CA2403243A1 - Nucleic acid based modulators of gene expression - Google Patents

Abstract

Nucleic acid molecules (antisenses or ribozymes) useful as inhibitors of gene expression, especially of HER2, BACE, TERT, PTP-1B, MetAP-2, HBV, phospholamban, presenilin-2 and PKC-alpha. The nucleic acid molecules can be modified in various ways on the sugar and/or base moieties and/or on the phosphate backbone. They are used in pharmaceutical formulations for the treatment of diseases involving increased expression of the target genes. Also disclosed is a method for the synthesis of a modified pyrimidine nucleotide triphosphate and its incorporation into an oligonucleotide.

Description

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NUCLEIC ACID BASED MODULATORS OF GENE EXPRESSION
Background of the Invention 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-s related diseases, and/or hepatitis B infections and related conditions.
Summary,of the Invention 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 (fox 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 In a preferred embodiment, the invention features novel nucleic acid-based techniques [e.g., enzymatic nucleic acid molecules (ribozymes), antisense nucleic acids, 2-SA 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-1b (PTP-1B, 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. X03363), Phospholamban (PLN, Genbank accession No.
NM 002667), Telomerase (TERT, Genbank accession No. NM_003219) and Hepatitis B
virus genes (HBV, Genbank accession No. AF100308.1). Such 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.
Thus, in an additional preferred embodiment, 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-1B), 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. In particular, applicant describes the selection and function of nucleic acid molecules capable of cleaving RNAs encoded by these genes and their use to reduce levels of PTP-1B, 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.
In a preferred embodiment, the invention features the use of one or more of the nucleic acid-based techniques independently or in combination to inhibit the expression of the genes encoding PTP-1B, MetAP-2, BACE, ps-1, ps-2, HER2, PLN, TERT, and/or HBV. Specifically, the invention features the use of nucleic acid-based techniques to specifically inhibit the expression of PTP-1B, MetAP-2, BACE, ps-1, ps-2, HER2, PLN, TERT, PKC alpha, and/or HBV genes.
In yet another preferred embodiment, 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-1B, MetAP-2, BALE, ps-1, ps-2, HER2, PLN, TERT, PKC alpha and/or HBV RNA.
Applicant indicates that these nucleic acid molecules are able to inhibit expression of PTP-1B, MetAP-2, BACE, ps-1, ps-2, HER2, PLN, TERT, PKC alpha, and/or HBV
genes. Those of ordinary skill in the art, will find that it is clear from the examples described that other nucleic acid molecules that inhibit target PTP-1B, MetAP-2, BACE, ps-1, ps-2, HER2, PLN, TERT, and/or HBV encoding mRNAs may be readily designed and are within the scope of the invention.
By "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). In one embodiment, 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. In another embodiment, inhibition with nucleic acid molecules, including enzymatic nucleic acid and antisense molecules, is preferably greater than that observed in the presence of, for example, an oligonucleotide with scrambled sequence or with mismatches. In another embodiment, inhibition of target genes with the nucleic acid molecule of the instant invention is greater than in the presence of the nucleic acid molecule than in its absence.
According to the invention, the activity of telomerase enzyme or the level of RNA encoding one or more portein subunits of the 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.
By "enzymatic nucleic acid molecule" it is meant a nucleic acid molecule which has complementarity in a substrate binding region to a specified gene target, and also has an enzymatic activity which is active to specifically cleave target RNA. That is, the enzymatic nucleic acid molecule is able to intermolecularly cleave RNA and thereby inactivate a target RNA molecule. These complementary regions allow sufficient hybridization of the enzymatic nucleic acid molecule to the target RNA and thus permit cleavage. One hundred percent complementarity is preferred, but complementarity as low as 50-75% may also be useful in this invention. The nucleic acids may be modified at the base, sugar, and/or phosphate groups. The term 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. The specific enzymatic nucleic acid molecules described in the instant application are not meant to be limiting and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it have a specific substrate binding site which is complementary to one or more of the target nucleic acid regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart a nucleic acid cleaving activity to the molecule (Cech et al., U.S. Patent No.
4,987,071;
Cech et al., 1988, JAMA 260:20 3030-4).
By "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.
By "enzymatic portion" or "catalytic domain" is meant that portion/region of the enzymatic nucleic acid molecule essential for cleavage of a nucleic acid substrate (for example see Figures 1-5).
By "substrate binding arm" or "substrate binding domain" is meant that portion/region of a ribozyme which is complementary to (i.e., able to base-pair with) a portion of its substrate. Generally, such complementarity is 100%, but can be less if desired. For example, as few as 10 bases out of 14 may be base-paired. Such arms are shown generally in 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 of the invention may have binding arms that are contiguous or non-contiguous and may be of varying lengths. The length of the binding arms) are preferably greater than or equal to four nucleotides and of sufficient length to stably interact with the target RNA; specifically 12-100 nucleotides; more specifically 14-24 nucleotides long. If two binding arms are chosen, the design is such that the length of the binding arms are symmetrical (i.e., each of the binding arms is of the same length; e.g., five and five nucleotides, six and six nucleotides or seven and seven nucleotides long) or asymmetrical (i.e., the binding arms are of different length; e.g., six and three nucleotides;
three and six nucleotides long; four and five nucleotides long; four and six nucleotides long; four and seven nucleotides long; and the like). Binding arms can be complementary to the specified substrate, to a portion of the indicated substrate, to the indicated substrate sequence and additional adjacent sequence, or a portion of the indicated sequence and additional adjacent sequence.
By "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.
By "G-cleaver" motif is meant, an enzymatic nucleic acid molecule comprising a motif as described in Eckstein et al., International PCT publication No. WO
99/16871, incorporated by reference herein in its entirety, including the drawings.

By "zinzyme" motif is meant, 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, incorporated by reference herein in its entirety, including the drawings.
By "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, incorporated by reference herein in its entirety, including the drawings.
By 'DNAzyme' is meant, an enzymatic nucleic acid molecule lacking a ribonucleotide (2'-OH) group. In particular embodiments, the enzymatic nucleic acid molecule may have an attached linkers) 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.
By "sufficient length" is meant an oligonucleotide of greater than or equal to nucleotides that is of a length great enough to provide the intended function under the expected condition. For example, for binding arms of enzymatic nucleic acid "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.
By "stably interact" is meant, interaction of the oligonucleotides with target nucleic acid (e.g., by forming hydrogen bonds with complementary nucleotides in the target under physiological conditions).
By "equivalent" RNA to PTP-1B, 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-1B, MetAP-2, BACE, ps-1, ps-2, HER2, PLN, TERT, and/or HBV proteins or encoding for proteins with similar function as PTP-1B, 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. The equivalent RNA sequence also includes in addition to the coding region, regions such as 5'-untranslated region, 3'-untranslated region, introns, intron-exon junction and the like in HBV.

By "homology" is meant the nucleotide sequence of two or more nucleic acid molecules is partially or completely identical.
By "antisense nucleic acid", it is meant a non-enzymatic nucleic acid molecule that binds to target RNA by means of RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al., 1993 Nature 365, 566) interactions and alters the activity of the target RNA (for a review, see Stein and Cheng, 1993 Science 261, 1004 and Woolf et al., US
patent No. 5,849,902). Typically, antisense molecules will be complementary to a target sequence along a single contiguous sequence of the antisense molecule.
However, in certain embodiments, an antisense molecule may bind to substrate such that the substrate molecule forms a loop, and/or an antisense molecule may bind such that the antisense molecule forms a loop. Thus, the antisense molecule may be complementary to two (or even more) 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. For a review of current antisense strategies, see Schmajuk et al., 1999, J. Biol.
Chem., 274, 21783-21789, Delihas et al., 1997, Nature, 15, 751-753, Stein et al., 1997, Antisense N. A. Drug Dev., 7, 151, Crooke, 1998, Biotech. Genet. Eng. Rev., 15, 121-157, Crooke, 1997, Ad. Pharmacol., 40, 1-49. In addition, 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.
By "2-SA antisense chimera" it is meant, an antisense oligonucleotide containing a S'-phosphorylated 2'-5'-linked adenylate residue. These chimeras bind to target RNA in a sequence-specific manner and activate a cellular 2-SA-dependent ribonuclease which, in turn, cleaves the target RNA (Torrence et al., 1993 Proc. Natl. Acad. Sci. USA
90, 1300).
By "triplex DNA" it is meant an oligonucleotide that can bind to a double-stranded DNA in a sequence-specific manner to form a triple-strand helix. Formation of such triple helix structure has been shown to inhibit transcription of the targeted gene (Duval-Valentin et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 504).
By "gene" it is meant a nucleic acid that encodes a RNA.

By "complementarity" is meant that a nucleic acid can form hydrogen bonds) with another RNA sequence by either traditional Watson-Crick or other non-traditional types.
In reference to the nucleic molecules of the present invention, the binding free energy for a nucleic acid molecule with its target or complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., ribozyme cleavage, antisense or triple helix inhibition. Determination of binding free energies for nucleic acid molecules is well known in the art (see, e.g., Turner et al., 1987, CSH Symp. Quant. Biol. LII
pp.123-133;
Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377; Turner et al., 1987, J. Am.
Chem. Soc. 109:3783-3785). A percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Perfectly complementary" means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.
At least seven basic varieties of naturally-occurnng enzymatic RNAs are known presently. Each can catalyze the hydrolysis of RNA phosphodiester bonds in traps (and thus can cleave other RNA molecules) under physiological conditions. Table I
summarizes some of the characteristics of these ribozymes. In general, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA.
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. In addition, 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-1B, 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-1B, MetAP-2, BACE, ps-1, ps-2, HER2, PLN, TERT, and/or HBV in a cell or tissue.
In one of the preferred embodiments of the inventions described herein, the enzymatic nucleic acid molecule is formed in a hammerhead or hairpin motif, but may also be formed in the motif of a hepatitis delta virus, group I intron, group II
intron or RNase P
RNA (in association with an RNA guide sequence), Neurospora VS RNA, DNAzymes, NCH cleaving motifs, or G-cleavers. Examples of such hammerhead motifs are described by Dreyfus, supra, Rossi et al., 1992, AIDS Research and Human Retroviruses 8, 183.
Examples of hairpin motifs are described by Hampel et al., EP0360257, Hampel and Tritz, 1989 Biochemistry 28, 4929, Feldstein et al., 1989, Gene 82, 53, Haseloff and Gerlach, 1989, Gene, 82, 43, Hampel et al., 1990 Nucleic Acids Res. 18, 299; and Chowrira &
McSwiggen, US. Patent No. 5,631,359. The hepatitis delta virus motif is described by Perrotta and Been, 1992 Biochemistry 31, 16. The RNase P motif is described by Guerner-Takada et al., 1983 Cell 35, 849; Forster and Altman, 1990, Science 249, 783;
and Li and Altman, 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. 2, 761; Michels and Pyle, 1995, Biochemistry 34, 2965; and Pyle et al., International PCT Publication No. WO
96/22689.
The Group I intron is described by Cech et al., U.S. Patent 4,987,071.
DNAzymes are described by Usman et al., International PCT Publication No. WO 95/11304;
Chartrand et al., 1995, NAR 23, 4092; Breaker et al., 1995, Chem. Bio. 2, 655; and Santoro et al., 1997, PNAS 94, 4262. NCH cleaving motifs are described in Ludwig & Sproat, International PCT Publication No. WO 98/58058; and G-cleavers are described in Kore et al., 1998, Nucleic Acids Research 26, 4116-4120 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 (Beigelinan et al., International PCT
publication No. WO 99/55857), all these references are incorporated by reference herein in their totalities, including drawings and can also be used in the present invention. These specific motifs are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule (Cech et al., U.S. Patent No. 4,987,071).
In preferred embodiments of the present invention, a nucleic acid molecule, e.g., an antisense molecule, a triplex DNA, or a ribozyme, is 13 to 100 nucleotides in length, e.g., in specific embodiments 35, 36, 37, or 38 nucleotides in length (e.g., for particular ribozymes or antisense). In particular embodiments, the nucleic acid molecule is 15-100, 17-100, 20-100, 21-100, 23-100, 25-100, 27-100, 30-100, 32-100, 35-100, 40-100, 50-100, 60-100, 70-100, or 80-100 nucleotides in length. Instead of 100 nucleotides being the upper limit on the length ranges specified above, the upper limit of the length range can be, for example, 30, 40, 50, 60, 70, or 80 nucleotides. Thus, for any of the length ranges, the length range for particular embodiments has lower limit as specified, with an upper limit as specified which is greater than the lower limit. For example, in a particular embodiment, the length range can be 35-50 nucleotides in length. All such ranges are expressly included. Also in particular embodiments, a nucleic acid molecule can have a length which is any of the lengths specified above, for example, 21 nucleotides in length.
In a preferred embodiment, 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. For example, the enzymatic nucleic acid molecule is preferably targeted to a highly conserved sequence region of target RNAs encoding PTP-1B, MetAP-2, BACE, ps-l, ps-2, HER2, PLN, TERT, and/or HBV proteins (specifically PTP-1B, 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 of the invention. Such nucleic acid molecules can be delivered exogenously to 5 specific tissue or cellular targets as required. Alternatively, the nucleic acid molecules (e.g., ribozymes and antisense) can be expressed from DNA and/or RNA vectors that are delivered to specific cells.
As used in herein "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 10 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).
By "PTP-1B, MetAP-2, BACE, ps-l, ps-2, HER2, PLN, TERT, and/or HBV
proteins" is meant, a protein or a mutant protein derivative thereof, comprising sequence expressed and/or encoded by PTP-1B, MetAP-2, BACE, ps-1, ps-2, HER2, PLN, TERT, genes and/or the HBV genome respectively.
By "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-1B, MetAP-2, BACE, ps-l, ps-2, HER2, PLN, TERT, and/or HBV expression are useful for the prevention of the 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-1B, 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 related to the levels of PTP-1B, MetAP-2, BACE, ps-1, ps-2, HER2, PLN, TERT, and/or HBV in a cell or tissue.

By "related" is meant that the reduction of PTP-1B, MetAP-2, BACE, ps-1, ps-2, HER2, PLN, TERT, and/or HBV expression (specifically PTP-1B, MetAP-2, BACE, ps-1, ps-2, HER2, PLN, TERT, and/or HBV genes) RNA levels and thus reduction in the level of the respective protein will relieve, to some extent, the symptoms of the disease or condition.
The nucleic acid-based inhibitors of the invention are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells or tissues. The nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex vivo, or in vivo through injection, infusion pump or stmt, with or without their incorporation in biopolymers. In preferred embodiments, the enzymatic nucleic acid inhibitors comprise sequences, which are complementary to the substrate 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.
1n yet another embodiment, 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 of the enzymatic nucleic acid molecules in Tables 3-29, 31, 33, 34, 37-43, 56, 58, 59, 62, 63. Similarly, triplex molecules can be provided targeted to the corresponding DNA target regions, and containing the DNA equivalent of a target sequence or a sequence complementary to the specified target (substrate) sequence.
Typically, antisense molecules will be complementary to a target sequence along a single contiguous sequence of the antisense molecule. However, in certain embodiments, an antisense molecule may bind to substrate such that the substrate molecule forms a loop, and/or an antisense molecule may bind such that the antisense molecule forms a loop.
Thus, the antisense molecule may be complementary to two (or even more) 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.
In another aspect, 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.

By "consists essentially off' is meant that the active nucleic acid molecule of the invention, for example, an enzymatic nucleic acid molecule, contains an enzymatic center or core equivalent to those in the examples, and binding arms able to bind mRNA such that cleavage at the target site occurs. Other sequences may be present which do not interfere with such cleavage. Thus, a core region may, for example, include one or more loop or stem-loop structures, which do not prevent enzymatic activity. "X" in the sequences in Tables 3, 4, 9,10,13,14,18,19, 24, 25, 33, 34, 37, 38, 63 can be such a loop. A core sequence for a hammerhead ribozyme can be CUGAUGAG X CGAA where X=GCCGUUAGGC or other stem II region as specifically or generally known in the art.
In another aspect of the invention, ribozymes or antisense molecules that interact with target RNA molecules and inhibit PTP-1B, MetAP-2, BACE, ps-1, ps-2, HER2, PLN, TERT, and/or HBV (specifically PTP-1B, 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.
Preferably, the recombinant vectors capable of expressing the ribozymes or antisense are delivered as described above, and persist in target cells. Alternatively, 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.
Delivery of 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 reintroduction 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.
By RNA is meant a molecule comprising at least one ribonucleotide residue. By "ribonucleotide" is meant a nucleotide with a hydroxyl group at the 2' position of a (3-D-ribo-furanose moiety.
By "vectors" is meant any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.

By "patient" is meant an organism, which is a donor or recipient of explanted cells or the cells themselves. "Patient" also refers to an organism to which the nucleic acid molecules of the invention can be administered. Preferably, a patient is a mammal or mammalian cells. More preferably, a patient is a human or human cells.
The nucleic acid molecules of the instant invention, individually, or in combination or in conjunction with other drugs, can be used to treat diseases or conditions discussed above. For example, to treat a disease or condition associated with PTP-1B, MetAP-2, BACE, ps-1, ps-2, HER2, PLN, TERT, and/or HBV, 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.
In a further embodiment, the described molecules, such as antisense or ribozymes, can be used in combination with other known treatments to treat conditions or diseases discussed above. For example, 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 A>DS, age related diseases such as macular degeneration and skin ulceration, Alzheimer's disease, dementia, diabetes, and/or obesity.
In another preferred embodiment, 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-1B, MetAP-2, BACE, ps-l, 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, transplant rejection and autoimmune disease such as multiple sclerosis, lupus, and A)DS, age related diseases such as macular degeneration and skin ulceration, Alzheimer's disease, dementia, diabetes, and/or obesity.

In another preferred embodiment, 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-1B, MetAP-2, BACE, ps-1, ps-2, HER2, PLN, TERT, and/or HBV RNA expression.
By "comprising" is meant including, but not limited to, whatever follows the word "comprising". Thus, use of the term "comprising" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present.
By "consisting of is meant including, and limited to, whatever follows the phrase "consisting of'. Thus, the phrase "consisting of indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.
Description Of The Preferred Embodiments The drawings will first briefly be described.
Drawings:
Figure 1 shows the secondary structure model for seven different classes of enzymatic nucleic acid molecules. Arrow indicates the site of cleavage. --------- indicate the target sequence. Lines interspersed with dots are meant to indicate tertiary interactions.
- is meant to indicate base-paired interaction. Group I Intron: P1-P9.0 represent various stem-loop structures (Cech et al., 1994, Nature Struc. Bio., 1, 273). RNase P
(M1RNA):
EGS represents external guide sequence (Forster et al., 1990, Science, 249, 783; Pace et al., 1990, J. Biol. Chem., 265, 3587). Group II Intron: 5'SS means 5' splice site; 3'SS
means 3'-splice site; IBS means intron binding site; EBS means exon binding site (Pyle et al., 1994, Biochemistry, 33, 2716). VS RNA: I-VI are meant to indicate six stem-loop structures; shaded regions are meant to indicate tertiary interaction (Collins, International PCT Publication No. WO 96/19577). HDV Ribozyme: I-IV are meant to indicate four stem-loop structures (Been et al., US Patent No. 5,625,047). Hammerhead Ribozyme: I-5 III are meant to indicate three stem-loop structures; stems I-III can be of any length and may be symmetrical or asymmetrical (Usman et al., 1996, Curr. Op. Struct.
Bio., 1, 527).
Hairpin °bozyme: Helix 1, 4 and 5 can be of any length; Helix 2 is between 3 and 8 base-pairs long; Y is a pyrimidine; Helix 2 (H2) is provided with a least 4 base pairs (i.e., n is 1, 2, 3 or 4) and helix 5 can be optionally provided of length 2 or more bases 10 (preferably 3 - 20 bases, i. e., m is from 1 - 20 or more). Helix 2 and helix 5 may be covalently linked by one or more bases (i.e., r is >_ 1 base). Helix 1, 4 or S
may also be extended by 2 or more base pairs (e.g., 4 - 20 base pairs) to stabilize the ribozyme structure, and preferably is a protein binding site. In each instance, each N
and N' independently is any normal or modified base and each dash represents a potential base-15 pairing interaction. These nucleotides may be modified at the sugar, base or phosphate.
Complete base-pairing is not required in the helices, but is preferred. Helix 1 and 4 can be of any size (i.e., o and p is each independently from 0 to any number, e.g., 20) as long as some base-pairing is maintained. Essential bases are shown as specific bases in the structure, but those in the art will recognize that one or more may be modified chemically (abasic, base, sugar and/or phosphate modifications) or replaced with another base without significant effect. Helix 4 can be formed from two separate molecules, i.e., without a connecting loop. The connecting loop when present may be a ribonucleotide with or without modifications to its base, sugar or phosphate. "q" >_ is 2 bases. The connecting loop can also be replaced with a non-nucleotide linker molecule. H refers to bases A, U, or C. Y refers to pyrimidine bases. " " refers to a covalent bond. (Burke et al., 1996, Nucleic Acids & Mol. Biol., 10, 129; Chowrira et al., US Patent No.
5,631,359).
Figure 2 shows examples of chemically stabilized ribozyme motifs. HH Rz, represents hammerhead ribozyme motif (LJsman 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 al., 1998, Nucleic Acids Research, 26, 4116-4120). N or n, represent independently a nucleotide which may be same or different and have complementarity to each other; rI, represents ribo-Inosine nucleotide; arrow indicates the site of cleavage within the target. Position 4 of the HH Rz and the NCH Rz is shown as having 2'-C-allyl modification, but those skilled in the art will recognize that this position can be modified with other modifications well known in the art, so long as such modifications do not significantly inhibit the activity of the ribozyme.
Figure 3 shows an example of the Amberzyme ribozyme motif that is chemically stabilized (see, for example, Beigelman et al., International PCT publication No. WO
99/55857; 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.
Figure 4 shows an example of the 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.
Figure 6 is a diagrammatic representation of the hammerhead ribozyme motif known in the art and the NCH motif. Stem II 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-III are meant to indicate three stem-loop structures; stems I-III can be of any length and may be symmetrical or asymmetrical (Usman et al., 1996, Curr. Op. Struct. Bio., 1, 527); arrow indicates the site of cleavage in the target RNA; Rz refers to ribozyme; 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 (LJsman et al., 1996, Curr. Op. Struct.
Bio., 1, 527); NCH-Inosine ltz 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; xI
represent xylo-inosine; arrow indicates the site of cleavage within the target. Position 4 of the 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 of the ribozyme.
Figure 8 is a graphical representation of data showing inhibition of cell proliferation mediated by NCH and HH ribozymes targeted against HER2/neu/ErbB2 gene.
Untreated, refers to cells not treated with ribozymes; HH RZ refers to hammerhead ribozyme; NCX
RZ refers to the NCH ribozymes of the invention; IA refers to catalytically inactive or attenuated ribozyme used as a control.
Figure 9 is a schematic diagram of the 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 regions) 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 regions) 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.
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 of the nucleic acid molecules in the pool, respectively. The column is subjected to conditions sufficient to facilitate cleavage of the immobilized oligonucleotide target. The molecules in the pool that cleave the target (active molecules) have A' region of the 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 of the 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. To separate the contaminating inactive molecules from the active molecules, 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 of the 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 of the substrate 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. In addition, the guanosine (G) shown at the cleavage site of the substrate 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 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 II
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 (refernng to the nucleotides of the substrate 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. In addition, the guanosine (G) shown at the cleavage site of the substrate 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 substrate specificities for Class II
(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 S'-triphosphates.
Figure 23 is a synthetic scheme outlining the synthesis of carboxylate tethered uridine S'-triphosphoates.
Figure 24 is a synthetic scheme outlining the synthesis of S-(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 stems) and/or non-nucleotide linkers used in loop structures.
Figure 27 is a diagram of "no-ribo" class II 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 II ribozymes with varying ribo content and their relative rates of catalysis.
Figure 30 is a graph showing class II ribozyme (zinzyme) mediated reduction of HERZ RNA in SKBR3 breast carcinoma cells. Cells were treated with 100 nm, and run of zinzyme (RPI 18656) targeting site 972 of HER2 RNA and a corresponding scrambled attenuated control complexed with 2.5 p,g/ml of lipid. Active zinzymes and scrambled attenuated controls were compared to untreated cells after 24 hours post treatment.

Figure 31 is a graph showing class II ribozyme (zinzyme) mediated dose response anti-prolferation assay in SKBR3 breast carcinoma cells. Cells were treated with 100 nm, and 200 nm of zinzyme (RPI 18656) targeting site 972 of HER2 RNA and a corresponding scrambled attenuated control complexed with 2.0 ~g/ml of lipid. Active zinzymes and 5 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 10 inhibition of cellular proliferation in SKBR-3 cells treated with RPI 19293 from 0 to 400 nM with S.0 ~g/ml of cationic lipid.
Figure 34 shows a non-limiting example of the replacement of a 2'-O-methyl 5'-CA-3' with a ribo G in the class II (zinzyme) motif. The representative motif shown for the purpose of the figure is a "seven-ribo" zinzyme motif, however, the interchangeability 15 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. For instance, a 2'-O-methyl G can replace the 2'-O-methyl S'-CA-3' and vise versa.
Figure 35 is a graph which shows a screen of class II ribozymes (zinzymes) targeting site 972 of HER2 RNA which contain ribo-G reductions (RPI 19727 = no ribo, 20 RPI 19728 = one ribo, RPI 19293 = two ribo, RPI 19729 = three ribo, RPI
19730 = four ribo, 19731 = five ribo, and RPI 19292 = seven ribo) for anti-proliferative activity in SKBR3 cells.
Figure 36 summarizes the results of functional group modification studies in which various nucleoside analogs were tested for activity in the NCH ribozyme motif.
Krel values describe the cleavage values of a given substituent at position 15.1 relative the Inosine at position 15.1 (I-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.
Krel values describe the cleavage values of a given substituent at position 15.1 relative the adenosine at position 15.1 (A-15.1).

Mechanism of action of Nucleic Acid Molecules of the Invention Antisense: Antisense molecules may be modified or unmodified RNA, DNA, or mixed polymer oligonucleotides and primarily function by specifically binding to matching sequences resulting in inhibition of peptide synthesis (Wu-Pong, Nov 1994, BioPharm, 20-33). The antisense oligonucleotide binds to target RNA by Watson Crick base-pairing and blocks gene expression by preventing ribosomal translation of the bound sequences either by steric blocking or by activating RNase H enzyme. Antisense molecules may also alter protein synthesis by interfering with RNA processing or transport from the nucleus into the cytoplasm (Mukhopadhyay & Roth, 1996, Crit. Rev. in Oncogenesis 7, 151-190).
In addition, binding of single stranded DNA to RNA may result in nuclease degradation of the heteroduplex (Wu-Pong, supra; Crooke, supra). To date, the only backbone modified DNA chemistry which will act as substrates for RNase H are phosphorothioates, phosphorodithioates, and borontrifluoridates. Recently, it has been reported that 2'-arabino and 2'-fluoro arabino- containing oligos can also activate RNase H activity.
A number of antisense molecules have been described that utilize novel configurations of chemically modified nucleotides, secondary structure, and/or RNase H
substrate domains (Woolf et al., International PCT Publication No. WO
98/13526;
Thompson et al., International PCT Publication No. WO 99/54459 ; Hartmann et al., International PCT Publication No. WO 00/17346) all of these are incorporated 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.
Triplex Forming OIiQonucleotides (TFO): Single stranded DNA may be designed to bind to genomic DNA in a sequence specific manner. TFOs are comprised of pyrimidine-rich oligonucleotides which bind DNA helices through Hoogsteen Base-pairing (Wu-Pong, supra). The resulting triple helix composed of the DNA sense, DNA antisense, and TFO
disrupts RNA synthesis by RNA polymerase. The TFO mechanism may result in gene expression or cell death since binding may be irreversible (Mukhopadhyay &
Roth, supra) 2'-5' Oli-og adenylates: The 2-5 A system is an interferon-mediated mechanism for RNA degradation found in higher vertebrates (Mitra et al., 1996, Proc Nat Acad Sci USA
93, 6780-6785). - Two types of enzymes, 2-5A synthetase and RNase L, are required for RNA cleavage. The 2-5A synthetases require double stranded RNA to form 2'-5' oligoadenylates (2-5A). 2-5A then acts as an allosteric effector for utilizing RNase L
which has the ability to cleave single stranded RNA. The ability to form 2-5A
structures with double stranded RNA makes this system particularly useful for inhibition of viral replication.
(2'-5') oligoadenylate structures may be covalently linked to antisense molecules to form chimeric oligonucleotides capable of RNA cleavage (Torrence, supra).
These molecules putatively bind and activate a 2-5A dependent RNase, the oligonucleotide/enzyme complex then binds to a target RNA molecule which can then be cleaved by the RNase enzyme. 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 of the instant invention.
Enz~natic Nucleic Acid: Seven basic varieties of naturally-occurnng enzymatic RNAs are presently known. In addition, several in vitro selection (evolution) strategies (Orgel, 1979, Proc. R. Soc. London, B 205, 435) have been used to evolve new nucleic acid catalysts capable of catalyzing cleavage and ligation of phosphodiester linkages (Joyce, 1989, Gene, 82, 83-87; Beaudry et al., 1992, Science 257, 635-641;
Joyce, 1992, Scientific American 267, 90-97; Breaker et al., 1994, TIBTECH 12, 268; Bartel et a1.,1993, Science 261:1411-1418; Szostak, 1993, TIBS 17, 89-93; Kumar et al., 1995, FASEB J., 9, 1183; Breaker, 1996, Curr. Op. Biotech., 7, 442; Santoro et al., 1997, Proc.
Natl. Acad.
Sci., 94, 4262; Tang et al., 1997, RNA 3, 914; Nakamaye & Eckstein, 1994, supra; Long &
IJhlenbeck, 1994, supra; Ishizaka et al., 1995, supra; Vaish et al., 1997, Biochemistry 36, 6495; all of these are incorporated by reference herein). Each can catalyze a series of reactions including the hydrolysis of phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions.
In general, 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 of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. 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-1B, 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-1B, 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 of the ribozyme to act enzymatically. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA. In addition, the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base-pairing mechanism of binding to the target 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. Med., 6, 92; Haseloff and Gerlach, 334 Nature, 585, 1988; Cech, 260 JAMA, 3030, 1988; Jefferies et al., 17 Nucleic Acids Research, 1371, 1989; and Santoro et al., 1997 supra).
Because of their sequence specificity, trans-cleaving ribozymes show promise as therapeutic agents for human disease (Usman & McSwiggen, 1995 Ann. Rep. Med.
Chem.
30, 285-294; Christoffersen and Marr, 1995 J. Med. Chem. 38, 2023-2037).
Ribozymes can be designed to cleave specific RNA targets within the background of cellular RNA.
Such a cleavage event renders the RNA non-functional and abrogates protein expression from that RNA. In this manner, synthesis of a protein associated with a disease state can be selectively inhibited (Warashina et al., 1999, Chemistry and Biology, 6, 237-250.

The nucleic acid molecules of the instant invention are also referred to as GeneBlocTM reagents, which are essentially nucleic acid molecules (e.g.;
ribozymes, antisense) capable of down-regulating gene expression.
Tar-eg t sites 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 incorporated 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 incorporated by reference herein. Rather than repeat the guidance provided in those documents here, below are provided specific examples of such methods, not limiting to those in the art.
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-1B, MetAP-2, BACE, ps-1, ps-2, HER2, PLN, TERT, and/or HBV RNAs (for example, GenBank accession Nos. (PTP-1B,. 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 S' 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. 6,017,756 entitled "METHOD AND REAGENT FOR
INHIBITING HEPATITIS B VIRUS REPLICATION" and Draper et al., International PCT publication No. WO 93/23569, filed April 29, 1993, entitled "METHOD AND
REAGENT FOR INHIBITING VIRAL REPLICATION". While human sequences can be screened and enzymatic nucleic acid molecule and/or antisense thereafter designed, as discussed in Stinchcomb et al., WO 95/23225, mouse targeted ribozymes may be useful to test efficacy of action of the enzymatic nucleic acid molecule and/or antisense prior to testing in humans.
5 Antisense, hammerhead, DNAzyme, NCH (Inozyme), amberzyme, zinzyme or G-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 10 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 15 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.
Synthesis of Nucleic acid Molecules Synthesis of nucleic acids greater than 100 nucleotides in length is difficult using automated methods, and the therapeutic cost of such molecules is prohibitive.
In this invention, small nucleic acid motifs ("small refers to nucleic acid motifs no more than 100 nucleotides in length, preferably no more than 80 nucleotides in length, and most preferably no more than 50 nucleotides in length; e.g., antisense oligonucleotides, hammerhead or the NCH ribozymes) are preferably used for exogenous delivery.
The simple structure of these molecules increases the ability of the nucleic acid to invade targeted regions of RNA structure. Exemplary molecules of the instant invention are chemically synthesized, and others can similarly be synthesized.

Oligonucleotides (e.g.; antisense GeneBlocs) are synthesized using protocols known in the art as described in Caruthers et al., 1992, Methods in Enzymology 211, 3-19, Thompson et al., International PCT Publication No. WO 99/54459, Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, Methods Mol. Bio., 74, 59, Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45, and Brennan, US patent No.

6,001,311. All of these references are incorporated herein by reference. The synthesis of oligonucleotides makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end. In a non-limiting example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc.
synthesizer using a 0.2 ~mol scale protocol with a 2.5 min coupling step for 2'-O-methylated nucleotides and a 45 sec coupling step for 2'-deoxy nucleotides.
Table II
outlines the amounts and the contact times of the reagents used in the synthesis cycle.
Alternatively, syntheses at the 0.2 pmol 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. A 33-fold excess (60 pL of 0.11 M = 6.6 ~,mol) of 2'-O-methyl phosphoramidite and a 105-fold excess of S-ethyl tetrazole (60 pL of 0.25 M =
15 pmol) can be used in each coupling cycle of 2'-O-methyl residues relative to polymer-bound S'-hydroxyl. A 22-fold excess (40 p.L of 0.11 M = 4.4 ~mol) of deoxy phosphoramidite and a 70-fold excess of S-ethyl tetrazole (40 pL of 0.25 M =10 pmol) can be used in each coupling cycle of deoxy residues relative to polymer-bound 5'-hydroxyl.
Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, are typically 97.5-99%.
Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc.
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-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc. Alternately, for the introduction of phosphorothioate linkages, Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide, 0.05 M in acetonitrile) is used.

Deprotection of the antisense oligonucleotides is performed as follows: the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 40% aq. methylamine (1 mL) at 65 °C for 10 min. After cooling to -20 °C, the supernatant is removed from the polymer support.
The support is washed three times with 1.0 mL of EtOH:MeCN:H20/3:1:1, vortexed and the supernatant is then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, are dried to a white powder.
The method of synthesis used for normal RNA including certain enzymatic nucleic acid molecules follows the procedure as described in Usman et al., 1987, J.
Am. Chem.
Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res., 18, 5433; and Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684 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. In a non-limiting example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc.
synthesizer using a 0.2 pmol scale protocol with a 7.5 min coupling step for alkylsilyl protected nucleotides and a 2.5 min coupling step for 2'-O-methylated nucleotides. Table II outlines the amounts and the contact times of the reagents used in the synthesis cycle.
Alternatively, 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. A 33-fold excess (60 ~L of 0.11 M = 6.6 ~mol) of 2'-O-methyl phosphoramidite and a 75-fold excess of S-ethyl tetrazole (60 ~L of 0.25 M =15 pmol) can be used in each coupling cycle of 2'-O-methyl residues relative to polymer-bound S'-hydroxyl. A 66-fold excess (120 ~L of 0.11 M = 13.2 ~mol) of alkylsilyl (ribo) protected phosphoramidite and a 150-fold excess of S-ethyl tetrazole (120 ~L of 0.25 M =
30 pmol) can be used in each coupling cycle of ribo residues relative to polymer-bound 5'-hydroxyl.
Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, are typically 97.5-99%.
Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc.
synthesizer include the following: detritylation solution is 3% TCA in methylene chloride (ABI);
capping is performed with 16% N methyl imidazole in THF (ABn 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. Alternately, for the introduction of phosphorothioate linkages, Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide0.05 M in acetonitrile) is used.
Deprotection of the RNA is performed using either a two-pot or one-pot protocol.
For the two-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:H20/3:1:1, vortexed and the supernatant is then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, are dried to a white powder.
The base deprotected oligoribonucleotide is resuspended in anhydrous TEA/HF/NMP
solution (300 pL 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 NH4HC03.
Alternatively, for the 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 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 1 S
min. The sample is cooled at -20 °C and then quenched with 1.5 M
NH4HC03.
For purification of the trityl-on oligomers, the quenched NH4HC03 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 NaCI and washed with water again. The oligonucleotide is then eluted with 30% acetonitrile.
Inactive hammerhead ribozymes or binding attenuated control (BAC) oligonucleotides) are synthesized by substituting a U for GS 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 control.

The average stepwise coupling yields are typically >98% (Wincott et al., 1995 Nucleic Acids Res. 23, 2677-2684). Those of ordinary skill in the art will recognize that the scale of synthesis can be adapted to be larger or smaller than the example described above including but not limited to 96-well format, all that is important is the ratio of chemicals used in the reaction.
Alternatively, the nucleic acid molecules of the present invention can be synthesized separately and joined together 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).
The nucleic acid molecules of the present invention are modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-O-methyl, 2'-H (for a review see Usman and Cedergren, 1992, TIBS
17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163). Ribozymes are purified by gel electrophoresis using general methods or are purified by high pressure liquid chromatography (HPLC; see Wincott et al., supra, the totality of which is hereby incorporated herein by reference) and are re-suspended in water.
The sequences of the 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 of the 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 may be formed of ribonucleotides or other nucleotides or non-nucleotides. Such ribozymes with enzymatic activity are equivalent to the ribozymes described specifically in the Tables.
timizing_Activity of the nucleic acid molecule of the invention.
Chemically synthesizing nucleic acid molecules with modifications (base, sugar and/or phosphate) that prevent their degradation by serum ribonucleases may increase their potency (see e.g., Eckstein et al., International Publication No. WO 92/07065;
Perrault et al., 1990 Nature 344, 565; Pieken et al., 1991, Science 253, 314; Usman and Cedergren, 1992, Trends in Biochem. Sci. 17, 334; Usman et al., International Publication No. WO
93/15187; Rossi et al., International Publication No. WO 91/03162; Sproat, US
Patent No.

5,334,711; and Burgin et al., supra; all of these describe various chemical modifications that can be made to the base, phosphate and/or sugar moieties of the nucleic acid molecules herein and are all hereby incorporated by reference herein).
Modifications which enhance their efficacy in cells, and removal of bases from nucleic acid molecules to 5 shorten oligonucleotide synthesis times and reduce chemical requirements are desired.
There are several examples in the art describing sugar, base and phosphate modifications that can be introduced into nucleic acid molecules (e.g., enzymatic nucleic acid molecules) without significantly effecting catalysis and with significant enhancement in their nuclease stability and efficacy. Enzymatic nucleic acid molecules are modified to 10 enhance stability and/or enhance catalytic activity by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-fluoro, 2'-D-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 15 described in the art (see Eckstein et al., International Publication PCT
No. WO 92/07065;
Perrault et al. Nature 1990, 344, 565-568; Pieken et al. Science 1991, 253, 314-317;
Usman and Cedergren, Trends in Biochem. Sci. 1992, 17, 334-339; Usman et al.
International Publication PCT No. WO 93/15187; Sproat, US Patent No. 5,334,711 and Beigelman et al., 1995 J. Biol. Chem. 270, 25702; all of the references are hereby 20 incorporated in their totality by reference herein). Such publications describe general methods and strategies to determine the location of incorporation of sugar, base and/or phosphate modifications and the like into enzymatic nucleic acid molecules without inhibiting catalysis, and are incorporated by reference herein. The 2'-position of the sugar in a nucleotide present in the nucleic acid molecules of the instant invention which 25 tolerates substitution is selected from the group comprising -H, -OH, -COOH, -CONHz, -CONHR~, -CONR1R2, -NH2, -NHR~, -NR~R2, -NHCOR~, -SH, SRI, -F, -ONH2, -ONHRI, -ONR1R2, -NHOH, -NHORI, -NRzOH, -NRZORI, substituted or unsubstituted C~-Clo straight chain or branched alkyl, substituted or unsubstituted C2-Coo straight chain or branched alkenyl, substituted or unsubstituted CZ-Clo straight chain or branched alkynyl, 30 substituted or unsubstituted C1-Coo straight chain or branched alkoxy, substituted or unsubstituted Cz-Clo straight chain or branched alkenyloxy, and substituted or unsubstituted C2-Clo straight chain or branched alkynyloxy. The substituents for sugar 2' position preferably are independently halogen, cyano, amino, carboxy, ester, ether, carboxamide, hydroxy, or mercapto. R1 and RZ 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.
In view of such teachings, similar modifications can be used as described herein to modify the nucleic acid molecules of the instant invention. Such publications describe general methods and strategies to determine the location of incorporation of sugar, base and/or phosphate modifications and the like into ribozymes without inhibiting catalysis, and are incorporated by reference herein. In view of such teachings, similar modifications can be used as described herein to modify the nucleic acid molecules of the instant invention.
Some of the non-limiting examples of base modifications that can be introduced into enzymatic nucleic acids without significantly effecting their catalytic activity include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyluracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., S-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine) and others (Burgin et al., 1996, Biochemistry, 35, 14090). By "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 of the enzyme and/or in the substrate-binding regions.
The nucleic acid bases can be hypoxanthin-9-yl, or a functional equivalent thereof, in positionl5vof 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-y1, 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-S-yl, thymin-1-yl or 5-propynyluracil-1-yl in position 4;
cytosin-1-yl, 5-methylcytosin-1-yl or 5-propynylcytosin-1-yl in position 3; and adenin-9-yl, cytosin-1-yl, guanin-9-yl, uracil-1-yl, uracil-5-yl, hypoxanthin-9-yl, thymin-1-yl, 5-methylcytosin-1-yl, 2,6-diaminopurin-9-yl, purin-9-yl, 7-deaza adenin-9-yl, 7-deazaguanin-9-yl, 5-propynylcytosin-1-yl, S-propynyluracil-1-yl, isoguanin-9-yl, 2-aminopurin-9-yl, 6-methyluracil-1-yl, 4-thiouracil-1-yl, 2-pyrimidone-1-yl, quinazoline-2,4-dione-1-yl, xanthin-9-yl, N2-dimethylguanin-9-yl, or a functional equivalent thereof in position 7. 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 of the base and the 2-oxo group of 016.1.
Preferably, B is not guanin-9-yl in position 15.1.
In particular, 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.
While chemical modification of oligonucleotide internucleotide linkages with phosphorothioate, phosphorothioate, and/or S'-methylphosphonate linkages improves stability, too many of these modifications may cause some toxicity. Therefore, when designing nucleic acid molecules, the amount of these internucleotide linkages should be minimized. The reduction in the concentration of these linkages should lower toxicity resulting in increased efficacy and higher specificity of these molecules.
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 of the target RNA has been inhibited long enough to reduce the levels of the undesirable protein. This period of time varies between hours to days depending upon the disease state. Clearly, nucleic acid molecules must be resistant to nucleases in order to function as effective intracellular therapeutic agents.
Improvements in the chemical synthesis of RNA and DNA (Wincott et al., 1995 Nucleic Acids Res. 23, 2677; Caruthers et al., 1992, Methods in Enzymology 211,3-19 (all are incorporated by reference herein) have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability as described above.
Use of these the nucleic acid-based molecules of the invention will lead to better treatment of the disease progression by affording the possibility of combination therapies (e.g., multiple antisense or enzymatic nucleic acid molecules targeted to different genes, nucleic acid molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations of molecules (including different motifs) and/or other chemical or biological molecules). The treatment of patients with nucleic acid molecules may also include combinations of different types of nucleic acid molecules.

Therapeutic nucleic acid molecules (e.g., enzymatic nucleic acid molecules and antisense nucleic acid molecules) delivered exogenously must optimally be stable within cells until translation of the target RNA has been inhibited long enough to reduce the levels of the undesirable protein. This period of time varies between hours to days depending upon the disease state. Clearly, these nucleic acid molecules must be resistant to nucleases in order to function as effective intracellular therapeutic agents.
Improvements in the chemical synthesis of nucleic acid molecules described in the instant invention and in the art have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability as described above.
By "enhanced enzymatic activity" is meant to include activity measured in cells and/or in vivo where the activity is a reflection of both catalytic activity and ribozyme stability. In this invention, the product of these properties is increased or not significantly (less than 10-fold) decreased in vivo compared to an all RNA ribozyme or all DNA
enzyme.
In yet another preferred embodiment, 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.
As exemplified herein such ribozymes are useful in a cell and/or in vivo even if activity over all is reduced 10 fold (Burgin et al., 1996, Biochemistry, 35, 14090). Such ribozymes herein are said to "maintain" the enzymatic activity of an all RNA ribozyme.
In another aspect the nucleic acid molecules comprise a S' and/or a 3'- cap structure.
By "cap structure" is meant chemical modifications, which have been incorporated at either terminus of the oligonucleotide (see, for example, Wincott et al., WO 97/26270, incorporated by reference herein). These terminal modifications protect the nucleic acid molecule from exonuclease degradation, and may help in delivery and/or localization within a cell. The cap may be present at the 5'-terminus (5'-cap) or at the 3'-terminal (3'-cap) or may be present on both termini. In non-limiting examples: 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; threo-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 nucleotide moiety;
3'-2'-inverted abasic moiety; 1,4-butanediol phosphate; 3'-phosphoramidate;
hexylphosphate; aminohexyl phosphate; 3'-phosphate; 3'-phosphorothioate;
phosphorodithioate; or bridging or non-bridging methylphosphonate moiety (for more details, see Wincott et al., International PCT publication No. WO 97/26270, incorporated by reference herein).
In yet another preferred embodiment, the 3'-cap is selected from a group comprising, 4',5'-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4'-thin nucleotide, carbocyclic nucleotide; 5'-amino-alkyl phosphate; 1,3-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; threo-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; incorporated by reference herein).
An "alkyl" group refers to a saturated aliphatic hydrocarbon, including straight-chain, branched-chain, and cyclic alkyl groups. Preferably, 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 alkyl group may be substituted or unsubstituted. When substituted the substituted groups) is preferably, hydroxyl, cyano, alkoxy, =O, =S, N02 or N(CH3)2, amino, or SH. 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. Preferably, 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. The alkenyl group may be substituted or unsubstituted. When substituted the substituted groups) is preferably, hydroxyl, cyano, alkoxy, =O, =S, N02, halogen, N(CH3)2, amino, or SH. The term "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 5 has 1 to 12 carbons. More preferably it is a lower alkynyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkynyl group may be substituted or unsubstituted. When substituted the substituted groups) is preferably, hydroxyl, cyano, alkoxy, =O, =S, N02 or N(CH3)2, amino or SH.
Such alkyl groups may also include aryl, alkylaryl, carbocyclic aryl, heterocyclic 10 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. An "alkylaryl" group refers to an 15 alkyl group (as described above) covalently joined to an aryl group (as described above).
Carbocyclic aryl groups are groups wherein the ring atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted. Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms. Suitable heteroatoms include oxygen, 20 sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted. An "amide" refers to an -C(O)-NH-R, where R is either alkyl, aryl, alkylaryl or hydrogen. An "ester" refers to an -C(O)-OR', where R is either alkyl, aryl, alkylaryl or hydrogen.
By "nucleotide" as used herein is as recognized in the art to include .natural bases 25 (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 30 example, Usman and McSwiggen, supra; Eckstein et al., International PCT
Publication No. WO 92/07065; Usman et al., International PCT Publication No. WO 93/15187;

Uhlman & Peyman, supra, all are hereby incorporated by reference herein).
There are several examples of modified nucleic acid bases known in the art as summarized by Limbach et al., 1994, Nucleic Acids Res. 22, 2183. Some of the non-limiting examples of 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., S-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).
By "modified bases" in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at 1' position or their equivalents; such bases may be used at any position, for example, within the catalytic core of an enzymatic nucleic acid molecule and/or in the substrate-binding regions of the nucleic acid molecule. Such 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.
In a preferred embodiment, the invention features modified ribozymes with phosphate backbone modifications comprising one or more phosphorothioate, phosphorodithioate, methylphosphonate, morpholino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl, substitutions. For a review of oligonucleotide backbone modifications, see Hunziker and Leumann, 1995, Nucleic Acid Analogues: Synthesis and Properties, in Modern Synthetic Methods, VCH, 331-417, and Mesmaeker et al., 1994, Novel Backbone Replacements for Oligonucleotides, in Carbohydrate Modifications in Antisense Research, ACS, 24-39. These references are hereby incorporated by reference herein.
By "abasic" is meant 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).
By "unmodified nucleoside" or "unmodified nucleotide" is meant one of the bases adenine, cytosine, guanine, thymine, uracil joined to the 1' carbon of (3-D-ribo-furanose.

By "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.
In connection with 2'-modified nucleotides as described for the present invention, by "amino" is meant 2'-NHZ or 2'-O- NH2, which may be modified or unmodif ed.
Such modified groups are described, for example, in Eckstein et al., U.S. Patent 5,672,695 and Matulic-Adamic et al., WO 98/28317, which are both incorporated by reference in their entireties.
Various modifications to nucleic acid (e.g., antisense and ribozyme) structure 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.
Use of these molecules will lead to better treatment of the disease progression by affording the possibility of combination therapies (e.g., multiple ribozymes targeted to different genes, ribozymes coupled with known small molecule inhibitors, or intermittent treatment with combinations of ribozymes (including different ribozyme motifs) and/or other chemical or biological molecules). The treatment of patients with nucleic acid molecules may also include combinations of different types of nucleic acid molecules.
Therapies may be devised which include a mixture of ribozymes (including different ribozyme motifs), antisense and/or 2-SA chimera molecules to one or more targets to alleviate symptoms of a disease.
Administration of Nucleic Acid Molecules Methods for the delivery of nucleic acid molecules are described in Akhtar et al., 1992, Trends Cell Bio., 2, 139; and Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995 which are both incorporated herein by reference. Sullivan et al., PCT WO 94/02595, further describes the general methods for delivery of enzymatic RNA molecules. These protocols may be utilized for the delivery of virtually any nucleic acid molecule. Nucleic acid molecules may be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres. For some indications, nucleic acid molecules may be directly delivered ex vivo to cells or tissues with or without the aforementioned vehicles. Alternatively, the nucleic acidlvehicle combination is locally delivered by direct injection or by use of a catheter, infusion pump or stmt. Many examples in the art describe CNS delivery methods of oligonucleotides by osmotic pump, (see Chun et al., 1998, Neuroscience Letters, 257, 135-138, D'Aldin et al., 1998, Mol. Brain Research, 55, 151-164, Dryden et al., 1998, J. Endocrinol., 157, 169-175, Ghirnikar et al., 1998, Neuroscience Letters, 247, 21-24) or direct infusion (Broaddus et al., 1997, Neurosurg. Focus, 3, article 4). Other routes of delivery include, but are not limited to oral (tablet or pill form) and/or intrathecal delivery (Gold, 1997, Neuroscience, 76, 1153-1158). For a comprehensive review on drug delivery strategies including broad coverage of CNS delivery, see Jain, Drug Delivery Systems: Technologies and Commercial Opportunities, Decision Resources, 1998. Other routes of delivery include, but are not limited to, intravascular, intramuscular, subcutaneous or joint injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. More detailed descriptions of nucleic acid delivery and administration are provided in Sullivan et al., supra, Draper et al., PCT
W093/23569;
Beigelman et al., PCT W099/05094, and Klimuk et al., PCT W099/04819 all of which are incorporated by reference herein.
The molecules of the instant invention can be used as pharmaceutical agents.
Pharmaceutical agents prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state in a patient.
The negatively charged polynucleotides of the invention can be administered (e.g., RNA, DNA or protein) and introduced into a patient by any standard means, with or without stabilizers, buffers, and the like, to form a pharmaceutical composition. When it is desired to use a liposome delivery mechanism, standard protocols for formation of liposomes can be followed. The compositions of the present invention may also be formulated and used as tablets, capsules or elixirs for oral administration;
suppositories for rectal administration; sterile solutions; suspensions for injectable administration; and the other compositions known in the art.

The present invention also includes pharmaceutically acceptable formulations of the compounds described. These formulations include salts of the above compounds, e.g., acid addition salts, for example, salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid.
A pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, e.g., systemic administration, into a cell or patient, preferably a human. Suitable forms, in part, depend upon the use or the route of entry, for example, oral, transdermal, or by injection. Such forms should not prevent the composition or formulation 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.
By "systemic administration" is meant in vivo systemic absorption or accumulation of drugs in the blood stream followed by distribution throughout the entire body.
Administration routes which lead to systemic absorption include, without limitations:
intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular. Each of these administration routes expose the desired negatively charged polymers, e.g., nucleic acids, to an accessible diseased tissue. The rate of entry of a drug into the circulation has been shown to be a function of molecular weight or size. The use of a liposome or other drug Garner comprising the compounds of the instant invention can potentially localize the drug, for example, in certain tissue types, such as the tissues of the reticular endothelial system (RES). A liposome formulation which can facilitate the association of drug with the surface of cells, such as, lymphocytes and macrophages is also useful. This approach may provide enhanced delivery of the drug to target cells by taking advantage of the specificity of macrophage and lymphocyte immune recognition of abnormal cells, such as cancer cells.
By pharmaceutically acceptable formulation is meant, a composition or formulation that allows for the effective distribution of the nucleic acid molecules of the instant invention in the physical location most suitable for their desired activity.
Nonlimiting examples of agents suitable for formulation with the nucleic acid molecules of the instant invention include: P-glycoprotein inhibitors (such as Pluronic P85) which can enhance entry of drugs into the CNS (Jolliet-Riant and Tillement, 1999, Fundam. Clin.
Pharmacol., 13, 16-26); biodegradable polymers, such as poly (DL-lactide-coglycolide) microspheres for sustained release delivery after intracerebral implantation (Emerich, DF
et al, 1999, Cell Transplant, 8, 47-58) Alkermes, Inc. Cambridge, MA; and loaded nanoparticles, such 5 as those made of polybutylcyanoacrylate, which can deliver drugs across the blood brain burner and can alter neuronal uptake mechanisms (frog Neuropsychopharmacol Biol .
Psychiatry, 23, 941-949, 1999). Other non-limiting examples of delivery strategies for the nucleic acid molecules of the instant invention include material described in Boado et al., 1998, J. Pharm. Sci., 87, 1308-1315; Tyler et al., 1999, FEBS Lett., 421, 280-284;
10 Pardridge et al., 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv. Drug Delivery Rev., 15, 73-107; Aldrian-Herrada et al., 1998, Nucleic Acids Res., 26, 4910-4916;
and Tyler et al., 1999, PNAS USA., 96, 7053-7058.
The invention also features the use of the composition comprising surface-modified liposomes containing poly (ethylene glycol) lipids (PEG-modified, or long-circulating 15 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, 1005-20 1011 ). Such liposomes have been shown to accumulate selectively in tumors, presumably by extravasation and capture in the neovascularized target tissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et a1.,1995, Biochim. Biophys. Acta, 1238, 86-90).
The long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA
and RNA, particularly compared to conventional cationic liposomes which are known to 25 accumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 1995, 42, 24864-24870; Choi et al., International PCT Publication No. WO 96/10391; Ansell et al., International PCT
Publication No. WO 96/10390; Holland et al., International PCT Publication No.
WO
96/10392; all of which are incorporated herein by reference). Long-circulating liposomes are also likely to protect drugs from nuclease degradation to a greater extent compared to 30 cationic liposomes, based on their ability to avoid accumulation in metabolically aggressive MPS tissues such as the liver and spleen.

The present invention also includes compositions prepared for storage or administration which include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent. Acceptable Garners or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R.
Gennaro edit. 1985) hereby incorporated by reference herein. For example, preservatives, stabilizers, dyes and flavoring agents may be provided. These include sodium benzoate, sorbic acid and esters ofp-hydroxybenzoic acid. In addition, antioxidants and suspending agents may be used.
A pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state. The pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors which those skilled in the medical arts will recognize.
Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients is administered dependent upon potency of the negatively charged polymer.
The nucleic acid molecules of the present invention may also be administered to a patient in combination with other therapeutic compounds to increase the overall therapeutic effect. The use of multiple compounds to treat an indication may increase the beneficial effects while reducing the presence of side effects.
Alternatively, certain of the nucleic acid molecules of the instant invention can be expressed within cells from eukaryotic promoters (e.g., Izant and Weintraub, 1985, Science, -229, 345; McGarry and Lindquist, 1986, Proc. Natl. Acad. Sci., USA
83, 399;
Scanlon et al., 1991, Proc. Natl. Acad. Sci. USA, 88, 10591-5; Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Dropulic et al., 1992, J. Virol., 66, 1432-41;
Weerasinghe et al., 1991, J. Yirol., 65, 5531-4; Ojwang et al., 1992, Proc. Natl. Acad. Sci.
USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Sarver et al., 1990 Science, 247, 1222-1225; Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Good et al., 1997, Gene Therapy, 4, 45; all of these references are hereby incorporated herein, in their totalities, by reference). Those skilled in the art realize that any nucleic acid can be expressed in eukaryotic cells from the appropriate DNA/RNA vector. The activity of such nucleic acids can be augmented by their release from the primary transcript by a ribozyme (Draper et al., PCT WO 93/23569, and Sullivan et al., PCT WO 94/02595; Ohkawa et al., 1992, Nucleic Acids Symp. Ser., 27, 15-6; Taira et al., 1991, Nucleic Acids Res., 19, 5125-30; Ventura et al., 1993, Nucleic Acids Res., 21, 3249-55; Chowrira et al., 1994, J. Biol.
Chem., 269, 25856; all of these references are hereby incorporated in their totality by reference herein).
In another aspect of the invention, RNA molecules of the present invention are preferably expressed from transcription units (see, for example, Couture et al., 1996, TIG., 12, 510) inserted into DNA or RNA vectors. The recombinant vectors are preferably DNA
plasmids or viral vectors. Ribozyme expressing viral vectors could be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus.
Preferably, the recombinant vectors capable of expressing the nucleic acid molecules are delivered as described above, and persist in target cells. Alternatively, viral vectors may be used that provide for transient expression of nucleic acid molecules. Such vectors might be repeatedly administered as necessary. Once expressed, the nucleic acid molecule binds to the target mRNA. Delivery of nucleic acid molecule expressing vectors could be systemic, such as by intravenous or intra-muscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that would allow for introduction into the desired target cell (for a review see Couture et al., 1996, TIG., 12, 510).
In one aspect, the invention features an expression vector comprising a nucleic acid sequence encoding at least one of the nucleic acid molecules of the instant invention is disclosed. The nucleic acid sequence encoding the nucleic acid molecule of the instant invention is operably linked in a manner which allows expression of that nucleic acid molecule.
In another aspect the invention features an expression vector comprising: a) a transcription initiation region (e.g., eukaryotic pol I, II or III initiation region); b) a transcription termination region (e.g., eukaryotic pol I, II or III
termination region); c) a nucleic acid sequence encoding at least one of the nucleic acid catalyst of the instant invention; and wherein said sequence is operably linked to said initiation region and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule. The vector may optionally include an open reading frame (ORF) for a protein operably linked on the 5' side or the 3'-side of the sequence encoding the nucleic acid catalyst of the invention; and/or an intron (intervening sequences).
Transcription of the nucleic acid molecule sequences are driven from a promoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA
polymerase III
(pol 111). Transcripts from pol II or pol III promoters will be expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type will depend on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby.
Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA
polymerase enzyme is expressed in the appropriate cells (Ekoy-Stein and Moss, 1990, Proc.
Natl.
Acad. Sci. U S A, 87, 6743-7; Gao and Huang 1993, Nucleic Acids Res., 21, 2867-72;
Lieber et al., 1993, Methods Enzymol., 217, 47-66; Zhou et al., 1990, Mol.
Cell. Biol., 10, 4529-37). All of these references are incorporated by reference herein.
Several investigators have demonstrated that nucleic acid molecules, such as ribozymes expressed from such promoters can function in mammalian cells (e.g. Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Ojwang et al., 1992, Proc. Natl. Acad. Sci. U S
A, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Yu et al., 1993, Proc. Natl.
Acad. Sci. USA, 90, 6340-4; L'Huillier et al., 1992, EMBO.I., 11, 4411-8;
Lisziewicz et al., 1993, Proc. Natl. Acad. Sci. U. S. A, 90, 8000-4; Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Sullenger & Cech, 1993, Science, 262, 1566). More specifically, transcription units such as the ones derived from genes encoding U6 small nuclear (snRNA), transfer RNA (tRNA) and adenovirus VA RNA are useful in generating high concentrations of desired RNA molecules such as ribozymes in cells (Thompson et al., supra; Couture and Stinchcomb, 1996, supra; Noonberg et al., 1994, Nucleic Acid Res., 22, 2830; Noonberg et al., US Patent No. 5,624,803; Good et al., 1997, Gene Ther., 4, 45;
Beigelman et al., International PCT Publication No. WO 96/18736; all of these publications are incorporated by reference herein. The above ribozyme transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated virus vectors), or viral RNA vectors (such as retroviral or alphavirus vectors) (for a review see Couture and Stinchcomb, 1996, supra).

In yet another aspect, the invention features an expression vector comprising nucleic acid sequence encoding at least one of the nucleic acid molecules of the invention, in a manner which allows expression of that nucleic acid molecule. The expression vector comprises in one embodiment; a) a transcription initiation region; b) a transcription termination region; c) a nucleic acid sequence encoding at least one said nucleic acid molecule; and wherein said sequence is operably linked to said initiation region and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule. In another preferred embodiment 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. In yet another embodiment, the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) a nucleic acid sequence encoding at least one said nucleic acid molecule; and wherein said sequence is operably linked to said initiation region, said intron and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.
In another embodiment, the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) an open reading frame; e) a nucleic acid sequence encoding at least one said nucleic acid molecule, wherein said sequence is operably linked to the 3'-end of said open reading frame; and wherein said sequence is operably linked to said initiation region, said intron, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.
Examples:
The following are non-limiting examples showing the selection, isolation, synthesis and activity of nucleic acids of the instant invention.

Example 1: Telomerase 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 5 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. Additional conserved telomere elements include a terminal 3'-overhang in the G-rich strand and non-histone structural 10 proteins that are complexed with telomeric DNA in the nucleus. (Blackburn, "E., 1990, JBC., 265, 5919-5921.). 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.
15 Conventional DNA polymerises are unable to fully replicate the ends of linear chromosomes (Watson, J. D., 1972, Nature, 239, 197-201). This inability stems from the 3' G-rich overhang that is a product of ribonuclease cleavage of the RNA
primer used in DNA replication. The overhang prevents DNA polymerise replication since the recessed C-rich parent strand cannot be used as a template. Telomerase overcomes this limitation 20 by extending the 3' end of the 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, 25 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, 30 Science, 269, 1267-1270). These studies showed that telomerase activity parallels TR
expression in protozoa, yeast and mice. However, the expression of human telomerase RNA (hTR) does not correlate well with telomerase activity in mammalian cells.
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).
The first catalytically active subunit of telomerase (p123) 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 (TRTl).
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).
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 transfection has resulted in the extension of life span in these cells. Such findings indicate that 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).
Expression of telomerase is observed in germ cell and most cancer cell lines.
These "immortal" cell lines continue to divide without shortening of their telomeres (Kim, N.
W., 1994, Science, 266, 2011-2015). A model of tumor progression has evolved from these findings, suggesting a role for telomerase expression in malignant transformation.
Successful malignant transformation in human cells was accomplished for the first time by ectopic expression of hTERT in combination with two oncogenes, SV40 large-T
and H-ras. Injection of nude mice with cells expressing these oncogenes and hTERT
resulted in rapid growth of tumors. These observations indicate that hTERT mediated telomere maintenance is essential for the formation of human tumor cells (Hahn, W. C., 1999, Nature, 400, 464-468).
Various methods have been developed to assay telomerase activity in vitro. The most widely used method to characterize telomerase activity is the telomeric repeat amplification protocol (TRAP). TRAP utilizes RT-PCR of cellular extracts to measure telomerase activity by making the amount of PCR target dependant upon the biochemical activity of the enzyme (Kim, N. W., 1997, Nucleic Acids Research, 25, 2595-2597, which is incorporated by reference herein).
A method based on Kim is as follows. Briefly, for the telomerase assay, 2ltg of protein extract is used. The extract is assayed in 501 of reaction mixture containing 0.1 ~g TS substrate primer (5~-AATCCGTCGAGCAGAGTT-3', end-labeled using alpha-32P-ATP and T4 polynucleotide kinase), 0.1 wg ACX return primer(5'-GCGCGG[CTTACC]3 CTAACC-3'), 0.1 ~g NT internal control primer (5'-ATCGCTTCTCGGCCTTTT-3'), 0.01 micromol TSNT internal control template (5'-AATCCGTCGAGCAGAGTTAAAAGGCCGAGAACGAT-3~), 50 pM each deoxynucleoside triphosphate, 2 U of Taq DNA polymerase, and 2 w1 CHAPS
protein extract, all in 1X TRAP buffer (20 mM Tris (pH 8.3), 68 mM KCI, 1.5 mM MgCl2, 1 mM
EGTA, 0.05% Tween 20). Each reaction is placed in a thermocycler block preheated to 30 C and incubated at 30 C for 10 minutes, then cycled for 27 cycles of 94 degrees C for 30 seconds, 60 degrees C for 30 seconds. Reaction products are separated on a denaturing 8%
polyacrylamide gel, followed by drying of the gel and autoradiography. The internal control (to control for possible Taq polymerase inhibition) generates a band of 36 nt.
Comparison of radioactive signal integrated (e.g., by phorphorimager analysis) for telomerase-extended bands with the radioactive signal from a reaction performed with a known amount of quantification standard template (termed R8; 5'-AATCCGTCGAGCAGAGTTAG [GGTTAG]~-3~) allows expression of telomerase activity as an absolute value. The absolute value = TPG (total product generated) =[(TP-TPi)/TI]/[(R8-B)/RI)] x 100, where TP = telomerase products from test extract, TPi =
telomerase products from a heat-inactivated (75 C, 10 minutes) extract reaction, TI = the signal from the internal control, R8 = the signal from the R8 qualification standard template reaction, B = signal from a lysis buffer-only blank reaction, and RI
= the internal control value for the reaction containing R8 template and NT and TSNT control primers.

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, 1mM MgCl2, 1mM EGTA, 0.1 mM PMSF, SmM -mercaptoethanol, 1mM DTT, 0.5% 3-[(3-cholamidopropyl)-dimethyl-amino]-1- 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. 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 extract, 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. Extracts may be stored frozen at -80 degrees C until assayed.
A variety of animal models have been designed to assay telomerase activity in vivo.
Inhibition of telomerase 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).
Human cell culture studies have been established to assay inhibition of telomerase activity in human carcinomas responding to various therapeutics. A human breast cancer model for studying telomerase inhibitors is described (Raymond, E., 1999, Br.
J. Cancer, 80, 1332-1341). Human studies of telomerase 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. Sci., 161, 124-134), lung carcinoma (Yashima, K., 1997, Cancer Reseach, 57, 2372-2377), testicular cancer in response to cisplatin (Burger, A. M., 1997, Eur. J. Cancer, 33, 638-644), and ovarian .
carcinoma (Counter, C. M., 1994, Proc. Natl. Acad. Sci., 91, 2900-2904).
Particular degenerative and disease states that can be associated with telomerase expression modulation include but are not limited to:
Cancer: Almost all human tumors have detectable telomerase activity (Shay, J.
W., 1997, Eur. J. Cancer, 33, 787-791). 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.
Restinosis: Telomerase inhibition in vascular smooth muscle cells may inhibit restinosis by limiting proliferation of these cells.
Infectious disease: 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.
Transplant re'ec~ tion: 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) of the instant invention. Those skilled in the art will recognize that other drugs such as anti-cancer compounds and therapies can be similarly be readily combined with the nucleic acid molecules of the instant invention (e.g. ribozymes and antisense molecules) and are hence within the scope of the 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. Lippincott Company, Philadelphia, USA;
5 incorporated herein by reference) and include, without limitations, antifolates;
fluoropyrimidines; cytarabine; purine analogs; adenosine analogs; amsacrine;
topoisomerase I inhibitors; anthrapyrazoles; retinoids; antibiotics such as bleomycin, anthacyclins, mitomycin C, dactinomycin, and mithramycin; hexamethylmelamine;
dacarbazine; 1-asperginase; platinum analogs; alkylating agents such as nitrogen mustard, 10 melphalan, chlorambucil, busulfan, ifosfamide, 4-hydroperoxycyclophosphamide, nitrosoureas, thiotepa; plant derived compounds such as vinca alkaloids, epipodophyllotoxins, taxol; Tomaxifen; radiation therapy; surgery; nutritional supplements; gene therapy; radiotherapy such as 3D-CRT; immunotoxin therapy such as ricin, monoclonal antibodies herceptin; and the like. For combination therapy, the nucleic 15 acids of the invention are prepared in one of two ways. First, the agents are physically combined in a preparation of nucleic acid and chemotherapeutic agent, such as a mixture of a nucleic acid of the 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/mz/day and 20 liposome-associated nucleic acid of the invention in the same solution to deliver 0.1-100 mg/kg/day). Alternatively, the agents are administered separately but simultaneously in their respective effective doses (e.g., 1000-1250 mg/m2/d ifosfamide and 0.1 to 100 mg/kg/day nucleic acid of the invention).
Gaeta et al., US patents No. 5,760,062; 5,767,278; 5,770,613 have described small 25 molecule inhibitors of human telomerase RNA (hTR) subunit.
Blasco et al., 1995, Science, 269, 1267-1270 describe the synthesis and testing of antisense oligonucleotides targeted against a specific region of the mouse telomerase RNA
(mTR) subunit and reported reduction in telomerase activity in mice.
Bisoffi et al., 1998, Eur. J. Cancer, 34, 1242-1249 have studied the down 30 regulation of human telomerase activity by a retrovirus vector expressing antisense RNA
targeted against the hTR RNA.

Norton et al., 1996, Nature Biotechnology, 14, 615-619 have reported the use of a peptide nucleic acid (PNA) molecule targeting hTR RNA to down regulate telomerase activity in human immortal breast epithelial cells.
Yokoyama et al., 1998, Cancer Research, 58, 5406-5410 have reported the synthesis and testing of hammerhead ribozyme constructs targeting hTR RNA
resulting in a decrease in the telomerase activity in Ishikawa cells.
Henderson, European Patent Application No. 666,313-A2 describes methods of identifying and cloning hTR gene for use in gene therapy approaches for creating aberrant telomeric sequences in transfected human tumor cells. A ribozyme based gene therapy approach to inhibit the expression of hTR gene is described as well. The intended result of such therapies involves incurred genetic instability based on non-native telomeric sequences resulting in rapid cell death of the treated cells.
West et al., US patent No. 5,489,508 describe methods for determining telomere length and telomerase activity in cells. Inhibitors of hTR RNA, including oligonucleotides and/or small molecules are described.
These foregoing approaches of targeting the telomerase RNA subunit (TR) 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.
Collins et al., International PCT publication No. WO 98/01542 describes assays for the detection of telomerase activity. Four human telomerase subunit proteins are described called p140, p105, p48 and p43. In addition, hybridization probes and primers are described as inhibitors of telomerase gene function. Antibody based inhibitors of telomerase protein subunits are described.
A more attractive approach to telomerase regulation would involve the regulation of human telomerase by modulating the expression of the protein subunits of the enzyme, preferably the reverse transcriptase (hTERT) subunit. Based of reconstitution experiments, 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. A study in which the three major subunits of telomerase (hTR, TP1, and hTERT were assayed in normal and malignant endometrial tissues determined that 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 of the telomerase enzyme. As such, hTERT is one of the 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. The 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.
Identification of Potential Target Sites in Human TERT RNA
The sequence of human TERT was screened for accessible sites using a computer folding algorithm. Regions of the 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.
Selection of Enzymatic Nucleic Acid Cleavage Sites in Human TERT RNA
To test whether the sites predicted by the computer-based RNA folding algorithm corresponded to accessible sites in TERT RNA, 10 hammerhead ribozyme and three G-Cleaver ribozyme sites were selected for further analysis (Table 17). Ribozyme target sites were chosen by analyzing sequences of Human TERT (Nakamura et al., 1997 Science 277, 955-959; Genbank sequence accession number: I~1M-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.
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 S bases on each arm are able to bind to, or otherwise interact with, the target RNA.
Chemical Synthesis and Purification of 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. 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 polyrnerase (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 incorporated herein by reference) and were resuspended in water. The sequences of the chemically synthesized ribozymes used in this study are shown below in Table 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.
Cleavage Reactions: Full-length or partially full-length, internally-labeled target RNA for ribozyme cleavage assay is prepared by in vitro transcription in the presence of [a-32p] CTP, passed over a G 50 Sephadex column by spin chromatography and used as substrate RNA without further purification. Alternately, substrates are 5'-32P-end labeled using T4 polynucleotide kinase enzyme. Assays are performed by pre-warming 15 ~1 of 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 (15 ~l) of substrate RNA (maximum of 1-5 nM; 5 x 105 to 1 x 10' cpm) that was also pre-warmed in cleavage buffer. As an initial screen, assays are carried out for 1 hour at 37°C using a final concentration of either 40 nM or 1 mM
ribozyme, i.e., ribozyme excess. The reaction is quenched by the addition of an equal volume (30 w1) of 95% formamide, 20 mM EDTA, 0.05% bromophenol blue and 0.05%
xylene cyanol after which the sample is heated to 95°C for 2 minutes, quick chilled and loaded onto a denaturing polyacrylamide gel. Substrate RNA and the specific RNA
cleavage products generated by. ribozyme cleavage are visualized on an autoradiograph of the gel. The percentage of cleavage is determined by Phosphor Imager~
quantitation of bands representing the intact substrate and the cleavage products.
Example 2: PTP-1B
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 of the 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 of the tyrosine kinase portion of the PTK in the cytoplasmic domain. Receptor-like transmembrane PTPs function through extracellular ligand binding that modulates dephosphorylation of intracellular 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 1B, a cytoplasmic PTP, 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 5 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 (Guar, K., 1991, J. Biol. Chem., 266, 17026-17030).
The crystal structure of human PTP1B defined the phosphate binding site of the enzyme as a 10 glycine rich cleft at the surface of the molecule with cysteine 215 positioned at the base of this cleft. The location of cysteine 215 and the shape of the 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 15 role in the regulation of phosphotyrosine mediated signal transduction. PTP-1B activity and specificity against a panel of receptor tyrosine kinases demonstrated clear differences between substrates, suggesting that cellular compartmentalization is a determinant in defining the activity and function of the enzyme (Lammers, 8.,1993, J. Biol.
Chem., 268, 22456-22462). Experiments have indicated that PTP-1B is localized predominantly in the 20 endoplasmic reticulum via its 35 amino acid carboxyterminal sequence. PTP-1B 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-1B has been identified as a negative regulator of the insulin response.

is widely expressed in insulin sensitive tissues (Goldstein, B. J., 1993, Receptor, 3, 1-15).
25 Isolated PTP-1B dephosphorylates the insulin receptor in vitro (Tonks, N.
K., 1988, J.
Biol. Chem., 263, 6731-6737). PTP-1B dephosphorylation of multiple phosphotyrosine residues of the 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). In addition to insulin receptor dephosphorylation, PTP-1B
30 also dephosphorylates the insulin related subtrate 1 (IRS-1), a principal substrate of the insulin receptor (Lammers, R., 1993, J. Biol. Chem., 268, 22456-22462).

Microinjection of PTP1B into Xenopus oocytes results in the inhibition of insulin stimulated tyrosine phosphorylation of endogenous proteins, including the ~3-subunit of the 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. Sci., 87, 5514-5518). Inactivation of recombinant rat PTP-1B with antibody immunoprecipitation results in the dramatic increase in insulin stimulated DNA synthesis and phosphatidylinositol 3'-kinase activity.
Insulin stimulated receptor autophosphorylation and insulin receptor substrate 1 tyrosine phosphorylation are increased dramatically as well through PTP-1B inhibition (Ahmad, F., 1995, J. Biol. Chem., 270, 20503-20508).
Increased PTP-1B expression correlates with insulin resistance in hyperglycemic cultured fibroblasts. In this study, desensitized insulin receptor function was observed via impaired insulin-induced autophosphorylation of the receptor. Treatment with insulin sensitivity normalizing thiazolidine derivatives resulted in the amelioration of the hyperglycemic insulin resistance via a normalization in PTP-1B expression (Maegawa, H., 1995, J. Biol. Chem., 270, 7724-7730). A marine model of insulin resistance with a knockout of the hetrerotrimeric GTP-binding protein subunit Gia2 provides a type 2 diabetis phenotype that correlates with the increased expression of PTP-1B
(Moxam, C.
M., 1996, Nature, 379, 840-844).
PTP-1B interacts directly with the activated insulin receptor ~i-subunit. An inactive homolog of PTP-1B was used to precipitate the activated insulin receptor in both purified receptor preparations and whole-cell lysates. Phosphorylation of the insulin receptor's triple tyrosine residues in the kinase domain is necessary for PTP-1B
interaction.
Furthermore, insulin stimulates tyrosine phosphorylation of PTP-1B (Seely, B.
L., 1996, Diabetes, 45, 1379-1385). A similar study confirmed the direct interaction of with the insulin receptor (3-subunit as well as the required multiple phosphorylation sites within the receptor and PTP-1B (Bandyopadhyay, D., J. Biol. Chem., 272, 1639-1645).
Knockout mice lacking the PTP-1B gene (both homozygous PTP-1B-/- and heterozygous PTP-1B+/-) have been used to study the specific role of PTP-1B
relating to insulin action in vivo. The resulting PTP-1B 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-1B deficient mice demonstrated enhanced insulin sensitivity in glucose and insulin tolerance tests. At the physiological level, the PTP-1B deficient mice showed increased phosphorylation of the insulin receptor after insulin administration. When fed a high fat diet, the PTP-1B deficient mice were resistant to weight gain and remained insulin sensitive as opposed to normal PTP-1B
expressing mice, who rapidly gained weight and become insulin resistant (Elchebly, M., 1999, Science, 283, 1544-1548). As such, modulation of PTP-1B expression could be used to regulate autophosphorylation of the insulin receptor and increase insulin sensitivity in vivo.
This modulation could prove beneficial in the treatment of insulin related disease states.
In light of the above findings, particular disease states that involve PTP-1B
expression include but are not limited to:
Diabetes: Both type 1 and type 2 diabetes may be treated by modulation of PTP-expression. Type 2 diabetes correlates to desensitized insulin receptor function (White et al., 1994). Disruption of the PTP-1B dephosphorylation of the 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 modulation through increased insulin sensitivity.
Obesity: Elchebly et al., 1999, demonstrated that PTP-1B deficient mice were resistant to weight gain when fed a high fat diet compared to normal PTP-1B expressing mice. This finding suggests that PTP-1B modulation may be beneficial in the treatment of obesity.
Ahmad et al., 1997, Metab. Clin. Exp., 46, 1140-1145, describe reduced PTPs in adipose tissue and improved insulin sensitivity in obese subjects following weight loss.
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) of the instant invention. Those skilled in the art will recognize that other drugs such as anti-diabetes and anti-obesity compounds and therapies can be similarly be readily combined with the nucleic acid molecules of the instant invention (e.g. ribozymes and antisense molecules) are hence within the scope of the instant invention.
Methods have been developed to assay PTP-1B activity.
Maegawa et al., 1995, J. Biol. Chem., 270, 7724-7730, describe a tissue culture model in which Rat 1 fibroblasts expressing human insulin receptors can be used to model hyperglycemia induced insulin resistance. Maegawa et al. also describe assays to measure PTPase activity using labeled phosphorylated insulin receptors and by immunoenzymatic techniques.
Moxham et al., 1996, Nature, 379, 840-844, describe a marine animal and tissue culture model employing Gia2 deficiency to study hyperinsulinaemia, impaired glucose tolerance and resistance to insulin in vivo. Assays for PTPase activity and tyrosine phosphorylation of insulin-receptor substrate 1 are described.
Khandelwal et al., 1995, Molecular and Cellular Biochemistry, 153, 87-94, describe four different animal models for studying insulin dependent and insulin resistant diabetes mellitus. These models were used to study the effect of vanadate, an insulin mimetic and PTPase inhibitor, on the insulin-stimulated phosphorylation of the insulin receptor and its tyrosine kinase acitivity.
Wang et al., 1999, Biochim. Biophys. Acta, 1431, 14-23, describe fluorescein monophosphates as fluorogenic substrates for PTPs.
Various methods and compounds have been developed to inhibit protein tyrosine phosphatase activity.
Wrobel et al., 1999, J. Med. Chem., 42, 3199-3202, describe PTP-1B inhibition and antihyperglycemic activity in the ob/ob mouse model by 11-arylbenzo[b]naphtho[2,3-d]furans and arylbenzo[b]naphtho[2,3-d]thiophenes.
Andersen et al., International PCT publication No. WO 98/DK407 describe the preparation of thienopyridzinones and thienochromenones as modulators of PTPases.
Taing et al., 1999, Biochemistry, 38, 3793-3803, describe potent and highly selective inhibitors of PTP-1B comprising an array of bis(aryldifluorophosphonates).
Ham et al., 1999, Bioorg. Med. Chem. Lett., 9, 185-186, describe selective inactivation of PTP-1B by a sulfone analog of naphthoquinone.
Desmarais et al., 1999, Biochem, J., 337, 219-223, describe [Difluro(phosphono)methyl]phenylalanine-containing peptide inhibitors of PTPs.
Taylor et al., 1998, Bioorg. Med. Chem., 6, 2235, describe potent non-peptidyl inhibitors of PTP-1B.
Kotoris et al., 1998, Bioorg. Med. Chem. Lett., 8, 3275-3280, describe novel phosphate mimetics for the design of non-peptidyl inhibitors of PTPs.

Groves et al., 1998, Biochemistry, 37, 17773-17783, describe the structural basis for PTP-1B inhibition by the phosphotyrosine peptide mimetics (difluoronaphthylmethyl)phosphoric acid and the fluoromalonyl tyrosines with complexed crystal structures.
Yao et al., 1998, Bioorgl Med. Chem., 6, 1799-1810, describe the structure-based design and synthesis of small molecule PTP-1B inhibitors comprising novel naphthyldifluoromethyl phosphoric acids 1 and 2.
Taylor et al., 1998, Bioorg. Med. Chem., 6, 1457-1468, describe potent non-peptidyl inhibitors of PTP-1B.
Desmarais et al., 1998, Arch. Biochem. Biophys., 354, 225-231, describe inhibition of PTP-1B and CD45 by sulfotyrosyl peptides.
Mjalli et al., application US 96-766114, cont. in part of US patent No.
543,630, describe the preparation of heterocyclic compounds as modulators of proteins with phosphotyrosine recognition units.
Wang et al., 1998, Bioorg. Med. Chem. Lett., 8, 345-350, describe naphthalenebis[a,a-difluoromethylenephosphonates] as potent inhibitors of PTPs.
Rice et al., 1997, Biochemistry, 36, 15965-15974, describe a targeted library of small molecule tyrosine and dual-specificity phosphatase inhibitors with random side chain variation from a rational core design.
Olefsky, International PCT publication No. WO 97/LJS2752 describes a method and phosphopeptides used for the treatment of insulin resistance based on the association of PTP-1B with the activated insulin receptor. Also included is a method for determining whether a compound inhibits PTP-1B binding to the insulin receptor.
Huyer et al., 1997, J. Biol. Chem., 272, 843-851, describe the mechanism of inhibition of PTPases by vanadate and pervanadate.
Burke et al., 1996, Biochemistry, 35, 15989-15996, describe the structure-based design of PTP-1B inhibitors.
Tonks et al., International PCT publication No. WO 97/U5 13016, describe substrate-trapping 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-1B
activity specifically by covalent binding to or modification of PTP-1B have the potential for multiple side effects. Conventional drug substances that will potently suppress PTP-1B
5 activity with few or no side effects from interaction with other PTPs are difficult to envision. A more attractive approach to PTP-1B modulation would involve the specific regulation of PTP-1B expression with oligonucleotides.
Identification of Potential Target Sites in Human PTP-1B RNA
10 The sequence of human PTP-1B was screened for accessible sites using a computer folding algorithm. Regions of the 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.
15 Selection of Enzymatic Nucleic Acid Cleavage Sites in Human PTP-1B RNA
To test whether the sites predicted by the computer-based RNA folding algorithm corresponded to accessible sites in PTP-1B RNA, 10 hammerhead ribozyme, five NCH
and three G-Cleaver ribozyme sites were selected for further analysis (Table 8).
Ribozyme target sites were chosen by analyzing sequences of Human PTP-1B
(Genbank 20 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. USA, 86, 7706) to assess whether the ribozyme sequences fold into the appropriate secondary structure. Those ribozymes with unfavorable intramolecular 25 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.

Chemical Synthesis and Purification of Ribozymes for Efficient Cleavage of PTP-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 incorporated 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.
Ribozyme Cleavage of PTP-1B RNA Target in vitro Ribozymes targeted to the human PTP-1B 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-1B RNA are given in Tables 3-8.
Cleavage Reactions: Full-length or partially full-length, internally-labeled target RNA for ribozyme cleavage assay is prepared by in vitro transcription in the presence of [a-32p] CTP, passed over a G 50 Sephadex column by spin chromatography and used as substrate RNA without fiuther purification. Alternately, substrates are 5'-32P-end labeled using T4 polynucleotide kinase enzyme. Assays are performed by pre-warming a 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 37°C using a final concentration of either 40 nM or 1 mM ribozyme, i.e., ribozyme excess.

The reaction is quenched by the addition of an equal volume of 95% formamide, 20 mM
EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol after which the sample is heated to 95~C for 2 minutes, quick chilled and loaded onto a denaturing polyacrylamide gel. Substrate RNA and the specific RNA cleavage products generated by ribozyme cleavage are visualized on an autoradiograph of the gel. The percentage of cleavage is determined by Phosphor Imager~ quantitation of bands representing the intact substrate and the cleavage products.
Example 3: MetAP-2 Methionyl aminopeptidases are metalloproteases that are known to possess post-translational 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 of the two classes are similar (Liu, S., 1998, Science, 282, 1324-1327). Methionine aminopeptidase expression appears to be involved in the control of cellular proliferation.
Deletion of the MetAP gene from E. Coli is lethal (Chang, S. Y., 1989, J.
Bacteriol., 171, 4071-4072). In Saccharomyces cerevisiae, deletion of the gene that codes for either MetAP-1 or 2 results in a slow growth phenotype while deletion of both genes is lethal (Li, X., 1995, Proc. Natl. Acad. Sci., 92, 12357-12361). (Human methionine aminopeptidase-1, MetAP-1, accession No. P53582).
The aminopeptidase function of this class of enzymes may serve a regulatory role in activating signal peptides in conjunction with N-myristoyl transferase (NMT) activity.
NMT is expressed from a lethal gene in yeast (Duronio, R. J., 1989, Science, 243, 796-800). NMT is responsible for amino-terminal ligation of myristic acid onto nascent peptides and cannot act on peptides with an amino-terminal methionine residue (Resh, M.
D., 1996, Cell. Signal., 8, 403-412). Myristoylation of proteins correlates to intracellular localization events that may determine why certain signaling proteins are dependent on NMT for activity (Taunton, J., 1997, Chemistry & Biology, 4, 493-496). Protein tyrosine kinase Src is dependant on myristoylation for activity and has been identified as an upstream regulator of human vascular endothelial growth factor (VEGF) expression through hypoxic induction in solid tumors (Mukhopadhyay, D., 1995, Nature, 375, 577-581). MetAPs may therefore regulate the activation of signal peptides (such as VEGF) through cotranslational modification of nascent peptides with NMT. Disruption of protein myristoylation by MetAP inhibition could result in the improper localization of signaling proteins resulting in inhibition of cell growth. (Human N-myristoyltransferase, hNMT, accession No. AF043324.) Fumagillin, a sesquiterpene diepoxide metabolite of the fungus Aspergillus fumigates, 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 fumigates in an endothelial cell culture dish in which cells closest to the fungal colony displayed growth inhibition.
Synthetic analogs of fiunagillin 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). Treatment of endothelial cells with these compounds results in late G1 phase arrest. 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. The use of affinity chromatography with a fumagillin-biotin conjugate resulted in the isolation of a 67-kDa mammalian protein through covalent interaction with the bound substrate. Analysis of digested peptide fragments from the isolated protein revealed MetAP-2 as the covalently bound substrate. Subsequent growth inhibition studies in yeast utilizing MetAP-1 and MetAP-2 deletion strains determined that MetAP-2 is selectively inhibited by fumagillin in vivo (Sin, N., 1997, Proc. Natl. Acad. Sci., 94, 6099-6103). A
similar study with TNP-470 and ovalicin, another potent inhibitor of neovascularization, determined that 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 of the 67-kDa MetAP-2 protein by immunoassay. MetAP-2 has been shown to affect translational initiation by association with eukaryotic initiation factor 2a (eIF-2a) (Ray, M. K., 1992, Proc. Natl. Acad. Sci., 89, 539-543). The binding of MetAP-2 with eIF-2a inhibits the heme-regulated inhibitor kinase (HRI) phosphorylation of eIF-2a in vitro in reticulocyte lysates (Datta, B., 1988, Proc. Natl. Acad.
Sci., 85, 3324-3328). MetAP-2/eIF-2a 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. also determined that covalent binding of TNP-470 and ovalicin, while potently inhibiting methionine aminopeptidase type 2 activity specifically, did not affect the regulatory activity of MetAP-2 on eIF-2a.
This finding by Griffith et al. rules out the possibility that control of eIF-2a phosphorylation by MetAP-2 is responsible for the inhibition of endothelial cell proliferation by fumagillin related compounds.
Particular 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 treat a wide variety of cancers.
Diabetic retinopathy and aye 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). The requirement of protein kinase Src in hypoxia induced VEGF expression (Mukhopadhyay, D., 1995, Nature, 375, 577-581) indicates that MetAP modulation of aminopeptidase activity can potentially be used to treat 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 treat arthritis.

Psoriasis: Angiogenesis has been implicated in psoriasis due to overexpression of the angiogenic polypeptide interleukin-8 and decreased expression of the angiogenesis inhibitor thrombospondin (Nickoloff, B. J., 1994, Amer. J. Pathol. 44, 820-828).
Angiogenesis inhibition through MetAP modulation can potentially be used to treat 5 psoriasis.
Female reproduction: Angiogenesis in the female reproductive system has been implicated in several disorders of the reproductive tract (Reynolds, L. P., 1992, FASEB, 6, 886-892). Modulation of angiogenesis through control of MetAP may have various applications in the area of female reproduction and fertility.
10 Various methods have been developed to assay MetAP activity.
Griffith et al., 1998, Proc. Natl. Acad. Sci., 95, 15183-15188, describe an enzymatic assay for MetAP-2 activity in vitro and an endothelial cell culture proliferation assay for MetAP-2 activity in vivo.
Weber et al., 1999, International PCT publication No. WO 98/US-21231 describe 15 novel fluorescent reporter molecules and an enzymatic assay that can be used for determining the activity of MetAP-2 for drug screening and determining the chemosensitivity of human cancer cells to treatment with chemotherapeutic drugs.
Larrabee, J. A. et al., 1999, Anal. Biochem, 269, 194-198, describe the use of a high-pressure liquid chromatographic (HPLC) method for assaying MetAP-2 activity with 20 application to the study of enzymic inactivation.
Quantitative methods have been developed to assay the efficacy of antiangiogenic therapies.
Wantanabe et al., 1992, Molec. Biol. Cell, 3, 324a, describe the quantitation of angiogenic peptides (bFGF) in human serum as a prognostic test for breast cancer.
25 Nguyen et al., 1994, J. Natn. 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 30 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.
Griffith et al., International PCT publication No. WO 9856372 describe small molecule inhibitors of MetAP2 and uses thereof.
D'Amato et al., International PCT publication No. WO 9805293 describe the use of AGM-1470 (TNP-470) as an angiogenesis inhibitor for use in regulating the female reproductive system and for treating diseases of the reproductive tissue.
Davidson et al., US patent No. 5,801,146 describe a compound and method for inhibiting angiogenesis using mammalian kringle 5 protein.
Cao et al., US patent No. 5,854,221 describe a protein-based endothelial cell proliferation inhibitor and its method of 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.
Wang et al., 1998, Proc. Am. Assoc. Cancer Res., 39, 98 (abstr.) describe blocked proliferation of human endothelial cells by human MetAP-2 antisense oligonucleotides.
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). A similar study employing MetAP-2 inhibition could be used to study ribozyme based inhibition of MetAP-2 induced angiogenesis in vivo.
Identification of Potential Target Sites in Human MetAP-2 RNA
The sequence of human MetAP-2 was screened for accessible sites using a computer-folding algorithm. Regions of the 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.
Selection of Enzymatic Nucleic Acid Cleava~~e Sites in Human MetAP-2 RNA
To test whether the sites predicted by the computer-based RNA folding algorithm corresponded to accessible sites in MetAP-2 RNA, 11 hammerhead ribozyme, 4 NCH
and three G-Cleaver ribozyme sites were selected for further analysis (Table 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.
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.
Chemical Synthesis and Purification of Ribozymes for Efficient Cleavage of MetAP-2 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 S'-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, S 1 ).
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 incorporated herein by reference) and were resuspended in water. The sequences of the chemically synthesized ribozymes used in this study are shown below in Table 9-12.
Riboz~me 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.
Cleavage Reactions: Full-length or partially full-length, internally-labeled target RNA for ribozyme cleavage assay is prepared by in vitro transcription in the presence of [a-32p] CTP, passed over a G 50 Sephadex column by spin chromatography and used as substrate RNA without further purification. Alternately, substrates are S'-32P-end labeled using T4 polynucleotide kinase enzyme. Assays are performed by pre-warming a 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 37°C using a final concentration of either 40 nM or 1 mM ribozyme, i.e., ribozyme excess.
The reaction is quenched by the addition of an equal volume of 95% formamide, 20 mM
EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol after which the sample is heated to 95°C for 2 minutes, quick chilled and loaded onto a denaturing polyacrylamide gel. Substrate RNA and the specific RNA cleavage products generated by ribozyme cleavage are visualized on an autoradiograph of the gel. The percentage of cleavage is determined by Phosphor Imager~ quantitation of bands representing the intact substrate and the cleavage products.
Example 4: BACE, ps-1, ps-2 Alzheimer's disease (AD) is a progressive, degenerative disease of the 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 of the next century if no cure or definitive prevention of the disease is found. Nearly one out of ten 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 of the 4 kD amyloid ~3 peptide (A~i).
These plaques are characterized by dystrophic neurites that show profound synaptic loss, neurofibrillary tangle formation, and gliosis. A~3 arises from the proteolytic cleavage of the large type I transmembrane protein, ~3-amyloid precursor protein (APP) (Kang et al., 1987, Nature, 325, 733). Processing of APP to generate A(3 requires two sites of cleavage by a (3-secretase and a y-secretase. ~3-secretase cleavage of APP results in the cytoplasmic release of a 100 kD soluble amino-terminal fragment, APPs(3, leaving behind a 12 kD
transmembrane carboxy-terminal fragment, C99. Alternately, APP can be cleaved by a a-secretase to generate cytoplasmic APPsa and transmembrane C83 fragments. Both remaining transmembrane fragments, C99 and C83, can be further cleaved by a y-secretase, leading to the release and secretion of Alzheimer's related A(3 and a non-pathogenic peptide, p3, respectively (Vassar et al., 1999, Science, 286, 735-741). Early onset familial Alzheimer's disease is characterized by mutant APP protein with a Met to Leu substitution at position P1, characterized as the "Swedish" familial mutation (Mullan et al., 1992, Nature Genet., 1, 345). This APP mutation is characterized by a dramatic enhancement in (3-secretase cleavage (Citron et al., 1992, Nature, 360, 672).
The identification of (3-secretase, and y-secretase constituents involved in the release of (3-amyloid protein is of primary importance in the development of treatment strategies for Alzheimer's disease. Characterization of a-secretase is also important in this regard since a-secretase cleavage may compete with (3-secretase cleavage resulting in non-pathogenic vs. pathogenic protein production. Involvement of the two metalloproteases, ADAM 10, and TACE has been demonstrated in a-cleavage of AAP (Buxbaum et al., 1999, J. Biol. Chem., 273, 27765, and Lammich et al., 1999, Proc. Natl. Acad.
Sci. U.S.A., 96, 3922). Studies of y-secretase activity have demonstrated presenilin dependence (De Stooper et al., 1998, Nature, 391, 387, and De Stooper et al., 1999, Nature, 398, S 18), and as such, presenilins have been proposed as y-secretase even though presenilin does not present proteolytic activity (Wolfe et al., 1999, Nature, 398, 513).

Recently, Vassar et al., 1999, supra reported (3-secretase cleavage of AAP by the transmembrane aspartic protease beta site APP cleaving enzyme, BACE. While other potential candidates for ~i-secretase have been proposed (for review see Evin et al., 1999, Proc. Natl. Acad. Sci. U.S.A., 96, 3922), none have demonstrated the full range of 5 characteristics expected from this enzyme. Vassar et al, supra, demonstrate that BACE
expression and localization are as expected for ~3-secretase, that BACE
overexpression in cells results in increased ~i-secretase cleavage of APP and Swedish APP, that isolated BACE demonstrates site specific proteolytic activity on APP derived peptide substrates, and that antisense mediated endogenous BACE inhibition results in dramatically reduced 10 ~3-secretase activity.
Current treatment strategies for Alzheimer's disease rely on either the prevention or the alleviation of symptoms and/or the slowing down of disease progression.
Two drugs approved in the treatment of Alzheimer's, donepezil (Aricept~) and tacrine (Cognex~), both cholinomimetics, attempt to slow the loss of cognitive ability by increasing the 15 amount of acetylcholine available to the brain. Antioxidant therapy through the use of antioxidant compounds such as alpha-tocopherol (vitamin E), melatonin, and selegeline (Eldepryl~) attempt to slow disease progression by minimizing free radical damage.
Estrogen replacement therapy is thought to incur a possible preventative benefit in the development of Alzheimer's disease based on limited data. The use of anti-inflammatory 20 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 25 enhancement of cognitive and memory function based on cellular metabolism.
Whereby the above treatment strategies may all improve quality of life in 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, 30 therapeutics that can eliminate and/or reverse the deposition of amyloid (3 peptide. The use of compounds to modulate the expression of proteases that are instrumental in the release of amyloid ~3 peptide, namely (3-secretase (BACE), and y-secretase (presenilin), is of therapeutic significance.
Tsai et al., 1999, Book of Abstrasts, 218th ACS National Meeting, New Orleans, Aug 22-26, describe substrate-based alpha-aminoisobutyric acid derivatives of difluoro ketone peptidomimetic inhibitors of amyloid ~i peptide through y-secretase inhibition.
Czech et al., International PCT publication No. W0/9921886, describe peptides capable of inhibiting the interaction between presenilins and the (3-amyloid peptide or its precursor for therapeutic use.
Fournier et al., International PCT publication No. W0/9916874, describe human brain proteins capable of interacting with presenilins and cDNAs encoding them toward therapeutic use.
St. George-Hyslop et al., International PCT publication No. W0/9727296, describe genes for proteins that interact with presenilins and their role in Alzheimer's disease toward therapeutic use.
Vassar et al., 1999, Science, 286, 735-741, describe specific antisense oligonucleotides targeting BACE, used for inhibition studies of endogenous BACE
expression in 101 cells and APPsw cells via lipid mediated transfection.
Vassar et al., 1999, Science, 286, 735-741, describe a cell culture model for studying BACE inhibition. Specific antisense nucleic acid molecules targeting BACE mRNA
were used for inhibition studies of endogenous BACE expression in 101 cells and APPsw (Swedish type amyloid precursor protein expressing) cells via lipid mediated transfection.
Antisense treatment resulted in dramatic reduction of both BACE mRNA by Northern blot analysis, and APPs~3sw ("Swedish" type (3-secretase cleavage product) by ELISA, with maximum inhibition of both parameters at 75-80%. This model was also used to study the effect of BACE inhibition on amyloid (3-peptide production in APPsw cells.
Games et al., 1995, Nature, 373, 523-527, describe a transgenic mouse model in which mutant human familial type APP (Phe 717 instead of Val) is overexpressed. This model results in mice that progressively develop many of the pathological hallmarks of Alzheimer's disease, and as such, provides a model for testing therapeutic drugs.
Particular degenerative and disease states that can be associated with BACE
expression modulation include but are not limited to Alzheimer's disease and dementia.

Donepezil, tacrine, selegeline, and acetyl-L-carnitine 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) of the instant invention. Those skilled in the art will recognize that other drugs such as diuretic and antihypertensive compounds and therapies can be similarly be readily combined with the nucleic acid molecules of the instant invention (e.g. ribozymes and antisense molecules) are hence within the scope of the instant invention.
Identification of Potential Target Sites in Human BACE RNA
The sequence of human BACE was screened for accessible sites using a computer-folding algorithm. Regions of the 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.
Selection of Enzymatic Nucleic Acid Cleavage Sites in Human BACE RNA
Ribozyme target sites were chosen by analyzing sequences of Human BACE
(Genbank sequence accession number: AF190725) 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.
Chemical Synthesis and Purification of Riboz~~nes and Antisense for Efficient Cleavage and/or blocking_of BACE RNA
Ribozymes and antisense constructs were designed to anneal to various sites in the RNA message. The binding arms of the ribozymes are complementary to the target site sequences described above, while the antisense constructs are fully complimentary to the target site sequences described above. The ribozymes and antisense constructs were chemically synthesized. The method of synthesis used followed the procedure for normal RNA synthesis as described above and in Usman et al., (1987 J. Am. Chem. 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 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 incorporated herein by reference) and were resuspended in water. The sequences of the chemically synthesized ribozymes and antisense constructs used in this study are shown below in Table 18-23.
RibozXme Cleavage of BACE RNA Target in vitro 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.
Cleavage Reactions: Full-length or partially full-length, internally-labeled target RNA for ribozyme cleavage assay is prepared by in vitro transcription in the presence of [a-32p] CTP, passed over a G SO Sephadex column by spin chromatography and used as substrate RNA without further purification. Alternately, substrates are 5'-32P-end labeled using T4 polynucleotide kinase enzyme. Assays are performed by pre-warming a 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 37°C using a final concentration of either 40 nM or 1 mM ribozyme, i.e., ribozyme excess.
The reaction is quenched by the addition of an equal volume of 95% formamide, 20 mM

EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol after which the sample is heated to 95~C for 2 minutes, quick chilled and loaded onto a denaturing polyacrylamide gel. Substrate RNA and the specific RNA cleavage products generated by ribozyme cleavage are visualized on an autoradiograph of the gel. The percentage of cleavage is determined by Phosphor Imager~ quantitation of bands representing the intact substrate and the cleavage products.
Example 5: Phospholamban Cardiac disease leading to heart failure is the leading cause of combined morbidity and mortality in the developed world. Nearly twenty million people worldwide suffer from heart failure related disease. An estimated five million Americans are afflicted with congestive heart failure (CHF), with 400,000 new cases diagnosed each year. In the US, cardiac disease associated failure results in approximately 40,000 deaths per year, and is associated with an additional 250,000 deaths (Harnish, 1999, Drug & Market Development, 10, 114-119). 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 (supply, 3). 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 of the 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 (myocardial hypertrophy) 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 of the 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 5 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 of the level of ventricular dysfunction (Davidson et al., 1996, Am. J.
Cardiol., 77, 828).
10 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. Digoxin is another popular choice for treating cardiac disease as an ionotropic agent, however, doubts remain concerning the long-term efficacy and 15 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.
Lastly, heart transplants have been effective in the treatment of patients with advanced stages of cardiac 20 disease, however, the limited supply of donor hearts greatly limits the scope of this treatment to the broad population (Harnish, 1999, Drug & Market Development, 10, 114-119).
Whereby the above treatment strategies can all improve morbidity and mortality associated with cardiac disease, the only existing definitive approach to curing the diseased 25 heart is replacement by transplant. Even a healthy, transplanted heart can become diseased in response to the various stresses of mechanical, hemodynamic, hormonal, and pathological stimuli associated with extrinsic risk factors. As such there exists the need for therapeutics effective in reversing the physiological changes associated with cardiac disease.
30 Myocardial hypertrophy and apoptosis are the underlying degenerative process associated with cardiac hypertrophy and failure. A variety of signaling pathways are involved in the progression of myocardial hypertrophy and myocardial apoptosis. Genetic studies have been instrumental in elucidating these pathways and their involvement in cardiac disease through in vitro assays of cardiac muscle cells and in vivo studies of genetically engineered animals: . .
Studies in which the expression of specific genes have been altered in cardiac myocytes have shown that specific peptide hormones, growth factors, and cytokines can activate various features of the hypertrophic response (Hunter and Chien, 1999, New England J of Medicine, 99, 313-322). Particular substances that have been characterized from these studies include potential therapeutic and molecular targets involved in heart failure. Hunter et al., in Chien, KR, ed. Molecular basis of heart disease: a companion to Braunwald's Heart Disease, Philadelphia: W.B. Saunders, 1999:211-250, describe classes of therapeutic and molecular targets involved in heart failure including:
1. Endothelin 1 and angiotensin II receptor antagonists, and antagonists of ras, p38, and c-jun N-terminal kinase (JNK) for inhibition of pathologic hypertrophy.
2. Insulin like growth factor I and growth hormone receptor stimulation for promotion of physiologic hypertrophy.
3. beta-1-adrenergic receptor blockers for inhibition of neurohumoral over stimulation.
4. Phospholamban and Sarcolipin small molecule inhibitors for relief of sarcoplasmic reticulum calcium ATPase inhibition to provide enhancement of myocardial contractile and relaxation responses.
5. Small molecule inhibitors of (3-adrenergic receptor kinase to counteract the desensitization of G protein coupled receptor kinases in order to provide enhancement of myocardial contractile and relaxation responses.
6. Enhancement of angiogenic growth factors (VEGF, FGF-5) for relief of energy deprivation in cardiac tissues.
7. Promoters of myocyte survival including gp 130 ligands (cardiotrophin 1), and Neuregulin for the inhibition of apoptosis of myocytes.
8. Inhibitors of apoptosis such as Caspase inhibitors for the inhibition of apoptosis of myocytes.

9. Inhibitors of cytokines such as TNF-alpha for the inhibition of apoptosis of myocytes.
Congestive heart failure, heart failure, dilated cardiomyopathy and pressure overload hypertrophy are nonlimiting examples of disorders and disease states that can be associated with the above classes of molecular targets.

The failure of cardiac contractile performance leading to cardiac disorders and disease, governed by impairment of cardiac excitation/contraction coupling, points to the importance of the signaling pathways involved in this process. The release and uptake of cytosolic Ca2+ by the sarcoplasmic reticulum plays an integral role in each cycle of cardiac contraction and excitation (Minamisawa et al., 1999, Cell, 99, 313-322). The process of Ca2+ reuptake is mediated by the cardiac sarcoplasmic reticulum Ca2+ ATPase (SERCA2a). 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.
This phosphorylation event is predominantly responsible for the proportional increase in the rate of Ca2+ 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).
Since a proportional decrease in Ca2+ uptake is a hallmark feature of heart failure (Sordahl et al., 1973, Am. J. Physiol., 224, 497-502) and since an increase in the relative ratio of phospholamban to SERCA2a is an important determinant of sarcoplasmic reticulum dysfunction in heart failure (Hasenfuss, 1998, Cardiovasc. Res., 37, 279-289), 'the targeting of phospholamban and related regulatory factors as therapeutic targets for heart disorders should prove valuable for cardiac indications.
Pystynen et al., International PCT publication No. WO 99/00132, describe bisethers of 1-oxa, aza and thianaphthalen-2-ones as small molecule inhibitors of phospholamban for increasing coronary flow via direct dilation of the coronary arteries.
Pystynen et al., International PCT publication No. WO 99/15523, describe bisethers of 1-oxa, aza and thianaphthalen-2-ones as small molecule inhibitors of phospholamban that are useful for treating heart failure.
The efficacy of the above mentioned treatment strategies is limited. Small molecule inhibition of a molecular target is often limited by toxicity, which can restrict dosing and overall efficacy.

He et al., 1999, Circulation, 100, 974-980, describe endogenous expression of mutant phospholamban and phospholamban antisense RNA to investigate the corresponding effect on SERCA2a activity and cardiac myocyte contractility.
A more attractive approach to the treatment of heart disease would involve the use of ribozymes and/or antisense constructs to modulate the expression of target molecules involved in heart failure. The use of nucleic acid molecules of the instant invention permits highly specific regulation of the molecular targets of interest, including phospholamban (PLN) (GenBank accession No.1VM-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. AF092535), 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), (31-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), cardiotrophin 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).
Various methods have been developed to assay phospholamban activity in vitro and in vivo. Holt et al., 1999, J. Mol. Cell. Cardiol., 31, 645-656, describe a cell culture model in which thyroid hormone control of contraction and the Ca2+-ATPase/phospholamban complex is studied in adult rat ventricular myocytes. Slack et al. 1997, J.
Biol. Chem., 272, 18862-18868, describe studies in which the ectopic expression of phospholamban in mouse fast-twitch skeletal muscle cells alters sarcoplasmic reticulum Ca2+
transport and muscle relaxation. MacLennan et al., 1996, Soc. Gen. Physiol. Ser., 51, 89-103, in a review of regulatory interactions between calcium ATPases and phospholamban describe phospholamban/ Ca2+-ATPase interactions in protein expressed in heterologous cell culture experiments. Cornwell et al., 1991, Mol. Pharmacol., 40,923-931, describe the regulation of sarcoplasmic reticulum protein phosphorylation by localized cyclic GMP-dependent protein kinase in vascular smooth muscle cells.
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 transgenic rat model for the study of altered expression of calcium regulatory proteins, including phospholamban, and their effect on myocyte contractile response. LekanneDeprez et al., 1998, J. Mol.
Cell.
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 Illal, and calsequestrin (CSQ). 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 .l. 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 of the physiological role of phospholamban. Arai et al., 1996, Saishin Igaku, S 1, 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. Animal models investigating the major calcium handling myocardial proteins, including phospholamban, are described. These models, as well as others, may be used to evaluate the effect of treatment with nucleic acid molecules of the instant invention on cardiac function. 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) of the instant invention. Those skilled in the art will recognize that other drugs such as diuretic and antihypertensive 5 compounds and therapies can be similarly be readily combined with the nucleic acid molecules of the instant invention (e.g. ribozymes and antisense molecules) are hence within the scope of the instant invention.
Identification of Potential Target Sites in Human phospholamban RNA

10 The sequence of human phospholamban was screened for accessible sites using a computer folding algorithm. Regions of the 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.
15 Selection of Enzymatic Nucleic Acid Cleavage Sites in Human phospholamban RNA
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.
20 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 25 to bind to, or otherwise interact with, the target RNA.
Chemical Synthesis and Purification of Ribozymes and Antisense for Efficient Cleavage and/or blocking of phospholamban RNA
Ribozymes and antisense constructs were designed to anneal to various sites in the 30 RNA message. The binding arms of the ribozymes are complementary to the target site sequences described above, while the antisense constructs are fully complimentary to the target site sequences described above. The ribozymes and antisense constructs were chemically synthesized. The method of synthesis used followed the procedure for normal RNA synthesis as described above and in Usman et al., (1987 J. Am. Chem. 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 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 0 electrophoresis using general methods or were purified by high pressure liquid chromatography (HPLC; see Wincott et al., supra; the totality of which is hereby incorporated herein by reference) and were resuspended in water. The sequences of the chemically synthesized ribozymes and antisense constructs used in this study are shown below in Table 24-30.

Ribozyme Cleavage of phospholamban RNA Target in vitro 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 0 location within the phospholamban RNA are given in Tables 24-30.
Cleavage Reactions: Full-length or partially full-length, internally-labeled target RNA for ribozyme cleavage assay is prepared by in vitro transcription in the presence of [a-32p] CTP, passed over a G 50 Sephadex column by spin chromatography and used as substrate RNA without further purification. Alternately, substrates are 5'-32P-end labeled 5 using T4 polynucleotide kinase enzyme. Assays are performed by pre-warming a 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 carned out for 1 hour at 0 37°C using a final concentration of either 40 nM or 1 mM ribozyme, i.e., ribozyme excess.
The reaction is quenched by the addition of an equal volume of 95% formamide, 20 mM

EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol after which the sample is heated to 95~C for 2 minutes, quick chilled and loaded onto a denaturing polyacrylamide gel. Substrate RNA and the specific RNA cleavage products generated by ribozyme cleavage are visualized on an autoradiograph of the gel. The percentage of cleavage is determined by Phosphor Imager~ quantitation of bands representing the intact substrate and the cleavage products.
Tissue distribution of BrdU-labeled antisense in mice 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 substrate. Visualization of the colored product by microscopy indicated nuclear staining, demonstrating effective distribution of antisense oligonucleotide in cardiac tissue.
Tissue distribution of BrdU-labeled ribozymes in monkey Rhesus monkeys were dosed with BrdU-labeled ribozyme by intravenous bolus ~0 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 substrate. Significant quantities of chemically modified ribozyme are detected in cardiac tissue following this dosing regimen.
?5 Example 6: HBV
Chronic hepatitis B is caused by an enveloped virus, commonly known as the hepatitis B virus or HBV. HBV is transmitted via infected blood or other body fluids, especially saliva and semen, during delivery, sexual activity, or sharing of needles 30 contaminated by infected blood. Individuals may be "carners" and transmit the infection to others without ever having experienced symptoms of the disease. Persons at highest risk are those with multiple sex partners, those with a history of sexually transmitted diseases, parenteral drug users, infants born 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, and acupuncture; the virus is also stable on razors, toothbrushes, baby bottles, eating utensils, and some hospital equipment such as respirators, scopes and instruments. There is no evidence that HBsAg positive food handlers pose a health risk in an occupational setting, nor should they be excluded from work. Hepatitis B has never been documented as being a food-borne disease. The average incubation period is 60 to 90 days, with a I 0 range of 45 to 180; the number of days appears to be related to the amount of virus to which the person was exposed. However, determining the length of incubation is difficult, since onset of symptoms is insidious. Approximately 50% of patients develop symptoms of acute hepatitis that last from 1 to 4 weeks. Two percent or less of these individuals develop fulminant hepatitis resulting in liver failure and death.
5 The determinants of severity include: (1) The size of the dose to which the person was exposed; (2) the person's age with younger patients experiencing a milder form of the disease; (3) the status of the 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. In symptomatic !0 cases, clinical signs include loss of appetite, nausea, vomiting, abdominal pain in the right upper quadrant, arthralgia, and tiredness/loss of energy. Jaundice is not experienced in all cases, however, jaundice is more likely to occur if the infection is due to transfusion or percutaneous serum transfer, and it is accompanied by mild pruritus in some patients.
Bilirubin elevations are demonstrated in dark urine and clay-colored stools, and liver !5 enlargement may occur accompanied by right upper-quadrant pain. The acute phase of the 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 of the virus .0 leads to production of large amounts of HBsAg in the blood. 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 (HBV) infects over 300 million people worldwide (Imperial, 1999, Gastroenterol. Hepatol., 14 (supply, S1-5). 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 of the 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 0 hepatocellular carcinoma (HCC) is common, and liver transplantation is the only treatment option for patients with end-stage liver disease from HBV.
The natural progression of chronic HBV infection over a 10 to 20 year period leads to cirrhosis in 20-to-50% of patients and progression of HBV infection to hepatocellular carcinoma has been well documented. There have been no studies that have determined 5 sub-populations that are most likely to progress to cirrhosis and/or hepatocellular carcinoma, thus all patients have equal risk of progression.
It is important to note that the survival for patients diagnosed with hepatocellular carcinoma is only 0.9 to 12.8 months from initial diagnosis (Takahashi et al., 1993, American Journal of Gastroenterology, 88, 240-243). Treatment of hepatocellular .0 carcinoma with chemotherapeutic agents has not proven effective and only 10% of patients will benefit from surgery due to extensive tumor invasion of the liver (Trinchet et al., 1994, Presse Medicine, 23, 831-833). Given the aggressive nature of primary hepatocellular carcinoma, the only viable treatment alternative to surgery is liver transplantation (Pichlmayr et al., 1994, Hepatology., 20, 33S-40S).
5 Upon progression to cirrhosis, patients with chronic HCV infection present with clinical features, which are common to clinical cirrhosis regardless of the initial cause (D'Amico et al., 1986, Digestive Diseases and Sciences, 31, 468-475). These clinical features may include: bleeding esophageal varices, ascites, jaundice, and encephalopathy (Zakim D, Boyer TD. Hepatology a textbook of liver disease, Second Edition Volume 1.
0 1990 W.B. Saunders Company. Philadelphia). In the early stages of cirrhosis, patients are classified as compensated, meaning that although liver tissue damage has occurred, the patient's liver is still able to detoxify metabolites in the blood-stream. In addition, most patients with compensated liver disease are asymptomatic and the minority with symptoms report only minor symptoms such as dyspepsia and weakness. In the later stages of cirrhosis, patients are classified as decompensated meaning that their ability to detoxify metabolites in the bloodstream is diminished and it is at this stage that the clinical features 5 described above will present.
In 1986, D'Amico et al. described the clinical manifestations and survival rates in 1155 patients with both alcoholic and viral associated cirrhosis (D'Amico supra). Of the 1155 patients, 435 (37%) had compensated disease although 70% were asymptomatic at the beginning of the study. The remaining 720 patients (63%) had decompensated liver I 0 disease with 78% presenting with a history of ascites, 31% with jaundice, 17% had bleeding and 16% had encephalopathy. Hepatocellular carcinoma was observed in six (0.5%) patients with compensated disease and in 30 (2.6%) patients with decompensated disease.
Over the course of six years, the patients with compensated cirrhosis developed I 5 clinical features of decompensated disease at a rate of 10% per year. In most cases, ascites was the first presentation of decompensation. In addition, hepatocellular carcinoma developed in 59 patients who initially presented with compensated disease by the end of the six-year study.
With respect to survival, the D'Amico study indicated that the five-year survival rate !0 for all patients on the study was only 40%. The six-year survival rate for the patients who initially had compensated cirrhosis was 54% while the six-year survival rate for patients who initially presented with decompensated disease was only 21 %. There were no significant differences in the survival rates between the patients who had alcoholic cirrhosis and the patients with viral related cirrhosis. The major causes of death for the !5 patients in the D'Amico study were liver failure in 49%; hepatocellular carcinoma in 22%;
and, bleeding in 13% (D'Amico supra).
Hepatitis B virus is a double-stranded circular DNA virus. It is a member of the 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) .0 and is 42 nm in diameter. It also contains an a antigen (HBeAg) which, along with HBcAg and HBsAg, is helpful in identifying this disease In HBV virions, the genome is found in an incomplete double-stranded form. 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 polymerise 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.
From the free-floating form, the virus must first attach itself specifically to a host cell membrane. Viral attachment is one of the 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.
After attachment, fusion of the viral envelope and host membrane must occur to allow the viral core proteins containing the genome and polymerise to enter the cell. Once inside, the genome is translocated to the nucleus where it is repaired and cyclized.
5 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 polymerise. A subclass of this transcript with a 5'-end extension codes for the precore protein that, after processing, is secreted as HBV a !0 antigen. The 2.4-kb RNA encompasses the pre-S 1 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 of the HBV genome begins within the nucleus of an infected cell.
!5 RNA polymerise II 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.
The packaging of pregenomic RNA into core particles is triggered by the binding of .0 the HBV polymerise to the 5' epsilon stem-loop. RNA encapsidation is believed to occur as soon as binding occurs. The HBV polymerise also appears to require associated core protein in order to function. The HBV polymerise initiates reverse transcription from the 5' epsilon stem-loop three to four base pairs at which point the polymerise and attached nascent DNA are transferred to the 3' copy of the DR1 region. Once there, the (-)DNA is extended by the HBV polymerise while the RNA template is degraded by the HBV
polymerise RNAse H activity. When the HBV polymerise 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 of the (-)DNA.
It is used as a primer for the (+)DNA synthesis which is also generated by the HBV
polymerise. It appears that the reverse transcription as well as plus strand synthesis may I 0 occur in the completed core particle.
Since 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 of the pregenomic RNA
I 5 template would be expected to result in a similar inhibition of HBV
replication. Further, targeting the 3'-end of the 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.
As previously mentioned, HBV does not infect cells in culture. However, !0 transfection 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. Thus, HBV replication competent DNA
would be co-transfected with ribozymes in cell culture. Such an approach has been used to report intracellular ribozyme activity against HBV (zu Putlitz, et al., 1999, J.
Yirol., 73, 5381-!5 5387, and Kim et al., 1999, Biochem. Biophys. Res. Commun., 257, 759-765).
In addition, 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.
Intracellular HBV gene expression can be assayed by a Taqman~ assay for HBV
.0 RNA or by ELISA for HBV protein. Extracellular 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.
There are several small animal models to study HBV replication. One is the transplantation of HBV-infected liver tissue into irradiated mice. Viremia (as evidenced by measuring HBV DNA by PCR) is first detected 8 days after transplantation and peaks between 18 - 25 days (Ilan et al., 1999, Hepatology, 29, 553-562).
Transgenic mice that express HBV have also been used as a model to evaluate potential anti-virals. HBV DNA is detectable in both liver and serum (Morrey et al., 1999, Antiviral Res., 42, 97-108).
I 0 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, I 5 genetic organization, and mechanism of replication. As with HBV in humans, persistent WHV infection is common in natural woodchuck populations and is associated with chronic hepatitis and hepatocellular carcinoma (HCC). Experimental studies have established that WHV causes HCC in woodchucks and woodchucks chronically infected with WHV have been used as a model to test a number of anti-viral agents. For example, !0 the nucleoside analogue 3T3 was observed to cause dose dependent reduction in virus (5O% reduction after two daily treatments at the highest dose) (Hurwitz et al., 1998.
Antimicrob. Agents Chemother., 42, 2804-2809).
Current therapeutic goals of treatment are three-fold: to eliminate infectivity and transmission of HBV to others, to arrest the progression of liver disease and improve the !5 clinical prognosis, and to prevent the development of hepatocellular carcinoma (HCC).
Interferon alpha use is the most common therapy for HBV; however, recently Lamivudine (3TC) has been approved by the FDA. Interferon alpha (IFN-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
DNA negative HBeAg negative) occurs in approximately 25% of patients. Several factors have been identified that predict a favorable response to therapy including:
High ALT , low HBV DNA , being female, and heterosexual orientation.

There is also a risk of reactivation of the hepatitis B virus even after a successful response, this occurs in around 5% of responders and normally occurs within 1 year.
Side effects resulting from treatment with type 1 interferons can be divided into four general categories including: Influenza-like symptoms, neuropsychiatric, laboratory abnormalities, and other miscellaneous side effects. Examples of 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, .lournal of Viral Hepatitis, 1, 3-5). Neuropsychiatric side effects include irritability, apathy, mood changes, insomnia, I 0 cognitive changes, and depression. Laboratory abnormalities include the reduction of myeloid cells, including granulocytes, platelets and to a lesser extent, red blood cells.
These changes in blood cell counts rarely lead to any significant clinical sequellae. In addition, increases in triglyceride concentrations and elevations in serum alaine and aspartate aminotransferase concentration have been observed. Finally, thyroid I 5 abnormalities have been reported. These thyroid abnormalities are usually reversible after cessation of interferon therapy and can be controlled with appropriate medication while on therapy. Miscellaneous side effects include nausea, diarrhea, abdominal and back pain, pruritus, alopecia, and rhinorrhea. In general, most side effects will abate after 4 to 8 weeks of therapy (Dushieko et al., supra ).
!0 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.
!5 Phase III studies with 3TC, showed that treatment for one year was associated with reduced liver inflammation and a delay in scarnng of the liver. In addition, patients treated with Lamivudine (100mg 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. However, stopping of therapy resulted in a reactivation of HBV
replication in most patients. In addition recent reports have documented 3TC
resistance in approximately 30% of patients.

Particular degenerative and disease states that can be associated with HBV
expression modulation include but are not limited to, HBV infection, hepatitis, cancer, tumorigenesis, cirrhosis, liver failure and others.
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) of the 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 of the I 0 instant invention (e.g. ribozymes and antisense molecules) and are, therefore, within the scope of the instant invention.
Current therapies for treating HBV infection, including interferon and nucleoside analogues, are only partially effective. In addition, drug resistance to nucleoside analogues is now emerging, making treatment of chronic Hepatitis B more difficult. Thus, a need I 5 exists for effective treatment of this disease which utilizes antiviral inhibitors which work by mechanisms other than those currently utilized in the treatment of both acute and chronic hepatitis B infections.
Draper, US patent No. 6,017,756, describes the use of ribozymes for the inhibition of Hepatitis B Virus.
?0 Passman et al., 2000, Biochem. Biophys. Res. Commun., 268(3), 728-733.; Gan et al., 1998, J. Med. Coll. PLA, 13(3), 157-159.; Li et al., 1999, Jiefangjun Yixue Zazhi, 24(2), 99-101.; Putlitz et al., 1999, J. Virol., 73(7), 5381-5387.; Kim et al., 1999, Biochem. Biophys. Res. Commun., 257(3), 759-765.; Xu et al., 1998, Bingdu Xuebao, 14(4), 365-369.; Welch et al., 1997, Gene Ther., 4(7), 736-743.; Goldenberg et al., 1997, ?5 International PCT publication No. WO 97/08309, Wands et al., 1997, J. of Gastroenterology and Hepatology, 12(suppl.), 5354-5369.; Ruiz et al., 1997, BioTechniques, 22(2), 338-345.; Gan et al., 1996, J. Med. Coll. PLA, 11(3), 171-175.;
Beck and Nassal, 1995, Nucleic Acids Res., 23(24), 4954-62.; Goldenberg, 1995, International PCT publication No. WO 95/22600.; Xu et al., 1993, Bingdu Xuebao, 9(4), 30 331-6.; Wang et al., 1993, Bingdu Xuebao, 9(3), 278-80, all describe ribozymes that are targeted to cleave a specific HBV target site.

The enzymatic nucleic acid molecules of the instant invention exhibit a high degree of specificity for only the viral mRNA in infected cells. Nucleic acid molecules of the instant invention targeted to highly conserved sequence regions allow the treatment of many strains of human HBV with a single compound. No treatment presently exists which specifically attacks expression of the viral genes) that are responsible for transformation of hepatocytes by HBV.
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 of the HBV
pregenomic mRNAs. For sequence references, see Renbao et al., 1987, Sci. Sin., 30, 507.
This region controls the translational expression of the core protein (C), X
protein (X) and DNA polymerase (P) genes and plays a role in the replication of the 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' of the encapsidation site can result in the inclusion of the disrupted 3' RNA within the core virion structure and targeting sequences 3' of the encapsidation site can result in the reduction in protein expression from both the 3' and 5' fragments.
Alternative regions outside of the 5' most 1500 nucleotides of the pregenomic mRNA also make suitable targets of enzymatic nucleic acid mediated inhibition of HBV
replication. Such targets include the mRNA regions that encode the viral S
gene.
Selection of particular target regions will depend upon the secondary structure of the pregenomic mRNA. 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 of the 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 of the HBV

pregenomic RNA carnes a cis-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 transcription 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 transcription proceeds from that point. The 3' stem-loop, and especially the DR1 primer binding site, I 0 appear to be highly effective targets for ribozyme intervention.
Sequences of the pregenomic RNA are shared by the mRNAs for surface, core, polymerase, and X proteins. Due to the overlapping nature of the HBV
transcripts, all share a common 3'-end. Ribozyme targeting this common 3'-end will thus cleave the pregenomic RNA as well as all of the mRNAs for surface, core, polymerase and X
5 proteins.
In preferred embodiments, 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) control proteins to determine the efficacy of agents !0 used to modulate HBV expression.
The method consists of coating a micro-titer plate with an antibody such as anti-HBsAg Mab (for example, Biostride B88-95-3lad,ay) at 0.1 to 10 p,g/ml in a buffer (for example, carbonate buffer, such as NaZC03 15 mM, NaHC03 35 mM, pH 9.5) at 4°C
overnight. The microtiter wells are then washed with PBST or the equivalent thereof, (for '.5 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 (for example, Accurate YVS1807) 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
~0 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). After washing as above, a substrate (for example, p-nitrophenyl phosphate substrate, Pierce 37620) is added to the wells, which are then incubated (for example, 1 hr. at 37° C).
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 concentrations may be varied to achieve optimum results, a non-limiting example is described in Example 6.
Comparison of this HBsAg ELISA method to a commercially available assay from World Diagnostics, Inc. 15271 NW 60'h Ave, #201, Miami Lakes, FL 33014 (305) 3304 (Cat. No. EL10018) demonstrates an increase in sensitivity (signal:noise) of 3-20 fold.
Identification of Potential Target Sites in Human HBV RNA
The sequence of human HBV was screened for accessible sites using a computer-folding algorithm. Regions of the RNA that did not form secondary folding structures and I 5 contained potential ribozyme and/or antisense binding/cleavage sites were identified. The sequences of these cleavage sites are shown in Tables 36-43.
Selection of Enzymatic Nucleic Acid Cleavage Sites in Human HBV RNA
Ribozyme target sites were chosen by analyzing sequences of Human HBV
!0 (accession number: AF100308.1) 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, 31 l, 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 !5 intramolecular interactions between the binding arms and the catalytic core were eliminated from consideration. As noted herein, 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.

Chemical Synthesis and Purification of Ribozymes and Antisense for Efficient Cleavage and/or blocking of HBV RNA
Ribozymes and antisense constructs were designed to anneal to various sites in the RNA message. The binding arms of the ribozymes are complementary to the target site sequences described above, while the antisense constructs are fully 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. Soc., 109, 7845), Scaringe et al., (1990 Nucleic Acids Res., 18, 5433) and Wincott et al., supra, and 0 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 typically >98%.
Ribozymes and antisense constructs were also synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods 5 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 incorporated herein by reference) and were resuspended in water. The sequences of the chemically synthesized ribozymes used in this study are shown below in Table 43.
'.0 Ribozyme Cleavage of HBV RNA Target in vitro 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.
Cleavage Reactions: Full-length or partially full-length, internally-labeled target RNA for ribozyme cleavage assay is prepared by in vitro transcription in the presence of [a-32p] CTP, passed over a G 50 Sephadex~ column by spin chromatography and used as substrate RNA without further purification. Alternately, 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 37°C using a final concentration of either 40 nM or 1 mM ribozyme, i.e., ribozyme excess.
5 The reaction is quenched by the addition of an equal volume of 95%
formamide, 20 mM
EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol after which the sample is heated to 95°C for 2 minutes, quick chilled and loaded onto a denaturing polyacrylamide gel. Substrate RNA and the specific RNA cleavage products generated by ribozyme cleavage are visualized on an autoradiograph of the gel. The percentage of cleavage is 0 determined by Phosphor Imagei~ quantitation of bands representing the intact substrate and the cleavage products.
Transfection of HepG2 Cells with psHBV-1 and Ribozymes The human hepatocellular carcinoma cell line Hep G2 was grown in Dulbecco's 5 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 transfection 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 '.0 to generate a replication competent cDNA). This was done with an EcoRI and Hind III
restriction digest. Following completion of the digest, a ligation was performed under dilute conditions (20 pg/ml) to favor intermolecular ligation. The total ligation mixture was then concentrated using Qiagen spin columns.
Secreted alkaline phosphatase (SEAP) was used to normalize the HBsAg levels to 'S control for transfection variability. The pSEAP2-TK control vector was constructed by ligating a Bgl II-Hind III fragment of the pRL-TK vector (Promega), containing the herpes simplex virus thymidine kinase promoter region, into Bgl IIlHind III digested pSEAP2-Basic (Clontech). Hep G2 cells were plated (3 x 104 cells/well) in 96-well microtiter plates and incubated overnight. A lipid/DNA/ribozyme complex was formed containing (at final concentrations) cationic lipid (15 pg/ml), prepared psHBV-1 (4.5 pg/ml), pSEAP2-TK
(0.5 pg/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-transfection for HBsAg and SEAP analysis.
Transfection of the human hepatocellular carcinoma cell line, Hep G2, with replication competent HBV DNA results in the expression of HBV proteins and the production of virions. To investigate the potential use of ribozymes for the treatment of chronic HBV infection, a series of ribozymes that target the 3' terminus of the HBV
genome have been synthesized. Ribozymes targeting this region have the potential to cleave all four major HBV RNA transcripts as well as the potential to block the production of HBV DNA by cleavage of the pregenomic RNA. To test the efficacy of these HBV
ribozymes, they 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. To control for variability in transfection efficiency, a control vector which expresses secreted alkaline phosphatase (SEAP), was also co-transfected. The efficacy of the HBV
ribozymes was determined by comparing the ratio of HBsAg:SEAP and/or HBeAg:SEAP
to that of a scrambled attenuated control (SAC) ribozyme. Twenty-five ribozymes (RPI18341, RPI18356, RPI18363, RPI18364, RPI18365, RPI18366, RPI18367, RPI18368, RPI18369, RPI18370, RPI18371, RPI18372, RPI18373, RPI18374, RPI18303, RPI18405, RPI18406, RPI18407, RPI18408, RPI18409, RPI18410, RPI18411, RPI18418, RPI18419, and RPI18422) have been identified which cause a reduction in the levels of HBsAg and/or HBeAg as compared to the corresponding SAC ribozyme.
Example 6: Analysis of HBsA~ and SEAP Levels Following Ribozyme Treatment Itnmulon 4 (Dynax) microtiter wells were coated overnight at 4° C with anti-HBsAg Mab (Biostride B88-95-3lad,ay) at 1 pg/ml in Carbonate Buffer (Na2C03 15 mM, NaHC03 35 mM, pH 9.5). The wells were then washed 4x with PBST (PBS, 0.05%
Tween~ 20) and blocked for 1 hr at 37° C with PBST, 1% BSA. Following washing as above, the wells were dried at 37° C for 30 min. Biotinylated goat ant-HBsAg (Accurate YVS1807) was diluted 1:1000 in PBST and incubated in the wells for 1 hr. at 37° C. The wells were washed 4x with PBST. Streptavidin/Alkaline Phosphatase Conjugate (Pierce 21324) was diluted to 250 ng/ml in PBST, and incubated in the wells for 1 hr.
at 37° C.
After washing as above, 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- exporter Assay The effect of ribozyme treatment on the level of transactivation of a SV40 promoter driven firefly luciferase gene by the HBV X-protein was analyzed in transfected Hep G2 cells. As a control 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 104 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 p,g/ml), the firefly reporter pSV40HCVluc (0.5 pg/ml), the Renilla luciferase control vector pRL-TK
(0.5 pg/ml), and ribozyme (100 pM). 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 transactivation of a firefly luciferase gene driven by the SV40 promoter in transfected Hep G2 cells. As a control for transfection 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 of the HBV ribozymes was determined by comparing the ratio of firefly luciferase: Renilla luciferase to that of a scrambled attenuated control (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 transactivation of a reporter gene by the X protein, as compared to the corresponding SAC ribozyme.
Example 8: HBV trans~enic mouse study A transgenic mouse strain (founder strain 1.3.32 with a C57B1/6 background) that expresses HBV RNA and forms HBV viremia (Money 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 of the 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 materials) 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, 1P). Baseline blood samples (200 p1) were obtained from each animal via a retro-orbital bleed. A 2 cm area near the base of the tail was shaved and cleansed with betadine 0 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 of the tail. Forceps were used to open a pocket rostrally (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 5 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. Animals were then deeply anesthetized with the ketamine/xylazine cocktail (150 mg/kg and 10 mg/kg, respectively; 0.5 ml, IP) on day 14 post pump implantation. A midline '.0 thoracotomy/ laparatomy was performed to expose the abdominal cavity and the thoracic cavity. The left ventricle was cannulated at the base and animals exsanguinated using a 23G needle and 1 ml syringe. Serum was separated, frozen and analyzed for HBV
DNA
and antigen levels. Experimental groups were compared to the saline control group in respect to percent change from day 0 to day 14. HBV DNA was assayed by quantitative 'S PCR
Results Table 44 is a summary of the group designation and dosage levels used in the HBV
transgenic mouse study. Baseline blood samples were obtained via a retroorbital bleed and .0 animals (N=10/group) received anti-HBV ribozymes (100 mg/kg/day) as a continuous SC
infusion. After 14 days, animals treated with a ribozyme targeting site 273 (RPL18341) of the HBV RNA showed a significant reduction in serum HBV DNA concentration, compared to the saline treated animals as measured by a quantitative PCR
assay. More specifically, the saline treated animals had a 69% increase in serum HBV DNA
concentrations over this 2-week period while treatment with the 273 ribozyme (RPL18341) resulted in a 60% decrease in serum HBV DNA concentrations.
Ribozymes directed against sites 1833 (RPI.18371), 1873 (RPI.18418), and 1874 (RPI.18372) decreased serum HBV DNA concentrations by 49%, 15% and 16%, respectively.
Example 7: Activity of NCH Ribozyme to inhibit HER2 e~ ne expression 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 of the 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).
5 Furthermore, overexpression of HER2 in malignant breast tumors has been correlated with increased metastasis, chemoresistance and poor survival rates (Slamon et al., 1987 Science 235: 177-182). Because 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).
!0 The greatest HER2 specific effects have been observed in cancer cell lines that express high levels of HER2 protein (as measured by ELISA). Specifically, in one study that treated five human breast cancer cell lines with the HER2 antibody (anti-erbB2-sFv), the greatest inhibition of cell growth was seen in three cell lines (MDA-MB-361, SKBR-3 and BT-474) that express high levels of HER2 protein. No inhibition of cell growth was !5 observed in two cell lines (MDA-MB-231 and MCF-7) that express low levels of HERZ
protein (Wright et al., 1997). Another group successfully used SKBR-3 cells to show HER2 antisense oligonucleotide-mediated inhibition of HER2 protein expression and HER2 RNA knockdown (Vaughn et al., 1995). Other groups have also demonstrated a decrease in the levels of HER2 protein, HERZ mRNA and/or cell proliferation in cultured .0 cells using anti-HER2 ribozymes or antisense molecules (Suzuki, T. et al., 1997; Weichen, et al., 1997; Czubayko, F. et al., 1997; Colomer, et al., 1994; Betram et al., 1994).
Because cell lines that express higher levels of HER2 have been more sensitive to anti-HER2 agents, we are pursuing several medium to high expressing cell lines, including SKBR-3 and T47D, for ribozyme screens in cell culture.
A variety of endpoints have been used in cell culture models to look at HER2-mediated effects after treatment with anti-HER2 agents. Phenotypic endpoints include 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) 0 after which either the cell viability, the incorporation of [3H] 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).
5 As a secondary, confirmatory endpoint a ribozyme-mediated decrease in the level of HER2 protein expression can be evaluated using a HER2-specific ELISA.
Validation of Cell Lines and Ribozyme Treatment Conditions Two human breast cancer cell lines (T47D and SKBR-3) that are known to express ;0 medium to high levels of HER2 protein, respectively, were considered for ribozyme screening. In order to validate these cell lines for HER2-mediated sensitivity, both cell lines were treated with the HER2 specific antibody, Herceptin~ (Genentech) and its effect on cell proliferation was determined. Herceptin was added to cells at concentrations ranging from 0-8 ~,M in medium containing either no serum (OptiMem), 0.1% or 0.5%
'5 FBS and efficacy was determined via cell proliferation. Maximal inhibition of proliferation (~50%) in both cell lines was observed after addition of Herceptin at 0.5 nM
in medium containing 0.1 % or no FBS. The fact that both cell lines are sensitive to an anti-HER2 agent (Herceptin) supports their use in experiments testing anti-ribozymes.
.0 Prior to ribozyme screening, the choice of the optimal lipids) and conditions for ribozyme delivery was determined empirically for each cell line. Applicant has established a panel of proprietary lipids that can be used to deliver ribozymes to cultured cells and are very usefizl for cell proliferation assays that are typically 3-5 days in length. Initially, this panel of proprietary lipid delivery vehicles was screened in SKBR-3 and T47D
cells using previously established control oligonucleotides. Specific lipids and conditions for optimal delivery were selected for each cell line based on these screens. These conditions were used to deliver HER2 specific ribozymes to cells for primary (inhibition of cell proliferation) and secondary (decrease in HER2 protein) efficacy endpoints.
Primar~Screen: Inhibition of Cell Proliferation Although optimal ribozyme delivery conditions were determined for two cell lines, I 0 the SKBR-3 cell line were be used for the initial screen because it has the higher level of HER2 protein, a.nd thus should be most susceptible to a HER2-specific ribozyme. Follow-up studies can be carned 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 S-day treatment period 5 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 Controls (SAC; or IA; 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 !0 determine non-specific inhibition of cell growth caused by ribozyme chemistry (i.e.
multiple 2' Q-Me modified nucleotides, a single 2'C-allyl uridine, 4 phosphorothioates and a 3' inverted abasic). Lead ribozymes are chosen from the primary screen based on their ability to inhibit cell proliferation in a specific manner. Dose response assays are carned out on these leads and a subset was advanced into a secondary screen using the !5 level of HER2 protein as an endpoint.
Secondary Screen: Decrease in HER2 Protein A secondary screen that measures the effect of anti-HER2 ribozymes on HER2 protein levels is used to support preliminary findings. A robust HER2 ELISA
for both .0 T47D and SKBR-3 cells has been established and is available for use as an additional endpoint.

Ribozvme Mechanism Assays 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 mRNA, thus leading to a decrease in cell surface HER2 protein receptors and a subsequent decrease in tumor cell proliferation.
0 Animal Models Evaluating the efficacy of anti-HER2 agents in animal models is an important prerequisite to human clinical trials. As in cell culture models, the most HER2 sensitive mouse tumor xenografts are those derived from human breast carcinoma cells that express high levels of HER2 protein. In a recent study, nude mice bearing BT-474 xenografts 5 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 treated in this manner (Baselga et al., 1998). This same study compared the efficacy of Herceptin alone or in combination with the commonly used chemotherapeutics, paclitaxel or doxorubicin. Although, all three anti-'.0 HER2 agents caused modest inhibition of tumor growth, the greatest antitumor activity was produced by the combination of Herceptin and paclitaxel (93% inhibition of tumor growth vs 35% with paclitaxel alone). The above studies provide proof that inhibition of HER2 expression by anti-HER2 agents causes inhibition of tumor growth in animals.
Lead anti-HER2 ribozymes chosen from in vitro assays are further tested in mouse 'S xenograft models. Ribozymes are first tested alone and then in combination with standard chemotherapies.
Animal Model Development 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 SC>I7 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 lines) 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 of the 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 treated with saline alone. Because the growth of 0 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. SO-100 mm3) in the presence or absence of ribozyme treatment.
Clinical Summary 5 Breast cancer is a common cancer in women and also occurs in men to a lesser degree. The incidence of breast cancer in the United States is 180,000 cases per year and 46,000 die each year of the disease. In addition, 21,000 new cases of ovarian cancer per year lead to 13,000 deaths (data from Hung et al., 1995 and the Surveillance, Epidemiology and End Results Program, NCI). Ovarian cancer is a potential secondary '0 indication for anti-HER2 ribozyme therapy.
A full review of breast cancer is given in the NCI PDQ for Breast Cancer. A
brief overview is given here. Breast cancer is evaluated or "staged" on the basis of tumor size, and whether it has spread to lymph nodes and/or other parts of the body. In Stage I breast cancer, the cancer is no larger than 2 centimeters and has not spread outside of the breast.
5 In Stage II, the patient's tumor is 2-5 centimeters but cancer may have spread to the axillary lymph nodes. By Stage III, metastasis to the lymph nodes is typical, and tumors are S centimeters. Additional tissue involvement (skin, chest wall, ribs, muscles etc.) may also be noted. Once cancer has spread to additional organs of the body, it is classed as Stage IV.
0 Almost all breast cancers (>90%) are detected at Stage I or II, but 31% of these are already lymph node positive. The S-year survival rate for node negative patients (with standard surgery/radiation/chemotherapy /hormone regimens) is 97%; however, involvement of the lymph nodes reduces the 5-year survival to only 77%.
Involvement of other organs ( Stage III) drastically reduces the overall survival, to 22% at 5 years. Thus, chance of recovery from breast cancer is highly dependent on early detection.
Because up to 10% of breast cancers are hereditary, those with a family history are considered to be at high risk for breast cancer and should be monitored very closely.
Breast cancer is highly treatable and often curable when detected in the early stages.
(For a complete review of breast cancer treatments, see the NCI PDQ for Breast Cancer.) Common therapies include surgery, radiation therapy, chemotherapy and hormonal therapy. Depending upon many factors, including the tumor size, lymph node involvement 0 and location of the lesion, surgical removal varies from lumpectomy (removal of the tumor and some surrounding tissue) to mastectomy (removal of the breast, lymph nodes and some or all of the underlying chest muscle). Even with successful surgical resection, as many as 21% of the patients may ultimately relapse (10-20 years). Thus, once local disease is controlled by surgery, adjuvant radiation treatments, chemotherapies and/or 5 hormonal therapies are typically used to reduce the rate of recurrence and improve survival. The therapy regimen employed depends not only on the stage of the 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 of the patient and if other organs are involved.
'.0 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 '.5 with certain of the combinations, e.g. doxorubin and paclitaxel, but are less common.
Testing for estrogen and progesterone receptors helps to determine whether certain anti-hormone therapies might be helpful in inhibiting tumor growth. If either or both receptors are present, therapies to interfere with the action of the hormone ligands, can be given in combination with chemotherapy and are generally continued for several years.
~0 These adjuvant therapies are called SERMs, selective estrogen receptor modulators, and they can give beneficial estrogen-like effects on bone and lipid metabolism while antagonizing estrogen in reproductive tissues. 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. However, 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 treat osteoporosis. In additional studies, removal of the ovaries and/or drugs to keep the ovaries from working are being tested.
Bone marrow transplantation is being studied in clinical trials for breast cancers that 0 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 "transplant" involves the exogenous treatment of peripheral blood stem cells with drugs to kill cancer cells prior to replacing the treated cells in the bloodstream.
5 One biological treatment, a humanized monoclonal anti-HER2 antibody, Herceptin (Genentech) has been approved by the FDA as an additional treatment for HER2 positive tumors. Herceptin binds with high affinity to the extracellular 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 0 Phase III studies, Herceptin significantly improved the response rate to chemotherapy as well as improving the time to progression (Ross & Fletcher, 1998). 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 (paclitaxel) can lead to cardiotoxicity (Sparano, 1999), 5 leukopenia, anemia, diarrhea, abdominal pain and infection.
HER2 Protein Levels for Patient Screenine and as a Potential Endpoint Because elevated 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 0 clinical trials testing an anti-HERZ ribozyme. Initial HER2 levels can be deterniined (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 of the anti-HER2 ribozyme treatment period could be useful in determining early indications of efficacy. In fact, the clinical course of Stage IV breast cancer was correlated with shed HER2 protein fragment following a dose-intensified paclitaxel monotherapy. In all responders, the HER2 serum level decreased below the detection limit (Luftner et al.).
Two cancer-associated antigens, CA27.29 and CA15.3, can also be measured in the serum. Both of these glycoproteins have been used as diagnostic markers for breast cancer. CA27.29 levels are higher than CA15.3 in breast cancer patients; the reverse is I 0 true in healthy individuals. Of these two markers, CA27.29 was found to better discriminate primary cancer from healthy subjects. In addition, 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). Moreover, both cancer antigens were found to be suitable for the detection of possible metastases during follow-up (Rodriguez I 5 de Paterna et al., 1999). Thus, blocking breast tumor growth may be reflected in lower CA27.29 and/or CA15.3 levels compared to a control group. FDA submissions for the use of 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.
!0 References Baselga, J., Norton, L. Albanell, J., Kim, Y.M. and Mendelsohn, J. (1998) Recombinant humanized anti-HER2 antibody (Herceptin) enhances the antitumor activity of paclitaxel and doxorubicin against HER2/neu overexpressing human breast cancer !5 xenografts. Cancer Res. 15: 2825-2831.
Berchuck, A. Kamel, A., Whitaker, R. et al. (1990) Overexpression of her-2/neu is associated with poor survival in advanced epithelial ovarian cancer. Cancer Research S0:
4087-4091.
Bertram, J. Killian, M., Brysch, W., Schlingensiepen, K.-H., and Kneba, M.
(1994) .0 Reduction of erbB2 gene product in mamma carcinoma cell lines by erbB2 mRNA-specific and tyrosine kinase consensus phosphorothioate antisense oligonucleotides.
Biochem. BioPhys. Res. Comm. 200: 661-667.

Beveridge, R.A. (1999) Review of clinical studies of CA27.29 in breast cancer management. Int. J. Biol. Markers 14: 36-39.
Colomer, R., Lupu, R., Bacus, S.S. and Gelmann, E.P. (1994) erbB-2 antisense oligonucloetides inhibit the proliferation of breast carcinoma cells with erbB-2 oncogene amplification. British J. Cancer 70: 819-825.
Czubayko, F., Downing, S.G., Hsieh, S.S., Goldstein, D.J., Lu P.Y., Trapnell, B.C.
and Wellstein, A. (1997) Adenovirus-mediated transduction of ribozymes abrogates HER-2/neu and pleiotrophin expression and inhibits tumor cell proliferation. Gene Ther. 4:
943-949.
0 Gion, M., Mione, R., Leon, A.E. and Dittadi, R. (1999) Comparison of the diagnostic accuracy of CA27.29 and CA15.3 in primary breast cancer. Clin.
Chem. 45:
630-637.
Hung, M.-C., Matin, A., Zhang, Y., Xing, X., Sorgi, F., Huang, L. and Yu, D.
(1995) HER-2/neu-targeting gene therapy - a review. Gene 159: 65-71.
5 Luftner, D., Schnabel. S. and Possinger, K. (1999) c-erbB-2 in serum of patients receiving fractionated paclitaxel chemotherapy. Int. J. Biol. Markers 14: 55-59.
McGuire, H.C. and Greene, M.I. (1989) The neu (c-erbB-2) oncogene. Semin.
Oncol. 16: 148-155.
NCI PDQ/Treatment/Health ProfessionalsBreast Cancer:
'0 http://cancernet.nci.nih.gov/clinpdq/soaBreast cancer Physician.html NCI PDQ/Treatrnent/PatientsBreast Cancer:
httn://cancernet.nci.nih. ovg /clinpdq/pif/Breast cancer Patient.html Pegram, M.D., Lipton, A., Hayes, D.F., Weber, B.L., Baselga, J.M., Tripathy, D., Baly, D., Baughman, S.A., Twaddell, T., Glaspy, J.A. and Slamon, D.J. (1998) Phase II
5 study of receptor-enhanced chemosensitivity using recombinant humanized anti-p185HER2/neu monoclonal antibody plus cisplatin in patients with HER2/neu-overexpressing metastatic breast cancer refractory to chemotherapy treatment.
J. Clin.
Oncol. 16: 2659-2671.
Rodriguez de Paterna, L., Arnaiz, F., Estenoz, J. Ortuno, B. and Lanzos E.
(1999) 0 Study of serum tumor markers CEA, CA15.3, CA27.29 as diagnostic parameters in patients with breast carcinoma. Int. J. Biol. Markers 10: 24-29.

Ross, J.S. and Fletcher, J.A. (1998) The HER-2/neu oncogene in breast cancer:
Prognostic factor, predictive factor and target for therapy. Oncologist 3:
1998.
Slamon, D.J., Clark, G.M., Wong, S.G., Levin, W.J., Ullrich, A. and McGuire, W.L.
(1987) Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 235: 177-182.
Sparano, J.A. (1999) Doxorubicin/taxane combinations: Cardiac toxicity and pharmacokinetics. Semin. Oncol. 26: 14-19.
Surveillance, Epidemiology and End Results Program (SEER) Cancer Statistics Review: http://www.seer.ims.nci.nih.gov/Publications/CSR1973_1996/
0 Suzuki T., Curcio, L.D., Tsai, J. and Kashani-Sabet M. (1997) Anti-c-erb-B-2 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 of the ERBB2 oncogene measured by 5 a flow cytometric assay. Proc Natl Acad Sci USA 92: 8338-8342.
Weichen, K., Zimmer, C. and Dietel, M. (1997) Selection of a high activity c-erbB-2 ribozyme using a fusion gene of c-erbB-2 and the enhanced green fluorescent protein.
Cancer Gene Therapy S: 45-51.
Wright, M., Grim, J., Deshane, J., Kim, M., Strong, T.V., Siegel, G.P., Curiel, D.T.
0 (1997) An intracellular anti-erbB-2 single-chain antibody is specifically cytotoxic to human breast carcinoma cells overexpressing erbB-2. Gene Therapy 4: 317-322.
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.

Proliferation assay:
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 treatment period. To calculate cell density for proliferation assays, the FIPS (fluoro-imaging 0 processing system) method well 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 pg/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. Of the 15 lead Rzs chosen from primary screens, 4 NCH and 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 I 0 scrambled control Rz (IA; RPI No. 17263). Of the five lead Rzs, the most efficacious is the NCH Rz (RPI No. 17251) against site 2939 of HER2 RNA. 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 5 capable of inhibiting HER2 gene expression in mammalian cells.
Example 8: Activity of Class II (Zinzvme) nucleic acid catalysts to inhibit HER2 gene e~ression Applicant has designed, synthesized and tested several class II (zinzyme) ribozymes !0 targeted against HER2 RNA (see, for example, Tables 58, 59, and 62) in cell proliferation RNA reduction assays.
Proliferation assay:
The model proliferation assay used in the study requires a cell-plating density of '.5 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). To calculate cell density for proliferation assays, the FIPS (fluoro-imaging processing system) method well known in the art was used. This method allows for cell density measurements after nucleic acids are stained ~0 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 pg/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 II
(zinzyme) ribozymes against sites, 314 (RPI No. 18653), 443 (RPI No. 18680), 597 (RPI
No. 18697), 659 (RPI No. 18682), 878 (RPI Nos. 18683 and 18654), 881 (RPI Nos.

and 18685) 934 (RPI No. 18651), 972 (RPI No. 18656, 19292, 19727, 19728, and 19293), 1292 (RPI No. 18726), 1541 (RPI No. 18687), 2116 (RPI No. 18729), 2932 (RPI
No.
18678), 2540 (RPI No. 18715), and 3504 (RPI No. 18710) caused inhibition of proliferation ranging from 25-80% as compared to a scrambled control ribozyme.
An 0 example of results from a cell culture assay is shown in Figure 20.
Referring to 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.
5 RNA assay:
RNA was harvested 24 hours post-treatment using the Qiagen RNeasy~ 96 procedure. 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 '.0 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 RNA targeting site 972 vs a scrambled attenuated control.
Dose response assays:
.5 Active ribozyme was mixed with binding arm-attenuated control (BAC) ribozyme to a final oligonucleotide concentration of either 100, 200 or 400 nM and delivered to cells in the presence of cationic lipid at 5.0 pg/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 II ribozyme targeting site 972 of HER2 RNA (RPI 19293).
Example 9: Compositions having RNA cleavin. activity Hammerhead ribozymes are an example of catalytic RNA molecules which are able to recognize and cleave a given specific RNA substrate (Hutchins et a1.,1986, Nucleic Acids Res. 14:3627; Keese and Symons, in Yiroids and viroid-like pathogens (J.J.
Semanchik, publ., CRC-Press, Boca Raton, Florida, 1987, pages 1-47). The catalytic 0 center of hammerhead ribozymes is flanked by three stems and can be formed by adjacent sequence regions of the 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, II and III. The nucleotides are numbered according to the standard nomenclature for hammerhead ribozymes (Hertel et al., 1992, 5 Nucleic Acids Res. 20:3252). In this nomenclature, bases are denoted by a number, which relates their position relative to the S' side of the cleavage site.
Furthermore, 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'S'' would indicate that this base is involved in a paired 0 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 of the enzyme to always have the same number relative to all of the other nucleotides. The size of the stems involved in substrate binding or core formation can be any size and of any sequence, and the position of A9, for example, will 5 remain the same relative to all of the other core nucleotides. Nucleotides designated, for example, N~'2 or N9~ represent an inserted nucleotide where the position of the caret (~) relative to the number denotes whether the insertion is before or after the indicated nucleotide. Thus, N~' 2 represents a nucleotide inserted before nucleotide position 12, and N9~ represents a nucleotide inserted after nucleotide position 9.
0 The consensus sequence of the catalytic core structure is described by Ruffner and LThlenbeck, 1990, Nucleic Acids Res. 18:6025-6029. Perriman et al., 1992, Gene 113:157-163, have meanwhile shown that this structure can also contain variations, for example, naturally occurring nucleotide insertions such as N9~ and N~'2. Thus, the positive strand of the satellite RNA of the tobacco ring-spot virus does not contain any of the two nucleotide insertions while the +RNA strand of the virusoid of the lucerne transient streak virus (vLTSV) contains a N9~ = U insertion which can be mutated to C or G
without loss of activity (Sheldon and Symons, 1989, Nucleic Acids Res. 17:5679-5685).
Furthermore, in this special case, N' = A and R's.' = A. On the other hand, the minus strand of the carnation stunt associated viroid (-CarSV) is quite unusual since it contains both nucleotide insertions, that is N~'2 = A and N9~ = C (Hernandez et a1.,1992, Nucleic Acids Res. 20:6323-6329). In this viroid N' = A and R's'' = A. In addition, this special 0 hammerhead structure exhibits a very effective self catalytic cleavage despite the more open central stem.
Possible uses of hammerhead ribozymes include, for example, generation of RNA
restriction enzymes and the specific inactivation of the expression of genes in, for example, animal, human or plant cells and prokaryotes, yeasts and plasmodia. A
particular 5 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 control and eventually treat such diseases. Moreover there is a great need to develop antiviral, antibacterial, and antifungal pharmaceutical agents. Ribozymes have potential as such anti-infective agents '.0 since RNA molecules vital to the survival of the organism can be selectively destroyed.
In addition to needing the correct hybridizing sequences for substrate binding, substrates for hammerhead ribozymes have been shown to strongly prefer the triplet Ni6.zUi6.iHm (~) where N can be any nucleotide, U is uridine, and H is either adenosine, cytidine, or uridine (Koizumi et al., 1988, FEBS Lett. 228, 228-230; Ruffner et '.5 al., 1990, Biochemistry 29, 10695-10702 ; Pernman et al., 1992, Gene 113, 157-163).
NUH is sometimes designated as NUX. The fact that changes to this general rule for substrate specificity result in non-functional substrates implies that there are "non core compatible" structures which are formed when substrates are provided which deviate from the stated requirements. Evidence along these lines was recently reported by Uhlenbeck ~0 and co-workers (Uhlenbeck et al., 1997, Biochemistry 36:1108-1114) when they demonstrated that the substitution of a G at position 17 caused a functionally catastrophic base pair between G" and C3 to form, both preventing the correct orientation of the scissile bond for cleavage and the needed tertiary interactions of C3 (Murray et al., 1995, Biochem. J. 311:487-494). The strong preference for a U at position 16.1 may exist for similar reasons. Many experiments have been done in an attempt to isolate ribozymes which are able to efficiently relieve the requirement of a U at position 16.1, however, attempts to find hammerhead type ribozymes which can cleave substrates having a base other than a U at position 16.1 have proven impossible (Perriman et al., 1992, Gene 113, 157-163).
Efficient catalytic molecules with reduced or altered requirements in the cleavage region are highly desirable because their isolation would greatly increase the number of I 0 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 Nl6.zUi6.iHm since this would increase the number of potential target cleavage sites.
Chemically modified oligonucleotides which contain a block of 5 deoxyribonucleotides in the middle region of the molecule have potential as pharmaceutical agents for the specific inactivation of the 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 of the RNA and so cannot be !0 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.
Since, unmodified ribozymes are sensitive to degradation by RNases, chemically !5 modified active substances have to be used in order to administer hammerhead ribozymes exogenously (discussed, for example, by Heidenreich et al., 1994, J. Biol.
Chem.
269:2131-2138; Kiehntopf et al., 1994, EMBO J. 13:4645-4652; Paolella et al., 1992, EMBO J. 11:1913-1919; and Usman et al., 1994, Nucleic Acids Symp. Ser. 31:163-164).
Sproat et al., U.S. Pat. No. 5,334,711, describe such chemically modified active .0 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 of the ribose. These oligonucleotides contain modified nucleotide building blocks and form a structure resembling a hammerhead structure. These oligonucleotides are able to cleave specific RNA substrates.
Usman et al., U.S. Patent No. 5,891,684, describe enzymatic nucleic acid molecules with one or more nucleotide base modifications) in a substrate binding arm.
Thompson et al., US Patent No. 5,599,704 describe enzymatic RNA molecules targeted against ErbB2/neu/Her2 RNA.
Sullivan et al., US Patent No. 5,616,490 describe enzymatic RNA molecules targeted against protein kinase C (PKC) RNA.
0 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.
5 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 substrate. Preferred compositions form, in 0 combination with an RNA substrate, a structure resembling a hammerhead structure. The active center of the disclosed compositions is characterized by the presence of h5-1 which allows cleavage of RNA substrates having 016.1. 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.
5 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 of the instant invention are distinct from other nucleic acid catalysts known in the art. Specifically, nucleic acid catalysts of the instant invention are capable of catalyzing an intermolecular or intramolecular endonuclease reaction.
0 It is another object of the present invention to provide compositions that cleave RNA
substrates having a cleavage site triplet other than N'6.2U16.1Hm (~~ Figure 6), where N
is a nucleotide, U is uridine and H is adenosine, uridine or cytidine. H is used interchangably with X. Specifically, the enzymatic nucleic acid molecule of the instant invention has an endonuclease activity to cleave RNA substrates having a cleavage triplet NI6.2C16.1H17 (NCH; Figure 6),. where N is a nucleotide, C is cytidine and H
is adenosine, uridine or cytidine. H is used interchangeably with X. In another aspect the invention features an enzymatic nucleic acid molecule of the instant invention has an endonuclease activity to cleave RNA substrates having a cleavage triplet N16.2C16.1N17 (NCN; Figure 6), where N is a nucleotide, C is cytidine.
In a preferred embodiment, the invention features an enzymatic nucleic acid molecule having formula 1:
- G-A-A-I
L
\~)n-~)p A-G-N-A -G-U-C-E-5' where 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 5 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, 1 S, 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 (I~o and (l~n are nucleotides, (I~o and (l~n are optionally able to interact by hydrogen bond interaction, in particular if n =1 !0 and o=1 then (N)n is preferably a purine (e.g., G, and A) and (I~o is preferably a pyrimidine (e.g., C and U) and (I~n preferably forms; ~ indicates base-paired interaction;
L is a linker which may be present or absent (i.e., the molecule may be assembled from two separate oligonucleotides), but when present, is a nucleotide and/or a non-nucleotide linker, which may be a single-stranded and/or double-stranded region; p is an integer 0 or !5 1, when p=l, (I~p is preferably A or U; and represents a chemical linkage (e.g. a phosphate ester linkage, amide linkage, phosphorothioate linkage or others known in the art). A, U, I, C and G represent adenosine, uridine, inosine, cytidine and guanosine nucleotides, respectively. The N in S'-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.
In a preferred embodiment, the invention features an enzymatic nucleic acid molecule having formula 2:
/ C G_ A_ A_ I D
L
~ CN) n G CN) p _a- G_ N_ A_ G- U- C _ E _ 5' where 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 0 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 (I~o and (I~n are nucleotides, (loo 5 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., the molecule may be assembled from two separate oligonucleotides), but when present, is a nucleotide and/or a non-nucleotide linker, which may be a single-stranded and/or double-stranded region; p is an integer 0 or 1, when p=1, (I~p is preferably A, C or U; and represents a 0 chemical linkage (e.g. a phosphate ester linkage, amide linkage, phosphorothioate linkage or others known in the art). 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.
5 In a preferred embodiment, the I (inosine) in formula l and 2 is preferably a ribo-inosine or a xylo-inosine.

In yet another embodiment, 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). 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. In a preferred embodiment L
0 has the sequence 5'-GAAA-3' or 5'-GUUA-3'.
In yet another embodiment, the non-nucleotide linker (L) is as defined herein.
The term "non-nucleotide", as used herein, includes either abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, or polyhydrocarbon compounds. Specific examples include those described by Seela and Kaiser, Nucleic 5 Acids Res. 1990, 18:6353 and Nucleic Acids Res. 1987, 15:3113; Cload and Schepartz, J.
Am. Chem. Soc. 1991, 113:6324; Richardson and Schepartz, J. Am. Chem. Soc.
1991, 113:5109; Ma et al., Nucleic Acids Res. 1993, 21:2585 and Biochemistry 1993, 32:1751;
Durand et al., Nucleic Acids Res. 1990,18:6353; McCurdy et al., Nucleosides &
Nucleotides 1991,10:287; Jschke et al., Tetrahedron Lett. 1993, 34:301; Ono et al., '0 Biochemistry 1991, 30:9914; Arnold et al., International Publication No. WO
89/02439;
Usman et al., International Publication No. WO 95/06731; Dudycz et al., International Publication No. WO 95/11910 and Ferentz and Verdine, J. Am. Chem. Soc. 1991, 113:4000, all hereby incorporated by reference herein. Non-nucleotide linkers can be any molecule, which is not an oligomeric sequence, that can be covalently coupled to an 5 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. Anu.
Chem. Soc. 115:8483-8484 (1993)) and Fu et al. , (J. Am. Chem. Soc. 116:4591-(1994)). Preferred non-nucleotide linkers, or subunits for non-nucleotide linkers, include 0 substituted or unsubstituted C1-Clo straight chain or branched alkyl, substituted or unsubstituted CZ-Clo straight chain or branched alkenyl, substituted or unsubstituted Cz-Clo straight chain or branched alkynyl, substituted or unsubstituted C1-Clo straight chain or branched alkoxy, substituted or unsubstituted CZ-Clo straight chain or branched alkenyloxy, and substituted or unsubstituted Cz-Clo straight chain or branched alkynyloxy.
The substituents for these preferred non-nucleotide linkers (or subunits) can be halogen, cyano, amino, carboxy, ester, ether, carboxamide, hydroxy, or mercapto. Thus, in a preferred embodiment, 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. By the term "non-nucleotide" is meant any group or compound which can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to 0 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. The terms "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.
5 In a preferred embodiment, the invention features modified ribozymes with phosphate backbone modifications comprising one or more phosphorothioate, phosphorodithioate, methylphosphonate, morpholino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl, substitutions. For a review of oligonucleotide backbone modifications '.0 see Hunziker and Leumann, 1995, Nucleic Acid Analogues: Synthesis and Properties, in Modern Synthetic Methods, VCH, 331-417, and Mesmaeker et al., 1994, Novel Backbone Replacements for Oligonucleotides, in Carbohydrate Modifications in Antisense Research, ACS, 24-39.
In a further preferred embodiment of the instant invention, an inverted deoxy abasic .5 moiety is utilized at the 3' end of the enzymatic nucleic acid molecule.
By "pyrimidines" is meant nucleotides comprising modified or unmodified derivatives of a six membered pyrimidine ring. An example of a pyrimidine is modified or unmodified uridine.
In a preferred embodiment, the nucleosides of the 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
histidyl) amino uridine; 2'-deoxy-2'-amino-5-methyl cytidine; 2'-(N ~i-carboxamidine-beta-alanyl)amino-2'-deoxy-uridine; 2'-deoxy-2'-(N-beta-alanyl)-guanosine; 2'-O-amino-adenosine; 2'-(N lysyl)amino -2'-deoxy-cytidine; 2'-Deoxy -2'-(L-histidine) amino Cytidine; and 5-Imidazoleacetic acid 2'-deoxy-5'-triphosphate uridine.
By "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 0 combinations thereof.
In a preferred embodiment, the enzymatic nucleic acid molecule of formula 1 or include at least three ribonucleotide residues, preferably 4, 5, 6, 7, 8, 9, and 10 ribonucleotide residues.
In preferred embodiments, the enzymatic nucleic acid of the instant invention 5 includes one or more stretches of RNA, which provide the enzymatic activity of the molecule, linked to the non-nucleotide moiety. The necessary RNA components are known in the art (see for e.g., Usman et al., supra).
Thus, in one preferred embodiment, the invention features enzymatic nucleic acid molecules that inhibit gene expression and/or cell proliferation in vitro or in vivo (e.g. in 0 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. Upon binding, the enzymatic nucleic acid molecules cleave the target molecules, preventing for example, translation and protein accumulation. In the absence of the 5 expression of the target gene, cell proliferation, for example, is inhibited.
In another preferred embodiment, catalytic activity of the 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 of the ribozyme binding arms, or chemically synthesizing ribozymes with modifications (base, sugar and/or phosphate) that 0 prevent their degradation by serum ribonucleases and/or enhance their enzymatic activity (see e.g., Eckstein et al., International Publication No. WO 92/07065;
Perrault et al., 1990 Nature 344, 565; Pieken et al., 1991 Science 253, 314; Usman and Cedergren, Trends in Biochem. Sci. 17, 334; Usman et al., International Publication No.
WO 93/15187; and Rossi et al., International Publication No. WO 91/03162;
Sproat, US
Patent No. 5,334,711; and Burgin et al., supra; all of these describe various chemical modifications that can be made to the base, phosphate and/or sugar moieties of enzymatic RNA molecules). Modifications which enhance their efficacy in cells, and removal of bases from stem loop structures to shorten RNA synthesis times and reduce chemical requirements are desired. (All these publications are hereby incorporated by reference herein.).
By "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. Such a molecule with endonuclease activity may have complementarity in a substrate binding region to a specified gene target, and also has an enzymatic activity that specifically cleaves RNA or DNA in that target. That is, the nucleic acid molecule with endonuclease activity is able to intramolecularly or intermolecularly cleave RNA or DNA and thereby inactivate a target RNA or DNA molecule. This complementarity functions to allow sufficient hybridization of the enzymatic RNA molecule to the target RNA or DNA to allow the cleavage to occur. 100% complementarity is preferred, but complementarity as low as 50-75% may also be useful in this invention. The nucleic acids may be modified at the base, sugar, and/or phosphate groups. The term enzymatic nucleic acid 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 of the instant invention with enzymatic activity. The specific examples of enzymatic nucleic acid molecules described in the instant application are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target nucleic acid regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart a nucleic acid cleaving activity to the molecule (Cech et al., U.S. Patent No. 4,987,071; Cech et al., 1988, 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.
By "chimeric nucleic acid molecule" or "mixed polymer" is meant that, the molecule may be comprised of both modified or unmodified nucleotides.
In yet another preferred embodiment, the 3'-cap is selected from a group comprising, 4',5'-methylene nucleotide; I-(beta-D-erythrofuranosyl) nucleotide; 4'-thio nucleotide, carbocyclic nucleotide; 5'-amino-alkyl phosphate; 1,3-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; threo-pentofuranosyl nucleotide;
acyclic 3',4'-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, S'-5'-inverted nucleotide moiety; 5'-5'-inverted abasic moiety; 5'-phosphoramidate;
5'-phosphorothioate; 1,4-butanediol phosphate; S'-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; incorporated by reference herein). By the term "non-nucleotide" is meant any group or compound which can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity. The group or compound is abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine. The terms "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.
In a preferred embodiment, the invention features 1-(beta-D-xylofuranosyl)-xypoxanthine phosphoramidite and a process for the synthesis thereof and incorporation into oligonucleotides, such as enzymatic nucleic acid molecule.
In yet another preferred embodiment, the invention features enzymatic nucleic acid molecules targeted against HERZ RNA, specifically, ribozymes in the hammerhead and NCH motifs.
In a preferred embodiment, 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.. 5,525,468 and 5,646,042, all are hereby incorporated by reference herein in their totality. Other examples include the following PCT
applications, which concern inactivation of expression of disease-related genes: WO
95/23225, WO 95/13380, WO 94/02595, all incorporated by reference herein.
The specific enzymatic nucleic acid molecules described in the instant application are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific 0 substrate binding site (e.g., D and E of Formula 1 above) which is complementary to one or more of the target nucleic acid regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart a nucleic acid cleaving activity to the molecule.
All naturally occurring hammerhead ribozymes have an A'SV-Ui6.i base pair. In 5 addition, it is known that substrates for ribozymes based on the consensus hammerhead sequence strongly prefer a substrate that contains an N'6.zUi6.iHm triplet in which H" is not a guanosine (Koizumi et al., FEBS Lett. 228, 228-230 (1988); Ruffner et al., Biochemistry 29, 10695-10702 (1990); Pernman et al., Gene 113, 157-163 (1992)). Many experiments have been done in an attempt to isolate ribozymes which are able to .0 efficiently relieve the requirement of a U at position 16.1, however, attempts to fmd ribozymes which can cleave substrates having a base other than a U at position 16.1 have proven largely unsuccessful (Perriman et al., Gene 113, 157-163 1992, Singh et al., Antisense and Nucleic Acid Drug Development 6:165-168 (1996)).
However, examination of the recently published X-ray crystal structures (Pley et al., 5 Nature 372:68-74 (1994), Scott et al., Cell 81:991-1002 (1995), and Scott et al., Science 274:2065-2069 (1996)) led to the realization that the A'S.'-U16.1 interaction is a non standard base pair with a single hydrogen bond between the exocyclic amine (N6) of the adenosine and the 4-oxo group of the uridine. Modeling studies (based on the crystal structure) then led to the discovery that the interaction of the wild-type A'S.i-Ui6.i base pair 0 can be spatially mimicked by replacement with an I'S''-C'6.' base pair that adopts an isostructural orientation and which preserves the required contact of the 2-keto group of C'6'' with A6 of the uridine turn. In the model, the polarity of the stabilizing hydrogen bond between positions 15.1 and 16.1 is reversed in the hs.i-Cib.i interaction, but the correct orientation of the bases around this bond is maintained.
It has been discovered that hammerhead ribozyme analogues containing an inosine at position 15.1 readily cleave RNA substrates containing an N'6.zC16.iH1~
triplet. Based on this, disclosed are compositions, preferably synthetic oligomers, which cleave a nucleic acid target sequence containing the triplet N16.2Ci6.iHp It is preferred that Hl' is not guanosine, however, under certain circumstances, NCG triplet containing RNA
can be cleaved by the ribozymes of the instant invention. The ability to cleave substrates having N16.2C16.1x17 mplets effectively doubles the number of targets available for cleavage by 0 compositions of the type disclosed.
Example 10: Synthesis of 1-(beta-D-xylofuranosyl)-xypoxanthine phosphoramidite Referring to Figure 9, Inosine (1) was 5'-O-monomethoxytritylated and 2'-O-silylated under standard conditions to afford 2 (Charubala, R; Pfleiderer, W.
Heterocycles 5 1990, 30, 1141). Oxidation/reduction procedure afforded 3 in moderate yield (Matulic-Adamic, J.; Daniher, A.T.; Gonzalez, C.; Beigelman, L. Bioorg. Med. Chem.
Lett.. 1999, 9, 157): 1H NMR (CDCl3) 8 12.80 (br s, 1H, NH), 8.11 (s, 1H, H-8), 8.08 (s, 1H, H-2), 7.45-6.80 (m, 14H, trityl), 5.85 (d, J~.~2~= 1.6, 1H, H-1'), 4.83 (d, J2y3~=7.2, 1H, H-2'), 4.46 (br s, 1H, 3'-OH), 4.34 (m, 1H, H-4'), 4.06 (m, 1H, H-3'), 3.77 (s, 6H, 2 x OMe), 0 3.60 (app d, 2H, H-5', H-5"), 0.89 (s, 9H, t-Bu), 0.07 (s, 3H, Me), 0.06 (s, 3H, Me).
Standard phosphitylation of 3 afforded the desired phosphoramidite 4.
More acid stable 5'-O-MMT group is used in this particular case because applicant found that 5'-O-DMT protection is more labile in xylo nucleoside series than in ribo nucleoside series.
5 The xylo-inosine was incorporated into oligonucleotides using the standard procedures known in the art and as described herein.
Example 11: Activity of the xylo-Inosine-modified NCH Ribozyme Several NCH ribozymes with xylo-inosine at position 15.1 were designed (Figure 7) 0 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).
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 purified by gel electrophoresis using general methods or were I 0 purified by high pressure liquid chromatography (HPLC; See Wincott et al., supra; the totality of which is hereby incorporated herein by reference) and were resuspended in water. The sequences of the chemically synthesized ribozymes used in this study are shown below in Table 33.
Cleavage Reactions: Full-length or partially full-length, internally-labeled target 5 RNA for ribozyme cleavage assay is prepared by in vitro transcription in the presence of [alpha-32p] CTP, passed over a G 50 Sephadex column by spin chromatography and used as substrate RNA without further purification. Alternately, 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
!0 7.5 at 37°C, 10 mM MgCl2) and the cleavage reaction was initiated by adding the 2X
ribozyme mix to an equal volume of substrate RNA (maximum of 1-5 nM) that was also pre-warmed in cleavage buffer. As an initial screen, assays are carried out for 1 hour at 37°C using a final concentration of 40 nM or 1 mM ribozyine, i.e., ribozyme excess. The reaction is quenched by the addition of an equal volume of 95% formamide, 20 mM
!5 EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol after which the sample is heated to 95°C for 2 minutes, quick chilled and loaded onto a denaturing polyacrylamide gel. Substrate RNA and the specific RNA cleavage products generated by ribozyme cleavage are visualized on an autoradiograph of the gel. The percentage of cleavage is determined by Phosphor Imager~ quantitation of bands representing the intact substrate .0 and the cleavage products.

The results of the experiments are summarized in Table 32, which shows that NCH-xylo ribozymes are catalytically active to cleave target RNA.
Example 12: Activity of NCH Ribozyme variants The nucleic acid molecules of the 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. Additional measurements of multiple turnover parameters of the all 0 ribo ribozymes performed under non-saturating conditions using SnM ribozyme and changing the substrate concentration from 50 to 500 nM at pH 7.4 with 10 mM Mg ++ at 37 °C gave Km= 100 nM and kcat=6.5 min -~ for GCA vs Km =30 nM and kcat =2.0 min -1 for GUA cleaving all ribo ribozymes. These data verify that the ribozymes with an I~C
base pair are efficient catalysts in multiple turnover reactions and the relative order of 5 activity between NCH and NUH cleavers established at pH 6 (Ludwig et al., 1998, Nucleic Acids Res., 26, 2279-2285) remains unchanged.
To gain more insight into the structural requirements of the 15.1- 16.1 base pair of the ribozymes of the instant invention, applicant synthesized several variants of the active I-15.1 ~C-16.1 structure and tested these ribozyme analogues with their corresponding .0 substrates. The influence of several core stabilization strategies on the activity of the NCH
cleaving ribozymes was also investigated.
Various nucleoside analogs were incorporated at position 1 S.1 of the 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 5 conditions). The modified oligonucleotides were synthesized by standard oligonucleotide synthesis procedures. Xanthosine was protected using O-2 ,O-4 pivaloyloxymethyl groups;
N,N-dimethylguanosine with 6-O-( 2-nitrophenyl-)ethyl and 6-thio-inosine with S-cyanoethyl protecting groups. The cleavage activity of the ribozymes containing the 15.1 analogs is summarized in Figure 36. For comparison Figure 37 summarizes reported 0 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 N1 and/or C6 positions (Figure 36 A, B, C) In the 6-thio-inosine (A) (s1) 15.1 substituted ribozyme, the original (I-15.1) position 6 O~H-N (C-16.1) bonds are replaced by weaker (sI-15.1) position 6 S~H-N (C-16.1) hydrogen bonds while all other functional groups remain unchanged. Ribozymes with an adenosine (B) at position 15.1 (A-15.1) are inactive with C-16.1 substrates since the ribozyme geometry requires the [A-1 S.1 ] position 6 amino group and the [C-16.1 ] position 4 amino group hydrogen-bond donor functional groups to be in close proximity.
Similarly, low activity is observed with I-15.1 ribozymes and U-16.1 substrates, where the [I-15.1]
position 6 keto and [C-16.1] position 4 keto hydrogen-bond acceptor groups are opposed (Figure 37, B). Although inosine can form stable mismatch pairs with uridine in RNA
duplexes or in tRNA anticodon-mRNA interactions, these results suggest that the geometry in the I~U mismatches differ from that of the A~U (or I~C) base pair in the active NUH ribozyme. Substitution of N1-Methyl-inosine (C) in place of inosine at position 15.1 leads to complete loss of cleavage activity.
Modifications at the purine 1 S.1 C2 and/or N3 position (Figure 36 D, E, F) The extremely low activity observed with the G-15.1 (D) substituted analog may be explained by the formation of a G-C Watson-Crick base pair. The replacement of the I~C
pair with a G~C pair can significantly distort the geometry at the 15.1-16.1 position. G-15.1 N2-alkylation (E) gives only minimal recovery of catalytic activity compared to G-15. l, suggesting that the steric problems introduced by the bulky N-methyl groups may interfere with stacking interactions. The activity of this construct is significantly less than that of iso-G-15.1 (Figure 37, E) containing ribozymes in the standard A-U
context.
Xanthosine 15.1 (F) contains the same functional groups as inosine at the N1 and C6 sites but contains an additional hydrogen-bond donor site at position N3 along with a C2 carbonyl group. The complete lack of activity seen with this construct reinforces the importance of the purine N3 acceptor functionality in transition state formation. Similarly, 3-deaza-adenosine (Figure 37, F) containing ribozymes were also inactive. The carbonyl of the 15.1 purine shows no significant negative interference in iso-guanosine containing 15.1 ribozymes.

Activity of modified core variants To complete the characterization of the I~C pair containing ribozymes, the acceptance of various core substitution patterns was tested. Short substrates containing GCH and GUH (H= non G) triplets were compared using 3 different modified ribozymes.
The acceptance of the U-4 2'-O-alkyl substituent is the greatest with GCA
triplets while U-4= 2'-deoxy-2'-amino uridine and U-4= ribo uridine substituted ribozymes show a similar level of activity with NCH and NUH triplets. The results of this comparison are summarized in Table 64. In addition, a ribozyme construct in which ribo inosine replaces adenosine at positions 14 and 15.1 was tested which demonstrated cleavage activity.
Apart from the A-15.1 ~U-16.1 to I-15 .1 ~C-16.1 change that reverses the polarity of an important H-bond in the ribozyme structure, no other functional group changes at the 15.1 purine residue seem to be compatible with the requirements of efficient catalysis. The I-15.1 and A-15.1 ribozymes are equally suitable for practical applications because there are only minor differences in the acceptance of stabilizing residues.
Example 13 : Activity of NCH Ribozyme to inhibit HERZ eg ne 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.
Proliferation assay: 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 S-day treatment period. To calculate cell density for proliferation assays, the FIPS (fluoro-imaging processing system) method well 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 ~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. Of the 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 control Rz (IA; RPI No. 17263). Of the five lead Rzs, the most efficacious is the NCH Rz (RPI No. 17251) against site 2939 of HER2 RNA. An example of results from cell culture assay is shown in Figure 3. Referring to 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.

Example 14: Activity of NCH Ribozyme to inhibit PKC alpha ene expression The Protein Kinase C family contains twelve currently known isozymes divided into three classes: the classic, Cap dependent (PKCa, ~iI, ~3II, y), the novel, non-Cap dependent (PKCB, E, p, r), 0) and the atypical (PKC ~, i/~,); all of which are 5 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). These second messengers include diacylglyceral (DAG), inositol-triphosphate (IP3), lysophospholipids, free fatty acids, and phosphatidate '.0 which act directly or in addition to changes in the Cap concentration. A
simple model for PKCa activation follows a two step mechanism. First, membrane association of PKCa is through Cap and phospholipid interactions and second, the kinase is activated by interaction with DAG. An example of a signal cascade subsequent to PKC
activation is PKC's phosphorylation of c-Raf, which phosphorylates MEK, which phosphorylates '5 MAP, which phosphorylates transcription factors such as Jun and thereby activates a mitogenic program in the nucleus. There are numerous substrates for the various PKC's, one which for PKCa ultimately stimulates transcription 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).

Cell Culture Review 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 demonstrated. 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, 0 Surgery, 124, 218-223), and human gastric cancer (Dean et al., 1996, Cancer Research, 56, 3499-3507). Mounting evidence suggests PKCa can stimulate adhesion molecule expression and can directly act on these membrane bound species as substrates, thereby modulating cellular adhesion to the extracellular matrix and increasing metastic potential.
Furthermore, human surgical specimens have demonstrated elevated PKC in breast 5 tumors, thyroid carcinomas and melanomas (Becker et al., 1990, Oncogene, 5, 1139).
Utz et al., 1994, Int. J. Cancer, 57, 104-110, describe a cell proliferation assay in which small molecule inhibitors of PKC demonstrate anti-proliferative activity in CCRF-VCR 1000 and KB-8511 cells with the multidrug resistant (MDR) phenotype. PKCa is .0 overexpressed in tumor tissues that express the MDR phenotype. This phenotype is associated with the expression of a 170 kDa broad specificity drug efflux pump, P-gp.
PKCa phosphorylation of P-gp has been shown in vitro. In addition, PKC
expression correlates 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
5 phenotype and decrease phosphorylation of P-gp (Caponigro et al., 1997, Anti-Cancer Drugs, 8, 26-33).
Dean et al., 1994, Journal of Biological Chemistry, 269, 16416-24, describe cell culture studies in which antisense targeting of PKC a resulted in the potent inhibition of mRNA and protein expression in human lung carcinoma (A549) cells. In this study, PKC
0 a inhibition resulted in the reduced induction of intercellular adhesion molecule 1 (ICAM-1) mRNA by phorbol esters.

Yano et al., 1999, Endocrinology, 140, 4622-4632, describe a cell proliferation study in which down regulation of different PKC isoforms, including PKCa, results in the inhibition of insulin like growth factor I induced vascular smooth muscle cell proliferation, migration, and gene expression.
Wang et al., 1999, Experimental Cell Research, 250, 253-263, describe cell culture studies in which antisense inhibition of PKCa results in the reversal of the transformed phenotype in human lung carcinoma (LTEPa-2) cells. In this study, the amounts of PKCa protein and total PKC activity were decreased when compared to control cells.
Sioud and Sorensen, 1998, Nature Biotechnology, 16, 556-561, describe 0 hammerhead ribozyme inhibition of PKCa in rat glioma cell lines (BT4C and BT4Cn).
This study demonstrated inhibition of malignant glioma cell proliferation along with the inhibition of regulatory Bcl-xL protein expression. Bcl-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 5 suppression of PKCa and Bcl-xL (Leirdal and Sioud, 1999, British J. of Cancer, 80, 1558-1564).
Animal Models Evaluating the efficacy of anti-PKCa agents in animal models is an important prerequisite to human clinical trials. A variety of mouse xenograft models using human :0 tumor cell lines have been developed using cell lines which express high levels of PKCa protein. McGraw et al, 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. Antisense oligonucleotides targeting PKCa administered 5 intravenously following s.c. transplanted tumor cells resulted in dose dependant decreases in tumor size when compared to controls in most cases. Similar studies using T-bladder carcinoma, non-small cell lung carcinoma (A549), and Colo 205 colon carcinoma mouse xenografts are described in Dean et al, 1996, Biochemical Society Transactions, 24, 623. Sioud and Sorensen, 1998, Nature Biotechnology, 16, 556-561, describe a rat model in which inbred syngeneic BDIX rats were inoculated subcutaneously with BT4Cn glioma cells. After approximately three weeks, rats were treated with a single injection of ribozyme targeting PKCa resulting in inhibition of tumor growth as determined by tumor size and/or weight when compared to controls. The above studies provide proof that inhibition of PKCa expression by anti-PKCa agents causes inhibition of tumor growth in animals. Lead anti-PKCa 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 SC1D 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 PKCa can also be used. The tumor cell lines) and implantation method that supports the most consistent and reliable tumor growth can be used in animal studies to test promising PKCa 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 of the 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 treated 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 mm3) in the presence or absence of ribozyme treatment.
Clinical Summary Overview Ribozymes targeting PKCa 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. Approximately 158,000 die each year of lung cancer, accounting for 28% of all cancer deaths.
Numerous other indications exist including cancers of the bladder, colon, breast, prostate, and ovary in addition to melanoma and glioblastoma.
McGraw et al., 1997, Anti-Cancer Drug Design, 12, 315-326, describe a Phase I
trial for ISIS 3521/CGP 64128A, a PKC alpha antisense construct. In this trial, ISIS

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. In patients receiving the two-hour i.v.
infusion schedule, the post-infusion plasma concentration of the compound increased proportional to the dose, and metabolites were determined to have been cleared rapidly from plasma with a half life of thirty to forty-five minutes. These metabolites were composed of chain-shortened oligonucleotides, consistent with exonuclease-mediated degradation.
No evidence of accumulation, induction, or inhibition of metabolism was found after the administration of repetitive doses.
Therapy Treatment options for lung cancer are determined by the type and stage of the cancer and include surgery, radiation therapy, and chemotherapy. For many localized cancers, 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.
Common chemotherapies include various combinations of 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 of the combinations, e.g. doxorubin and paclitaxel, but are less common.
Applicant has designed several NCH ribozymes targeted against PKCa 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.
Proliferation assay: 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 treatment period. To calculate cell density for proliferation assays, the FIPS (fluoro-imaging processing system) method well known in the art can be used. This method allows I 0 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 pg/mL
and inhibition of proliferation is determined on day 5 post-treatment. Two full ribozyme I 5 screens are usually completed and lead ribozymes are chosen for further testing. Of the lead ribozymes chosen from primary screens, ribozymes which continue to inhibit cell proliferation in subsequent experiments are selected for PKCa RNA and protein inhibition studies.
!0 Example 15: Nucleoside Triphosphates and their incorporation into olig_onucleotides The synthesis of nucleotide triphosphates and their incorporation into nucleic acids using polymerise enzymes has greatly assisted in the advancement of nucleic acid research. The polymerise enzyme utilizes nucleotide triphosphates as precursor molecules to assemble oligonucleotides. Each nucleotide is attached by a phosphodiester bond !5 formed through nucleophilic attack by the 3' hydroxyl group of the oligonucleotide's last nucleotide onto the 5' triphosphate of the next nucleotide. Nucleotides are incorporated 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.
Most natural polymerise enzymes incorporate standard nucleotide triphosphates into .0 nucleic acid. For example, a DNA polymerise incorporates dATP, dTTP, dCTP, and dGTP into DNA and an RNA polymerise generally incorporates ATP, CTP, UTP, and GTP into RNA. There are however, certain polymerises that are capable of incorporating non-standard nucleotide triphosphates into nucleic acids (Joyce, 1997, PNAS
94, 1619-1622, Huang et al., Biochemistry 36, 8231-8242).
Before nucleosides can be incorporated into RNA transcripts using polymerise enzymes they must first be converted into nucleotide triphosphates which can be recognized by these enzymes. Phosphorylation of unblocked nucleosides by treatment with POC13 and trialkyl phosphates was shown to yield nucleoside 5'-phosphorodichloridates (Yoshikawa et al., 1969, Bull. Chem. Soc. (Japan) 42, 3505).
Adenosine or 2'-deoxyadenosine S'-triphosphate was synthesized by adding an additional step consisting of treatment with excess tri-n-butylammonium pyrophosphate in DMF
followed by hydrolysis (Ludwig, 1981, Acta Biochim. et Biophys. Acid. Sci.
Hung. 16, 131-133).
Non-standard nucleotide triphosphates are not readily incorporated into RNA
transcripts by traditional RNA polymerises. Mutations have been introduced into RNA
polymerise to facilitate incorporation 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).
McGee et al., International PCT Publication No. WO 95/35102, describes the incorporation of 2'-NHZ-NTP's, 2'-F-NTP's, and 2'-deoxy-2'-benzyloxyamino UTP
into RNA using bacteriophage T7 polymerise.
Wieczorek et al., 1994, Bioorganic ~c Medicinal Chemistry Letters 4, 987-994, describes the incorporation of 7-deaza-adenosine triphosphate into an RNA
transcript using bacteriophage T7 RNA polymerise.
Lin et al., 1994, Nucleic Acids Research 22, 5229-5234, reports the incorporation of 2'-NHZ-CTP and 2'-NHz-UTP into RNA using bacteriophage T7 RNA polymerise and polyethylene glycol containing buffer. The article describes the use of the polymerise synthesized RNA for in vitro selection of aptamers to human neutrophil elastase (HNE).
This invention relates to novel nucleotide triphosphate (NTP) molecules, and their incorporation into nucleic acid molecules, including nucleic acid catalysts.
The NTPs of the instant invention are distinct from other NTPs known in the art. The invention further relates to incorporation of these nucleotide triphosphates into oligonucleotides using an RNA polymerise; the invention further relates to novel transcription conditions for the incorporation of modified (non-standard) and unmodified NTP's, into nucleic acid molecules. Further, the invention relates to methods for synthesis of novel NTP's In a first aspect, the invention features NTP's having the formula triphosphate-OR, for example the following formula 3:
O O O
-O-P -fl -P -~ -P OR
0- o- o-where 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 [3-alanyl) amino ; 2'-deoxy-2'-(lysiyl) amino uridine; 2'-C-allyl uridine; 2'-O-amino-uridine;
0 2'-O-methylthiomethyl adenosine; 2'-O-methylthiomethyl cytidine ; 2'-O-methylthiomethyl guanosine; 2'-O-methylthiomethyl-uridine; 2'-deoxy-2'-(N
histidyl) amino uridine; 2'-deoxy-2'-amino-5-methyl cytidine; 2'-(N [i-carboxamidine-[3-alanyl)amino-2'-deoxy-uridine; 2'-deoxy-2'-(N-(3-alanyl)-guanosine; 2'-O-amino-adenosine; 2'-(N lysyl)amino-2'-deoxy-cytidine; 2'-Deoxy -2'-(L-histidine) amino 5 Cytidine; 5-Imidazoleacetic acid 2'-deoxy uridine, 5-[3-(N-4-imidazoleacetyl)aminopropynyl]-2'-O-methyl uridine, 5-(3-aminopropynyl)-2'-O-methyl uridine, 5-(3-aminopropyl)-2'-O-methyl uridine, 5-[3-(N-4-imidazoleacetyl)aminopropyl]-2'-O-methyl uridine, 5-(3-aminopropyl)-2'-deoxy-2-fluoro uridine, 2'-Deoxy-2'-((3-alanyl-L-histidyl)amino uridine, 2'-deoxy-2'-(3-alaninamido-uridine, 3-(2'-deoxy-2'-fluoro-[i-D-ribofuranosyl)piperazino[2,3-D]pyrimidine-2-one, 5-[3-(N-4-imidazoleacetyl)aminopropyl]-2'-deoxy-2'-fluoro uridine, 5-[3-(N-4-imidazoleacetyl)aminopropynyl]-2'-deoxy-2'-fluoro uridine, 5-E-(2-carboxyvinyl-2'-deoxy-2'-fluoro uridine, 5-[3-(N-4-aspartyl)aminopropynyl-2'-fluoro uridine, S-(3-aminopropyl)-2'-deoxy-2-fluoro cytidine, and S-[3-(N-4-succynyl)aminopropyl-2'-deoxy-5 2-fluoro cytidine.
In a second aspect, the invention features inorganic and organic salts of the nucleoside triphosphates of the instant invention.

In a third aspect, 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-triazolides, phospho-tris-triimidazolides and the like), trialkyl phosphate (such as triethylphosphate or trimethylphosphate or the like) and a hindered base (such as dimethylaminopyridine, DMAP and the like) under conditions suitable for the formation of pyrimidine monophosphate; and pyrophosphorylation where the pyrimidine monophosphate is contacted with a pyrophosphorylating reagent (such as I 0 tributylammonium pyrophosphate) under conditions suitable for the formation of pyrimidine triphosphates.
By "nucleotide triphosphate" or "NTP" is meant a nucleoside bound to three inorganic phosphate groups at the 5' hydroxyl group of the modified or unmodified ribose or deoxyribose sugar where the 1' position of the sugar may comprise a nucleic acid base I 5 or hydrogen. The triphosphate portion may be modified to include chemical moieties which do not destroy the functionality of the group (i.e., allow incorporation into an RNA
molecule).
In another preferred embodiment, nucleotide triphosphates (NTPs) of the instant invention are incorporated into an oligonucleotide using an RNA polymerase enzyme.
'.0 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 of the present invention can be incorporated into oligonucleotides using certain DNA
polymerases, such as Taq polymerase.
In yet another preferred embodiment, the invention features a process for '.5 incorporating modified NTP's into an oligonucleotide including the step of incubating a mixture having a DNA template, RNA polymerase, NTP, and an enhancer of modified NTP incorporation under conditions suitable for the incorporation of the modified NTP
into the oligonucleotide.
By "enhancer of modified NTP incorporation" is meant a reagent which facilitates SO the incorporation of modified nucleotides into a nucleic acid transcript by an RNA
polymerase. Such reagents include, but are not limited to, methanol, LiCI, polyethylene glycol (PEG), diethyl ether, propanol, methyl amine, ethanol, and the like.

In another preferred embodiment, the modified nucleotide triphosphates can be incorporated by transcription into a nucleic acid molecules including enzymatic nucleic acid, antisense, 2-SA antisense chimera, oligonucleotides, triplex forming oligonucleotide (TFO), aptamers and the like (Stun et al., 1995 Pharmaceutical Res. 12, 465).
By "triplex forming oligonucleotides (TFO)" it is meant an oligonucleotide that can bind to a double-stranded DNA in a sequence-specific manner to form a triple-strand helix. Formation of such triple helix structure has been shown to inhibit transcription of the targeted gene (Duval-Valentin et al., 1992 Proc. Natl. Acad. Sci. USA 89, 504).
In yet another preferred embodiment, the modified nucleotide triphosphates of the 0 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.
In another preferred embodiment, the invention features nucleic acid based techniques (e.g., enzymatic nucleic acid molecules), antisense nucleic acids, 5 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.
In yet another preferred embodiment, the invention features enzymatic nucleic acid molecules targeted against HER2 RNA, specifically including ribozymes in the class II
'.0 (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 incorporated by .5 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 incorporated by reference herein.
In yet another preferred embodiment, the invention features a process for incorporating a plurality of compounds of formula 3.
In yet another embodiment, the invention features a nucleic acid molecule with catalytic activity having formula 4:

W
J i ' (Y'Y' )m i (X)o ~ Domain A
In the formula shown above 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; Y' is a nucleotide complementary to Y; 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; m is an integer greater than 1 and preferably less than 10, more specifically 2, 3, 4, 5, 6, or 7; 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 15;1 and o may be the same length (1= o) or different lengths (1 ~ o); each X(1) 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); W is a linker of >_ 2 nucleotides in length or may be a non-nucleotide linker; A, U, C, and G
represent the nucleotides; G is a nucleotide, preferably 2'-O-methyl or ribo; A is a nucleotide, preferably 2'-O-methyl or ribo; U is a nucleotide, preferably 2'-amino (e.g., 2'-NHZ or 2'-O- NHz), 2'-O-methyl or ribo; C represents a nucleotide, preferably 2'-amino (e.g., 2'-NHZ or 2'-O-(Z'Z' )n II
W

NHZ), and represents a chemical linkage (e.g. a phosphate ester linkage, amide linkage, phosphorothioate, phosphorodithioate or others known in the art).
In yet another embodiment, the invention features a nucleic acid molecule with catalytic activity having formula 5:
(X)o W ' ~Z'1 G
Z %~ C
G C U (X)~
A U
G UG
U G
\ Y A i In the formula shown above 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 15;1 and o may be the same length (1= o) or different lengths (1 ~ o); each X(1~ and X(ot 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); X~o~ preferably has a G at the 3'-end, X(t~ 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'-OH; A is a nucleotide, preferably 2'-O-methyl, 2'-deozy-2'-fluoro, or 2'-OH; U is a nucleotide, preferably 2'-O-methyl, 2'-deozy-2'-fluoro, or 2'-OH;
C represents a nucleotide, preferably 2'-amino (e.g., 2'-NH2 or 2'-O- NH2, 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.
In yet another preferred embodiment, the 3'-cap is selected from a group comprising, 4',5'-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4'-thio nucleotide;
carbocyclic nucleotide; 5'-amino-alkyl phosphate; 1,3-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; threo-pentofuranosyl nucleotide;
acyclic 3',4'-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide; 5'-5'-inverted nucleotide moiety; S'-5'-inverted abasic moiety; S'-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 S'-mercapto moieties (for more details, see Beaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporated by reference herein).
In another aspect, 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.
Nucleotide Synthesis Addition of dimethylaminopyridine (DMAP) to the phosphorylation protocols known in the art can greatly increase the yield of nucleotide monophosphates while decreasing the reaction time. Synthesis of the nucleosides of the invention have been described in several publications and Applicants previous applications (Beigelman et al., International PCT publication No. WO 96/18736; Dudzcy et al., Int. PCT Pub.
No. WO
95/11910; Usman et al., Int. PCT Pub. No. WO 95/13378; Matulic-Adamic et al., 1997, Tetrahedron Lett. 38, 203; Matulic-Adamic et al., 1997, Tetrahedron Lett. 38, 1669; all of which are incorporated herein by reference). These nucleosides are dissolved in triethyl phosphate and chilled in an ice bath. Phosphorus oxychloride (POC13) is then added followed by the introduction of DMAP. The reaction is then warmed to room temperature and allowed to proceed for S 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 stirred 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 of the reaction, and purification methods can easily be alternated with substitutes and equivalents and still obtain the desired product.
Nucleotide Tri~hosphates 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.
Incorporation of modified nucleotides into DNA or RNA oligonucleotides can alter the properties of the molecule. For example, modified nucleotides can hinder binding of nucleases, thus increasing the chemical half life of the 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 of the molecules (Burgin et al., 1996, Biochemistry 35, 14090-14097; Usman et al., 1996, Curr. Opin. Struct. Biol. 6, 527-533).
Modified nucleotides are incorporated using either wild type or mutant polymerases.
For example, mutant T7 polymerase is used in the presence of modified nucleotide triphosphate(s), DNA template and suitable buffers. Those skilled in the art will recognize that other polymerases and their respective mutant versions can also be utilized for the incorporation of NTP's of the invention. Nucleic acid transcripts were detected by incorporating radiolabelled nucleotides (a-32P NTP). The radiolabeled NTP
contained the same base as the modified triphosphate being tested. The effects of methanol, PEG and LiCI were tested by adding these compounds independently or in combination.
Detection and quantitation of the nucleic acid transcripts was performed using a Molecular Dynamics PhosphorImager. Efficiency of transcription was assessed by comparing modified nucleotide triphosphate incorporation with all-ribonucleotide incorporation control. Wild-type polymerise was used to incorporate NTP-'s using the manufacturer's buffers and instructions (Boehringer Mannheim).
Transcription Conditions Incorporation rates of modified nucleotide triphosphates into oligonucleotides can be increased by adding to traditional buffer conditions, several different enhancers of modified NTP incorporation. Applicant has utilized methanol and LiCI in an attempt to increase incorporation rates of dNTP using RNA polymerise. These enhancers of modified NTP incorporation 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 incorporation 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.
Synthesis of purine nucleotide triphosphates: 2'-O-methyl-~uanosine-5'-triphosphate 2'-O-methyl guanosine nucleoside (0.25 grams, 0.84 mmol) was dissolved in triethyl phosphate (5.0) ml by heating to 100°C for 5 minutes. The resulting clear, colorless solution was cooled to 0°C using an ice bath under an argon atmosphere.
Phosphorous oxychloride (1.8 eq., 0.141 ml) was then added to the reaction mixture with vigorous stirring. The reaction was monitored by HPLC, using a sodium perchlorate gradient.
After 5 hours at 0°C, tributylamine (0.65 ml) was added followed by the addition of anhydrous acetonitrile (10.0 ml), and after S 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 stirred overnight at room temperature, the mixture was evaporated in vacuo with methanol co-evaporation (4x) then diluted in 50 ml O.OSM TEAB. DEAF sephadex purification was used with a gradient of 0.05 to 0.6 M TEAB to obtain pure triphosphate (0.52 g, 66.0%
yield) (elutes around 0.3M TEAB); the purity was confirmed by HPLC and NMR analysis.
Smthesis of Pyrimidine nucleotide triphosphates: 2'-O-methylthiomethyl-uridine-5'-trinhosphate 2'-O-methylthiomethyl uridine nucleoside (0.27 grams, 1.0 mmol) was dissolved in triethyl phosphate (5.0 ml). The resulting clear, colorless solution was cooled to 0°C with an ice bath under an argon atmosphere. Phosphorus oxychloride (2.0 eq., 0.190 ml) was then added to the reaction mixture with vigorous stirring.
Dimethylaminopyridine (DMAP, 0.2eq., 25 mg) was added, the solution warmed to room temperature and the reaction was monitored by HPLC, using a sodium perchlorate gradient. After 5 hours at 20°C, tributylamine (1.0 ml) was added followed by anhydrous acetonitrile (10.0 ml), and after 5 minutes tributylammonium pyrophosphate (4.0 eq., 1.8 g) was added. The reaction mixture was quenched with 20 ml of 2M TEAB after 15 minutes at 20°C
(HPLC analysis with above conditions showed consumption of monophosphate at 10 minutes) then stirred overnight at room temperature. The mixture was evaporated in vacuo with methanol co-evaporation (4x) then diluted in SO ml O.OSM TEAB. DEAF fast flow Sepharose purification with a gradient of 0.05 to 1.0 M TEAB was used to obtain pure triphosphate (0.40 g, 44% yield) (elutes around 0.3M TEAB) as determined by HPLC and NMR
analysis.
Utilization of DMAP in Uridine 5'-Triphosphate Synthesis The reactions were performed on 20 mg aliquots of nucleoside dissolved in 1 ml of triethyl phosphate and 19 u1 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 (SOmM & 100mM 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. These conditions doubled the product yield and resulted in a 10-fold improvement in the reaction time to maximum yield (1200 minutes down to 120 minutes for a 90% yield). Selectivity for 5'-monophosphorylation was observed for all reactions. Subsequent triphosphorylation occurred in nearly quantitative yield.
Materials Used in Bacteriopha~e T7 RNA Polymerase Reactions Buffer 1: Reagents are mixed together to form a l OX stock solution of buffer (400 mM Tris-Cl [pH 8.1], 200 mM MgCl2, 100 mM DTT, 50 mM spermidine, and 0.1%
triton~ X-100). Prior to initiation of the polymerise reaction methanol, LiCI
is added and the buffer is diluted such that the final reaction conditions for condition 1 consisted of 40mM tris (pH 8.1), 20mM MgCl2, 10 mM DTT, 5 mM spermidine, 0.01% triton~ X-100, 10% methanol, and 1 mM LiCI.
BUFFER 2: Reagents are mixed together to form a l OX stock solution of buffer (400 mM Tris-Cl [pH 8.1 ], 200 mM MgCl2, 100 mM DTT, 50 mM spermidine, and 0.1 triton~ X-100). Prior to initiation of the polymerise reaction PEG, LiCI is added and the buffer is diluted such that the final reaction conditions for buffer 2 consisted of : 40mM
tris (pH 8.1), 20mM MgCl2, 10 mM DTT, 5 mM spermidine, 0.01% triton~ X-100, 4%
PEG, and 1 mM LiCI.
BUFFER 3: Reagents are mixed together to form a l OX stock solution of buffer (400 mM Tris-Cl [pH 8.0], 120 mM MgCl2, 50 mM DTT, 10 mM spermidine and 0.02%
triton~ X-100). Prior to initiation of the polymerise 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 MgCl2, 5 mM DTT, 1 mM spermidine, 0.002% triton~ X-100, and 4%
PEG.
BUFFER 4: Reagents are mixed together to form a l OX stock solution of buffer (400 mM Tris-Cl [pH 8.0], 120 mM MgCl2, 50 mM DTT, 10 mM spermidine and 0.02%
triton~ X-100). Prior to initiation of the polymerise reaction PEG, methanol is added and the buffer is diluted such that the final reaction conditions for buffer 4 consisted of 40mM tris (pH 8.0), 12 mM MgCl2, 5 mM DTT, 1 mM spermidine, 0.002% triton~ X-100, 10% methanol, and 4% PEG.
BUFFER 5: Reagents are mixed together to form a l OX stock solution of buffer (400 mM Tris-Cl [pH 8.0], 120 mM MgCl2, 50 mM DTT, 10 mM spermidine and 0.02%
triton~ X-100). Prior to initiation of the polymerise reaction PEG, LiCI 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 MgCl2, 5 mM DTT, 1 mM spermidine, 0.002% triton~ X-100, 1 mM LiCI and 4% PEG.
BUFFER 6: Reagents are mixed together to form a l OX stock solution of buffer (400 mM Tris-Cl [pH 8.0], 120 mM MgCl2, SO mM DTT, 10 mM spermidine and 0.02%
triton~ X-100). Prior to initiation of the polymerise 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 MgCl2, 5 mM DTT, 1 mM spermidine, 0.002% triton~ X-100, 10% methanol, and 4% PEG.
BUFFER 7: Reagents are mixed together to form a l OX stock solution of buffer (400 mM Tris-Cl [pH 8.0], 120 mM MgCl2, 50 mM DTT, 10 mM spermidine and 0.02%
triton~ X-100). Prior to initiation of the polymerise reaction PEG, methanol and LiCI 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 MgCl2, 5 mM DTT, 1 mM spermidine, 0.002%
triton~
X-100, 10% methanol, 4% PEG, and 1 mM LiCI.
Screening of Modified nucleotide triphosphates with Mutant T7 RNA Polymerise 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 polymerise (Sousa and Padilla, supra) (0.3-2 mg/20 ml reaction), NTP's (2 mM each), DNA template (10 pmol), inorganic pyrophosphatase (SU/ml) and oc-32P 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 electrophoresis. Using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA), the amount of full-length transcript was quantified and compared with an all-RNA
control 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 control was run with the mutant T7 polymerise using commercially available polymerise buffer (Boehringer Mannheim, Indianapolis,1N).

Incorporation of Modified NTP's using Wild-type T7 RNA polymerise Bacteriophage T7 RNA polymerise was purchased from Boehringer Mannheim at 0.4 U/pL concentration. Applicant used the commercial buffer supplied with the enzyme and 0.2 pCi alpha-32P NTP in a 50 pL reaction with nucleotides triphosphates at 2 mM
each. The template was a double-stranded PCR fragment, which was used in previous screens. Reactions were carned out at 37°C for 1 hour. Ten pL of the sample was run on a 7.5% analytical PAGE and bands were quantitated using a PhosphorImager.
Results are calculated as a comparison to an "all ribo" control (non-modified nucleotide triphosphates) and the results are in Table 49.
Incorporation of Multiple Modified nucleotide triphosphates Into Oli~onucleotides Combinations of modified nucleotide triphosphates were tested with the transcription protocol described above, to determine the rates of incorporation of two or more of these triphosphates. Incorporation of 2'-Deoxy-2'-(L-histidine) amino uridine (2'-his-NH2-UTP) was tested with unmodified cytidine nucleotide triphosphates, rATP and rGTP in reaction condition number 9. The data is presented as a percentage of incorporation of modified NTP's compared to the all rNTP control and is shown in Table 50a.
Two modified cytidines (2'-NHZ-CTP or 2'dCTP) were incorporated along with 2'-his-NHZ-UTP with identical efficiencies. 2'-his-NH2-UTP and 2'-NHZ-CTP were then tested with various unmodified and modified adenosine triphosphates in the same buffer (Table 50b). The best modified adenosine triphosphate for incorporation with both 2'-his-NHZ-UTP and 2'-NHZ-CTP was 2'-NHZ-DAPTP.
Outimization of Reaction conditions for Incorporation of Modified Nucleotide Triphosphate The combination of 2'-his-NHZ-UTP, 2'-NH2-CTP, 2'-NHZ-DAP, and rGTP was tested in several reaction conditions (Table 51) using the incorporation protocol described above. The results demonstrate that of the buffer conditions tested, incorporation of these modified nucleotide triphosphates occur in the presence of both methanol and LiCI.

Selection of Novel Enzymatic nucleic acid molecule Motifs using 2'-deoxv-2'amino Modified GTP and CTP
For selection of new enzymatic nucleic acid molecule motifs, pools of enzymatic nucleic acid molecules were designed to have two substrate binding arms (5 and nucleotides long) and a random region in the middle. The substrate has a biotin on the 5' end, S nucleotides complementary to the short binding arm of the pool, an unpaired G (the desired cleavage site), and 16 nucleotides complementary to the long binding arm of the pool. The substrate 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.
Construction of Libraries:
The oligonucleotides listed below were synthesized by Operon Technologies (Alameda, CA). Templates were gel purified and then run through a Sep-PakTM
cartridge (Waters, Millford, MA) using the manufacturers protocol. Primers (MST3, MST7c, MST3del) were used without purification.
Primers:
MST3 (30 mer): 5'- CAC TTA GCA TTA ACC CTC ACT AAA GGC CGT-3' MST7c (33 mer): S'-TAA TAC GAC TCA CTA TAG GAA AGG TGT GCA ACC-3' MST3del (18 mer): 5'-ACC CTC ACT AAA GGC CGT-3' Templates:
MSN60c (93 mer): 5'-ACC CTC ACT AAA GGC CGT (I~6o GGT TGC ACA CCT
TTG-3' MSN40c (73 mer): 5'-ACC CTC ACT AAA GGC CGT (N'4o GGT TGC ACA CCT
TTG-3' MSN20c (53 mer): 5'-ACC CTC ACT AAA GGC CGT (1~2o 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 MST3de1/MST7c as primers.

Single-stranded templates were converted into double-stranded 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. Products were checked on agarose gel to confirm the length of each fragment (N60=123 bp, N40=91 bp, N20=71 bp) and then were phenol/chloroform extracted and ethanol precipitated.
The concentration of the double-stranded product was 25 wM.
Transcription of the initial pools was performed in a 1 ml volume comprising:

pmol double-stranded template (3 x 10'4 molecules), 40 mM tris-HCl (pH 8.0), 12 mM
MgCl2, 1 mM spermidine, 5 mM DTT, 0.002% triton X-100, 1 mM LiCI, 4% PEG 8000, 10% methanol, 2 mM ATP (Pharmacia), 2 mM GTP (Pharmacia), 2 mM 2'-deoxy-2'-amino-CTP (USB), 2 mM 2'-deoxy-2'-amino-UTP (LTSB), 5 U/ml inorganic pyrophosphatase (Sigma), S U/pl T7 RNA polymerase (USB; Y639F mutant was used in some cases at 0.1 mg/ml (Sousa and Padilla, supra)), 37°C, 2 hours.
Transcribed libraries were purified by denaturing PAGE (N60=106 ntds, N40=74, N20=54) and the resulting product was desalted using Sep-PakTM columns and then ethanol precipitated.
Initial column-Selection:
The following biotinylated substrate was synthesized using standard protocols (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):
5'-biotin-C18 spacer-GCC GUG GGU UGC ACA CCU UUC C-C18 spacer-thiol-modifier C6 S-S-inverted abasic-3' Substrate was purified by denaturing PAGE and ethanol precipitated. 10 nmol of substrate was linked to a NeutrAvidinTM column using the following protocol:
400 p1 UltraLink Immobilized NeutrAvidinTM slurry (200 ~.1 beads, Pierce, Rockford, IL) were loaded into a polystyrene column (Pierce). The column was washed twice with 1 ml of binding buffer (20 mM NaP04 (pH 7.5), 150 mM NaCI) and then capped off (i.e., a cap was put on the bottom of the column to stop the flow). 200 p1 of the substrate 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 of the 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
NaCI, 50 mM KCl). The column was then ready for use and capped off. 1 nmol of the initial pool RNA was loaded on the column in a volume of 200 ~1 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 p1 of elution buffer (50 mM tris-HCl (pH
8.5), 100 mM NaCI, 50 mM KCI, 25 mM MgCl2) was applied to the column followed by 30 minute incubation at room temperature with occasional vortexing. The cap was removed and four 200 p1 fractions were collected using elution buffer.
Second column (counter selection):
A diagram for events in the second column is generally shown in Figure 12 and substrate oligonucleotide used is shown below:
5'-GGU UGC ACA CCU UUC C-C18 spacer-biotin-inverted abasic-3' This column substrate was linked to UltraLink NeutrAvidinTM resin as previously described (40 pmol) which was washed twice with elution buffer. The eluent from the first column purification was then run on the second column. The use of this column allowed for binding of RNA that non-specifically diluted from the first column, while RNA that performed a catalytic event and had product bound to it, flowed through the second column. The fractions were ethanol precipitated using glycogen as carrier and rehydrated in sterile water for amplification.
Amplification:
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): 1X PCR buffer (Boerhinger Mannheim), 1 mM
dNTP's (for PCR, Boerhinger Mannheim), 2 U/~1 RNase-Inhibitor (Boerhinger Mannheim), 10 U/pl SuperscriptTM II 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. The following reagents were then added to the tube to increase the volume five-fold for the PCR step (final concentrations/amounts given): MST7c primer (10-100 pmol, same amount as in RT step), 1X PCR buffer, taq DNA polymerase (0.025-0.05 U/~1, 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, Sminutes; 30°C, 30 minutes. Cycle number and annealing temperature were decided on a round by round basis. In cases where heteroduplex was observed, the reaction was diluted five-fold with fresh reagents and allowed to progress through 2 more amplification cycles.
Resulting products were analyzed for size on an agarose gel (N60=123 bp, N40=103 bp, N20=83 bp) and then ethanol precipitated.
Transcriptions:
Transcription of amplified products was done using the conditions described above with the following modifications: 10-20% of the amplification reaction was used as template, reaction volume was 100-500 p1, and the products sizes varied slightly (N60=106 ntds, N40=86, N20=66). A small amount of 32P-GTP was added to the reactions for quantitation purposes.
Subsequent rounds:
Subsequent rounds of selection used 20 pmols of input RNA and 40 pmol of the nucleotide substrate on the column.
Activity of pools:
Pools were assayed for activity under single turnover conditions every three to four rounds. Activity assay conditions were as follows: 50 mM tris-HCl (pH 8.5), 25 mM
MgCl2, 100 mM NaCI, 50 mM KCI, trace 32P-labeled substrate, 10 nM RNA pool. 2X
pool in buffer and, separately, 2X substrate in buffer were incubated at 90°C for 3 minutes, then at 37°C for 3 minutes. Equal volume 2X substrate was then added the 2X pool tube (t=0). Initial assay time points were taken at 4 and 24 hours: 5 ~.1 was removed and quenched in 8 ~1 cold Stop buffer (96% formamide, 20 mM EDTA, 0.05% bromphenyl blue/xylene cyanol). Samples were heated 90°C, 3 minutes, and loaded on a 20%

sequencing gel. Quantitation was performed using a Molecular Dynamics Phosphorimager and ImageQuaNTTM software. The data is shown in Table 52.
Samples from the pools of oligonucleotide were cloned into vectors and sequenced using standard protocols (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press). The enzymatic nucleic acid molecules were transcribed from a representative number of these clones using methods described in this application. Individuals from each pool were tested for RNA cleavage from N60 and N40 by incubating the enzymatic nucleic acid molecules from the clones with S/16 substrate in 2mM MgCl2, pH 7.5, l OmM KCl at 37°C. The data in Table 54 shows that the enzymatic nucleic acid molecules isolated from the pool are individually active.
Kinetic Activity:
Kinetic activity of the 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 MgClz, 100 mM NaCI, 50 mM KC1) at 37°C.
Magnnesium Dependence:
Magnesium dependence of round 15 of N20 was tested by varying MgCl2 while other conditions were held constant (50 mM tris [pH 8.0], 100 mM NaCI, 50 mM
KCI, single turnover, 10 nM pool). The data is shown in Table 55, which demonstrates increased activity with increased magnesium concentrations.
Selection of Novel Enzymatic nucleic acid molecule Motifs using 2'-Deoxy-2'-(N
histidyl~ amino UTP, 2'-Fluoro-ATP, and 2'-deoxy-2'-amino CTP and GTP
The method used for selection of novel enzymatic nucleic acid molecule motifs using 2'-deoxy-2'amino modified GTP and CTP was repeated using 2'-Deoxy-2'-(N
histidyl) amino UTP, 2'-Fluoro-ATP, and 2'-deoxy-2'-amino CTP and GTP.
However, rather than causing cleavage on the initial column with MgClz, the initial random modified-RNA pool was loaded onto substrate-resin in the following buffer; 5 mM
NaOAc pH 5.2, 1 M NaCI 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 KCI, 10 mM NaCI, 1 mM CaCl2, 1 mM MgCl2. In one selection of N60 oligonucleotides, no divalent canons (MgCl2, CaCl2) 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 1-3% of the present substrate even in the absence of divalent cations, the background (in the absence of modified pools) was 0.2 - 0.4 %.
Synthesis of 5-substituted 2'-modified nucleosides 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 substrates for RNA
polymerases.
(Aurup, H.; Williams, D.M.; Eckstein, F. Biochemistry 1992, 31, 9637; and Padilla, R.;
Sousa, R. Nucleic Acids Res. 1999, 27, 1561.) On the other hand it was shown that variety of substituents at pyrimidine 5-position is well tolerated by T7 RNA
polymerase (Tarasow, T.M.; Eaton, B.E. Biopolymers 1998, 48, 29), most likely because the natural hydrogen-bonding pattern of these nucleotides is preserved. We have chosen 2'-fluoro and 2'-D-methyl pyrimidine nucleosides as starting materials for attachment of different functionalities to the 5-position of the base. Both rigid (alkynyl) and flexible (alkyl) spacers are used. The choice of imidazole, amino and carboxylate pendant groups is based on their ability to act as general acids, general bases, nucleophiles and metal ligands, all of which can improve the catalytic effectiveness of selected nucleic acids.
Figures 21 - 24 relate to the synthesis of these compounds.
2'-O-methyluridine was 3',5'-bis-acetylated using acetic anhydride in pyridine and then converted to its 5-iodo derivative 1 a using I2/ceric ammonium nitrate reagent (Asakura, J.; Robins, M.J. J. Org. Chem. 1990, 55, 4928) (Scheme 1). Both reactions proceeded in a quantitative yield and no chromatographic purifications were needed.
Coupling between 1 and N trifluoroacetyl propargylamine using copper(I) iodide and tetrakis(triphenylphosphine)palladium(0) catalyst as described by Hobbs (Hobbs, F.W., Jr.
J. Org. Chem. 1989, 54, 3420) yielded 2a in 89% yield. Selective O-deacylation with aqueous NaOH afforded 3a which was phosphorylated with POCl3/triethylphosphate (TEP) in the presence of 1,8-bis(dimethylamino)naphthalene (Proton-Sponge) (Method A) (Kovacz, T; Otvos, L. Tetrahedron Lett. 1988, 29, 4525). The intermediate nucleoside phosphorodichloridate was condensed in situ with tri-n-butylammonium pyrophosphate.
At the end, the N TFA group was removed with concentrated ammonia. 5'-Triphosphate was purified on Sephadex~ DEAF 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 Na-salt was achieved by passing the aqueous solution of triphosphate through Dowex SOWX8 ion exchange resin in Na+ 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 S-aminopropyl derivative Sa which was phosphorylated under conditions identical to those described for propynyl derivative 3a to afford triphosphate 6a in SO% yield.
For the preparation of imidazole derivatized triphosphates 9a and 11a, we developed an efficient synthesis of N diphenylcarbamoyl 4-imidazoleacetic acid (~DPC): Transient protection of carboxyl group as TMS-ester using TMS
Cl/pyridine followed by DPC-Cl allowed for a clean and quantitative conversion of 4 imidazoleacetic acid (ImAA) to its N DPC protected derivative.
Complete deacylation of 2a afforded 5-(3-aminopropynyl) derivative 8a which was condensed with 4-imidazoleacetic acid in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) to afford 9a in 68% yield.
Catalytic hydrogenation of 8a yielded S-(3-aminopropyl) derivative 10a which was condensed with ImAADP~ to yield conjugate lla in 32% yield. Yields in these couplings were greatly improved when S'-OH was protected with DMT group (not shown) thus efficiently preventing undesired 5'-O-esterification. Both 9a and 11 a failed to yield triphosphate products in reaction with POC13/TEP/Proton-Sponge.
On the contrary, phosphorylation of 3'-O-acetylated derivatives 12a and 13a using 2-chloro-4H 1,3,2-benzodioxaphosphorin-4-one followed by pyrophosphate addition and oxidation (Method B, Scheme 2; Ludwig, J., Eckstein, F., J. Org. Chem.
1989, 54, 631) afforded the desired triphosphates 14a and 15a in 57% yield, respectively.

2'-Deoxy-2'-fluoro nucleoside 5'-triphosphates containing amino- (4b, 6b) and imidazole- (14b,15b) linked groups were synthesized in a manner analogous to that described for the preparation of 2'-O-methyl nucleoside 5'-triphosphates (Schemes 1 and 2). Again, only Ludwig-Eckstein's phosphorylation worked for the preparation of 4-imidazoleacetyl derivatized triphosphates.
It is worth noting that when "one-pot-two-steps" phosphorylation reaction (Kovacz, T; Otvos, L. Tetrahedron Lett. 1988, 29, 4525) of 5b was quenched with 40%
aqueous methylamine instead of TEAB or H20, the y-amidate 7b was generated as the only detectable product. Similar reaction was reported recently for the preparation of the y-amidate of pppA2'p5'A2'p5'A.12 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 reactionl3 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
1,3,2-benzodioxa-phosphorin-4-one followed by pyrophosphate addition and oxidation (Ludwig, J.; Eckstein, F. J. Org. Chem. 1989, 54, 631) afforded the desired triphosphate in 54% yield. On the other hand, 5-(3-aminopropyl)uridine 5'-triphosphate 6b was coupled with N hydroxysuccinimide ester of Fmoc-Asp-OFm to afford, after removal of Fmoc and Fm groups with diethylamine, the desired aminoacyl conjugate 20 in 50% yield.
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 S% overall yield into 3'-acetylated cytidine derivative 25. This synthesis was plagued by poor solubility of intermediates and formation of the N4-cyclized byproduct during ammonia treatment of the 4-triazolyl intermediate. Phosphorylation of 25 as described in reference 11 yielded triphosphate 26 and N4-cyclized product 27 in 1:1 ratio.
They were easily separated on Sephadex DEAF A-25 ion exchange column using 0.1-0.8M TEAB
gradient. It appears that under basic conditions the free primary amine can displace any remaining intact 4-NHBz group leading to the cyclized product. This is similar to displacement of 4-triazolyl group by primary amine as mentioned above.

We reasoned that utilization of N4-unprotected cytidine will solve this problem.
This lead to an improved synthesis of 26: Iodination of 2'-deoxy-2'-fluorocytidine (28) provided the S-iodo derivative 29 in 58% yield. This compound was then smoothly converted into S-(3-aminopropynyl) derivative 30. Hydrogenation afforded 5-(3-aminopropyl) derivative 31 which was phosphorylated directly with POC13/ PPi to afford 26 in 37% yield. Coupling of the 5'-triphosphate 26 with succinic anhydride yielded succinylated derivative 32 in 36% yield.
Synthesis of 5-Imidazoleacetic acid 2'-deoxy-5'-triphosphate uridine 5-dintrophenylimidazoleacetic acid 2'-deoxy uridine nucleoside (80 mg) was dissolved in 5 ml of triethylphosphate while stirnng 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 MeCN (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). After 45 minutes, the reaction was quenched with 10 ml aq. methyl amine for four hours. After co-evaporation with MeOH (3x), purified material on DEAF Sephadex followed by RP chromatography to afford 15 mg of triphosphate.
Synthesis of 2'-(N lysyll-amino-2'-deoxy-cytidine Triphosphate 2'-(N-lysyl)-amino-2'-deoxy cytidine (0.180 g, 0.22 mmol) was dissolved in triethyl phosphate (2.00 ml) under Ar. The solution was cooled to 0 °C in an ice bath.
Phosphorus oxychloride (99.999%, 3 eq., 0.0672 mL) was added to the solution and the reaction was stirred 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 stirred at room temperature for 2 hours. A biphasic mixture appeared (little beads at the bottom of the flask). TLC (7:1:2 iPrOH:NH40H:H20) showed the appearance of triphosphate material. The solution was concentrated, dissolved in water and loaded on a newly prepared DEAF 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 90-95. The fractions were analyzed by ion exchange HPLC. Each fraction showed one triphosphate peak that eluted at 4.000 minutes. The fractions were combined and pumped down from methanol to remove buffer salt to yield 15.7 mg of product.
thesis of 2'-deoxy-2'-(L-histidine)amino Cytidine Triphosphate 2'-[N Fmoc, lVimid _dinitrophenyl-histidyl]amino-2'-cytidine (0.310 g, 4.04 mmol) was dissolved in triethyl phosphate (3 ml) under Ar. The solution was cooled to 0 °C.
Phosphorus oxychloride (1.8 eq., 0.068 mL) was added to the solution and stored overnight in the freezer. The next morning TLC (10% MeOH in CH2C12) showed significant starting material, one more equivalent of POC13 was added. After two hours, TLC still showed starting material. 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 stirnng continued for 2 hours. TLC (7:1:2 iPrOH:NH40H:H20) showed the appearance of triphosphate material. The solution was concentrated, dissolved in water and loaded on a DEAF 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.
Screening for Novel Enzymatic nucleic acid molecule Motifs Using Modified NTPs (Class I Moti Our initial pool contained 3 x 1014 individual sequences of 2'-amino-dCTP/2'-amino-dUTP RNA. We optimized transcription conditions in order to increase the amount of RNA product by inclusion of methanol and lithium chloride. 2'-amino-2'-deoxynucleotides do not interfere with the reverse transcription and amplification steps of selection and confer nuclease resistance. We designed the pool to have two binding arms complementary to the substrate, separated by the random 40 nucleotide region.
The 16-mer substrate had two domains, S and 10 nucleotides long, that bind the pool, separated by an unpaired guanosine. On the 5' end of the substrate was a biotin attached by a C18 linker. This enabled us to link the substrate to a NeutrAvidinTM resin in a column format.

The desired reaction would be cleavage at the unpaired G upon addition of magnesium cofactor followed by dissociation from the column due to instability of the S
base pair helix. A detailed protocol follows:
Enzymatic nucleic acid molecule Pool Prep: The initial pool DNA was prepared by converting the following template oligonucleotides into double-stranded DNA by filling in with taq polymerise. (template=5'-ACC CTC ACT AAA GGC CGT (I~4o GGT TGC
ACA CCT TTC-3'; primer 1=S'- CAC TTA GCA TTA ACC CTC ACT AAA GGC
CGT-3'; primer 2=5'-TAA TAC GAC TCA CTA TAG GAA AGG TGT GCA ACC-3'.) All DNA oligonucleotides were synthesized by Operon technologies. Template oligos were purified by denaturing PAGE and Sep-pak chromatography columns (Waters).
RNA
substrate 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-32P-ATP and T4 polynucleotide kinase followed by denaturing PAGE purification and ethanol precipitation.
5 nmole of template, 10 nmole of each primer and 250 U taq polymerise were incubated in a 10 ml volume with 1X PCR buffer (10 mM tris-HCl (pH 8.3), 1.5 mM
MgCl2, 50 mM KCl) and 0.2 mM each dNTP as follows: 94°C, 4 minutes;
(94°C, 1 min;
42°C, 1 min; 72°C, 2 min) through four cycles; and then 72°C, for 10 minutes. The product was analyzed on 2% SeparideTM agarose gel for size and then was extracted twice with buffered phenol, then chloroform-isoamyl alcohol, and ethanol precipitated. The initial RNA pool was made by transcription of 500 pmole (3 x 10'4 molecules) of this DNA as follows. Template DNA was added to 40 mM tris-HCl (pH 8.0), 12 mM
MgCl2, S mM dithiothreitol (DTT), 1 mM spermidine, 0.002% triton X-100, 1 mM LiCI, 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/pl T7 RNA polymerise at room temperature for a total volume of 1 ml. A separate reaction contained a trace amount of alpha-32P-GTP for detection. Transcriptions were incubated at 37°C for 2 hours followed by addition of equal volume STOP buffer (94% formamide, ~20 mM EDTA, 0.05%
bromophenol blue). The resulting RNA was purified by 6% denaturing PAGE gel, Sep-pakTM chromatography, and ethanol precipitated.
INITIAL SELECTION: 2 nmole of 16 mer 5'-biotinylated substrate (S'-biotin-C18 linker-GCC GUG GGU UGC ACA C-3') was linked to 200 p1 UltraLink Immobilized NeutrAvidinTM resin (400 ~1 slurry, Pierce) in binding buffer (20 mM NaP04 (pH
7.5), 150 mM NaCI) for 30 minutes at room temperature. The resulting substrate column was washed with 2 ml binding buffer followed by 2 ml column buffer (50 mM tris-HCl (pH
8.5), 100 mM NaCI, 50 mM KCl). The flow was capped off and 1000 pmole of initial pool RNA in 200 ~1 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. 200 p1 elution buffer (=column buffer + 25 mM MgCl2) was added to the column and allowed to incubate 30 minutes at room temperature. The column was uncapped and eluent collected followed by three 200 u1 elution buffer washes.
The eluent/washes were ethanol precipitated using glycogen as Garner and rehydrated in SO ~1 sterile HzO. The eluted RNA was amplified by standard reverse transcription/PCR
amplification techniques. 5-31 p1 RNA was incubated with 20 pmol of primer 1 in 14 w1 volume 90° for 3 min then placed on ice for 1 minute. The following reagent were added (final concentrations noted): 1X PCR buffer, 1 mM each dNTP, 2 U/~1 RNase Inhibitor, 10 U/pl SuperScriptTM II 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 ~1 by adding water and reagents for PCR: 1X 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 of the PCR DNA was used to transcribe the next generation, in 100 p1 volume, as described above. Subsequent rounds used 20 pmol RNA for the column with 40 pmol substrate.
TWO COL UMN SELECTION: At generation 8 (G8), the column selection was changed to the two column format. 200 pmoles of 22 mer 5'-biotinylated substrate (S'-biotin-C 18 linker-GCC GUG GGU UGC ACA CCU UUC C-C 18 linker-thiol modifier C6 S-S-inverted abasic-3') was used in the selection column as described above.
Elution was in 200 p1 elution buffer followed by a 1 ml elution buffer wash. The 1200 p.1 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 ~1 of the 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 (G11), there was visible activity in a single turnover cleavage assay. By generation 13, 45% of the substrate was cleaved at 4 hours;
k°bs of the pool was 0.037 miri' in 25 mM MgCl2. We subcloned and sequenced generation 13; the pool was still very diverse. Since our goal was a enzymatic nucleic acid molecule that would work in a physiological environment, we decided to change selection pressure rather than exhaustively catalog G13.
Reselection of the N40 pool was started from G12 DNA. Part of the G12 DNA was subjected to hypermutagenic PCR (Vartanian et al., 1996, Nucleic Acids Research 24, 2627-2631) to introduce a 10% per position mutation frequency and was designated N40H. At round 19, part of the DNA was hypermutagenized again, giving N40M and N40HM (a total of 4 parallel pools). The column substrates remained the same;
buffers were changed and temperature of binding and elution was raised to 37°C.
Column buffer was replaced by physiological buffer (50 mM tris-HCl (pH 7.5), 140 mM KCI, 10 mM
NaCI) and elution buffer was replaced by 1 mM Mg buffer (physiological buffer + 1 mM
MgCl2). Amount of time allowed for the pool to bind the column was eventually reduced to 10 min and elution time was gradually reduced from 30 min to 20 sec.
Between rounds 18 and 23, k°bs for the N40 pool stayed relatively constant at 0.035-0.04 miri 1. Generation 22 from each of the 4 pools was cloned and sequenced.
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 MacVector 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 SO pmol each primer 1 and primer 2 in 1X
PCR
buffer, 0.2 mM each dNTP, and 2.5 U of taq polymerase in 100 ~1 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 p1 H20, and 10 ~1 was transcribed in 100 p1 volume and purified as previously described.
Thirty-six clones from each pool were sequenced and were found to be variations of the same consensus motif. Unique clones were assayed for activity in 1 mM
MgCl2 and physiological conditions; nine clones represented the consensus sequence and were used in subsequent experiments. There were no mutations that significantly increased activity;
most of the mutations were in regions believed to be duplex, based on the proposed secondary structure. In order to make the motif shorter, we deleted the 3'-terminal 25 nucleotides necessary to bind the primer for amplification. The measured rates of the full length and truncated molecules were both 0.04 miri'; thus we were able reduce the size of the motif from 86 to 61 nucleotides. 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. In addition, many of the ribonucleotides were replaced with 2-O-methyl modified nucleotides to stabilize the molecule. An example of the new motif is given in Figure 13. Those of ordinary skill in the art will recognize that the molecule is not limited to the chemical modifications shown in the figure and that it represents only one possible chemically modified molecule.
Kinetic Analysis:
Single turnover kinetics were performed with trace amounts of 5'-3zP-labeled substrate and 10-1000 nM pool of enzymatic nucleic acid molecule. 2X substrate in 1X
buffer and 2X pool/enzymatic nucleic acid molecule in 1X buffer were incubated separately 90° for 3 min followed by equilibration to 37° for 3 min. Equal volume of 2X
substrate was added to pool/enzymatic nucleic acid molecule at to and the reaction was incubated at 37°C. Time points were quenched in 1.2 vol STOP buffer on ice. Samples were heated to 90°C for 3 min prior to separation on 15% sequencing gels. Gels were imaged using a PhosphorImager and quantitated using ImageQuantTM software (Molecular Dynamics). Curves were fit to double-exponential decay in most cases, although some of the curves required linear fits.
STABILITY: Serum stability assays were performed as previously described (Beigelman et al., 1995, J. Biol. Chem. 270, 25702-25708). 1 wg of 5'-3zP-labeled synthetic enzymatic nucleic acid molecule was added to 13 ~1 cold and assayed for decay in human serum. Gels and quantitation were as described in kinetics section.
SUBSTRATE REQUIREMENTS: Table 60 outlines the substrate requirements for Class I motif. Substrates 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 KCI, 10 mM NaCI, mM MgCl2 , 100 nM ribozyme, 5 nM substrate, 37°C).
RANDOMREGIONMUTATIONALIGNMENT.~ Table 61 outlines the random region alignment of 134 clones from generation 22 (l .x = N40, 2.x = N40M, 3.x = N40H, 4.x = N40HM). The number of copies of each mutant is in parenthesis in the table, deviations from consensus are shown. Mutations that maintain base pair U19:A34 are shown in italic. Activity in single turnover kinetic assay is shown relative to the G22 pool rate (50 mM Tris-HCL pH 7.5, 140 mM KCI, 10 mM NaCI, 1 mM MgCl2 , 100 nM
ribozyme, trace substrate, 37°C).
STEM TRUNCATIONAND LOOP REPLACEMENT ANALYSIS: Figure 25 shows a representation of Class I ribozyme stem truncation and loop replacement analysis. The Kre1 is compared to a 61 mer Class I ribozyme measured as described above.
Figure 26 shows examples of Class I ribozymes with truncated stems) and/or non-nucleotide linker replaced loop structures.
Inhibition of HCV Using Class I (Amberzyme) Motif During HCV infection, 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. Although the association between the HCV initial ribosome entry site (IRES) and the translation apparatus is mimicked in the HCV 5'UTR/luciferase reporter system, 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. Moreover, 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.
Recently, Lu and Wimmer characterized an HCV-poliovirus chimera in which the poliovirus IRES was replaced by the IRES from HCV (Lu & Wimmer, 1996, Proc.
Natl.
Acad. Sci. USA. 93, 1412-1417). 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 of the 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). A number of enzymatic nucleic acid molecules having the new class I motif (Amberzyme) were designed and tested (Table 56). The Amberzyme ribozymes were targeted to the 5' HCV UTR region, which when cleaved, would prevent the translation of the transcript into luciferase. 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. A
plasmid containing T7 promoter expressing 5' HCV UTR and firefly luciferase (T7C1-341 (Wang et al., 1993, J. of Yirol. 67, 3338-3344)) was mixed with a pRLSV40 Renilla control plasmid (Promega Corporation) followed by enzymatic nucleic acid molecule, and cationic lipid to make a SX concentration of the reagents (T7C1-341 (4 pg/ml), pRLSV40 renilla luciferase control (6 pg/ml), enzymatic nucleic acid molecule (250 nM), transfection reagent (28.5 pg/ml).
The complex mixture was incubated at 37°C for 20 minutes. The media was removed from the cells and 120 p1 of Opti-mem media was added to the well followed by p,1 of the SX complex mixture. 1 SO p1 of Opti-mem was added to the wells holding the 25 untreated cells. The complex mixture was incubated on OST-7 cells for 4 hours, lysed with passive lysis buffer (Promega Corporation) and luminescent signals were quantified using the Dual Luciferase Assay Kit using the manufacturer's protocol (Promega Corporation). The data shown in Figure 15 is a dose curve of enzymatic nucleic acid molecule targeting site 146 of the HCV RNA and is presented as a ratio between the firefly 30 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 ICSO of approximately S 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 irrelevant (1RR) controls.
Cleavage of Substrates Using Completely Modified class I (Amberzyrnel enzymatic nucleic acid molecule The ability of an enzymatic nucleic acid, which is modified at every 2' position to cleave a target RNA was tested to determine if any ribonucleotide positions are necessary in the Amberzyme motif. Enzymatic nucleic acid molecules were constructed with 2'-O-methyl, and 2'-amino (NHz) 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
MgClz at physiological conditions (37°C). The Amberzyme with no ribonucleotide present in it has a Kre~ 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.

Substrate Recognition Rules for Class II (zinzyme) enzymatic nucleic acid molecules Class II (zinzyme) ribozymes were tested for their ability to cleave base-paired substrates 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 KCI, 10 mM
NaCI, 1 mM MgCl2, 1 mM CaCl2 - 37° C]. Figure 19 shows the results of this study. Base paired substrate 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 substrates. All positions outside of the cleavage triplet were found to tolerate any base pairings (data not shown).
All possible mispairs immediately 5' and 3' proximal to the bulged cleavage site G
were tested to a class II ribozyme designed to cleave a 5'-C-G-C -3'. It was observed the 5' and 3' proximal sites are as active with G:U wobble pairs, in addition, the 5' proximal site will tolerate a mismatch with only a slight reduction in activity [data not shown].
Screening for Novel Enzymatic nucleic acid molecule Motifs (Class II Motifs) The selections were initiated with pools of > 1014 modified RNA's of the following sequence: 5'-GGGAGGAGGAAGUGCCU (N)35 UGCCGCGCUCGCUCCCAGUCC-3'. The RNA
was enzymatically generated using the mutant T7 Y639F RNA polymerise prepared by Rui Souza. The following modified NTP's were incorporated: 2'-deoxy-2'-fluoro-adenine triphosphate, 2'-deoxy-2'-fluoro-uridine triphosphate or 2'-deoxy-2'-fluoro-5-[(N-imidazole-4acetyl)propyl amine] uridine triphosphate, and 2'-deoxy-2'-amino-cytidine triphosphate; natural guanidine triphosphate was used in all selections so that alpha 32P-GTP could be used to label pool RNA's. RNA pools were purified by denaturing gel electrophoresus 8% polyacrilamide 7 M Urea.
The following target RNA (resin A) was synthesized and coupled to Iodoacetyl UltralinkTM resin (Pierce) by the supplier's proceedurea' -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 NaCI) 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 NaCI, 1 mM MgCl2, 1 mM CaCl2). Incubation proceeded for 20 minutes in the first generation and was reduced progressively to 1 minute in the final generations; with 13 total generations. The reaction eluent was collected in 5 M NaCI to give a final concentration of 2 M NaCI. To this was added 100 p,1 of 50%
slurry Ultralink NeutraAvidinTM (Pierce). Binding of cleaved biotin product to the avidin resin was allowed by 20 minute incubation at 22° C. The resin was subsequently washed with 5 ml of 20 mM HEPES pH 7.4, 2 M NaCI. Desired RNA's were removed by a 1.2 ml denaturing wash 1M NaCI, 10 M Urea at 94° C over 10 minutes. RNA's were double precipitated in 0.3 M sodium acetate to remove Cl- ions inhibitory to reverse transcription.
Standard protocols of reverse transcription and PCR amplification were performed.
RNA's were again transcribed with the modified NTP's described above. After 13 generations cloning and sequencing provided 14 sequences which were able to cleave the target substrate. 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.
Nucleic Acid Catalyst En~,ineerin~
Sequence, chemical and structural variants of Class I and Class II enzymatic nucleic acid molecule can be engineered and re-engineered using the techniques shown in this application and known in the art. For example, 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; Ruffner et al., 1990, Biochem., 29, 10695; Beaudry et al., 1990, Biochem., 29, 6534; McCall et al., 1992, Proc. Natl. Acad. Sci., USA., 89, 5710;
Long et al., 1994, supra; Hendry et al., 1994, BBA 1219, 405; Benseler et al., 1993, JACS, 115, 8483; Thompson et al., 1996, Nucl. Acids Res., 24, 4401; Michels et al., 1995, Biochem., 34, 2965; Been et al., 1992, Biochem., 31, 11843; Guo et al., 1995, EMBO. J., 14, 368; Pan et al., 1994, Biochem., 33, 9561; Cech, 1992, Curr. Op. Struc.
Bio., 2, 605;
Sugiyama et al., 1996, FEBS Lett., 392, 215; Beigelman et al., 1994, Bioorg.
Med. Chem., 4, 171 S; Santoro et al., 1997, PNAS 94, 4262; all are incorporated in their totality by reference herein), to the extent that the overall catalytic activity of the ribozyme is not significantly decreased.
Further rounds of in vitro selection strategies described herein and variations thereof can be readily used by a person skilled in the art to evolve additional nucleic acid catalysts and such new catalysts are within the scope of the instant invention.
Example 16' Activit~r of Class II (zinzyme) nucleic acid catalysts to inhibit HER2 gene expression Applicant has designed, synthesized and tested several class II (zinzyme) ribozymes targeted against HER2 RNA (see, for example, Tables 58, 59, and 62) in cell proliferation RNA reduction assays.
Proliferation assay:
The model proliferation assay used in the study can require 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). To calculate cell density for proliferation assays, the FIPS (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 pg/mL and inhibition of proliferation was determined on day S post-treatment.
Two full ribozyme screens were completed resulting in the selection of 14 ribozymes.
Class II
(zinzyme) ribozymes against sites, 314 (RPI No. 18653), 443 (RPI No. 18680), 597 (RPI

No. 18697), 659 (RPI No. 18682), 878 (RPI Nos. 18683 and 18654), 881 (RPI Nos.

and 18685) 934 (RPI No. 18651), 972 (RPI No. 18656, 19292, 19727, 19728, and 19293), 1292 (RPI No. 18726), 1541 (RPI No. 18687), 2116 (RPI No. 18729), 2932 (RPI
No.
18678), 2540 (RPI No. 18715), and 3504 (RPI No. 18710) caused inhibition of proliferation ranging from 25-80% as compared to a scrambled control ribozyme.
An example of results from a cell culture assay is shown in Figure 20. Refernng to 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) ribozyrnes are capable of inhibiting HER2 gene expression in mammalian cells.
RNA assay:
RNA was harvested 24 hours post-treatment using the Qiagen RNeasy~ 96 procedure. 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 II ribozyme (zinzyme) mediated reduction in RNA targeting site 972 vs a scrambled attenuated control.
Dose response assays:
Active ribozyme was mixed with binding arm-attenuated control (BAC) ribozyme to a final oligonucleotide concentration of either 100, 200 or 400 nM and delivered to cells in the presence of cationic lipid at 5.0 ~tg/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 S 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 II ribozyme targeting site 972 of HER2 RNA (RPI 19293).

Example 17' Reduction of ribose residues in Class II (zinzyme) nucleic acid catalysts Class II (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 of the 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 fold).
The Kras zinzyme shown in Figure 27a was tested in physiological buffer with the divalent concentrations as indicated in the legend (high NaCI is an altered monovalent 10 condition shown) of Figure 28. The 1 mM Cap condition yielded a rate of 0.005 miri' while the 1 mM Mgr condition yielded a rate of 0.002 miri 1. The ribose containing wild type yields a rate of 0.05 miri' while substrate in the absence of zinzyme demonstrates less than 2% degradation at the longest time point under reaction conditions shown.
This illustrates a well-behaved cleavage reaction done by a non-ribose containing catalyst with only a 10-fold reduced cleavage as compared to ribonucleotide-containing zinzyme and vastly above non-catalyzed degradation.
A more detailed investigation into the role of ribose positions in the Class II
(zinzyme) motif was carried out in the context of the HER2 site 972 (Applicant has further designed a fully modified Zinzyme as shown in Figure 27b targeting the HER2 RNA site 972). Figure 29 is a diagram of the 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 (see Figure 34) was insignificant when assayed with the Kras target but showed a modest rate enhancement in the HER2 assays. The activity of all 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 demonstrated a greater extent of cleavage and profiles almost identical to the 2 ribose motif. Applicant has thus demonstrated that a Zinzyme with no ribonucleotides present at any position can catalyze efficient RNA
cleavage activity. Thus, Zinzyme enzymatic nucleic acid molecules do not require the presence of 2'-OH group within the molecule for catalytic activity.

Example 18: Activity of reduced ribose containin Class II zinzyme) nucleic acid cata~sts to inhibit HER2 gene expression A cell proliferation assay-for testing reduced ribo class II (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.
Applications The use of 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 incorporated 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 al.,1993, Science 261:1411-1418;
Szostak, 1993, TIBS 17, 89-93; Kumar et al., 1995, FASEB J., 9, 1183; Breaker, 1996, Curr.
Op. Biotech., 7, 442).
Additionally, these modified nucleotide triphosphates can be employed to generate modified oligonucleotide combinatorial chemistry libraries. Several 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 incorporated herein by reference.
Diagnostic uses 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 of the target RNA allows the detection of mutations in any region of the molecule which alters the base-pairing and three-dimensional structure of the target RNA. By using multiple 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. In this manner, other genetic targets may be defined as important mediators of the disease. These experiments will lead to better treatment of the disease progression by affording the possibility of combinational therapies (e.g., multiple enzymatic nucleic acid molecules targeted to different genes, enzymatic nucleic acid molecules coupled with known small molecule inhibitors, radiation or intermittent treatment with combinations of enzymatic nucleic acid molecules and/or other chemical or biological molecules). Other in vitro uses of enzymatic nucleic acid molecules of this invention are well known in the art, and include detection of the presence of mRNAs associated with related conditions. Such RNA is detected by determining the presence of a cleavage product after treatment with a enzymatic nucleic acid molecule using standard methodology.
In a specific example, enzymatic nucleic acid molecules which can cleave only wild type or mutant forms of the 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. As reaction controls, synthetic substrates 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 of the "non-targeted" RNA species. The cleavage products from the synthetic substrates will also serve to generate size markers for the analysis of wild type and mutant RNAs in the sample population. Thus each analysis can involve two enzymatic nucleic acid molecules, two substrates 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 of the desired phenotypic changes in target cells. The expression of mRNA whose protein product is implicated in the development of the phenotype is adequate to establish risk. If probes of comparable specific activity are used for both transcripts, then a qualitative comparison of RNA levels will be adequate and will decrease the cost of the initial diagnosis. Higher mutant form to wild-type ratios will be correlated with higher risk whether RNA levels are compared qualitatively or quantitatively.
Additional Uses Potential usefulness of sequence-specific enzymatic nucleic acid molecules of the instant invention can have many of the same applications for the study of RNA
that DNA
restriction endonucleases have for the study of DNA (Nathans et al., 1975 Ann.
Rev.
Biochem. 44:273). For example, the pattern of restriction fragments can be used to establish sequence relationships between two related RNAs, and large RNAs could be specifically cleaved to fragments of a size more useful for study. The ability to engineer sequence specificity of the enzymatic nucleic acid molecule is ideal for cleavage of RNAs of unknown sequence. Applicant describes the use of nucleic acid molecules to down-regulate gene expression of target genes in bacterial, microbial, fungal, viral, and eukaryotic systems including plant, or mammalian cells.
All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains.
All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually.
One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The methods and compositions described herein as presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art, which are encompassed within the spirit of the invention, are defined by the scope of the claims.
It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. Thus, such additional embodiments are within the scope of the present invention and the following claims.
The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising", "consisting essentially of and "consisting of may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the description and the appended claims.
In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.
Thus, additional embodiments are within the scope of the invention and within the following claims Table 1 Characteristics of naturally occurring ribozymes Group I Introns ~ Size: 150 to >1000 nucleotides.
~ Requires a U in the target sequence immediately 5' of the cleavage site.
~ Binds 4-6 nucleotides at the 5'-side of the cleavage site.
~ Reaction mechanism: attack by the 3'-OH of guanosine to generate cleavage products with 3'-OH and 5'-guanosine.
~ Additional protein cofactors required in some cases to help folding and maintainance of the active structure.
~ Over 300 known members of this class. Found as an intervening sequence in Tetrah~rnena thermophila rRNA, fungal mitochondria, chloroplasts, phage T4, blue-green algae,' and others.
~ Major structural features largely established through phylogenetic comparisons, mutagenesis, and biochemical studies (',i'].
~ Complete kinetic framework established for one ribozyme ("
;'°,",°'].
~ Studies of ribozyme folding and substrate docking underway ("i',""','XJ.
~ Chemical modification investigation of important residues well established (x,Xy_ ~ 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' [3-galactosidase message by the ligation of new (3-galactosidase sequences onto the defective message (X"].
RNAse P RNA (M1 RNA) ~ Size: 290 to 400 nucleotides.
~ RNA portion of a ubYquitous ribonucleoprotein enzyme.
~ Cleaves tRNA precursors to form mature tRNA (X"'].
~ Reaction mechanism: possible attack by MZ+-OH to generate cleavage products with 3'-OH and 5'-phosphate.
~ RNAse P is found throughout the prokaryotes and eukaryotes. The RNA subunit has been sequenced from bacteria, yeast, rodents, and primates.
~ Recruitment of endogenous RNAse P for therapeutic applications is possible through hybridization of an External Guide Sequence (EGS) to the target RNA
(Xi°,x°]
~ Important phosphate and 2' OH contacts recently identified (X°'~X""]
Group If Introns Size: >1000 nucleotides.
Trans cleavage of target RNAs recently demonstrated (X""i,XiX]

Table 1 ~ Sequence requirements not fully determined.
~ Reaction mechanism: 2'-OH of an internal adenosine generates cleavage products with 3'-OH and a "lariat" RNA containing a 3'-5' and a 2'-5' branch point.
~ Only natural ribozyme with demonstrated participation in DNA cleavage (XX~xx'] in addition to RNA cleavage and ligation.
~ Major structural features largely established through phylogenetic comparisons (xXllJ.
~ Important 2' OH contacts beginning to be identified (Xx"'~
~ Kinetic framework under development (xxi"]
Neurospora VS RNA
~ Size: ~144 nucleotides.
Trans cleavage of hairpin target RNAs recently demonstrated (XX°].
~ Sequence requirements not fully determined.
~ Reaction mechanism: attack by 2'-OH 5' to the scissile bond to generate cleavage products with 2';3'-cyclic phosphate and 5'-OH ends.
~ Binding sites and structural requirements not fully determined.
~ Oi-tly 1 known member of this class. Found in Neurospora VS RNA.
Hammerhead Ribozyme (see text for references) ~ Size: ~13 to 40 nucleotides.
~ Requires the target sequence UH immediately 5' of the cleavage site.
~ Binds a variable number nucleotides on both sides of the cleavage site.
~ Reaction mechanism: attack by 2'-OH 5' to the scissile bond to generate cleavage products with 2',3'-cyclic phosphate and 5'-OH ends.
~ 14 known members of this class. Found in a number of plant pathogens (virusoids) that use RNA as the infectious agent.
~ Essential structural features largely defined, including 2 crystal structures (xX°i~XX°ll~
~ Minimal ligation activity demonstrated (for engineering through in vitro selection) (XX°"']
~ Complete kinetic framework established for two or more ribozymes (XX'XJ.
~ Chemical modification investigation of important residues well established (XXX].
Hairpin Ribozyme ~ Size: ~50 nucleotides.
~ Requires the target sequence GUC immediately 3' of the cleavage site.
~ Binds 4-6 nucleotides at the 5'-side of the cleavage site and a variable number to the 3'-side of the cleavage site.
~ Reaction mechanism: attack by 2'-OH 5' to the scissile bond to generate cleavage products with 2',3'-cyclic phosphate and 5'-OH ends.
~ 3 known members of this class. Found in three plant pathogen (satellite RNAs of the tobacco ringspot virus, arabis mosaic virus and chicory yellow mottle virus) which uses RNA as the infectious agent.
~ Essential structural features largely defined (XXX' XXXu XXXiii XxX'°~

Table 1 Ligation activity (in addition to cleavage activity) makes ribozyme amenable to engineering through in vitro selection (XXX°) Complete kinetic framework established for one ribozyme (xXXVt].
Chemical modification investigation of important residues begun (xXx°° xXX"iii~.
Hepatitis Delta Virus (HDV) Ribozyme ~ Size: ~60 nucleotides.
~ Trans cleavage of target RNAs demonstrated (XXXiX].
~ Binding sites and structural requirements not fully determined, although no sequences 5' of cleavage site are required. Folded ribozyme contains a pseudoknot structure [x1].
~ Reaction mechanism: attack by 2'-OH 5' to the scissile bond to generate cleavage products with 2',3'-cyclic phosphate and 5'-OH ends.
~ Only 2 known members of this class. Found in human HDV.
~ Circular form of HDV is active and shows increased nuclease stability (Xy ' . Michel, Francois; Westhof, Eric. Slippery substrates. Nat. Struct. Biol.
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Purine Functional Groups in Essential Residues of the Hairpin Ribozyme Required for Catalytic Cleavage of RNA. Biochemistry Table 1 (1995), 34(12), 4068-76. . .
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*
d d ~

U U U U
C_ C U U N H N N U U N
. .

a ~ ~ O ~ = tn tn Q

Z ~f7Lf7N N t O Q ~ ~ ~ tnN O
tntnp Z

I~ f~~ InN '~Yc'O~Z ~ ~ ~ LnlO~ ~ M
~

N N

O

C

C_ C U U N I- O U U N
. . i' '= ~ ~ ~ ~ ~ N

~ a~a~t v o -E M M ~ ~ c O
n 7 O n ' ~ ~ N ~ r ~ O Q ' M M N N O ~ O
~

O N N tt7l()N ~YM Z ~ O N N lf)tf~r r M

C C

E z E z L O L D

V.I * .1-n d C ~ C E

U U
U U U U O ~ f- U U U U N

O O M O O O

M a c ~ N N c ~ o 4 t c ~ ~ t ~ O
n n n n ~ Q tn tnN N O ~ O
Z ~

~ LnN ~t Q ~ ~ ~ ~ r r r d d N

N j p ~ 3 J J J J J J J ~ () ~ J J J J J

'~ U p O- ~ 0.~ ~ ~ z N p ~ O.s ~ O.
~ l , _ M 00M M ~ ~ ~ d'N ~ N
(- ~

(D M M M M ~ ~f ~ N r r N N M d'M

r N N N N r (D CO = Q M M r r (~N N

w ~, C C

N

O .;~ O r Z d ~ d m 7 N >

N O ~ O COCON O O 7 ~ ~ ~tO O

~ M O 00i~r N Q ~ ~ 00InN O O
a m Ll~ CD N r r r r Z LLI r M COr I~N f~

O O
N O p ~ N O N N

_ ~O~ _ ;O ;~
O N

N
O F- ~ ~ I-Q j, ~ O Q ~, m t (~ . t t U

p~ Nt .V~ O~ O C7 tone v O 00 41 t ~ U ~ ~ U d ~ ~ U ~ ~
U N U N

a c~ a z ~ a ~ a cn a z ~
~ m ~ m *
d U U
O

N O O N N fn O O N N N N O
Q

CO (OO O ~ O O Z
M M ~ ~ M N

Z

N

d U C_ U
H >> ~ ~ N N

O O N N
N N N

O O O
op 00O O InO O Q
Z

r-- ~-~ M N

Q

Z

_ C *
O d E ~ U

O f- U U U U U U N

w N N N N N N N

c ~ Z O O O o ~nO o Q

CO COe-~ ~ M ~ Z

d z ' o d J >, ~

Z

y_~ t J O
E-~ ~t ~ In O J J z N t E O. ~ J J ~ J O v ' O O O ~ O

,. O O . . ~ 0 N

C ~ r l~l~O 0 ~
O N \ \ \ \ O 0 O
\

O O O O ~ O N
O O tf7lf~\ CO~ O

E Z O O _ _ ~ _ _ ~- d O O O O

Q D ~ ~ tn~ N 0000 >_ N d O
p1 V O 's;.

.a 3 t , , , wr , lf~N Lf7 t~
d C ~ N t~ct~ r0..

\ \ \ C
O O ~ ~ O

tnCOO I~O V

M O N ~ ~ C\O.- G7 ~ \ \ \ ~

N d0 \ -p N ~ N ~ N M

L1J t0 Z
D

V

C_ w Q7 N ~ O

y O O O N C

'a f9 f'9~ ~ O

O

Q = O I-Q ~, v~.'-, t _ N t V O ~ O ....

a ci~Q z H m Q
*

Table 3 Table 3: Human PTP-1B Hammerhead Ribozyme and Target Sequence Nt. Riboryme Seq. Substrate Seq.
Position Sequence ID Sequence ID
Nos. Nos.

72 AUCUCCAUCUGAUGAGX CGAA ACGGGCCA2 ' TGGCCCGTC ATGGAGAT530 CUGAUGAG

CUGAUGAG

CUGAUGAG

CUGAUGAG

Table 3 340. GCAUGUGU CUGAUGAG X CGAA AGGCAAAG46 CTTTGCCT A ACACATGC574 Table 3 684 UCCGGGCU CUGAUGAG X CGAA AGUGACCC107. GGGTCACT C AGCCCGGA635 -, Table 3 AAGACTGC

AAGGAAGA

CUGAUGAG AAATGCCG

AATGCCGC

CUGAUGAG AAGACACT

CUGAUGAG

CUGAUGAG

CUGAUGAG AACATGTG

CUGAUGAG AACAGCAA

Table 3 CUGAUGAG

.

CUGAUGAG

CUGAUGAG

CUGAUGAG

CUGAUGAG

CUGAUGAG

CUGAUGAG

1698 CCAGGAGGCUGAUGAGX CGAA AGCCCUGG232 ~ CCAGGGCTC CCTCCTGG760 I I

Table 3 Table 3 CUGAUGAG

CUGAUGAG

CUGAUGAG

CUGAUGAG AATAAGAA

CUGAUGAG

Table 3 AATCACTG

CUGAUGAG

CUGAUGAG

CUGAUGAG

2304. AACAGGCUCUGAUGAGX CGAA AGGAACCA356 TGGTTCCT A AGCCTGTT884 CUGAUGAG

Table 3 CUGAUGAG

CUGAUGAG

CUGAUGAG

Table 3 CUGAUGAG

CUGAUGAG

CUGAUGAG

CUGAUGAG

CUGAUGAG

CUGAUGAG

CUGAUGAG

CUGAUGAG

Table 3 CUGAUGAG

CUGAUGAG

CUGAUGAG

CUGAUGAG

CUGAUGAG

Table 3 AGUAAAUA

AUGAGUAA

ACCUGAUG

AUGACCUG

AAUGACCU

AUGGUUUA

Input Sequence = PTPN1 (Homo sapiens protein tyrosine phosphatase, non-receptor type 1 (PTPN1) 3215 bp) Table 4 Table 4: Human PTP-1 B NCH Ribozyme and Target Sequence Nt. Ribozyme Seq. Substrate Seq.
Position Sequence ID Sequence ID
Nos. Nos.

CUGAUGAG

' ICCGCCCA

CUGAUGAG

CUGAUGAG

CUGAUGAG IGAAGCUU

CUGAUGAG AAAACCGA

IACUGACG

CUGAUGAG

CUGAUGAG

Table 4 Table 4 CUGAUGAG CGAA

CGAA

CGAA

CGAA
IUUGUAAG

CUGAUGAG CGAA AAGAAACT

CGAA

CGAA

CGAA

CUGAUGAG CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA
ICCAUGUG

CGAA
IGCCAUGU

CUGAUGAG CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA
IGUGAUUC

CGAA

CGAA

CUGAUGAG CGAA
IAGGCUGG

CGAA
IAAUGAGG

CUGAUGAG CGAA

CUGAUGAG CGAA
IAAAGUUC

CUGAUGAG CGAA

CGAA

CGAA
IACUCUCG

CGAA
IACCCUGA

CGAA
IUGACCCU

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA
ICACUGCA

CGAA
ICCUGCAC

CGAA

Table 4 CUGAUGAG

~

CUGAUGAG

851 UCUGGAUCCUGAUGAGXCGAA.ICCCCAUC1214 GATGGGGC T GATCCAGA 1938 CUGAUGAG

CUGAUGAG AATTCATC

IAAGAGUC

CUGAUGAG ICUCCUUC

Table 4 CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA
IGGAUAUG

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA
IGGAGGUG

CGAA

CGAA

CGAA
IUGGCCGG

CGAA AAACGAAT

CGAA
IGGUGGCC

CGAA
IAUUCGUU

CGAA

CGAA

CGAA

CGAA

CGAA
IUGUGGCU

CGAA
ICAUUUCC

CGAA

CGAA

CUGAUGAG CGAA AAATCACC

CUGAUGAG CGAA AATCACCA

CGAA

CGAA

CUGAUGAG CGAA
IGUGAUUU

CGAA

CGAA
IGUCUCUU

CGAA

CGAA

CGAA
ICAGUCUU

CGAA

Table 4 CGAA

CGAA

CGAA

CGAA

CGAA

CUGAUGAG CGAA

CGAA

CGAA

CGAA

CGAA

CGAA
IGUGCGGC

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA .

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA
IGAGGCAG

CGAA

CGAA
IGGGAGGC

CGAA AAAGGGGA

CUGAUGAG CGAA AAGGGGAG

CGAA

CGAA

CGAA
IUGACGGC

CGAA

CGAA
IGCAGUGA

CGAA

CGAA

CGAA
ICAUGGUC

CGAA

CGAA

CGAA

CUGAUGAG CGAA

CGAA

CGAA

CGAA GGTCAACA

CGAA
IACCAGGA

CGAA

Table 4 CGAA
ICCACGCA

CGAA

CGAA

CGAA

CGAA
ICCGUGAG

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CUGAUGAG CGAA

CGAA

CGAA

CGAA

1372 CUAUGUGUCUGAUGAGX ICUGUUGA1350' TCAACAGC A ACACATAG 2074 CGAA

CGAA

CGAA

CGAA
ICUAUGUG

CGAA
IGCUAUGU

CGAA
IUCAGGCU

CGAA
IGUCAGGC

CGAA

CGAA

CGAA
IGAGGGUC

CGAA

CUGAUGAG CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA
IUGGAGGU

CGAA
IGUGGAGG

CGAA

CGAA
IUGGGUGG

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

Table 4 CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

1499 UCCGUCCGCUGAUGAGX IGCCCGGC1394 GC.CGGGCC C CGGACGGA 2118 CGAA

CGAA

CGAA

CGAA

CUGAUGAG CGAA AAAACCCA

CGAA

CGAA

CGAA

1533 UCCGGGGACUGAUGAGX IAUGGGUU1402 AACCCATC T TCCCCGGA 2126.
CGAA

CGAA

CGAA

CGAA

CGAA
IACACACA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CUGAUGAG CGAA

CGAA

CGAA

CGAA

CGAA

CUGAUGAG CGAA

CGAA

CUGAUGAG CGAA

CGAA

CUGAUGAG CGAA

CUGAUGAG CGAA AAATCCAC

CGAA AATCCACA

CGAA

CGAA

CUGAUGAG CGAA

CGAA

fable 4 CUGAUGAG CGAA

CUGAUGAG CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CUGAUGAG CGAA

CGAA

CGAA

CGAA

CGAA

CGAA
IGCGAGCC

CGAA

CGAA ' CGAA

CGAA

CGAA ' CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA
IAUGCUCC

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CUGAUGAG CGAA

CUGAUGAG CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA
IAGUUGCU

CGAA

Table 4 IGAGAGUU

IUGGAGAG

IAGUGGAG

IGAGUGGA

IUUUAAAU

IAAAAAAU

IGAAAAAA

IGGAAAAA

IGGGAAAA

ICCUUUGG

IAUGCCUU

IGAUGCCU

ICACUAUG

IUGCACUA

ICUAGUGC

IAAAAUGC

IUUCAAGA

IGUUCAAG

IACAUCAA

ICUGACAU

IGCUGACA

ICAAGGCU

IAUGCAAG

ICCCUUGA

IAUAAAGC

IUACUUUU

IAUUUAUU

IGAUUUAU

IAGGAUUU

IUACUACC

ICCUUCCA

ICAAAGCC

IGCAAAGC

ICCCAUGG

IGCCCAUG

ICAGGCCC

IACGCAGC

IUCUGACG

IGUCUGAC

IUACUGGU

ICUUACAA

IUUCUAUC

ICAUUGUU

IUUCAUUA

IUUUCUUA

IUAAUCUC

IACAAAGU

Table 4 CUGAUGAG AATGTGCC

CUGAUGAG AAGTCCAA

CUGAUGAG

CUGAUGAG

Table 4 CGAA

CGAA

CUGAUGAG CGAA

CGAA

CGAA

CGAA

CUGAUGAG CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CUGAUGAG CGAA

CGAA

CUGAUGAG CGAA AATTCCTG

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CUGAUGAG CGAA

CUGAUGAG CGAA AAGGGTAT

245.7 CAUACCCUCUGAUGAGX IGAAUGUC1610 GACATTCC A AGGGTATG 2334 CGAA

2472 GUGAAUAUCUGAUGAGX ICUUCCCA1611 TGGGAAGC C ATATT'CAC 2335 CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

Table 4 CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CUGAUGAG CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CUGAUGAG CGAA AAGTCGAC

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CUGAUGAG CGAA

CUGAUGAG CGAA

Table 4 CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CUGAUGAG CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CUGAUGAG CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CUGAUGAG CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CUGAUGAG CGAA

CUGAUGAG CGAA

CGAA

CGAA

CGAA

CGAA

2819 AGCUCGGG X ICUACAGC1709 GCTGTAGC T CCCGAGCT 2433' CUGAUGAG CGAA

CGAA

Table 4 CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA
IAAAAUGC

CUGAUGAG CGAA

CUGAUGAG CGAA
ICAAAAUG

CUGAUGAG CGAA
IGCAAAAU

CGAA
IAAAGGCA

CGAA
ICUUCUAC

CGAA

CGAA

CGAA

CUGAUGAG CGAA

CGAA
IACACCUC

CGAA

CGAA

CGAA
IGGUGACA

2922 CAUAGCUCCUGAUGAGX ICAGGGUG1733 CACCCTGC A GAGCTATG. 2457 CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CUGAUGAG CGAA

CGAA

CGAA

CGAA
ICCAGCUC

CGAA
IACUUAAA

CGAA
IGACUUAA

CUGAUGAG CGAA

CUGAUGAG CGAA AAATGGAC
IAAAUGCU

CGAA

CGAA

CGAA

CGAA

CGAA
IGAGGUUA

CUGAUGAG CGAA
IAUAGGAG

CGAA

CGAA
ICUCUCCA

CGAA GGCTCTCC

CGAA

CGAA

Table 4 CUGAUGAG

CUGAUGAG

CUGAUGAG

AATATTTA

-CUGAUGAG

N O r-IN M CWf1~Of~0001O r1N M C~1f1l0l~c001O ~-IN M VWf1\Ol~

O I~l~f~l~f~l~I~L~hI~W COODN ODW N ODODc00101Q1O~O~O~O~O~

Q,~D~O~O10~OlDlDtD~D~D~D~O~O1D~Ol0lO~Dl0l0~Ol0~D~O~O~O~O~O
~

y N N N N N N N N NN N N N N N N N NN N N N N N N N NN

C7C~a C7C7C7C~U Ua F F H U aU F U H U F a Ua H U F a F U FH a a C7 ~ H ~ t7a ~ ~ F a C7~ C~U U ~ a C7F U F a F C7U C7U U U C U C ~ ~
a C7a U U U U U~ U U F H U C7F FC7a F 7 ~ F 7 .

~ U aa y U a U U a a a a a a U a U U U aU U a a c C7C7C7C~C7C~C7C7C7U C7C7C7C7U C7C7C7C7C7C7C7U C7C7C7L7C7 ai U U U U U U U H HU F F U H F F F HF U F F H H F F UU

a~C7U C~U F F U a C7U F H F a a C7 C7a H H F U C7C7F

C7C7C7C7F a F U a H ~ ~ F H U U a N C7F F a F a HU
s.U C7a a C7C7a a U U H U C7U a F C7~ a U U U U C7 ,"''"a C7C~U a a F C7U U a H a U U C7a C7F F a a ~

C7F C~C7C7U a U C7 U C7a F U ~ U UF ~ a ~ a H U
a C7 a C7C7C7U U F F U C7 F F ~ C7U H C7a a /~U a ~ U a C7F ~ C7~ a C7C7H C7 C7F F a F F F F

C7U C7C7~ C7a a C7 a C7F U C7U F UU E-~ a U H

~ a U
C7C7a U U U F F U a U a a U C7HF C7 C7a C~

v7 A

~ ' ~ ' ~

Q,u11Dt N 01O r1N MV Lf1lOI W O~O r1NM V Lf110l N Q1O r1N
O

O O O O O r-1riv-1r1r1n-1r1r1r1r1N N NN N N N N N N M MM

t11,Lf1LfWf1U1l17Lf1Lf1Lf7Lf7f1t!1Ll1LfiLf7L11U~Ll11I1~f1UWf1Lf1Ll1Lf1Lf1LfWf1 N N N N N N N N NN N N N N N N N NN N N N N N N N NN

C~

U U ~ C7 C7C7a a C7~ C7a ~ C7U aa U U ~ ~ ~ aC7 U C7U U ~ U ~ U~ ~ U a 7 U 7 a 7 ~ C C C7C ~ C7 t7~ ~ C7 ~ U a ~C7U ~ ~ U U a U U ~ a ~ ~ a ~

~ U ~ U U U C7 a a C7U a U C7 U ~ ~U

U a U U U C7~ C7U C7U ~ a C7 C7C7 ~ U U C7~ ~ a U C7 C7~ a ~ C7C7U ~ U a y C7U ~ ~ U U ~ ~ C7 C7a C7U C7~ a U ~ C7 C7C7C7U
U U U U ~ a C7~ C7C7~~ a U ~ ~ a ~ aC~
U C7U C7~ a C7~ U7 ~ ~ ~ ~ ~ U U ~ ~ ~ C7U Ua >, ~ c~~ c~c~c~~ a a~ a ~ a a aa ~ a a a ~

C~C7C7C7C7C7C5C7C7C7C7C7C7C7C7C~C7C7U C7C~C7C7C7C7C7C7C7 U U U U U U U U UU U U U U U U U UU U U U U U U U UU

C7C7C7C7C7C7t7C7C7C7C7C7C7C7U C7ChC7C7C7C7C7C7C7C7C7C7C7 U U U U U U U U UU U U U U U U U UU U U U U U U U UU

y~ o C~C7C~C7C7C7C7C7C~C7C7C7C7C~C7C7U C7C7C7C7C7C7C7C7C7C~C7 y C ~ ~ ~ ~ ~ ~ ~ ~ ~~ ~ ~ ~ ~ ~ ~ ~ ~~ ~ 5 ~ ~ ~ ~ ~ 5 a a a a a a a a aa a a a a a a a aa a a a a a a a aa ~ a U U U U U U U U U U U U U U

U U U U U U U U U UU
E

a a a a a a a a aa a a a a a a a aa a a a a a a a aa U U U U U U U U U UU U U U U U U U UU U U U U U U U UU
' ~ c~c~~ c~ca~ c~~~ c~c~c~c~c~c~~ ~~ ~ ~ ~ ~ ~ ~ ~ c~
a ~

C'7 ~ a a a a a a a a aa a a a a a a a aa a a a a a a a aa CGU U U U U U U U UU U U U U U U U UU U U U U U U U UU

C7U C7C7C7C7C7C~UC7C7C7C7C7C7C7C7C7C7C7C~C7C~C7C7C~C7C7 C7C7C7C7C7C7C7C7C~C7C7C7C7C7C~C7C7UC7C7C~C7C7C7C7C7C7C7 a a a a a a a a aa a a a a a a a aa a a a a a a a aa ~ c~

~ U ~ C7U C7C~7~ C7 C~7~ a a C7U a aU ~ a ~ ~ a ~ U C~.7U

U U C7C7 ~ U C7a ~ a U C7UC7C7C~U ~~----''C7U
a C7a ~ a C7 a ~ ~ U ~ a U~ ~ a ~ C7UU

U U ~ U U U U C7C7~ a a a C7 ~C7 C7 C~a ~ C7 x r ~ t!11f11Dtf1OWD O M NIIIV'O N O r-1N V'~OO~r1~OM L~01~DN CM
w , Z N M V't!10001M ~'Ll101N 10I~N M ~'01ON M l0I~0001O M Mt i n .-Iri,--Ir1N N N N M M M ~V~V~V~V'V~V~1f1Ll11f1L!1 O

m O~O v-1N M ~ tnl0r m 01Or1N M V'~f110r m 01O v-IN M V'Lf110rm 0101O OO O O O O O O O r-1~-1r1r1~-ir1n-I,-Ir1r1N N N N N N N NN

~n~ r rr r r r r r r r rr r r r r r r r rr r r r r r r rr N N N NN N N N N N N N NN N N N N N N N NN N N N N N N NN

E-~a E~FC~C7U U H H a U E-~~ a C7F f-~a E~ aU E-~U C7a a a UU

U U ~ U H a U U U U H U U ~ Ua U H U U U U UU

H H ~ C ~ C C r.(C7C7 C C.C C C C a 7 .7.7 7 77 7 7 .7 r~r~ r~ r~ a r~
U U U U U U U U ~ U U U U
.

~ a U ~ ~ U~ ~ ~ U a ~ ~ 2 ~

C7C7C7C~C7C~C~C7C7C7C~C7C7C7C7C7C7C7C7C~C7C7C~C7C7C7C7C7C~C7C7 H H U F HH U U F H H U U H H F

U U H UC U C U C U N C U U C C H .7C~ C U UH
_7 7 -~ 7 7 _7 C 7 7 U H U U U ~ U U a UU ~ ~ U a ~ U

C U H tH C ~ H H U ~ C ~ ~ U L ~ ~ C C H
7 7 7 7 7 .77 C7E-~E-~ C7C7C7E-~E-~U E-~U C7C7U U C7C7E-~ E~U U U C7E E-~ C~

F C7a ~E~E-~E-~U a U U E-~C7a C7a U a C7C7C7E~E-~U U C7C7~ E-~ C7 a a U E-~C7U E-~C7C7N E-~ E-~E-~C7a U E-~H U UE U U C7a U U

U C7E-~UC7C7a C7H H U U ~E-~a a C7U U U E-'HE-~U a E-~U F U E-~a a C7U E~U F U E-~U U U H UC~C7U U a U C7a UU U U ~ U a U HH

U E-~U E-~U C7C7U C7U C7E-~E-~E-~C7U U t7a t7C7aC7C7U a C7U UU

M V'tf11Dr m O~O r1N M ~'t!1~Dr m 01O v-iN M ~tf11Dr m O~O '-INM
M M M MM M M V~V'V'~'~ ~'V'V'V'V'Lf1IfWf1t11If1Lf1t!1Lf1tf1t!1~O1D~D~O

u1InV1Lf1Lf1~f1If7f1U~Lf1tf1~1f1U1U1l11LOLf1Lf1111L11Lf1Ll1tf1t!1LJ1Lf1Lf1LOtf1 Lf1Lf1 N N N NN N N N N N N N NN N N N N N N N NN N N N N N N NN

C7a t7 C7U U C7U C7U aa U C7C7U ~ U U ~U U C7 ~ U C7C~C7 ~ U C7~C7a C7a C7C7C7~ C7U U C7C7~ C7.U ~ C7C7C7C7 C7~ C7 a C7U a C7U U ~ U a a C7C7 ~ ~ U C7C7C7a a C7~ a C7a C7 ~ ~ C7 U C7a U U a ~~ a U ~ C7a a C7C~ C7C7U ~ C7 C7 ~

a a C7~ C7C7~ U~ U ~ C7~ U U U C7C7U U U
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y C7 U U C7 a H H a C7 C7 C7 H a H C7 a U C7 C7 a U a U U H U
p a C7 U H C7 U ~ H U C7 H C7 U C7 a C7 U a a U U U C7 H C7 ~ U C7 C' H C7 C7 U U U H H H U C7 H H C7 C7 C7 U C7 U C7 U H U U C7 U U U
U U U U H U H U U U H U U H H H H U U H U U U U U H U U
ar U .a U U a H a a U U H H U a H a a U a U U U U H U H a H
C7 C7 C7~ C7 C7 C7 C7 C7 C7 C7 C7 C7 C7 C7 C7 C7 C7 C7 C7 C7 C7 C7 C7 U C7 C7 v C7 a a U a a C7 H a a U H H H U C7 H a U a F-~ H H C7 C7 H H H
U U U U U U U U U U U U U U U U U U U U U U U U U U U U
p C7 C7 C7 C7 C7 H H U U H U H U C7 H H C7 a C7 U a C7 a a a U U a V1 U C~ a a a C7 C7 C7 a U C7 H a H H H C7 C7 a a C7 C7 U H U H C7 U
a U V U C7 U a C7 U a C7 U H H U H C7 a U C7 a a H U H H a U
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a a a a a a a a a a a a a a a a a a a a a a a a a a a a ~ a ~
c~wc~~~~~~~c~~~~~~~~c~
c~ ~ c~ ~ c~ ~ ~ ~ ~ ~ ~ c~ c~ c~ ~ ~ c~ ~
U U U U U U U U U U U U U U U U U U U U U U U U U U U U
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a a a a a a a a a a a a a a a a a a a a a a a a a a a a U U U U U U U U U U U U U U U U U U U U U U U U U U U U
a a a a a a a a a a a a a a a a a a a a a a a a a a a a C7 C7 C7 C7 C7- C7 C7 C~ C7 C7 C~ C7 C7 C7 C7 C7 C7 C~ C~ C7 C7 C7 C7 C7 C~ C7 C~ C7 ~ " x x x x x x x x x x x x x x x x x x x x x x x x x x x x a a a a a a a a a a a a a a a a a a a a a a a a a a a a U U U
N
a C7 C7 C7 C7 C7 C7 C7 C7 C7 C7 C7 C7 C7 C7 C7 C7 C7 C7 C~ C7 C7 C7 C7 C7 C7 a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a o U U U U U U U U U U U U U U U U U U U U U U U U U U U U
U U U U U U U U U U U U U U U U U U U U U U U U U U U U
a a a a a a a a a a a a a a a a a a a a a a a a a a a a CC ~ C7 C7 C7 U C7 ~ U C7 ~ U C7 a ~ C7 U ~ C7 U ~ ~ a C~ a ~ C7 C7 U ~ ~ ~ U U U ~ C7 U ~ U U ~ U U C7 a C7 U C~
U U U U U a a C7 C7 a C7 ~ C7 U ~ ~ U ~ ~ C7 ~ U ~ ~ ~ C7 ~
C~ C7 C7 C7 C7 C7 C7 C7 C7 C7 C7 C7 C7 C7 V C7 C7 V C7 C7 C7 C7 C7 C7 C7 C7 C7 C~ C7 C7 C7 C7 C7 C7 C7 C7 C7 C7 C7 C7 C7 C7 C7 C7 C~ C7 C7 C7 C7 C7 C7 C7 C7 a a a a a a a a a a a a a a a a a a a a a a a a a a a a a U U C7 C7 C7 a a C7 U a a U U U C7 U C7 U C7 a C7 C7 U C7 C7 C7 ~ U Ch a U C7 ~ C7 U a U C7 U ~ U C~ ~ ~ U U U U ~ U ~ a C7 U C7 L7 U ~ ~ U U U ~ a U ~ C7 U U ~ C7 ~ C7 C7 ~ U C7 U C7 ~ ~ U ~ ~ U C7 ~ C7 ~ U U C7 C7 C7 a C7 U ~ U C7 C7 C~
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N M M M M r M M M M M M ' ' ' M M
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( 7,~U H C U C H 7 7 U7 ~ 7 7 H H H U VF U H 2 U H C7HU U 7 rU 7 F U K 7 C C H CC C C H 7 H C U U7 t 7 U 7 H H UU C U
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UU U ~CU U U U H U UU H U FCH H H H H'H H U H H H U UU U U
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7 C7C7C7C7O C7UU C~U C7U C~C7C7C7U C7C7U C7U C7C7C7C7C7 H H ~C~CHU FC~C

C7U C9C U C7C7C7C7U ~CFCH FC2 C7FCU H HH H H U.U U U UU U U
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UU U U d U U FCH U V U FCO U H r~U H U U C7U d ~C
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M~ ~ ~pr m 01O ~ N MV'U1\Or m ~ O ~ NM ~'~1lOr m 01O~ N M
mm'm m m m m 01~ ~ ~p~p~p~p1O~OvO O OO O O O O O O r1r1.-~~-1 N N N N N N N NN N N N N N M M MM M M M M M M MM M M
' ' V'C V'C'C~d'V'd' NN N V'V'V'C'C V'C'v1'd'V'd'V'd'~'d'V'V'C d d d'V'~' dd d d d a a d d d da a a a d d d d dd d d d a a d aa d d r ~~ ~ a ~ ~ ~ ~ ~ ~ ~~ ~ ~ ~ ~ ~ ~ ~ a~
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UU U U U U U U U U UU V U U U U U d ad d a a a a d dd a a dd d d d a d a a d da a a a a d a a ~
c~~ ~ c~~ ~ ~ ~ ~ ~ c~~ ~ O ~ ~ c~~ ~ ~c~c~c~~ c~~ ~ ~~ c~c~
x xx x x x x x x x xx x x x x x x xx x x xx x x x x x x x a ad a a a a d d a da d d d d a a ad a a dd a a a a d d d ~ ~ ~ c~~ ~ c~

c~~ ~ c~c~~ c~O c~O c~~ ~ O ~ ~ ~ c~~ OO ~ ~ ~ c a a da a a da a d d d a a d d ad a d a d d a a aa d a a a O ~ O
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aa d U U U U U U U UU U U U U U U U UU U U U U U U UU U U
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C7C7C7U C7C7U C7C7~~CU C7U U ~ U U ~Cd O 7 ~ U FCC7UC7~
U C7U U C7U C7~ UU U U ~ U d ~ U C7C7U C7C7C7FC

UC7C7U U C7C7~ C7C7~~CC~Q C7~ V C7C7~C7FCC C7C7U U C7C~C7U
dFCC~U O C~C7C7C7U C7U C7C7C7U C7C7 C7C7C7C7 OC7C7C~C7C7C7C7C7C7C7C7C7C7C7C7C7C7C7C7C7C7C7C7U U C7UC7U O
aa d a a a d a d d aa a a a a a a a ad a d a d a a aa d d FCC7FCU U C7U U U ~ C7~ U C~C7U U FCC7C7U ~CFCC7U ~ FCC7C7FCC7 C7U U U U U U FC C7 C7~ ~C~t ~ C7C7 ~ U ~ C7 U FCC7C7U
rC7U FCU C7~ ~ U U C7U U U U ~ C7C7 C7FC FCC7FCUC7C7FC
~ ~ ~ d C7~ rlU ~ U C7C7UC7U U

.~ C7~ C7U C7U FCU C7C~FCFCU C7~ ~ a U ~ ~ C~7~ U EC~C~7CU7 UU ~ U C7~CU FC~ U CU.7~ ~ C~7~
C7~ U U ~ FC
U

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r-IN V'l0r r O O M r Ml0C U'11Dr m OvO V'01M ~Dr O V'r1r1N d'OD
' ' V'V'V W 7 1f110rm O1O~01O101O r1r1r1N N N M M d~V~t!11f1Lf1 I f1 N N N N N NN N N

V C ,~,~,~r1r-iN N NN N

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c fw w r e f'r ~ v ' r ra < ' ~ v <

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C H C 7H U H H H U U U U~ H ~ H

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C H C HC7~ H ~ U C C7C7~C~CFCH H

H U U UU U H U H U ~CU C7C7C7C7C7 U H U 7 C~H C7. U C7 U U
U

C~C7C7C7C7C U H C7H H FCH~CH ~CH
H r.~(~r.~H U U U U U U U UU U U U

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,~'-1,-Ir1r1N N N N N N NN N N f'1 ''1M f"1f1f'1f~1f''1('1r'1f"1r'~1c'~1('~1f'~1M M t''1 V'C V'd'd'~PV'V'V'V'V'V'd'd'd'V' r' a d a da d d a a a a d da a a a c~~ c~c~

a ~ ~ ~~ ~ ~ a ~ ~ ~ ~ w~
H U U U UU U U U U U U U UU U U U
ECFCFCFC~C~Cd ~CFCFLFCFtr-~d a ~C~C

d a d da d d d a a d d dd d d a U U U UU U U U U U U U UU U U U
a d a dd a a d a d d d dd d d a ~5 C7C7C~C7C7C7U C7C7C7C7C7C7C7C7C7C7 x x x xx x x x x x x x xx x x x U U U UU U U U U U U U UU U U U

a d a da a d a a a a d dd d d d c~~
d a a da d d d a a d d dd a a a U U U UU U U U U U U U UU U U U
U U U UU U U U U U U U UU U U U
d d a aa a a a a a d a aa a d d C7 ~C~~ C7C7FCFCC7U C7C7~ U U U
U UU FCC7C~C7U U U ~U
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U C~C7C7FCU C7~ FC~ ~CU ~CC7U
U C7U rCU U C7C7U C7~ U FCC7 7 a C7C7C7C7U ~CC7a C7FCa Ua C C7 C7d ~CC7~ ~ ~C~
U U C7C7~C

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p m o ro0ovo ,~N M m ior w ov o ,~N

C~ N N NN N M M M M M M MM M M V'V'V' ~ ~ V~V~V~V~V'V~V~V~V~ C~V~V~V~V~ ~ V~V~
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t/ ~ w v~~ a~~rc~~ ~ w a ~~ra~~ w w w a U aU U a U ~ U U U UU ~ U

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O I~ODO~O H N M V'Lf1lD L~NO~O H N M V' O O OH H H H H H H' ~I~--W--AN N N N N

V~V'V'd'~'V~V'V'd'V' ~'V'C C V' ~'V'~' ~

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U U UU U U U U U U U UU U U a a a x ~ 7 C7C7C7C7C7 U U U

~a C7C7C7C7C~C7C7C7C7C C~C7C7C7C7 C~C7C7 x C~C7C~C7C7C7C7C~C7C7 N a a aa a a a a a a a aa a a ~

o c~~ c~c~~ ~ c~c~~ ~ c~c~~ c~~ a a a ~ ~ ~ ~ ~ ~ ~ ~~ ~ ~ U

a a U C~
a C7C7C~C7C~C7C7C7C~C7 C7C7C7C7C7 C7C7 ~.

U U UU ~ U U U U U U UU U U ~

~ a ~ ~ a a ~ ~ a 5~ ~ U ~ 5 ~

C ~ C ~ 7 7 ~ a a C7C7C7C7C7a U U U U C~C7C C U U U
C7a aC7C7U a a a L7aU C7 a ~ C7a ChC7U C7U a ~ Ua ~ ~ U a C7 ~ c~c~~ ~ a a c~c~c~ ~ ac~c~a ~

o ~ c~~~ ~ ~ c~a ~ ~ ~ c~~ a c~ c~c~c~

x ...

MODV'ODriM 00N 1f1Wt!1ODM SrI~V'e-1 . ~ ~ 01O~O r1OD07M Lf1 01O1f7I~H 47~ON V~

O MM VW-1'-I~-IN N M ~'lf1U1"~' N H N

~ y -i.-Ir1N N N N N ("~r1rdr-Ir1N ~ '-VN N

O x ~ .. . . . . . . , ~~ , ~ ~,~ , , ~ ~ ~ ~ ~ ~ _ ~ ~ ~

x ~~ ~ ~ ~ ~ ~ ~ ~ ~

E-~E-~E-~E~F E-~E-~F H EE-~E-~E''urW

GLCLCLW P~CLC4W C4C4CLC4W

Table 9 Table 9: Human, methionine aminopeptidase type 2 (Met AP-2) Hammerhead Ribozyme and Target Sequence Nt. Riboryme Sequence Seq Substrate Sequence Seq osition ID 1D
nos. nos.

368 AACUGAGG CUGAUGAG X CGAA AGGGUCUG22 CAGACCCT C.CCTCAGTT 434 393 CAUUAGGA CUGAUGAG X CGAA ACAGGUCA.27 TGACCTGT A TCCTAATG 439 527 UUCUCGAA CUGAUGAG X CGAA AUCAUUCC42 GGAATGAT T TTCGAGAA ~ 4541 Table 9 604 ~ CAGAUUUC CUGAUGAG X CGAA 52 ACAATGAT A GAAATCTG 464 AUCAUUGU

' Table 9 1031 AUGUAUUC CUGAUGAG X CGAA AUAUUGCC.133 GGCAATAT A GAATACAT 545 -Table 9 1362 UGUCACAU CUGAUGAG X CGAA AUGGUGGA181 TCCACCAT T ATGTGACA 593' Table 9 ' I

Table 9 1585 UCUUUUfIU CUGAUGAG X CGAA AACAUGGA233 TCCATGTT T AAAAAAGA 645 ,1648 AAAGUCCG CUGAUGAG X CGAA AAAGCUUU244 AAAGCTTT C CGGACTTT 656 1717, GUCAUUCC CUGAUGAG X CGAA AACUAAUU265 AATTAGTT A GGAATGAC 677 Table 9 i 1760 UAUCCAAA CUGAUGAG X CGAA AGUAUCUC278 GAGATACT T TTTGGATA 690 Table 9 Table 9 2377 UUUUUGAA CUGAUGAG X CGAA AGUUtJWCT386 AAAAAACT C TTCAAAAA 798 2379 AU<JUUUUG CUGAUGAG X CGAA AGAGUUUU387 AAAACTCT T CAAAAAAT 799 2380 GAUfJUUULT CUGAUGAG X CGAA AAGAGUUU388 AAACTCTT C AAAAAATC 800 2388 GAUUCAUU CUGAUGAG.X CGAA AUUUUUUG389 CAAAAAAT C AATGAATC 801 2450 , UUCUUUAU CUGAUGAG X CGAA AGUCUUGC400 GCAAGACT A ATAAAGAA 812 Input Sequence = HSU29607. Cut Site = UH/.
Stem Length = 8 . Core Sequence = CUGAUGAG X CGAA (X = GCCGUUAGGC or other stem II) Seq 1 = HSU29607 (Human methionine aminopeptidase mRNA, complete cds., 2569 bp) Table 10 Table 10: Human methionine aminopeptidase type 2 (MetAP-2) NCH Ribozyme and Target Sequence Nt. Ribozyme Seq. Substrate Seq.
osition Sequence ID Sequence ID
Nos. Nos.

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA
IGCUCCCG

CGAA

CGAA
IGUGGCUC

CGAA

CGAA

CGAA

CGAA

CGAA

CUGAUGAG CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CUGAUGAG CGAA
ICUGCUUC

128 WUUUIJCU CUGAUGAGX IGCUGCUU 849 AAGCAGCC A AGAAAA.AA1279 CGAA

CUGAUGAG CGAA

CUGAUGAG CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA

CGAA
IUCUUGCU

CGAA

CUGAUGAG CGAA

DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.

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Claims (294)

Claims:
We claim:

1. An enzymatic nucleic acid molecule having formula 4 namely:

wherein each X, Y, and Z represents independently a nucleotide which may be the same or different; 1 is an integer greater than or equal to 3; m is an integer greater than 1 ; n is an integer greater than 1; 0 is an integer greater than or equal to 3; Z' is a nucleotide complementary to Z; Y' is a nucleotide complementary to Y; each X(1) and X(o) are oligonucleotides which are of sufficient length to stably interact independently with a target nucleic acid sequence; W is a linker of >= 2 nucleotides; A, U, G, and C represent nucleotides; C is 2'-amino; and ~ represents a chemical linkage.

2. An enzymatic nucleic acid molecule having formula 5 namely:

wherein each X, Y, and Z represents independently a nucleotide which may be the same or different; l is an integer greater than or equal to 3; n is an integer greater than 1; 0 is an integer greater than or equal to 3; Z' is a nucleotide complementary to Z;
each X(l) and X(o) are oligonucleotides which are of sufficient length to stably interact independently with a target nucleic acid sequence; W is a linker of >= 2 nucleotides in length or may be a non-nucleotide linker; A, U, G, and C represent nucleotides; C is 2'-amino; and ~ represents a chemical linkage.

3. The enzymatic nucleic acid molecule of claims 1 or 2, wherein 1 is selected from the group consisting of 4, 5, 6, 7, 8, 9, 10, 11, 12, and 15.

4. The enzymatic nucleic acid molecule of claim 1, wherein m is selected from the group consisting of 2, 3, 4, 5, 6, and 7.

5. The enzymatic nucleic acid molecule of claims 1 or 2, wherein n is selected from the group consisting of 2, 3, 4, 5, 6, and 7.

6. The enzymatic nucleic acid molecule of claims 1 or 2, wherein o is selected from the group consisting of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 15.

7. The enzymatic nucleic acid molecule of claims 1 or 2, wherein l and o are of the same length.

8. The enzymatic nucleic acid molecule of claims 1 or 2, wherein l and o are of different length.

9. The enzymatic nucleic acid molecule of claims 1 or 2, wherein the target nucleic acid sequence is selected from the group consisting of an RNA, DNA and RNA/DNA mixed polymer.

10. The enzymatic nucleic acid molecule of claims 1 or 2, wherein said chemical linkage is selected from the group consisting of phosphate ester linkage, amide linkage, phosphorothioate, and phosphorodithioate.

11. The enzymatic nucleic acid molecule of claims 1 or 2, wherein said C is selected from the group consisting of 2'-deoxy-2'-NH2 and 2'-deoxy-2'-O-NH2.

12. A method for inhibiting expression of a gene in a cell, comprising the step of administering to said cell the enzymatic nucleic acid molecule of claims 1 or under conditions suitable for said inhibition.

13. A method of cleaving a separate RNA molecule comprising, contacting the enzymatic nucleic acid molecule of claims 1 or 2 with said separate RNA
molecule under conditions suitable for the cleavage of said separate RNA molecule.

14. The method of claim 13, wherein said cleavage is carried out in the presence of a divalent cation.

15. The method of claim 14, wherein said divalent canon is Mg2+.

16. The enzymatic nucleic acid molecule of claims 1 or 2, wherein said enzymatic nucleic acid molecule is chemically synthesized.

17. The enzymatic nucleic acid molecule of claims 1 or 2, wherein said enzymatic nucleic acid molecule comprises at least one ribonucleotide.

18. The enzymatic nucleic acid molecule of claims 1 or 2, wherein said enzymatic nucleic acid molecule comprises no ribonucleotide residues.

19. The enzymatic nucleic acid molecule of claims 1 or 2, wherein said enzymatic nucleic acid molecule comprises at least one 2-amino modification.

20. The enzymatic nucleic acid molecule of claims 1 or 2, wherein said enzymatic nucleic acid molecule comprises at least three phosphorothioate modifications.

21. The enzymatic nucleic acid molecule of claim 20, wherein said phosphorothioate modification is at the 5'-end of said enzymatic nucleic acid molecule.

22. The enzymatic nucleic acid molecule of claims 1 or 2, wherein said enzymatic nucleic acid molecule comprises a 5'-cap or a 3'-cap or both a 5'-cap and a 3'-cap.

23. The enzymatic nucleic acid molecule of claim 22, wherein said 5-cap is phosphorothioate modification.

24. The enzymatic nucleic acid molecule of claim 22, wherein said 3'-cap is an inverted abasic moiety.

25. A compound having the formula 3:

wherein R is independently any nucleoside selected from the group consisting of 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 histidyl) amino uridine; 2'-deoxy-2'-amino-5-methyl cytidine; 2'-(N .beta.-carboxamidine-.beta.-alanyl)amino-2'-deoxy-uridine; 2'-deoxy-2'-(N-.beta.-alanyl)-guanosine;
2'-O-amino-adenosine; 2'-(N lysyl)amino-2'-deoxy-cytidine; 2'-Deoxy -2'-(L-histidine) amino Cytidine; 5-Imidazoleacetic acid 2'-deoxy uridine, 5-[3-(N-4-imidazoleacetyl)aminopropynyl]-2'-O-methyl uridine, 5-(3-aminopropynyl)-2'-O-methyl uridine, 5-(3-aminopropyl)-2'-O-methyl uridine, 5-[3-(N-4-imidazoleacetyl)aminopropyl]-2'-O-methyl uridine, 5-(3-aminopropyl)-2'-deoxy-2-fluoro uridine, 2'-Deoxy-2'-(.beta.-alanyl-L-histidyl)amino uridine, 2'-deoxy-2'-.beta.-alaninamido-uridine, 3-(2'-deoxy-2'-fluoro-.beta.-D-ribofuranosyl)piperazino[2,3-D]pyrimidine-2-one, 5-[3-(N-4-imidazoleacetyl)aminopropyl]-2'-deoxy-2'-fluoro uridine, 5-[3-(N-4-imidazoleacetyl)aminopropynyl]-2'-deoxy-2'-fluoro uridine, (2-carboxyvinyl-2'-deoxy-2'-fluoro uridine, 5-[3-(N-4-aspartyl)aminopropynyl-2'-fluoro uridine, 5-(3-aminopropyl)-2'-deoxy-2-fluoro cytidine, and 5-[3-(N-4-succynyl)aminopropyl-2'-deoxy-2-fluoro cytidine.

26. A process for incorporation of the compounds of claim 25 into an oligonucleotide comprising the step of contacting said compound with a mixture comprising a nucleic acid template, an RNA polymerase enzyme, and an enhancer of modified nucleotide triphosphate incorporation, under conditions suitable for the incorporation of said compound into said oligonucleotide.

27. The process of claim 26, wherein said RNA polymerase is a T7 RNA
polymerase.

28. The process of claim 26, wherein said RNA polymerase is a mutant T7 RNA
polymerase.

29. The process of claim 26, wherein said RNA polymerase is a SP6 RNA
polymerase.

30. The process of claim 26, wherein said RNA polymerase is a mutant SP6 RNA
polymerase.

31. The process of claim 26, wherein said RNA polymerase is a T3 RNA
polymerase.

32. The process of claim 26, wherein said RNA polymerase is a mutant T3 RNA
polymerase.

33. The process of claim 26, wherein said enhancer of modified nucleotide triphosphate incorporation is selected from the group consisting of LiCl, methanol, polyethylene glycol, diethyl ether, propanol, methylamine, and ethanol.

34. A process for the synthesis of a pyrimidine nucleotide triphosphate comprising the steps of:
a. monophosphorylation, wherein a pyrimidine nucleoside is contacted with a mixture comprising a phosphorylating reagent, a trialkyl phosphate and dimethylaminopyridine, under conditions suitable for the formation of a pyrimidine nucleotide monophosphate; and b. pyrophosphorylation, wherein said pyrimidine monophosphate from step (a) is contacted with a pyrophosphorylating reagent under conditions suitable for the formation of said pyrimidine nucleotide triphosphate.

35. The process of claim 34, wherein said pyrimidine nucleoside triphosphate is uridine triphosphate.

36. The process of claim 34, wherein said uridine triphosphate has a 2'-sugar modification.

37. The process of claim 36, wherein said uridine triphosphate is 2'-O-methylthiomethyl uridine triphosphate.

38. The process of claim 34, wherein said phosphorylating agent is selected from the group consisting of phosphorus oxychloride, phospho-tris-triazolides and phospho-tris-triimidazolides.

39. The process of claim 34, wherein said trialkylphosphate is triethyl phosphate.

40. The process of claim 34, wherein said pyrophosphorylating reagent is tributyl ammonium pyrophosphate.

41. The process of claim 26, wherein said oligonucleotide is RNA.

42. The process of claim 26, wherein said oligonucleotide is an enzymatic nucleic acid molecule.

43. The process of claim 26, wherein said oligonucleotide is an aptamer.

44. A kit for synthesis of an oligonucleotide comprising an RNA polymerase, an enhancer of modified nucleotide triphosphate incorporation and at least one compound of claim 25.

45. A kit for synthesis of an oligonucleotide comprising a DNA polymerase, an enhancer of modified nucleotide triphosphate incorporation and at least one compound of claim 25.

46. The kit of claim 44, wherein said RNA polymerase is a bacteriophage T7 RNA
polymerase.

47. The kit of claim 44, wherein said RNA polymerase is a bacteriophage SP6 RNA
polymerase.

48. The kit of claim 44, wherein said RNA polymerase is a bacteriophage T3 RNA
polymerase.

49. The kit of claim 44, wherein said RNA polymerase is a mutant T7 RNA
polymerase.

50. The kit of claim 44 or 45, wherein said kit comprises at least two different compounds of claim 25.

51. A nucleic acid catalyst comprising a histidyl modification, wherein said nucleic acid catalyst is able to catalyze an endonuclease reaction in the absence of a metal ion co-factor.

52. The nucleic acid catalyst of claim 51, wherein said catalyst is able to cleave a separate nucleic acid molecule.

53. The nucleic acid catalyst of claim 52, wherein said separate nucleic acid molecule is an RNA molecule.

54. The nucleic acid catalyst of claim 52, wherein said separate nucleic acid molecule is a DNA molecule.

55. The nucleic acid catalyst of claim 51, wherein said nucleic acid catalyst comprises at least one ribonucleotide.

56. The enzymatic nucleic acid molecule of claim 2, wherein said nucleic acid molecule has an endonuclease activity to cleave RNA of HER2 gene.

57. The enzymatic nucleic acid molecule of claim 56, wherein said nucleic acid molecule comprises sequences complementary to any of substrate sequences defined as Target sequence in Tables 58, 59 and 62.

58. The enzymatic nucleic acid molecule of claim 56, wherein said nucleic acid molecule comprises any of ribozyme sequences defined as Ribozyme sequence in Tables 58, 59 and 62.

59. A method for treating cancer using the enzymatic nucleic acid molecule of claim 56.

60. The method of claim 59, wherein said cancer is breast cancer.

61. A method for treating conditions associated with the level of HER2 gene using the enzymatic nucleic acid molecule of claim 56.

62. The enzymatic nucleic acid molecule of claim 56, wherein said enzymatic nucleic acid molecule comprises a substrate binding region which has between 5 and 30 nucleotides complementary to the RNA.

63. The enzymatic nucleic acid molecule of claim 56, wherein said enzymatic nucleic acid molecule comprises a substrate binding region which has between 7 and 12 nucleotides complementary to the RNA.

64. A mammalian cell including the enzymatic nucleic acid molecule of claim 56.

65. The mammalian cell of claim 64, wherein said mammalian cell is a human cell.

66. A mammalian cell including the enzymatic nucleic acid molecule of claims 1 or 2.

67. The mammalian cell of claim 66, wherein said mammalian cell is a human cell.

68. A method for inhibiting expression of HER2 gene in a cell, comprising the step of administering to said cell the enzymatic nucleic acid molecule of claim 56 under conditions suitable for said inhibition.

69. A method of cleaving RNA derived from HER2 gene comprising, contacting the enzymatic nucleic acid molecule of claim 56 with said RNA molecule under conditions suitable for the cleavage of said RNA molecule.

70. A pharmaceutical composition comprising the enzymatic nucleic acid molecule of any of claims 1 or 2.

71. A pharmaceutical composition comprising the enzymatic nucleic acid molecule of claim 56.

72. A method of treatment of a patient having a condition associated with the level of HER2, wherein said patient is administered the enzymatic nucleic acid molecule of claim 56 under conditions suitable for said treatment.

73. The method of claim 72, wherein said method is performed in conjunction with one or more other therapies.

74. The method of claim 59, wherein said enzymatic nucleic acid molecule is used in conjunction with one or more other therapies.

75. The enzymatic nucleic acid molecule of claim 56, wherein said enzymatic nucleic acid molecule comprises at least one sugar modification.

76. The enzymatic nucleic acid molecule of claim 56, wherein sam enzymatic nucleic acid molecule comprises at least one nucleic acid base modification.

77. The enzymatic nucleic acid molecule of claim 56, wherein said enzymatic nucleic acid molecule comprises at least one phosphate backbone modification.

78. The enzymatic nucleic acid molecule of claim 56, wherein said phosphate backbone modification is selected from the group consisting of phosphorothioate, phosphorodithioate and amide.

79. An enzymatic nucleic acid molecule which down regulates expression of genes selected from the group consisting of beta site APP-cleaving enzyme (BACE) and telomerase reverse transciptase (TERT) genes.

80. The enzymatic nucleic acid molecule of claim 79, wherein said gene is the beta site APP-cleaving enzyme (BACE).

81. The enzymatic nucleic acid molecule of claim 79, wherein said gene is the telomerase reverse transcriptase (TERT).

82. A nucleic acid molecule which down regulates expression of genes selected from the group consisting of protein-tyrosine phosphatase-1B (PTP-1B), methionine aminopeptidase (MetAP-2), hepatitis B virus (HBV), phospholamban (PLN), and presenilin (ps-2) genes.

83. The nucleic acid molecule of claim 82, wherein said nucleic acid molecule is an enzymatic nucleic acid molecule.

84. The nucleic acid molecule of claim 82, wherein said nucleic acid molecule is an antisense nucleic acid molecule.

85. The nucleic acid molecule of any of claims 82-84, wherein said gene is the protein-tyrosine phosphatase-1B (PTP-1B).

86. The nucleic acid molecule of any of claims 82-84, wherein said gene is the methionine aminopeptidase (MetAP-2).

87. The nucleic acid molecule of any of claims 82-84, wherein said gene is the hepatitis B virus (HBV).

88. The nucleic acid molecule of any of claims 82-84, wherein said gene is the phospholamban (PLN).

89. The nucleic acid molecule of any of claims 82-84, wherein said gene is the presenilin (ps-2).

90. The enzymatic nucleic acid molecule of any of claims 79 or 83, wherein said enzymatic nucleic acid molecule is adapted for use to treat diseases and conditions related to the expression of genes selected from the group consisting of beta site APP-cleaving enzyme (BACE), telomerase reverse transciptase (TERT), protein-tyrosine phosphatase-1B (PTP-1B), methionine aminopeptidase (MetAP-2), hepatitis B virus (HBV), phospholamban (PLN), and presenilin (ps-2) genes.

91. The nucleic acid molecule of claim 82, wherein said nucleic acid molecule is adapted for use to treat diseases and conditions related to the expression of genes selected from the group consisting of protein-tyrosine phosphatase-1B (PTP-1B), methionine aminopeptidase (MetAP-2), hepatitis B virus (HBV), phospholamban (PLN), and presenilin (ps-2) genes.

92. The enzymatic nucleic acid molecule of any of claims 79 or 83, wherein said enzymatic nucleic acid molecule has an endonuclease activity to cleave RNA
encoded by said beta site APP-cleaving enzyme (BACE), telomerase reverse transciptase (TERT), protein-tyrosine phosphatase-1B (PTP-1B), methionine aminopeptidase (MetAP-2), hepatitis B virus (HBV), phospholamban (PLN), and presenilin (ps-2) genes.

93. The enzymatic nucleic acid of any of claims 79 or 83, wherein a binding arm of said enzymatic nucleic acid molecule comprise sequences complementary to any of the sequences defined as Target or Substrate sequence in Tables 3-30, and 43.

94. The enzymatic nucleic acid molecule of any of claims 79 or 83 wherein said enzymatic nucleic acid molecule comprises any of the sequences defined as Ribozyme or DNAzyme sequence in Tables 3-29, and 37-43.

95. The nucleic acid molecule of claim 84, wherein said antisense nucleic acid molecule comprises sequence complementary to any of the sequences defined as Target or Substrate sequence in Tables 3-12, 24-30, and 36-43.

96. The enzymatic nucleic acid molecule of any of claims 79 or 83, wherein said enzymatic nucleic acid molecule is in a hammerhead (HH) motif.

97. The enzymatic nucleic acid molecule of any of claims 79 or 83, wherein said enzymatic nucleic acid molecule is in a zinzyme (Class II) motif.

98. The enzymatic nucleic acid molecule of any of claims 79 or 83, wherein said enzymatic nucleic acid molecule is in a amberzyme (Class 1) motif.

99. The enzymatic nucleic acid molecule of any of claims 79 or 83, wherein said enzymatic nucleic acid molecule is in a hairpin, hepatitis Delta virus, group I
intron, VS nucleic acid, or RNAse P nucleic acid motif.

100. The enzymatic nucleic acid molecule of claim 97, wherein said zinzyme motif comprises sequences complementary to any of the substrate sequences shown in Tables 21, 27 and 40.

101. The enzymatic nucleic acid molecule of claim 98, wherein said amberzyme motif comprises sequences complementary to any of the substrate sequences shown in Tables 23, 29, and 42.

102. The enzymatic nucleic acid molecule of any of claims 79 or 83, wherein said enzymatic nucleic acid molecule is in a NCH motif.

103. The enzymatic nucleic acid molecule of any of claims 79 or 83, wherein said enzymatic nucleic acid molecule is in a G-cleaver motif.

104. The enzymatic nucleic acid molecule of any of claims 79 or 83, wherein said enzymatic nucleic acid molecule is a DNAzyme.

105. The enzymatic nucleic acid molecule of any of claims 79 or 83, wherein said enzymatic nucleic acid molecule comprises between 12 and 100 bases complementary to the RNA of genes selected from the group consisting of beta site APP-cleaving enzyme (BACE), telomerase reverse transciptase (TERT), protein-tyrosine phosphatase-1B (PTP-1B), methionine aminopeptidase (MetAP-2), hepatitis B virus (HBV), phospholamban (PLN), and presenilin (ps-2) genes.

106. The enzymatic nucleic acid of any of claims 79 or 83, wherein said enzymatic nucleic acid molecule comprises between 14 and 24 bases complementary to the RNA of genes selected from the group consisting of beta site APP-cleaving enzyme (BACE), telomerase reverse transciptase (TERT), protein-tyrosine phosphatase-1B (PTP-1B), methionine aminopeptidase (MetAP-2), hepatitis B
virus (HBV), phospholamban (PLN), and presenilin (ps-2) genes.

107. The enzymatic nucleic acid molecule of any of claims 79 or 83, wherein said enzymatic nucleic acid is chemically synthesized.

108. The enzymatic nucleic acid molecule of any of claims 79 or 83, wherein said enzymatic nucleic acid comprises at least one 2'-sugar modification.

109. The enzymatic nucleic acid molecule of any of claims 79 or 83, wherein said enzymatic nucleic acid comprises at least one nucleic acid base modification.

110. The enzymatic nucleic acid molecule of any of claims 79 or 83, wherein said enzymatic nucleic acid comprises at least one phosphate backbone modification.

111. A mammalian cell including the enzymatic nucleic acid molecule of any of claims 79 or 83, wherein said mammalian cell is not a living human.

112. The mammalian cell of claim 111, wherein said mammalian cell is a human cell.

113. The antisense nucleic acid molecule of claim 84, wherein said antisense nucleic acid is chemically synthesized.

114. The antisense nucleic acid molecule of claim 84, wherein said antisense nucleic acid comprises at least one 2'-sugar modification.

115. The antisense nucleic acid molecule of claim 84, wherein said antisense nucleic acid comprises at least one nucleic acid base modification.

116. The antisense nucleic acid molecule of claim 84, wherein said antisense nucleic acid comprises at least one phosphate backbone modification.

117. A mammalian cell including the antisense nucleic acid molecule of claim 84, wherein said mammalian cell is not a living human.

118. The mammalian cell of claim 117, wherein said mammalian cell is a human cell.

119. A method of reducing BACE activity in a cell, comprising the step of contacting said cell with the enzymatic nucleic acid molecule of claim 80, under conditions suitable for said inhibition.

120. A method of reducing TERT activity in a cell, comprising the step of contacting said cell with the enzymatic nucleic acid molecule of claim 81, under conditions suitable for said inhibition.

121. A method of reducing PTP-1B activity in a cell, comprising the step of contacting said cell with the nucleic acid molecule of claim 85, under conditions suitable for said inhibition.

122. A method of reducing MetAP-2 activity in a cell, comprising the step of contacting said cell with the nucleic acid molecule of claim 86, under conditions suitable for said inhibition.

123. A method of reducing HBV activity in a cell, comprising the step of contacting said cell with the nucleic acid molecule of claim 87, under conditions suitable for said inhibition.

124. A method of reducing phospholamban (PLN) activity in a cell, comprising the step of contacting said cell with the nucleic acid molecule of claim 88, under conditions suitable for said inhibition.

125. A method of reducing presenilin-2 (ps-2) activity in a cell, comprising the step of contacting said cell with the nucleic acid molecule of claim 89, under conditions suitable for said inhibition.

126. A method of treatment of a patient having a condition associated with the level of BACE, comprising contacting cells of said patient with the enzymatic nucleic acid molecule of claim 80, under conditions suitable for said treatment.

127. A method of treatment of a patient having a condition associated with the level of TERT, comprising contacting cells of said patient with the enzymatic nucleic acid molecule of claim 81, under conditions suitable for said treatment.

128. A method of treatment of a patient having a condition associated with the level of PTP-1B, comprising contacting cells of said patient with the nucleic acid molecule of claim 85, under conditions suitable for said treatment.

129. A method of treatment of a patient having a condition associated with the level of MetAP-2, comprising contacting cells of said patient with the nucleic acid molecule of claim 86, under conditions suitable for said treatment.

130. A method of treatment of a patient having a condition associated with the level of HBV, comprising contacting cells of said patient with the nucleic acid molecule of claim 87, under conditions suitable for said treatment.

131. A method of treatment of a patient having a condition associated with the level of phospholamban (PLN), comprising contacting cells of said patient with the nucleic acid molecule of claim 88, under conditions suitable for said treatment.

132. A method of treatment of a patient having a condition associated with the level of presenilin-2 (ps-2), comprising contacting cells of said patient with the nucleic acid molecule of claim 89, under conditions suitable for said treatment.

133. The method of any of claims 126-132 further comprising the use of one or more drug therapies under conditions suitable for said treatment.

134. A method of cleaving RNA of BACE gene, comprising, contacting the enzymatic nucleic acid molecule of claim 80, with said RNA under conditions suitable for the cleavage of said RNA.

135. A method of cleaving RNA of TERT gene, comprising, contacting the enzymatic nucleic acid molecule of claim 81, with said RNA under conditions suitable for the cleavage of said RNA.

136. A method of cleaving RNA of PTP-1B gene, comprising, contacting the enzymatic nucleic acid molecule of claim 85, with said RNA under conditions suitable for the cleavage of said RNA.

137. A method of cleaving RNA of MetAP-2 gene, comprising, contacting the enzymatic nucleic acid molecule of claim 86, with said RNA under conditions suitable for the cleavage of said RNA.

138. A method of cleaving RNA of HBV gene, comprising, contacting the enzymatic nucleic acid molecule of claim 87, with said RNA under conditions suitable for the cleavage of said RNA.

139. A method of cleaving RNA of phospholamban (PLN) gene, comprising, contacting the enzymatic nucleic acid molecule of claim 88, with said RNA
under conditions suitable for the cleavage of said RNA.

140. A method of cleaving RNA of presenilin-2 (ps-2) gene, comprising, contacting the enzymatic nucleic acid molecule of claim 89, with said RNA under conditions suitable for the cleavage of said RNA.

141. The method of any of claims 134-140, wherein said cleavage is carried out in the presence of a divalent cation.

142. The method of claim 141, wherein said divalent cation is Mg2+.

143. The enzymatic nucleic acid molecule of any of claims 79 or 83, wherein said enzymatic nucleic acid comprises a cap structure, wherein the cap structure is at the 5'-end or 3'-end or both the 5'-end and the 3'-end.

144. The antisense nucleic acid molecule of claim 84, wherein said antisense nucleic acid comprises a cap structure, wherein the cap structure is at the 5'-end or 3'-end or both the 5'-end and the 3'-end.

145. The enzymatic nucleic acid molecule of claim 96, wherein said hammerhead motif comprises sequences complementary to any of sequences defined as Target or Substrate sequences in Tables 3, 9, 13, 18, 24, and 37.

146. The enzymatic nucleic acid molecule of claim 102, wherein said NCH motif comprises sequences complementary to any of sequences defined as Target or Substrate sequences in Tables 4, 10, 14, 19, 25, and 38.

147. The enzymatic nucleic acid molecule of claim 103, wherein said G-cleaver motif comprises sequences complementary to any of sequences defined as Target or Substrate sequences in Tables 5, 11, 15, 20, 26, and 39.

148. The enzymatic nucleic acid molecule of claim 104, wherein said DNAzyme comprises sequences complementary to any of sequences defined as Target or Substrate sequences in Tables 6, 16, 22, 28, and 41.

149. The method of any of claims 119-125 or 133, wherein said enzymatic nucleic acid molecule is in a hammerhead motif.

150. The method of any of claims 119-125 or 133, wherein said nucleic acid molecule is a DNAzyme.

151. An expression vector comprising nucleic acid sequence encoding at least one enzymatic nucleic acid molecule of any of claims 79 or 83, in a manner which allows expression of that enzymatic nucleic acid molecule.

152. An expression vector comprising nucleic acid sequence encoding at least one antisense nucleic acid molecule of claim 84, in a manner which allows expression of that antisense nucleic acid molecule.

153. A mammalian cell including an expression vector of any of claims 151 or 152, wherein said mammalian cell is not a living human.

154. The mammalian cell of claim 153, wherein said mammalian cell is a human cell.

155. The expression vector of claim 151, wherein said enzymatic nucleic acid molecule is in a hammerhead motif.

156. The expression vector of claim 151, wherein said expression vector further comprises a sequence for an antisense nucleic acid molecule complementary to the RNA of genes selected from the group consisting of beta site APP-cleaving enzyme (BACE), telomerase reverse transciptase (TERT), protein-tyrosine phosphatase-1B (PTP-1B), methionine aminopeptidase (MetAP-2), hepatitis B
virus (HBV), phospholamban (PLN), and presenilin (ps-2) genes.

157. The expression vector of claim 151, wherein said expression vector comprises sequence encoding at least two said enzymatic nucleic acid molecules, which may be same or different.

158. The expression vector of claim 157, wherein one said expression vector further comprises sequence encoding antisense nucleic acid molecule complementary to the RNA of genes selected from the group consisting of beta site APP-cleaving enzyme (BACE), telomerase reverse transciptase (TERT), protein-tyrosine phosphatase-1B (PTP-1B), methionine aminopeptidase (MetAP-2), hepatitis B
virus (HBV), phospholamban (PLN), and presenilin (ps-2) genes.

159. A method for treatment of Alzheimer's disease comprising the step of administering to a patient the enzymatic nucleic acid molecule of claim 80 under conditions suitable for said treatment.

160. The method of claim 159, wherein said treatment of Alzheimer's disease is treatment of dementia.

161. A method for treatment of Alzheimer's disease comprising the step of administering to a patient the antisense nucleic acid molecule of claim 89 under conditions suitable for said treatment.

162. A method for treatment of diabetes comprising the step of administering to a patient the nucleic acid molecule of claim 85 under conditions suitable for said treatment.

163. The method of claim 162, wherein said diabetes is type I diabetes.

164. The method of claim 162, wherein said diabetes is type II diabetes.

165. A method for treatment of diabetes comprising the step of administering to a patient the antisense nucleic acid molecule of claim 85 under conditions suitable for said treatment.

166. A method for treatment of obesity comprising the step of administering to a patient the nucleic acid molecule of claim 85 under conditions suitable for said treatment.

167. A method for treatment of obesity comprising the step of administering to a patient the antisense nucleic acid molecule of claim 85 under conditions suitable for said treatment.

168. A method for treatment of heart disease comprising the step of administering to a patient the nucleic acid molecule of claim 88 under conditions suitable for said treatment.

169. The method of claim 168, wherein said heart disease is heart failure.

170. The method of claim 168, wherein said heart disease is congestive heart failure.

171. A method for treatment of pressure overload hypertrophy, or dilated cardiomyopathy, or both, comprising the step of administering to a patient the nucleic acid molecule of claim 88 under conditions suitable for said treatment.

172. A method for treatment of cancer comprising the step of administering to a patient the nucleic acid molecule of claim 86 under conditions suitable for said treatment.

173. A method for treatment of hepatitis comprising the step of administering to a patient the nucleic acid molecule of claim 87 under conditions suitable for said treatment.

174. A method for treatment of hepatocellular carcinoma comprising the step of administering to a patient the nucleic acid molecule of claim 87 under conditions suitable for said treatment.

175. The method of claim 159, wherein said enzymatic nucleic acid molecule is in a hammerhead motif.

176. The method of claim 159, wherein said method further comprises administering to said patient the enzymatic nucleic acid molecule in conjunction with one or more of other therapies.

177. The method of any of claims 162, 165-168, or 171-174, wherein said nucleic acid molecule is an enzymatic nucleic acid molecule.

178. The method of any of claims 162, 166-168, or 171-174, wherein said nucleic acid molecule is an antisense nucleic acid molecule.

179. The method of any of claims 162, 165-168, or 171-174, wherein said method further comprises administering to said patient the nucleic acid molecule in conjunction with one or more of other therapies.

180. The enzymatic nucleic acid molecule of any of claims 79 or 83, wherein said enzymatic nucleic acid molecule comprises at least five ribose residues; at least ten 2'-O-methyl modifications, and a 3'- end modification.

181. The enzymatic nucleic acid molecule of claim 180, wherein said enzymatic nucleic acid molecule further comprises phosphorothioate linkages on at least three of the 5' terminal nucleotides.

182. The enzymatic nucleic acid molecule of claim 180, wherein said 3'- end modification is 3'-3' inverted abasic moiety.

183. The enzymatic nucleic acid molecule of claim 104, wherein said DNAzyme comprises at least ten 2'-O-methyl modifications and a 3'-end modification.

184. The enzymatic nucleic acid molecule of claim 183, wherein said DNAzyme further comprises phosphorothioate linkages on at least three of the 5' terminal nucleotides.

185. The enzymatic nucleic acid molecule of claim 183, wherein said 3'- end modification is 3'-3' inverted abasic moiety.

186. An enzymatic nucleic acid molecule having formula 1:
wherein 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 with a target RNA molecule; o and n are integers independently greater than or equal to 1, wherein if (N)o and (N)n are nucleotides, (N)o and (N)n are optionally able to interact by hydrogen bond interaction;
.cndot. indicates base-paired interaction; L is a linker which may be present or absent, but when present, is a nucleotide linker, a non-nucleotide linker, or a combination of nucleotide and a non-nucleotide linker; p is an integer 0 or 1; represents a chemical linkage; and A, U, I, C and G represent adenosine, uridine, inosine, cytidine and guanosine nucleotides, respectively.

187. An enzymatic nucleic acid molecule having formula 2:
wherein 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 with a target RNA molecule; o and n are integers independently greater than or equal to 0, wherein if (N)o and (N)n are nucleotides, (N)o and (N)n are optionally able to interact by hydrogen bond interaction;
.cndot. indicates base-paired interaction; L is a linker which may be present or absent, but when present, is a nucleotide linker, a non-nucleotide linker, or a combination of nucleotide and a non-nucleotide linker; p is an integer 0 or 1; represents a chemical linkage; and A, U, I, C and G represent adenosine, uridine, inosine, cytidine and guanosine nucleotides, respectively.

188. The enzymatic nucleic acid molecule of claims 186 or 187, wherein said D
and E are independently of length selected from the group consisting of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, and 20 nucleotides.

189. The enzymatic nucleic acid molecule of claims 186 or 187, wherein said D
and E are of the same length.

190. The enzymatic nucleic acid molecule of claims 186 or 187, wherein said D
and E are of different length.

191. The enzymatic nucleic acid molecule of claim 186, wherein said o and n are independently integers selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, and 50.

192. The enzymatic nucleic acid molecule of claim 187, wherein said o and n are independently integers selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, and 50.

193. The enzymatic nucleic acid molecule of claims 186 or 187, wherein said (N)o and (N)n comprise nucleotides that are complementary to each other.

194. The enzymatic nucleic acid molecule of claims 186 or 187, wherein said (N)o and (N)n are of the same length.

195. The enzymatic nucleic acid molecule of claims 186 or 187, wherein said (N)o and (N)n are of different length.

196. The enzymatic nucleic acid molecule of claims 186 or 187, wherein said L
is a nucleotide linker.

197. The enzymatic nucleic acid molecule of claim 196, wherein said nucleotide linker is of length between 3-50 nucleotides.

198. The enzymatic nucleic acid molecule of claim 196, wherein said nucleotide linker is an aptamer.

199. The enzymatic nucleic acid molecule of claim 196 wherein said nucleotide linker is selected from the group consisting of 5'-GAAA-3' and 5'-GUUA-3'.

200. The enzymatic nucleic acid molecule of claims 186 or 187, wherein said L
is a non-nucleotide linker.

201. The enzymatic nucleic acid molecule of claims 186 or 187, wherein said chemical linkage is independently or in combination selected from the group consisting of phosphate ester linkage, amide linkage, phosphorothioate, arabino, arabinofluoro, and phosphorodithioate.

202. The enzymatic nucleic acid molecule of claims 186 or 187, wherein said p is 1.

203. The enzymatic nucleic acid molecule of claim 202, wherein said N of (N)p is independently selected from the group consisting of adenosine, uridine, and cytidine.

204. The enzymatic nucleic acid molecule of claims 186 or 187 wherein said enzymatic nucleic acid molecule is chemically synthesized.

205. The enzymatic nucleic acid molecule of claims 186 or 187, wherein said enzymatic nucleic acid molecule comprises at least three ribonucleotide residues.

206. The enzymatic nucleic acid molecule of claims 186 or 187, wherein said enzymatic nucleic acid molecule comprises at least four ribonucleotide residues.

207. The enzymatic nucleic acid molecule of claims 186 or 187, wherein said enzymatic nucleic acid molecule comprises at least five ribonucleotide residues.

208. The enzymatic nucleic acid molecule of claims 186 or 187, wherein said I
is selected from the group consisting of ribo-inosine and xylo-inosine.

209. The enzymatic nucleic acid molecule of claims 186 or 187, wherein said enzymatic nucleic acid molecule comprises at least one sugar modification.

210. The enzymatic nucleic acid molecule of claims 186 or 187, wherein said enzymatic nucleic acid molecule comprises at least nucleic acid base modification.

211. The enzymatic nucleic acid molecule of claims 186 or 187, wherein said enzymatic nucleic acid molecule comprises at least one phosphate backbone modification.

212. The enzymatic nucleic acid molecule of claim 209, wherein said sugar modification is selected from the group consisting of 2'-H, 2'-O-methyl, 2'-O-allyl, and 2'-deoxy-2'-amino.

213. The enzymatic nucleic acid molecule of claim 211, wherein said phosphate backbone modification is selected from the group consisting of phosphorothioate, phosphorodithioate and amide.

214. The enzymatic nucleic acid molecule of claims 186 or 187 wherein said enzymatic nucleic acid molecule comprises a 5'-cap or a 3'-cap or both a 5'-cap and a 3'-cap.

215. The enzymatic nucleic acid molecule of claim 214, wherein said 5'-cap is a phosphorothioate modification of at least one 5'-terminal nucleotide in said enzymatic nucleic acid molecule.

216. The enzymatic nucleic acid molecule of claim 214, wherein said 5'-cap is a phosphorothioate modification of at least two 5'-terminal nucleotide in said enzymatic nucleic acid molecule.

217. The enzymatic nucleic acid molecule of claim 214, wherein said 5'-cap is a phosphorothioate modification of at least three 5'-terminal nucleotide in said enzymatic nucleic acid molecule.

218. The enzymatic nucleic acid molecule of claim 214, wherein said 3'-cap is a 3'-3' inverted abasic moiety.

219. The enzymatic nucleic acid molecule of claim 214, wherein said 3'-cap is a 3'-3' inverted nucleotide moiety.

220. A method for inhibiting expression of a gene in a cell, comprising the step of administering to said cell the enzymatic nucleic acid molecule of claims 186 or 187 under conditions suitable for said inhibition.

221. A method of cleaving a separate RNA molecule comprising, contacting the enzymatic nucleic acid molecule of claims 186 or 187 with said separate RNA

molecule under conditions suitable for the cleavage of said separate RNA
molecule.

222. The method of claim 221, wherein said cleavage is carried out in the presence of a divalent cation.

223. The method of claim 222, wherein said divalent cation is Mg2+.

224. The enzymatic nucleic acid molecule of claims 186 or 187, wherein said enzymatic nucleic acid molecule has an endonuclease activity to cleave RNA
derived from HER2 gene.

225. The enzymatic nucleic acid molecule of claim 224, wherein said enzymatic nucleic acid molecule comprises sequences complementary to any of NCH
substrate sequence of Table 34.

226. The enzymatic nucleic acid molecule of claim 224, wherein said enzymatic nucleic acid molecule comprises any of the NCH ribozyme sequences shown in Table 34.

227. The enzymatic nucleic acid molecule of claim 224, wherein said enzymatic nucleic acid molecule is used to treat cancer.

228. The enzymatic nucleic acid molecule of claim 224, wherein said cancer is breast cancer.

229. The enzymatic nucleic acid molecule of claim 224, wherein said enzymatic nucleic acid molecule is used to treat conditions associated with the level of gene.

230. An enzymatic nucleic acid molecule, wherein said enzymatic nucleic acid molecule comprises any of sequence shown as NCH ribozyme sequence in Table 31.

231. The enzymatic nucleic acid molecule of claim 224, wherein said enzymatic nucleic acid molecule comprises a substrate binding region which has between 5 and 30 nucleotides complementary to the RNA.

232. The enzymatic nucleic acid molecule of claim 224, wherein said enzymatic nucleic acid molecule comprises a substrate binding region which has between 7 and 12 nucleotides complementary to the RNA.

233. A mammalian cell including the enzymatic nucleic acid molecule of claim 224, wherein said mammalian cell is not a living human.

234. The mammalian cell of claim 233, wherein said mammalian cell is a human cell.

235. A mammalian cell including the enzymatic nucleic acid molecule of claims or 187, wherein said mammalian cell is not a living human.

236. The mammalian cell of claim 235, wherein said mammalian cell is a human cell.

237. A method for inhibiting expression of HER2 gene in a cell, comprising the step of administering to said cell the enzymatic nucleic acid molecule of claim 224 under conditions suitable for said inhibition.

238. A method of cleaving RNA derived from HER2 gene comprising, contacting the enzymatic nucleic acid molecule of claim 224 with said RNA molecule under conditions suitable for the cleavage of said RNA molecule.

239. A pharmaceutical composition comprising the enzymatic nucleic acid molecule of any of claims 186 or 187.

240. A pharmaceutical composition comprising the enzymatic nucleic acid molecule of claim 224.

241. A method of treatment of a patient having a condition associated with the level of HER2, wherein said patient is administered the enzymatic nucleic acid molecule of claim 224 under conditions suitable for said treatment.

242. The method of claim 241, wherein said method is performed in conjunction with one or more other therapies.

243. The enzymatic nucleic acid molecule of claim 227, wherein said enzymatic nucleic acid molecule is used in conjunction with one or more other therapies.

244. The enzymatic nucleic acid molecule of claims 186 or 187, wherein said nucleic acid molecule comprises at least five ribose residues; a 2'-C-allyl modification at position No. 4 of said enzymatic nucleic acid; at least ten 2'-O-alkyl modifications, and a 3'- cap structure.

245. The enzymatic nucleic acid molecule of claim 244, wherein said 2'-O-alkyl modifications is selected from the group consisting of 2'-O-methyl and 2'-O-allyl.

246. The enzymatic nucleic acid molecule of claim 244, wherein said 3'-cap is 3'-3' inverted abasic moiety.

247. The enzymatic nucleic acid molecule of claim 244, wherein said 3'-cap is 3'-3' inverted nucleotide.

248. The enzymatic nucleic acid molecule of claim 244, wherein said enzymatic nucleic acid comprises phosphorothioate linkages in at least three of the 5' terminal nucleotides.

249. The enzymatic nucleic acid molecule of claims 186 or 187, wherein said nucleic acid molecule comprises at least five ribose residues; a 2'-deoxy-2'-amino modification at position Nos. 4 and 7 of said enzymatic nucleic acid; at least ten 2'-O-alkyl modifications, and a 3'- cap structure.

250. The enzymatic nucleic acid molecule of claim 249, wherein said 2'-O-alkyl modifications is selected from the group consisting of 2'-O-methyl and 2'-O-allyl.

251. The enzymatic nucleic acid molecule of claim 249, wherein said 3'-cap is 3'-3' inverted abasic moiety.

252. The enzymatic nucleic acid molecule of claim 249, wherein said 3'-cap is 3'-3' inverted nucleotide.

253. The enzymatic nucleic acid molecule of claim 249, wherein said enzymatic nucleic acid comprises phosphorothioate linkages in at least three of the 5' terminal nucleotides.

254. The enzymatic nucleic acid molecule of claim 224, wherein said enzymatic nucleic acid molecule comprises at least one sugar modification.

255. The enzymatic nucleic acid molecule of claim 224, wherein said enzymatic nucleic acid molecule comprises at least one nucleic acid base modification.

256. The enzymatic nucleic acid molecule of claim 224, wherein said enzymatic nucleic acid molecule comprises at least one phosphate backbone modification.

257. The enzymatic nucleic acid molecule of claim 224, wherein said phosphate backbone modification is selected from the group consisting of phosphorothioate, phosphorodithioate and amide.

258. The enzymatic nucleic acid molecule of claim 224, wherein said nucleic acid molecule comprises at least five ribose residues; a 2'-C-allyl modification at position No. 4 of said enzymatic nucleic acid; at least ten 2'-O-alkyl modifications, and a 3'- cap structure.

259. The enzymatic nucleic acid molecule of claim 258, wherein said 2'-O-alkyl modifications is selected from the group consisting of 2'-O-methyl and 2'-O-allyl.

260. The enzymatic nucleic acid molecule of claim 258, wherein said 3'-cap is 3'-3' inverted abasic moiety.

261. The enzymatic nucleic acid molecule of claim 258, wherein said 3'-cap is 3'-3' inverted nucleotide.

262. The enzymatic nucleic acid molecule of claim 258, wherein said enzymatic nucleic acid comprises phosphorothioate linkages in at least three of the 5' terminal nucleotides.

263. The enzymatic nucleic acid molecule of claim 224, wherein said nucleic acid molecule comprises at least five ribose residues; a 2'-deoxy-2'-amino modification at position Nos. 4 and 7 of said enzymatic nucleic acid; at least ten 2'-O-alkyl modifications, and a 3'- cap structure.

264. The enzymatic nucleic acid molecule of claim 263, wherein said 2'-O-alkyl modifications is selected from the group consisting of 2'-O-methyl and 2'-O-allyl.

265. The enzymatic nucleic acid molecule of claim 263, wherein said 3'-cap is 3'-3' inverted abasic moiety.

266. The enzymatic nucleic acid molecule of claim 263, wherein said 3'-cap is 3'-3' inverted nucleotide.

267. The enzymatic nucleic acid molecule of claim 263, wherein said enzymatic nucleic acid comprises phosphorothioate linkages in at least three of the 5' terminal nucleotides.

268. The enzymatic nucleic acid molecule of claim 186, wherein said enzymatic nucleic acid molecule is capable of down-regulating the expression of protein kinase C alpha (PKC alpha) gene.

269. A method for inhibiting expression of a PKC alpha gene in a cell, comprising the step of administering to said cell the enzymatic nucleic acid molecule of claim 268 under conditions suitable for said inhibition.

270. A method of cleaving a PKC alpha RNA molecule comprising, contacting the enzymatic nucleic acid molecule of claim 268 with said separate PKC alpha RNA
molecule under conditions suitable for the cleavage of said PKC alpha RNA
molecule.

271. The method of claim 270, wherein said cleavage is carried out in the presence of a divalent cation.

272. The method of claim 271, wherein said divalent cation is Mg2+.

273. The enzymatic nucleic acid molecule of claim 268, wherein said enzymatic nucleic acid molecule has an endonuclease activity to cleave RNA derived from PKC alpha gene.

274. The enzymatic nucleic acid molecule of claim 273, wherein said enzymatic nucleic acid molecule comprises sequences complementary to any of NCH
substrate sequence of Table 63.

275. The enzymatic nucleic acid molecule of claim 273 wherein said enzymatic nucleic acid molecule comprises any of the NCH ribozyme sequences shown in Table 63.

276. The enzymatic nucleic acid molecule of claim 268, wherein said enzymatic nucleic acid molecule is used to treat cancer.

277. The enzymatic nucleic acid molecule of claim 276, wherein said cancer is selected from the group consisting of lung, breast, colon, prostate, bladder, ovary, melanoma, and glioblastoma cancer.

278. The enzymatic nucleic acid molecule of claim 268, wherein said enzymatic nucleic acid molecule is used to treat conditions associated with the level of PKC
alpha gene.

279. The enzymatic nucleic acid molecule of claim 268, wherein said D and E
independently has between 5 and 30 nucleotides complementary to the RNA.

280. The enzymatic nucleic acid molecule of claim 268, wherein said D and E
independently has between 7 and 12 nucleotides complementary to the RNA.

281. A mammalian cell including the enzymatic nucleic acid molecule of claim 268, wherein said mammalian cell is not a living human.

282. The mammalian cell of claim 281, wherein said mammalian cell is a human cell.

283. A pharmaceutical composition comprising the enzymatic nucleic acid molecule of claim 238.

284. A pharmaceutical composition comprising the enzymatic nucleic acid molecule of claim 273.

285. A method of treatment of a patient having a condition associated with the level of PKC alpha, wherein said patient is administered the enzymatic nucleic acid molecule of claim 268 under conditions suitable for said treatment.

286. The method of claim 285, wherein said method is performed in conjunction with one or more other therapies.

287. The enzymatic nucleic acid molecule of claim 286, wherein said enzymatic nucleic acid molecule is used in conjunction with one or more other therapies.

288. An antisense nucleic acid molecule comprising sequence complementary to any of substrate sequence in Tables 13-23.

289. The antisense nucleic acid molecule of claim 288, wherein said enzymatic nucleic acid is chemically synthesized.

290. The antisense nucleic acid molecule of claim 288, wherein said antisense nucleic acid comprises at least one 2'-sugar modification.

291. The antisense nucleic acid molecule of claim 288, wherein said antisense nucleic acid comprises at least one nucleic acid base modification.

292. The antisense nucleic acid molecule of claim 288, wherein said antisense nucleic acid comprises at least one phosphate backbone modification.

293. A mammalian cell including the antisense nucleic acid molecule of claim 288, wherein said mammalian cell is not a living human.

294. The mammalian cell of claim 293, wherein said mammalian cell is a human cell.