-mel 1) carcinoma cells.
  • rats were freated with a single injection of ribozyme targeting PKC ⁇ resulting in inhibition of tumor growth as determined by tumor size and/or weight when compared to confrols.
  • the above studies provide proof that inhibition of PKC ⁇ expression by anti-PKC ⁇ agents causes inhibition of tumor growth in animals.
  • Lead anti-PKC ⁇ ribozymes chosen from in vitro assays can be further tested in mouse xenograft models. Ribozymes can be first tested alone and then in combination with standard chemotherapies. Animal Model Development
  • Human lung (A549, NCI H520) tumor and breast (MDA-MB 231) cell lines can be characterized to establish their growth curves in mice. These cell lines are been implanted into both nude and SCLD mice and primary tumor volumes are measured 3 times per week. Growth characteristics of these tumor lines using a Matrigel implantation format can also be established. In addition, the use of other cell lines that have been engineered to express high levels of PKC ⁇ can also be used. The tumor cell line(s) and implantation method that supports the most consistent and reliable tumor growth can be used in animal studies to test promising PKC ⁇ ribozyme(s).
  • Ribozymes can be administered by daily subcutaneous injection or by continuous subcutaneous infusion from Alzet mini osmotic pumps beginning 3 days after tumor implantation and continuing for the duration ofthe study. Group sizes of at least 10 animals are employed. Efficacy is determined by statistical comparison of tumor volume of ribozyme-freated animals to a control group of animals freated with saline alone. Because the growth of these tumors is generally slow (45-60 days), an initial endpoint will be the time in days it takes to establish an easily measurable primary tumor (i.e. 50-100 mm 3 ) in the presence or absence of ribozyme treatment.
  • Ribozymes targeting PKC ⁇ have strong potential to develop into useful therapeutics directed towards numerous cancer types.
  • Lung cancer is the leading cause of cancer deaths for both men and women in the USA.
  • the incidence of lung cancer in the United States is -172,000 cases per year, accounting for 14% of cancer diagnoses.
  • ISIS 3521/CGP 64128A a PKC alpha antisense construct.
  • ISIS 3521/CGP 64128A was administered as either a two-hour i.v. infusion three times per week for three consecutive weeks, or as a continuous i.v. infusion for twenty-one consecutive days.
  • the authors report that patients demonstrated excellent tolerance to the antisense compound when administered at doses of up to 2.5 mg/kg by the two-hour i.v. infusion and at 1.5 mg/kg/day by continuous i.v. infusion.
  • Treatment options for lung cancer are determined by the type and stage ofthe cancer and include surgery, radiation therapy, and chemotherapy.
  • surgery is usually the treatment of choice. Because the disease has usually spread by the time it is discovered, radiation therapy and chemotherapy are often needed in combination with surgery.
  • Chemotherapy alone or combined with radiation has replaced surgery as the treatment of choice for small cell lung cancer; on this regimen, a large percentage of patients experience remission, which in some cases is long-lasting.
  • the 1-year relative survival rates for lung cancer have increased from 32% in 1973 to 41% in 1994, largely due to improvements in surgical techniques.
  • the 5-year relative survival rate for all stages combined is only 14%. The survival rate is 50% for cases detected when the disease is still localized, but only 15% of lung cancers are discovered that early.
  • chemotherapies include various combinations of cytotoxic drugs to kill the cancer cells. These drugs include paclitaxel (Taxol), docetaxel, cisplatin, methofrexate, cyclophosphamide, doxorubin, fluorouracil etc. Significant toxicities are associated with these cytotoxic therapies. Well-characterized toxicities include nausea and vomiting, myelosuppression, alopecia and mucosity. Serious cardiac problems are also associated with certain ofthe combinations, e.g. doxorubin and paclitaxel, but are less common.
  • NCH ribozymes targeted against PKC ⁇ RNA (Genebank accession No NM_002737) (see, for example, Table 63). These ribozymes are used first in a proliferation assay that is used to select ribozyme leads.
  • the model proliferation assay useful in the study can require a cell plating density of 2000 cells/well in 96-well plates and at least 2 cell doublings over a 5-day freatment period.
  • the FLPS (fluoro- imaging processing system) method well known in the art can be used. This method allows for cell density measurements after nucleic acids are stained with CyQuant® dye, and has the advantage of accurately measuring cell densities over a very wide range 1,000-100,000 cells/well in 96-well format.
  • Ribozymes (50-200 nM) are delivered in the presence of cationic lipid at 2.0 ⁇ g/mL and inhibition of proliferation is determined on day 5 post-treatment. Two full ribozyme screens are usually completed and lead ribozymes are chosen for further testing. Ofthe lead ribozymes chosen from primary screens, ribozymes which continue to inhibit cell proliferation in subsequent experiments are selected for PKC ⁇ RNA and protein inhibition studies.
  • nucleotide triphosphates and their inco ⁇ oration into nucleic acids using polymerase enzymes has greatly assisted in the advancement of nucleic acid research.
  • the polymerase enzyme utilizes nucleotide triphosphates as precursor molecules to assemble oligonucleotides. Each nucleotide is attached by a phosphodiester bond formed through nucleophilic attack by the 3' hydroxyl group ofthe oligonucleotide 's last nucleotide onto the 5' triphosphate ofthe next nucleotide. Nucleotides are inco ⁇ orated one at a time into the oligonucleotide in a 5' to 3' direction. This process allows RNA to be produced and amplified from virtually any DNA or RNA templates.
  • RNA polymerase Most natural polymerase enzymes inco ⁇ orate standard nucleotide triphosphates into nucleic acid.
  • a DNA polymerase inco ⁇ orates dATP, dTTP, dCTP, and dGTP into DNA and an RNA polymerase generally inco ⁇ orates ATP, CTP, UTP, and GTP into RNA.
  • certain polymerases that are capable of inco ⁇ orating non-standard nucleotide triphosphates into nucleic acids (Joyce, 1997, PNAS 94, 1619- 1622, Huang et al., Biochemistry 36, 8231-8242).
  • nucleosides Before nucleosides can be inco ⁇ orated into RNA transcripts using polymerase enzymes they must first be converted into nucleotide triphosphates which can be recognized by these enzymes. Phosphorylation of unblocked nucleosides by treatment with POCl and trialkyl phosphates was shown to yield nucleoside 5'- phosphorodichloridates (Yoshikawa et al, 1969, Bull. Chem. Soc. (Japan) 42, 3505).
  • Adenosine or 2'-deoxyadenosine 5 '-triphosphate was synthesized by adding an additional step consisting of freatment with excess tri-n-butylammonium pyrophosphate in DMF followed by hydrolysis (Ludwig, 1981, Acta Biochim. et Biophys. Acad. Sci. Hung. 16, 131-133).
  • Non-standard nucleotide triphosphates are not readily inco ⁇ orated into RNA transcripts by traditional RNA polymerases. Mutations have been introduced into RNA polymerase to facilitate inco ⁇ oration of deoxyribonucleotides into RNA (Sousa & Padilla, 1995, EMBO J. 14,4609-4621, Bonner et al, 1992, EMBO J. 11, 3767-3775, Bonner et al., 1994, J. Biol. Chem. 42, 25120-25128, Aurup et al, 1992, Biochemistry 31, 9636-9641).
  • This invention relates to novel nucleotide triphosphate (NTP) molecules, and their inco ⁇ oration into nucleic acid molecules, including nucleic acid catalysts.
  • NTPs of the instant invention are distinct from other NTPs known in the art.
  • the invention further relates to inco ⁇ oration of these nucleotide triphosphates into oligonucleotides using an RNA polymerase; the invention further relates to novel transcription conditions for the inco ⁇ oration of modified (non-standard) and unmodified NTP's, into nucleic acid molecules. Further, the invention relates to methods for synthesis of novel NTP's
  • the invention features NTP's having the formula triphosphate-OR, for example the following formula 3:
  • R is any nucleoside; specifically the nucleosides 2'-O-methyl-2,6- diaminopurine riboside; 2'-deoxy-2'amino-2,6-diaminopurine riboside; 2'-(N-alanyl) amino-2'-deoxy-uridine; 2'-(N-phenylalanyl)amino-2'-deoxy-uridine; 2'-deoxy -2'-(N- ⁇ - alanyl) amino ; 2'-deoxy-2'-(lysiyl) amino uridine; 2'-C-allyl uridine; 2'-O-amino-uridine; 2'-O-methylthiomethyl adenosine; 2'-O-methylthiomethyl cytidine ; 2'-O- methylthiomethyl guanosine; 2'-O-methylthiomethyl-uridine; 2'-deoxy-2'-(N-histidyl) amino uridine
  • the invention features inorganic and organic salts ofthe nucleoside triphosphates ofthe instant invention.
  • the invention features a process for the synthesis of pyrimidine nucleotide triphosphate (such as UTP, 2'-O-MTM-UTP, dUTP and the like) including the steps of monophosphorylation where the pyrimidine nucleoside is contacted with a mixture having a phosphorylating agent (such as phosphorus oxychloride, phospho-tris-
  • a phosphorylating agent such as phosphorus oxychloride, phospho-tris-
  • nucleotide triphosphate or “NTP” is meant a nucleoside bound to three inorganic phosphate groups at the 5' hydroxyl group ofthe modified or unmodified ribose or deoxyribose sugar where the 1 ' position ofthe sugar may comprise a nucleic acid base
  • the triphosphate portion may be modified to include chemical moieties which do not destroy the functionality ofthe group (i.e., allow inco ⁇ oration into an RNA molecule).
  • nucleotide triphosphates (NTPs) ofthe instant invention are inco ⁇ orated into an oligonucleotide using an RNA polymerase enzyme.
  • RNA polymerases include but are not limited to mutated and wild type versions of bacteriophage T7, SP6, or T3 RNA polymerases. Applicant has also found that the NTPs ofthe present invention can be inco ⁇ orated into oligonucleotides using certain DNA polymerases, such as Taq polymerase.
  • the invention features a process for
  • modified nucleotides into a nucleic acid transcript by an RNA polymerase.
  • reagents include, but are not limited to, methanol, LiCl, polyethylene glycol (PEG), diethyl ether, propanol, methyl amine, ethanol, and the like.
  • the modified nucleotide triphosphates can be inco ⁇ orated by franscription into a nucleic acid molecules including enzymatic nucleic acid, antisense, 2-5A antisense chimera, oligonucleotides, triplex forming oligonucleotide (TFO), aptamers and the like (Stull et al, 1995 Pharmaceutical Res.
  • TFO triple forming oligonucleotides
  • the modified nucleotide triphosphates ofthe instant invention can be used for combinatorial chemistry or in vitro selection of nucleic acid molecules with novel function.
  • Modified oligonucleotides can be enzymatically synthesized to generate libraries for screening.
  • the invention features nucleic acid based techniques (e.g., enzymatic nucleic acid molecules), antisense nucleic acids, 2-5 A antisense chimeras, triplex DNA, antisense nucleic acids containing RNA cleaving chemical groups) isolated using the methods described in this invention and methods for their use to diagnose, down regulate or inhibit gene expression.
  • nucleic acid based techniques e.g., enzymatic nucleic acid molecules
  • antisense nucleic acids 2-5 A antisense chimeras, triplex DNA, antisense nucleic acids containing RNA cleaving chemical groups
  • the invention features enzymatic nucleic acid molecules targeted against HER2 RNA, specifically including ribozymes in the class II (zinzyme) motif.
  • Targets for example HER2 RNA, for useful ribozymes and antisense nucleic acids can be determined, for example, as described 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 Nos. 5,525,468 and 5,646,042, all are hereby inco ⁇ orated by reference herein in their totalities.
  • Other examples include the following PCT applications, which concern inactivation of expression of disease-related genes: WO 95/23225, and WO 95/13380; all of which are inco ⁇ orated by reference herein.
  • the invention features a process for inco ⁇ orating a plurality of compounds of formula 3.
  • the invention features a nucleic acid molecule with catalytic activity having formula 4:
  • the invention features a nucleic acid molecule with catalytic activity having formula 5:
  • X, Y, and Z represent independently a nucleotide or a non-nucleotide linker, which may be same or different; • indicates hydrogen bond formation between two adjacent nucleotides which may or may not be present; Z' is a nucleotide complementary to Z; 1 is an integer greater than or equal to 3 and preferably less than 20, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, or 15; n is an integer greater than 1 and preferably less than 10, more specifically 3, 4, 5, 6, or 7; o is an integer greater than or equal to 3 and preferably less than 20, more specifically 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or
  • each Xm and X( o) are oligonucleotides which are of sufficient length to stably interact independently with a target nucleic acid sequence (the target can be an RNA, DNA or RNA/DNA mixed polymers); preferably has a G at the 3 '-end, XQ) preferably has a G at the 5 '-end;
  • W is a linker of ⁇ 2 nucleotides in length or may be a non-nucleotide linker;
  • Y is a linker of > 1 nucleotides in length, preferably G, 5' -CA-3', or 5' -CAA-3', or may be a non-nucleotide linker;
  • A, U, C, and G represent nucleotides;
  • G is a nucleotide, preferably 2'-O-methyl, 2'-deozy-2'-fluoro, or 2
  • C represents a nucleotide, preferably 2'-amino (e.g., 2'-NH 2 or 2'-O- NH 2 , and represents a chemical linkage (e.g. a phosphate ester linkage, amide linkage, phosphorothioate, phosphorodithioate or others known in the art).
  • the enzymatic nucleic acid molecules of Formula 4 and Formula 5 may independently comprise a cap structure which may independently be present or absent.
  • the 3 '-cap is selected from a group comprising,
  • nucleotide 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 ⁇ reo-pentofuranosyl nucleotide; acyclic
  • 3',4'-seco nucleotide 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide; 5'-5'- inverted nucleotide moiety; 5'-5'-inverted abasic moiety; 5'-phosphoramidate; 5'- phosphorothioate; 1,4-butanediol phosphate 5'-amino; bridging and/or non-bridging 5'- phosphoramidate, phosphorothioate and/or hosphorodithioate; bridging or non bridging methylphosphonate and 5'-mercapto moieties (for more details, see Beaucage and Iyer,
  • 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.
  • nucleosides are dissolved in triethyl phosphate and chilled in an ice bath. Phosphorus oxychloride (POCl 3 ) is then added followed by the introduction of DMAP. The reaction is then warmed to room temperature and allowed to proceed for 5 hours. This reaction allows the formation of nucleotide monophosphates which can then be used in the formation of nucleotide triphosphates. Tributylamine is added followed by the addition of anhydrous acetonitrile and tributylammonium pyrophosphate. The reaction is then quenched with TEAB and sti ⁇ ed overnight at room temperature (about 20°C).
  • the triphosphate is purified using Sephadex® column purification or equivalent and/or HPLC and the chemical structure is confirmed using NMR analysis.
  • Those skilled in the art will recognize that the reagents, temperatures ofthe reaction, and purification methods can easily be alternated with substitutes and equivalents and still obtain the desired product.
  • the invention provides nucleotide triphosphates which can be used for a number of different functions.
  • the nucleotide triphosphates formed from nucleosides found in Table 45 are unique and distinct from other nucleotide triphosphates known in the art.
  • modified nucleotides can alter the properties ofthe molecule.
  • modified nucleotides can hinder binding of nucleases, thus increasing the chemical half-life ofthe molecule. This is especially important if the molecule is to be used for cell culture or in vivo. It is known in the art that the introduction of modified nucleotides into these molecules can greatly increase the stability and thereby the effectiveness ofthe molecules (Burgin et al, 1996, Biochemistry 35, 14090-14097; Usman et al, 1996, Curr. Opin. Struct. Biol. 6, 527-533).
  • Modified nucleotides are inco ⁇ orated using either wild type or mutant polymerases.
  • mutant T7 polymerase is used in the presence of modified nucleotide triphosphate(s), DNA template and suitable buffers.
  • modified nucleotide triphosphate(s) DNA template and suitable buffers.
  • Other polymerases and their respective mutant versions can also be utilized for the inco ⁇ oration of NTP's ofthe invention.
  • Nucleic acid transcripts were detected by inco ⁇ orating radiolabelled nucleotides ( ⁇ - 32 P NTP).
  • the radiolabeled NTP contained the same base as the modified triphosphate being tested.
  • the effects of methanol, PEG and LiCl were tested by adding these compounds independently or in combination.
  • Detection and quantitation ofthe nucleic acid transcripts was performed using a Molecular Dynamics Phosphorlmager. Efficiency of franscription was assessed by comparing modified nucleotide triphosphate inco ⁇ oration with all-ribonucleotide inco ⁇ oration control. Wild- type polymerase was used to inco ⁇ orate NTP's using the manufacturer's buffers and instructions (Boehringer Mannheim).
  • Inco ⁇ oration rates of modified nucleotide triphosphates into oligonucleotides can be increased by adding to traditional buffer conditions, several different enhancers of modified NTP inco ⁇ oration.
  • Applicant has utilized methanol and LiCl in an attempt to increase inco ⁇ oration rates of dNTP using RNA polymerase. These enhancers of modified NTP inco ⁇ oration can be used in different combinations and ratios to optimize transcription. Optimal reaction conditions differ between nucleotide triphosphates and can readily be determined by standard experimentation. Overall, however, Applicant has found that inclusion of enhancers of modified NTP inco ⁇ oration such as methanol or inorganic compound such as lithium chloride increase the mean transcription rates.
  • Applicant synthesized pyrimidine nucleotide triphosphates using DMAP in the reaction For purines, applicant utilized standard protocols previously described in the art (Yoshikawa et al supra;. Ludwig, supra). Described below is one example of a pyrimdine nucleotide triphosphate and one purine nucleotide triphosphate synthesis.
  • tributylamine (0.65 ml) was added followed by the addition of anhydrous acetonitrile (10.0 ml), and after 5 minutes (reequilibration to 0°C) tributylammonium pyrophosphate (4.0 eq., 1.53 g) was added.
  • the reaction mixture was quenched with 20 ml of 2M TEAB after 15 minutes at 0°C (HPLC analysis with above conditions showed consumption of monophosphate at 10 minutes) then sti ⁇ ed overnight at room temperature, the mixture was evaporated in vacuo with methanol co-evaporation (4x) then diluted in 50 ml 0.05M TEAB.
  • the reactions were performed on 20 mg aliquots of nucleoside dissolved in 1 ml of triethyl phosphate and 19 ul of phosphorus oxychloride. The reactions were monitored at 40 minute intervals automatically by HPLC to generate yield-of-product curves at times up to 18 hours.
  • a reverse phase column and ammonium acetate/ sodium acetate buffer system (50mM & lOOmM respectively at pH 4.2) was used to separate the 5', 3', 2' monophosphates (the monophosphates elute in that order) from the 5 '-triphosphate and the starting nucleoside. The data is shown in Table 46.
  • Buffer 1 Materials Used in Bacteriophage T7 RNA Polymerase Reactions Buffer 1: Reagents are mixed together to form a 10X stock solution of buffer 1
  • BUFFER 2 Reagents are mixed together to form a 10X stock solution of buffer 2 (400 mM Tris-Cl [pH 8.1], 200 mM MgCl 2 , 100 mM DTT, 50 mM spermidine, and 0.1% triton® X-100). Prior to initiation ofthe polymerase reaction PEG, LiCl is added and the buffer is diluted such that the final reaction conditions for buffer 2 consisted of : 40mM tris (pH 8.1), 20mM MgCl 2 , 10 mM DTT, 5 mM spermidine, 0.01% triton® X-100, 4% PEG, and 1 mM LiCl.
  • buffer 2 400 mM Tris-Cl [pH 8.1], 200 mM MgCl 2 , 100 mM DTT, 50 mM spermidine, and 0.1% triton® X-100.
  • BUFFER 3 Reagents are mixed together to form a 10X stock solution of buffer 3 (400 mM Tris-Cl [pH 8.0], 120 mM MgCl 2 , 50 mM DTT, 10 mM spermidine and 0.02% triton® X-100). Prior to initiation ofthe polymerase reaction PEG is added and the buffer is diluted such that the final reaction conditions for buffer 3 consisted of : 40mM tris (pH 8.0), 12 mM MgCl 2 , 5 mM DTT, 1 mM spermidine, 0.002% triton® X-100, and 4% PEG.
  • buffer 3 400 mM Tris-Cl [pH 8.0], 120 mM MgCl 2 , 50 mM DTT, 10 mM spermidine and 0.02% triton® X-100.
  • BUFFER 4 Reagents are mixed together to form a 10X stock solution of buffer 4 (400 mM Tris-Cl [pH 8.0], 120 mM MgCl 2 , 50 mM DTT, 10 mM spermidine and 0.02% triton® X-100). Prior to initiation ofthe polymerase reaction PEG, methanol is added and the buffer is diluted such that the final reaction conditions for buffer 4 consisted of :
  • BUFFER 5 Reagents are mixed together to form a 10X stock solution of buffer 5 (400 mM Tris-Cl [pH 8.0], 120 mM MgCl 2 , 50 mM DTT, 10 mM spermidine and 0.02% triton® X-100). Prior to initiation ofthe polymerase reaction PEG, LiCl is added and the buffer is diluted such that the final reaction conditions for buffer 5 consisted of : 40mM tris (pH 8.0), 12 mM MgCl 2 , 5 mM DTT, 1 mM spermidine, 0.002% triton® X-100, 1 mM LiCl and 4% PEG.
  • buffer 5 400 mM Tris-Cl [pH 8.0], 120 mM MgCl 2 , 50 mM DTT, 10 mM spermidine and 0.02% triton® X-100.
  • BUFFER 6 Reagents are mixed together to form a 10X stock solution of buffer 6 (400 mM Tris-Cl [pH 8.0], 120 mM MgCl 2 , 50 mM DTT, 10 mM spermidine and 0.02% triton® X-100). Prior to initiation ofthe polymerase reaction PEG, methanol is added and the buffer is diluted such that the final reaction conditions for buffer 6 consisted of : 40mM tris (pH 8.0), 12 mM MgCl 2 , 5 mM DTT, 1 mM spermidine, 0.002% triton® X- 100, 10% methanol, and 4% PEG.
  • buffer 6 400 mM Tris-Cl [pH 8.0], 120 mM MgCl 2 , 50 mM DTT, 10 mM spermidine and 0.02% triton® X-100.
  • buffer 6 400 mM Tris-Cl [pH 8.0], 120
  • BUFFER 7 Reagents are mixed together to form a 10X stock solution of buffer 6 (400 mM Tris-Cl [pH 8.0], 120 mM MgCl 2 , 50 mM DTT, 10 mM spermidine and 0.02% triton® X-100). Prior to initiation ofthe polymerase reaction PEG, methanol and LiCl is added and the buffer is diluted such that the final reaction conditions for buffer 6 consisted of : 40mM tris (pH 8.0), 12 mM MgCl 2 , 5 mM DTT, 1 mM spermidine, 0.002% triton® X-100, 10% methanol, 4% PEG, and 1 mM LiCl.
  • buffer 6 400 mM Tris-Cl [pH 8.0], 120 mM MgCl 2 , 50 mM DTT, 10 mM spermidine and 0.02% triton® X-100.
  • Modified nucleotide triphosphates were tested in buffers 1 through 6 at two different temperatures (25 and 37°C). Buffers 1-6 tested at 25°C were designated conditions 1-6 and buffers 1-6 tested at 37°C were designated conditions 7-12 (Table 47). In each condition, Y639F mutant T7 polymerase (Sousa and Padilla, supra) (0.3-2 mg/20 ml reaction), NTP's (2 mM each), DNA template (10 pmol), inorganic pyrophosphatase (5U/ml) and ⁇ - 32 P NTP (0.8 mCi/pmol template) were combined and heated at the designated temperatures for 1-2 hours. The radiolabeled NTP used was different from the modified triphosphate being testing.
  • the samples were resolved by polyacrylamide gel elecfrophoresis. Using a Phosphorhnager (Molecular Dynamics, Sunnyvale, CA), the amount of full-length transcript was quantified and compared with an all-RNA confrol reaction. The data is presented in Table 48; results in each reaction are expressed as a percent compared to the all-ribonucleotide triphosphate (rNTP) control.
  • the confrol was run with the mutant T7 polymerase using commercially available polymerase buffer (Boehringer Mannheim, Indianapolis, LN). Inco ⁇ oration of Modified NTP's using Wild-type T7 RNA polymerase
  • Bacteriophage T7 RNA polymerase was purchased from Boehringer Mannheim at 0.4 U/ ⁇ L concentration. Applicant used the commercial buffer supplied with the enzyme and 0.2 ⁇ Ci alpha- 32 P NTP in a 50 ⁇ L reaction with nucleotides triphosphates at 2 mM each. The template was a double-stranded PCR fragment, which was used in previous screens. Reactions were carried out at 37°C for 1 hour. Ten ⁇ L ofthe sample was run on a 7.5%o analytical PAGE and bands were quantitated using a Phosphorhnager. Results are calculated as a comparison to an "all ribo" control (non-modified nucleotide triphosphates) and the results are in Table 49.
  • Two modified cytidines (2'-NH 2 -CTP or 2'dCTP) were inco ⁇ orated along with 2'- his-NH 2 -UTP with identical efficiencies.
  • 2'-his-NH 2 -UTP and 2'-NH 2 -CTP were then tested with various unmodified and modified adenosine triphosphates in the same buffer (Table 50b).
  • the best modified adenosine triphosphate for inco ⁇ oration with both 2'-his- NH 2 -UTP and 2'-NH 2 -CTP was 2'-NH 2 -DAPTP.
  • pools of enzymatic nucleic acid molecules were designed to have two substrate binding arms (5 and 16 nucleotides long) and a random region in the middle.
  • the subsfrate has a biotin on the 5' end, 5 nucleotides complementary to the short binding arm ofthe pool, an unpaired G (the desired cleavage site), and 16 nucleotides complementary to the long binding arm ofthe pool.
  • the subsfrate was bound to column resin through an avidin-biotin complex.
  • the general process for selection is shown in Figure 11.
  • the protocols described below represent one possible method that may be utilized for selection of enzymatic nucleic acid molecules and are given as a non-limiting example of enzymatic nucleic acid molecule selection with combinatorial libraries.
  • MST7c (33 mer): 5'-TAA TAC GAC TCA CTA TAG GAA AGG TGT GCA ACC-3'
  • MSN60c (93 mer): 5'-ACC CTC ACT AAA GGC CGT (N) 60 GGT TGC ACA CCT TTG-3'
  • MSN20c (53 mer): 5'-ACC CTC ACT AAA GGC CGT (N) 20 GGT TGC ACA CCT
  • TTG-3' N60 library was constructed using MSN60c as a template and MST3/MST7c as primers.
  • N40 and N20 libraries were constructed using MSN40c (or MSN20c) as template and MST3del/MST7c as primers.
  • Single-stranded templates were converted into double-sfranded DNA by the following protocol: 5 nmol template, 10 nmol each primer, in 10 ml reaction volume using standard PCR buffer, dNTP's, and taq DNA polymerase (all reagents from Boerhinger Mannheim). Synthesis cycle conditions were 94°C, 4 minutes; (94°C, 1 minute; 42°C, 1 minute; 72°C, 2 minutes) x 4; 72°C, 10 minutes.
  • the column was washed twice with 1 ml of binding buffer (20 mM NaPO 4 (pH 7.5), 150 mM NaCl) and then capped off (i.e., a cap was put on the bottom ofthe column to stop the flow). 200 ⁇ l ofthe subsfrate suspended in binding buffer was applied and allowed to incubate at room temperature for 30 minutes with occasional vortexing to ensure even linking and distribution ofthe solution to the resin. After the incubation, the cap was removed and the column was washed with 1 ml binding buffer followed by 1 ml column buffer (50 mM tris-HCL (pH 8.5), 100 mM NaCl, 50 mM KC1). The column was then ready for use and capped off.
  • 1 ml of binding buffer (20 mM NaPO 4 (pH 7.5), 150 mM NaCl)
  • 200 ⁇ l ofthe subsfrate suspended in binding buffer was applied and allowed to incubate at room temperature for 30 minutes with occasional vortexing to ensure even
  • RNA 1 nmol ofthe initial pool RNA was loaded on the column in a volume of 200 ⁇ l column buffer. It was allowed to bind the substrate by incubating for 30 minutes at room temperature with occasional vortexing. After the incubation, the cap was removed and the column was washed twice with 1 ml column buffer and capped off. 200 ⁇ l of elution buffer (50 mM tris-HCl (pH 8.5), 100 mM NaCl, 50 mM KC1, 25 mM MgCl 2 ) was applied to the column followed by 30 minute incubation at room temperature with occasional vortexing. The cap was removed and four 200 ⁇ l fractions were collected using elution buffer.
  • elution buffer 50 mM tris-HCl (pH 8.5), 100 mM NaCl, 50 mM KC1, 25 mM MgCl 2
  • RNA and primer MST3 (10-100 pmol) were denatured at 90°C for 3 minutes in water and then snap-cooled on ice for one minute.
  • the following reagents were added to the tube (final concentrations given): IX PCR buffer (Boerhinger Mannheim), 1 mM dNTP's (for PCR, Boerhinger Mannheim), 2 U/ ⁇ l RNase-Inhibitor (Boerhinger Mannheim), 10 U/ ⁇ l SuperscriptTM LI Reverse Transcriptase (BRL).
  • the reaction was incubated for 1 hour at 42°C, then at 95 °C for 5 minutes in order to destroy the SuperscriptTM.
  • MST7c primer (10-100 pmol, same amount as in RT step)
  • IX PCR buffer taq DNA polymerase (0.025-0.05 U/ ⁇ l, Boerhinger Mannheim).
  • the reaction was cycled as follows: 94°C, 4minutes; (94°C, 30s; 42-54°C, 30s; 72°C, lminute) x 4-30 cycles; 72°C, 5minutes; 30°C, 30 minutes. Cycle number and annealing temperature were decided on a round by round basis.
  • Subsequent rounds used 20 pmols of input RNA and 40 pmol ofthe 22 nucleotide substrate on the column.
  • Kinetic activity ofthe enzymatic nucleic acid molecule shown in Table 54 was determined by incubating enzymatic nucleic acid molecule (10 nM) with substrate in a cleavage buffer (pH 8.5, 25 mM MgCl 2 , 100 mM NaCl, 50 mM KCl) at 37°C.
  • a cleavage buffer pH 8.5, 25 mM MgCl 2 , 100 mM NaCl, 50 mM KCl
  • the initial random modified-R ⁇ A pool was loaded onto substrate-resin in the following buffer; 5 mM ⁇ aOAc pH 5.2, 1 M ⁇ aCl at 4° C. After ample washing, the resin was moved to 22 ° C and the buffer switch 20 mM HEPES pH 7.4, 140 mM KCl, 10 mM ⁇ aCl, 1 mM CaCl 2 , 1 mM MgCl 2 . In one selection of ⁇ 60 oligonucleotides, no divalent cations (MgCl 2 , CaCl 2 ) was used. The resin was incubated for 10 minutes to allow reaction and the eluant collected.
  • the enzymatic nucleic acid molecule pools were capable of cleaving l-3%> ofthe present subsfrate even in the absence of divalent cations, the background (in the absence of modified pools) was 0.2 - 0.4 %.
  • RNA polymerases When designing monomeric nucleoside triphosphates for selection of therapeutic catalytic RNAs, one has to take into account nuclease stability of such molecules in biological sera. A common approach to increase RNA stability is to replace the sugar 2'- OH group with other groups like 2'-fluoro, 2'-O-methyl or 2'-amino. Fortunately such 2'- modified pyrimidine 5 'triphosphates are shown to be subsfrates for RNA polymerases. (Aurup, H.; Williams, D.M.; Eckstein, F. Biochemistry 1992, 57, 9637; and Padilla, R.; Sousa, R. Nucleic Acids Res.
  • 2'-O-methyluridine was 3',5'-bis-acetylated using acetic anhydride in pyridine and then converted to its 5-iodo derivative la using ceric ammonium nitrate reagent
  • 5'- Triphosphate was purified on Sephadex® DEAE A-25 ion exchange column using a linear gradient of 0.1-0.8M triethylammonium bicarbonate (TEAB) for elution. Traces of contaminating inorganic pyrophosphate are removed using C-18 RP HPLC to afford analytically pure material. Conversion into ⁇ a-salt was achieved by passing the aqueous solution of triphosphate through Dowex 50WX8 ion exchange resin in ⁇ a + form to afford 4a in 45% yield. When Proton-Sponge was omitted in the first phosphorylation step, yields were reduced to 10-20%. Catalytic hydrogenation of 3a yielded 5-aminopropyl derivative 5a which was phosphorylated under conditions identical to those described for propynyl derivative 3a to afford triphosphate 6a in 50% yield.
  • TEAB triethylammonium bicarbonate
  • Carboxylate group was introduced into 5-position of uridine both on the nucleoside level and post-synthetically (Method C) (Scheme 3).
  • 5-Iodo-2'-deoxy-2'-fluorouridine (16) was coupled with methyl acrylate using modified Heck reaction 13 to yield 17 in 85% yield.
  • 5'-O-Dimethoxytritylation, followed by in situ 3'-O-acetylation and subsequent detritylation afforded 3 '-protected derivative 18.
  • Phosphorylation using 2-chloro-4H- l,3,2-benzodioxa-phosphorin-4-one followed by pyrophosphate addition and oxidation Lidwig, J.; Eckstein, F. J. Org.
  • Cytidine derivatives comprising 3-aminopropyl and 3(N-succinyl)aminopropyl groups were synthesized according to Scheme 4.
  • Peracylated 5-(3-aminopropynyl)uracil derivative 2b is reduced using catalytic hydrogenation and then converted in seven steps and 5% ⁇ overall yield into 3'-acetylated cytidine derivative 25.
  • This synthesis was plagued by poor solubility of intermediates and formation ofthe ⁇ 4 -cyclized byproduct during ammonia freatment ofthe 4-triazolyl intermediate.
  • Phosphorylation of 25 as described in reference 11 yielded triphosphate 26 and N -cyclized product 27 in 1 : 1 ratio.
  • 5-dintrophenylimidazoleacetic acid 2'-deoxy uridine nucleoside (80 mg) was dissolved in 5 ml of triethylphosphate while stirring under argon, and the reaction mixture was cooled to 0°C. Phosphorous oxychloride (1.8 eq, 22 ml) was added to the reaction mixture at 0°C, three more aliquots were added over the course of 48 hours at room temperature. The reaction mixture was then diluted with anhydrous MeC ⁇ (5 ml) and cooled to 0°C, followed by the addition of tributylamine (0.65 ml) and tributylammonium pyrophosphate (4.0 eq, 0.24 g).
  • Phosphorus oxychloride (99.999%, 3 eq., 0.0672 mL) was added to the solution and the reaction was sti ⁇ ed for two hours at 0 °C.
  • Tributylammonium pyrophosphate (4 eq., 0.400 g) was dissolved in 3.42 mL of acetonitrile and tribuytylamine (0.165 mL). Acetonitrile (1 mL) was added to the monophosphate solution followed by the pyrophosphate solution which was added dropwise. The resulting solution was clear. The reaction was allowed to warm up to room temperature. After stirring for 45 minutes, methylamine (5 mL) was added and the reaction and sti ⁇ ed at room temperature for 2 hours.
  • Tributylamine (0.303 mL) and Tributylammonium pyrophosphate (4 eq., 0.734 g) dissolved in 6.3 mL of acetonitrile (added dropwise) were added to the monophosphate solution.
  • the reaction was allowed to warm up to room temperature. After stirring for 15 min, methylamine (10 mL) was added at room temperature and stirring continued for 2 hours.
  • TLC (7:1:2 iPrOH: ⁇ H4 ⁇ H:H2 ⁇ ) showed the appearance of triphosphate material.
  • the solution was concenfrated, dissolved in water and loaded on a DEAE Sephadex A-25 column.
  • the column was washed with a gradient up to 0.6 M TEAB buffer and the product eluted off in fractions 170-179.
  • the fractions were analyzed by ion exchange HPLC. Each fraction showed one triphosphate peak that eluted at ⁇ 6.77 minutes.
  • the fractions were combined and pumped down from methanol to remove buffer salt to afford 17 mg of product.
  • Our initial pool contained 3 x IO 14 individual sequences of 2'-amino-dCTP/2'- amino-dUTP RNA.
  • 2'-amino-2'- deoxynucleotides do not interfere with the reverse transcription and amplification steps of selection and confer nuclease resistance.
  • the 16- mer subsfrate had two domains, 5 and 10 nucleotides long, that bind the pool, separated by an unpaired guanosine.
  • On the 5' end ofthe substrate was a biotin attached by a C18 linker.
  • Enzymatic nucleic acid molecule Pool Prep The initial pool DNA was prepared by converting the following template oligonucleotides into double-sfranded DNA by filling in with taq polymerase.
  • All DNA oligonucleotides were synthesized by Operon technologies.
  • Template oligos were purified by denaturing PAGE and Sep-pak chromatography columns (Waters). RNA subsfrate oligos were using standard solid phase chemistry and purified by denaturing PAGE followed by ethanol precipitation. Substrates for in vitro cleavage assays were 5'- end labeled with gamma- 32 P-ATP and T4 polynucleotide kinase followed by denaturing PAGE purification and ethanol precipitation.
  • RNA pool was made by franscription of 500 pmole (3 x IO 14 molecules) of this DNA as follows.
  • Template DNA was added to 40 mM tris-HCl (pH 8.0), 12 mM MgCl 2 , 5 mM dithiothreitol (DTT), 1 mM spermidine, 0.002% triton X-100, 1 mM LiCl, 4% PEG-8000, 10% methanol, 2 mM ATP, 2 mM GTP, 2 mM 2'-amino-dCTP, 2 mM 2'- amino-dUTP, 5 U/ml inorganic pyrophosphatase, and 5 U/ ⁇ l T7 RNA polymerase at room temperature for a total volume of 1 ml.
  • a separate reaction contained a trace amount of
  • the flow was capped off and 1000 pmole of initial pool RNA in 200 ⁇ l column buffer was added to the column and incubated 30 minutes at room temperature.
  • the column was uncapped and washed with 2 ml column buffer, then capped off.
  • the column was uncapped and eluent collected followed by three 200 ⁇ l elution buffer washes.
  • the eluent washes were ethanol precipitated using glycogen as carrier and rehydrated in 50 ⁇ l sterile H O.
  • the eluted RNA was amplified by standard reverse transcription/PCR amplification techniques.
  • RNA was incubated with 20 pmol of primer 1 in 14 ⁇ l volume 90° for 3 min then placed on ice for 1 minute.
  • the following reagent were added (final concentrations noted): IX PCR buffer, 1 mM each dNTP, 2 U/ ⁇ l RNase Inhibitor, 10 U/ ⁇ l SuperscriptTM IL reverse transcriptase.
  • the reaction was incubated 42° for 1 hour followed by 95° for 5 min in order to inactivate the reverse transcriptase.
  • the volume was then increased to 100 ⁇ l by adding water and reagents for PCR: IX PCR buffer, 20 pmol primer 2, and 2.5 U taq DNA polymerase.
  • the reaction was cycled in a Hybaid thermocycler: 94°, 4 min; (94°C, 30 sec; 54°C, 30 sec; 72°C, 1 min) X 25; 72°C, 5 min. Products were analyzed on agarose gel for size and ethanol precipitated. One-third to one- fifth ofthe PCR DNA was used to transcribe the next generation, in 100 ⁇ l volume, as described above. Subsequent rounds used 20 pmol RNA for the column with 40 pmol substrate.
  • TWO COLUMN SELECTION At generation 8 (G8), the column selection was changed to the two column format. 200 pmoles of 22 mer 5 '-biotinylated subsfrate (5'- biotin-C18 linker-GCC GUG GGU UGC ACA CCU UUC C-C18 linker-thiol modifier C6 S-S-inverted abasic-3') was used in the selection column as described above. Elution was in 200 ⁇ l elution buffer followed by a 1 ml elution buffer wash. The 1200 ⁇ l eluent was passed through a product trap column by gravity.
  • the product trap column was prepared as follows: 200 pmol 16 mer 5 '-biotinylated "product" (5'-GGU UGC ACA CCU UUC C-C18 linker-biotin-3') was linked to the column as described above and the column was equilibrated in elution buffer. Eluent from the product column was precipitated as previously described. The products were amplified as above only with 2.5-fold more volume and 100 pmol each primer. 100 ⁇ l ofthe PCR reaction was used to do a cycle course; the remaining fraction was amplified the minimal number of cycles needed for product. After 3 rounds (Gl 1), there was visible activity in a single turnover cleavage assay.
  • CLONING AND SEQUENCING Generations 13 and 22 were cloned using Novagen's Perfectly BluntTM Cloning kit (pT7Blue-3 vector) following the kit protocol. Clones were screened for insert by PCR amplification using vector-specific primers. Positive clones were sequenced using ABI Prism 7700 sequence detection system and vector-specific primer. Sequences were aligned using Mac Vector software; two- dimensional folding was performed using Mulfold software ( Zuker,, 1989, Science 244, 48-52; Jaeger et al, 1989, Biochemistry 86, 7706-7710; Jaeger et al, 1989, R. F. Doolittle ed., Methods in Enzymology, 183, 281-306).
  • Individual clone transcription units were constructed by PCR amplification with 50 pmol each primer 1 and primer 2 in IX PCR buffer, 0.2 mM each dNTP, and 2.5 U of taq polymerase in 100 ⁇ l volume cycled as follows: 94°C, 4 min; (94°C, 30 sec; 54°C, 30 sec; 72°C, 1 min) X 20; 72°C, 5 min. Transcription units were ethanol precipitated, rehydrated in 30 ⁇ l H2O, and 10 ⁇ l was transcribed in 100 ⁇ l volume and purified as previously described.
  • the molecule was shortened even further by truncating base pairs in the stem loop structures as well as the substrate recognition arms to yield a 48 nucleotide molecule.
  • many ofthe ribonucleotides were replaced with 2-O-methyl modified nucleotides to stabilize the molecule.
  • An example ofthe new motif is given in Figure 13.
  • Table 60 outlines the substrate requirements for Class I motif. Subsfrates maintained Watson-Crick or wobble base pairing with mutant Class I constructs. Activity in single turnover kinetic assay is shown relative to wild type Class I and 22 mer substrate (50 mM Tris-HCL (pH 7.5), 140 mM KCl, 10 mM NaCl, 1 mM MgCl 2 , 100 nM ribozyme, 5 nM substrate, 37°C).
  • Figure 25 shows a representation of Class I ribozyme stem truncation and loop replacement analysis. The K re j is compared to a 61 mer Class I ribozyme measured as described above.
  • Figure 26 shows examples of Class I ribozymes with truncated stem(s) and/or non-nucleotide linker replaced loop structures.
  • viral RNA is present as a potential target for enzymatic nucleic acid molecule cleavage at several processes: uncoating, translation, RNA replication and packaging.
  • Target RNA may be more or less accessible to enzymatic nucleic acid molecule cleavage at any one of these steps.
  • LRES initial ribosome entry site
  • these other viral processes are not represented in the OST7 system.
  • the resulting RNA/protein complexes associated with the target viral RNA are also absent.
  • these processes may be coupled in an HCV-infected cell which could further impact target RNA accessibility. Therefore, we tested whether enzymatic nucleic acid molecules designed to cleave the HCV 5'UTR could effect a replicating viral system.
  • Poliovirus (PV) is a positive strand RNA virus like HCV, but unlike HCV is non-enveloped and replicates efficiently in cell culture.
  • the HCV-PV chimera expresses a stable, small plaque phenotype relative to wild type PV.
  • the capability ofthe new enzymatic nucleic acid molecule motifs to inhibit HCV RNA intracellularly was tested using a dual reporter system that utilizes both firefly and Renilla luciferase ( Figure 14).
  • OST-7 cells were plated at 12,500 cells per well in black walled 96-well plates (Packard) in medium DMEM containing 10%> fetal bovine serum, 1%> pen/strep, and 1% L-glutamine and incubated at 37°C overnight.
  • T7C1-341 Wang et al, 1993, J. of Virol. 67, 3338-33414
  • pRLSV40 Renilla confrol plasmid Promega Co ⁇ oration
  • the complex mixture was incubated at 37 C for 20 minutes. The media was removed from the cells and 120 ⁇ l of Opti-mem media was added to the well followed by 30 ⁇ l ofthe 5X complex mixture. 150 ⁇ l of Opti-mem was added to the wells holding the untreated cells. The complex mixture was incubated on OST-7 cells for 4 hours, lysed with passive lysis buffer (Promega Co ⁇ oration) and luminescent signals were quantified using the Dual Luciferase Assay Kit using the manufacturer's protocol (Promega Co ⁇ oration).
  • the data shown in Figure 15 is a dose curve of enzymatic nucleic acid molecule targeting site 146 ofthe HCV RNA and is presented as a ratio between the firefly and Renilla luciferase fluorescence.
  • the enzymatic nucleic acid molecule was able to reduce the quantity of HCV RNA at all enzymatic nucleic acid molecule concentrations yielding an IC 50 of approximately 5 nM.
  • Other sites were also efficacious (Figure 16), in particular enzymatic nucleic acid molecules targeting sites 133, 209, and 273 were also able to reduce HCV RNA compared to the i ⁇ elevant (LRR) confrols.
  • LRR i ⁇ elevant
  • Enzymatic nucleic acid molecules were constructed with 2'-O- methyl, and 2'-amino (NH 2 ) nucleotides and included no ribonucleotides (Table 56; gene name: no ribo) and kinetic analysis was performed as described in example 13. 100 nM enzymatic nucleic acid was mixed with trace amounts of substrate in the presence of 1 mM MgCl 2 at physiological conditions (37°C).
  • the Amberzyme with no ribonucleotide present in it has a K re ⁇ of 0.13 compared to the enzymatic nucleic acid with a few ribonucleotides present in the molecule shown in Table 56 (ribo). This shows that Amberzyme enzymatic nucleic acid molecule may not require the presence of 2' -OH groups within the molecule for activity.
  • Class II (zinzyme) enzymatic nucleic acid molecules Class LI (zinzyme) ribozymes were tested for their ability to cleave base-paired subsfrates with all sixteen possible combinations of bases immediately 5' and 3' proximal to the bulged cleavage site G. Ribozymes were identical in all remaining positions of their 7 base pair binding arms. Activity was assessed at two and twenty- four hour time points under standard reaction conditions [20 mM HEPES pH 7.4, 140 mM KCl, 10 mM NaCl, 1 mM MgCl 2 , 1 mM CaCl 2 - 37° C]. Figure 19 shows the results of this study.
  • Base paired subsfrate UGG (not shown in the figure) cleaved as poorly as CGG shown in the figure.
  • the figure shows the cleavage site substrate triplet in the 5'- 3' direction and 2 and 24 hour time points are shown top to bottom respectively.
  • the results indicate the cleavage site triplet is most active with a 5'- Y-G-H -3' (where Y is C or U and H is A, C or U with cleavage between G and H); however, activity is detected particularly with the 24 hour time point for most paired subsfrates. All positions outside ofthe cleavage triplet were found to tolerate any base pairings (data not shown).
  • RNA was enzymatically generated using the mutant T7 Y639F RNA polymerase prepared by Rui Souza.
  • RNA pools were purified by denaturing gel elecfrophoresus 8% polyacrilamide 7 M Urea.
  • target RNA (resin A) was synthesized and coupled to Iodoacetyl
  • UlfralinkTM resin (Pierce) by the supplier's proceedure:5' -b-L- GGACUGGGAGCGAGCGCGGCGCAGGCACU GAAG-L-S-B-3'; where b is biotin (Glenn Research cat# 10-1953-nn), L is polyethylene glycol spacer (Glenn Research cat# 10-1918- nn), S is thiol-modifier C6 S-S (Glenn Research cat# 10-1936-nn), B is a standard inverted deoxy abasic.
  • RNA pools were added to 100 ⁇ l of 5 uM Resin A in the buffer A (20 mM HEPES pH 7.4, 140 mM KCL, 10 mM NaCl) and incubated at 22°C for 5 minutes. The temperature was then raised to 37°C for 10 minutes. The resin was washed with 5 ml buffer A. Reaction was triggered by the addition of buffer B(20 mM HEPES pH 7.4, 140 mM KCL, 10 mM NaCl, 1 mM MgCl 2 , 1 mM CaCl 2 ). Incubation proceeded for 20 minutes in the first generation and was reduced progressively to 1 minute in the final generations; with 13 total generations.
  • buffer A (20 mM HEPES pH 7.4, 140 mM KCL, 10 mM NaCl, 1 mM MgCl 2 , 1 mM CaCl 2 .
  • RNA's were removed by a 1.2 ml denaturing wash IM NaCl, 10 M Urea at 94° C over 10 minutes. RNA's were double precipitated in 0.3 M sodium acetate to remove CI " ions inhibitory to reverse franscription.
  • RNA's were again transcribed with the modified NTP's described above.
  • 13 generations cloning and sequencing provided 14 sequences which were able to cleave the target subsfrate.
  • Six sequences were characterized to determine secondary structure and kinetic cleavage rates.
  • the structures and kinetic data are given in Figure 17.
  • the sequences of eight other enzymatic nucleic acid molecule sequences are given in Table 57.
  • the size, sequence, and chemical compositions of these molecules can be modified as described below or using other techniques well known in the art.
  • Class I and Class LI enzymatic nucleic acid molecule can be engineered and re-engineered using the techniques shown in this application and known in the art.
  • the size of class I and class II enzymatic nucleic acid molecules can, be reduced or increased using the techniques known in the art (Zaug et al, 1986, Nature, 324, 429; Ruffher et al, 1990, Biochem., 29, 10695; Beaudry et al, 1990, Biochem., 29, 6534; McCall et al, 1992, Proc. Natl. Acad.
  • Example 16 Activity of Class TJ (zinzyme) nucleic acid catalysts to inhibit HER2 gene expression
  • Applicant has designed, synthesized and tested several class LI (zinzyme) ribozymes targeted against HER2 RNA (see, for example, Tables 58, 59, and 62) in cell proliferation
  • the model proliferation assay used in the study can require a cell-plating density of
  • Ribozymes (50-200 nM) were delivered in the presence of cationic lipid at 2.0-5.0 ⁇ g/mL and inhibition of proliferation was determined on day 5 post-treatment. Two full ribozyme screens were completed resulting in the selection of 14 ribozymes.
  • Class LI
  • FIG. 20 An example of results from a cell culture assay is shown in Figure 20.
  • Class II ribozymes targeted against HER2 RNA are shown to cause significant inhibition of proliferation of cells. This shows that ribozymes, for instance the Class ⁇ (zinzyme) ribozymes are capable of inhibiting HER2 gene expression in mammalian cells.
  • Real time RT-PCR (TaqMan® assay) was performed on purified RNA samples using separate primer/probe sets specific for either target HER2 RNA or confrol actin RNA (to normalize for differences due to cell plating or sample recovery). Results are shown as the average of triplicate determinations of HER2 to actin RNA levels post- treatment.
  • Figure 30 shows class II ribozyme (zinzyme) mediated reduction in HER2 RNA targeting site 972 vs a scrambled attenuated control.
  • Active ribozyme was mixed with binding arm-attenuated control (BAC) ribozyme to a final oligonucleotide concenfration of either 100, 200 or 400 nM and delivered to cells in the presence of cationic lipid at 5.0 ⁇ g/mL. Mixing active and BAC in this manner maintains the lipid to ribozyme charge ratio throughout the dose response curve.
  • BAC binding arm-attenuated control
  • Figure 33 shows a combined dose response plot of both anti- proliferation and RNA reduction data for a class LI ribozyme targeting site 972 of HER2 RNA (RPI 19293).
  • Example 17 Reduction of ribose residues in Class LI (zinzyme) nucleic acid catalysts
  • Class LI (zinzyme) nucleic acid catalysts were tested for their activity as a function ribonucleotide content.
  • a Zinzyme having no ribonucleotide residue (ie., no 2'-OH group at the 2' position ofthe nucleotide sugar) against the K-Ras site 521 was designed. This molecules were tested utilizing the chemistry shown in Figure 27a. The in vitro catalytic activity zinzyme construct was not significantly effected (the cleavage rate reduced only 10 fold).
  • the Kras zinzyme shown in Figure 27a was tested in physiological buffer with the divalent concentrations as indicated in the legend (high NaCl is an altered monovalent condition shown) of Figure 28.
  • the 1 mM Ca* * condition yielded a rate of 0.005 min "1 while the 1 mM Mg " " " condition yielded a rate of 0.002 min "1 .
  • the ribose containing wild type yields a rate of 0.05 min while subsfrate in the absence of zinzyme demonstrates less than 2% degradation at the longest time point under reaction conditions shown.
  • FIG. 29 is a diagram ofthe alternate formats tested and their relative rates of catalysis.
  • the effect of substitution of ribose G for the 2'-O-methyl C-2'-O-methyl A in the loop of Zinzyme was insignificant when assayed with the Kras target but showed a modest rate enhancement in the HER2 assays.
  • Zinzyme motifs including the fully stabilized "0 ribose” (RPI 19727) are well above background noise level degradation. Zinzyme with only two ribose positions (RPI 19293) are sufficient to restore "wild-type” activity. Motifs containing 3 (RPI 19729), 4 (RPI 19730) or 5 ribose (RPI 19731) positions demonsfrated a greater extent of cleavage and profiles almost identical to the 2 ribose motif. Applicant has thus demonsfrated that a Zinzyme with no ribonucleotides present at any position can catalyze efficient RNA cleavage activity.
  • Zinzyme enzymatic nucleic acid molecules do not require the presence of 2'- ⁇ H group within the molecule for catalytic activity.
  • Example 18 Activity of reduced ribose containing Class II (zinzyme) nucleic acid catalysts to inhibit HER2 gene expression
  • a cell proliferation assay for testing reduced ribo class LI (zinzyme) nucleic acid catalysts (50-400 nM) targeting HER2 site 972 was performed as described in example 19. The results of this study are summarized in Figure 35. These results indicate significant inhibition of HER2 gene expression using stabilized Class II (zinzyme) motifs, including two ribo (RPI 19293), one ribo (RPI 19728), and non-ribo (RPI 19727) containing nucleic acid catalysts.
  • NTP's described in this invention have several research and commercial applications. These modified nucleotide triphosphates can be used for in vitro selection (evolution) of oligonucleotides with novel functions. Examples of in vitro selection protocols are inco ⁇ orated herein by reference (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 , 9, 1183; Breaker, 1996, Curr. Op. Biotech., 7, 442).
  • modified nucleotide triphosphates can be employed to generate modified oligonucleotide combinatorial chemistry libraries.
  • references for this technology exist (Brenner et al, 1992, PNAS 89, 5381-5383, Eaton, 1997, Curr. Opin. Chem. Biol. 1, 10-16), which are all inco ⁇ orated herein by reference.
  • Enzymatic nucleic acid molecules of this invention may be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of specific RNA in a cell.
  • the close relationship between enzymatic nucleic acid molecule activity and the structure ofthe target RNA allows the detection of mutations in any region ofthe molecule which alters the base-pairing and three-dimensional structure ofthe target RNA.
  • By using multiple enzymatic nucleic acid molecules described in this invention one may map nucleotide changes which are important to RNA structure and function in vitro, as well as in cells and tissues. Cleavage of target RNAs with enzymatic nucleic acid molecules may be used to inhibit gene expression and define the role (essentially) of specified gene products in the progression of disease.
  • enzymatic nucleic acid molecules of this invention include detection ofthe presence of mRNAs associated with related conditions. Such RNA is detected by determimng the presence of a cleavage product after treatment with a enzymatic nucleic acid molecule using standard methodology.
  • enzymatic nucleic acid molecules which can cleave only wild- type or mutant forms ofthe target RNA are used for the assay.
  • the first enzymatic nucleic acid molecule is used to identify wild-type RNA present in the sample and the second enzymatic nucleic acid molecule will be used to identify mutant RNA in the sample.
  • synthetic subsfrates of both wild-type and mutant RNA will be cleaved by both enzymatic nucleic acid molecules to demonstrate the relative enzymatic nucleic acid molecule efficiencies in the reactions and the absence of cleavage ofthe "non- targeted" RNA species.
  • the cleavage products from the synthetic subsfrates will also serve to generate size markers for the analysis of wild type and mutant RNAs in the sample population.
  • each analysis can involve two enzymatic nucleic acid molecules, two subsfrates and one unknown sample which can be combined into six reactions.
  • the presence of cleavage products can be determined using an RNAse protection assay so that full-length and cleavage fragments of each RNA can be analyzed in one lane of a polyacrylamide gel. It is not absolutely required to quantify the results to gain insight into the expression of mutant RNAs and putative risk ofthe desired phenotypic changes in target cells.
  • the expression of mRNA whose protein product is implicated in the development ofthe phenotype is adequate to establish risk.
  • RNA levels are compared qualitatively or quantitatively.
  • sequence-specific enzymatic nucleic acid molecules ofthe instant invention can have many ofthe 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 ofthe enzymatic nucleic acid molecule is ideal for cleavage of RNAs of unknown sequence.
  • 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" ⁇ -galactosidase message by the ligation of new ⁇ -galactosidase sequences onto the defective message [ xii ].
  • RNAse P RNA M1 RNA
  • Size -290 to 400 nucleotides.
  • RNA portion of a ubiquitous ribonucleoprotein enzyme • RNA portion of a ubiquitous ribonucleoprotein enzyme.
  • Reaction mechanism possible attack by M -OH to generate cleavage products with 3'- OH and 5' -phosphate.
  • RNAse P is found throughout the prokaryotes and eukaryotes.
  • the RNA subunit has been sequenced from bacteria, yeast, rodents, and primates.
  • Reaction mechanism 2'-OH of an internal adenosine generates cleavage products with 3'- OH and a "lariat" RNA containing a 3' -5' and a 2' -5' branch point.
  • Reaction mechanism attack by 2' -OH 5' to the scissile bond to generate cleavage products with 2',3'-cyclic phosphate and 5' -OH ends.

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