CA2397813A1 - Method and reagent for the inhibition of grid - Google Patents

Abstract

The present invention relates to nucleic acid molecules, including antisense and enzymatic nucleic acid molecules, such as hammerhead ribozymes, DNAzymes , and antisense, which modulate the expression of the GRID (Grb2-related with Insert Domain) gene.

Description

DESCRIPTION
METHOD AND REAGENT FOR THE INHIBITION OF GRID
Background Of The Invention This invention claims priority from Jarvis et al., USSN (60/181,594), filed February 24, 2000, entitled "METHOD AND REAGENT FOR THE INHIBITION OF GRID". This application is hereby incorporated by reference herein in its entirety including the drawings.
The present invention concerns compounds, compositions, and methods for the study, diagnosis, and treatment of conditions and diseases related to the expression of the T-cell co-stimulatory adapter protein GRID (Grb2-related with Insert Domain).
The following is a brief description of the current understanding of GRID. The discussion is not meant to be complete and is provided only for understanding the invention that follows. The summary is not an admission that any of the work described below is prior art to the claimed invention.
One of the emerging paradigms for signal transduction in lymphocytes is that receptors and other signaling molecules do not operate in isolation, but through the recruitment of a complex of other proteins (Pawson and Scott, 1997; Science, 278, 2075;
Rudd, 1999, Cell, 96, 5). These other proteins sexve to amplify and diversify the signal into a number of biochemical cascades. The archetypal adapter protein is Grb2, which serves to regulate downstream pathways such as Ras activation and Ca2+ mobilization (Lowenstein et al., 1992, Cell, 70, 431), and is ultimately responsible for modulating gene expression required for proliferation and differentiation. Grb2 is recruited to LAT and SLP-76 which are downstream targets in the signaling cascade initiated by ligation of the T-cell receptor by MHC-antigen. These functions are mediated by specialized domains which bind specific motifs and include the phosphotyrosine binding SH2 (Src homology) domain and SH3 domain which are associated with proline-rich PXXP motifs. Grb2, whose sole function appears to be the formation of bridges between other proteins, is entirely comprised of such domains having an SH3-SH2-SH3 structure (Peterson et al., 1998, CzsYf-.
Opin. Imnaunol., 10, 337; Koretzky, 1997, Immunol Today, 18, 401).
A novel member of the Grb2 family of adapter proteins termed GRID (Grb2-related with Insert Domain) has recently been identified (Asada et al, 1999, J. Exp.
Med., 189, 1383; Liu et al., 1999, Cunr. Biol., 9, 67; Liu et al., 1998, Oncogene, 17, 3073; Law et al., 1999, J. Exp. 'Med., 189, 1243; Qiu et al., 1998, Biochem. Biophys. Res.
Comrnun., 253, 443; Bourette et al., 1998, Ernbo. J., 17, 7273). GRID is recruited to the T
cell co-stimulatory receptor CD28 upon activation of this receptor by cross-linking antibodies.
Although GRID shares significant similarity at the protein level with Grb2, possessing an SH3-SH2-SH3 domain structure, GRID also contains a unique proline-glutamine rich domain situated between the SH2 and C-terminal SH3 domain. The association of GRID
with activated CD28 is absolutely dependent upon the integrity of the SH2 domain and phosphorylation of residue Y173 in the cytoplasmic tail of CD28. Although GRID
has been shown to associate with other T cell signaling proteins including SLP-76 and LAT
(Asada et al., supra; Liu et al., supra; Law et al., supra), it's role in T
cell signaling pathways is not well defined.
Tari et al.,, 1999, Oncogene, 18(6), 1325-1332, describe the antisense inhibition of Grb2 in breast cancer cells in order to investigate the role of Grb2 in the proliferation of breast cancer cells. The resulting Grb2 inhibition led to MAP kinase inactivation in EGFR
but not in ErbB2 expressing breast cancer cells.
Tari et al., 1998, J. Liposorne Res., 8(2), 251-264, describe P-ethoxy antisense oligonucleotides targeting Bcr-Abl, Grb2, Crkl, and Bcl-2 mRNA. Delivery of these antisense oligonucleotides via liposome transfection results in the inhibition of corresponding proteins, thereby inducing growth inhibition in leukemia and lymphoma cell lines.
Lopez-Berestein et al., 1998, International PCT publication No. WO 98/01547, describe inhibition of chronic myelogenous leukemic cell growth by liposomal-antisense oligodeoxynucleotides targeting Grb2 and Crkl.
Tari et al., 1997, Biochenz. Biophys. Res. Comrnun., 235(2), 383-388, describe the antisense-based inhibition of Grb2 and Crkl proteins results in growth inhbition of Philadelphia chromosome positive leukemic cells.

Summary Of The Invention The invention features novel nucleic acid-based techniques [e.g., enzymatic nucleic acid molecules (for example, ribozymes or DNAzymes), antisense nucleic acids, antisense chimeras, triplex DNA, antisense nucleic acids containing RNA
cleaving chemical groups] and methods for their use to modulate the expression of GRID
(Grb2-related with Insert Domain).
The description below of the various aspects and embodiments is provided with reference to the exemplary gene GRID. However, the various aspects and embodiments are also directed to other genes which express GRID-like adapter proteins involved in T-cell co-activation. Those additional genes can be analyzed for target sites using the methods described for GRID. Thus, the inhibition and the effects of such inhibition of the other genes can be performed as described herein.
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 GRID. For example, the nucleic acid-based techniques of the present invention can be used to inhibit the expression of GRID gene sequences found at GenBank Accession NOS. AJ011736, NM 004810, Y18051, AF121002, AF042380, AF129476, AF090456).
In another preferred embodiment, the invention features the use of an enzymatic nucleic acid molecule, preferably in the hammerhead, NCH (Inozyme), G-cleaver, amberzyme, zinzyme andlor DNAzyme motif, to inhibit the expression of GRID
gene.
By "inhibit" it is meant that the activity of GRID or level of GRID RNAs or equivalent RNAs encoding one or more protein subunits of GRID or GRID-like proteins is xeduced below that observed in the absence of the nucleic acid molecules of the invention.
In one embodiment, the inhibition with enzymatic nucleic acid molecule preferably is below that level observed in the presence of an enzymatically inactive or attenuated molecule that is able to bind to the same site on the target RNA, but is unable to cleave that RNA. In another embodiment, inhibition with antisense oligonucleotides is preferably below that level observed in the presence of, for example, an oligonucleotide with scrambled sequence or with mismatches. In another embodiment, inhibition of GRID or GRID-like 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.
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% can also be useful in this invention (see for example Werner and Uhlenbeclc, 1995, Nucleic Acids Research, 23, 2092-2096; Hammann et al., 1999, Antisense and Nucleic Acid Df~ug Dev., 9, 25-31). The nucleic acids can 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 limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target nucleic acid regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart a nucleic acid cleaving and/or ligation activity to the molecule (Cech et al., U.S. Patent No. 4,987,071; Cech et al., 1988, 260 JAMA
3030).
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.
By "enzymatic portion" or "catalytic domain" is meant that portion or 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 or region of a enzymatic nucleic acid which is able to interact, for example, via complementarity (i.e., able to base-pair with), with a portion of its substrate. Preferably, such complernentarity is 100%, but can be less if desired. For example, as few as 10 bases out of 14 can be base-paired (see for example Werner and Uhlenbeck, 1995, Nucleic Acids Research, 23, 2092-2096; Hammann et al., 1999, Antisense and Nucleic Acid Drug Dev., 9, 25-31). Examples of such anus are shown generally in Figures 1-5. That is, these arms contain sequences within an enzymatic nucleic acid which are intended to bring enzymatic nucleic acid and target RNA together through complementary base-pairing interactions.
The enzymatic nucleic acid of the invention can have binding arms that are contiguous or non-contiguous and can 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. Preferably, the binding arms) are 12-100 nucleotides in length. More preferably, the binding arms are 14-24 nucleotides in length (see, for 5 example, Werner and Uhlenbeclc, supra; Hamman et al., supYa; Hampel et al., EP0360257;
Berzal-Herrance et al., 1993, EMBO J., 12, 2567-73). 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, or six and six nucleotides, or seven and seven nucleotides long) or asymmetrical (i. e., the binding arms are of different length; e.g., six and three nucleotides; three and six nucleotides long; four and five nucleotides long; four and six nucleotides long; four and seven nucleotides long; and the like).
By "Inozyme" or "NCH" motif is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described as NCH Rz in Figure 2. Inozymes possess endonuclease activity to cleave RNA substrates having a cleavage triplet NCH/, where N is a nucleotide, C is cytidine and H is adenosine, uridine or cytidine, and /
represents the cleavage site. H is used interchangeably with X. Inozymes can also possess endonuclease activity to cleave RNA substrates having a cleavage triplet NCNI, where N is a nucleotide, C is cytidine, and / represents the cleavage site. "I" in Figure 2 represents an Inosine nucleotide, preferably a ribo-Inosine or xylo-Inosine nucleoside.
By "G-cleaver" motif is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described as G-cleaver in Figure 2. G-cleavers possess endonuclease activity to cleave RNA substrates having a cleavage triplet NYN/, where N is a nucleotide, Y is uridine or cytidine and / represents the cleavage site. G-cleavers may be chemically modified as is generally shown in Figure 2.
By "amberzyme" motif is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described in Figure 3. Amberzymes possess endonuclease activity to cleave RNA substrates having a cleavage triplet NG/N, where N is a nucleotide, G is guanosine, and / represents the cleavage site. Amberzymes can be chemically modified to increase nuclease stability through substitutions as are generally shown in Figure 3. In addition, differing nucleoside and/or non-nucleoside linkers can be used to substitute the 5'-gaaa-3' loops shown in the figure. Amberzymes represent a non-limiting example of an enzymatic nucleic acid molecule that does not require a ribonucleotide (2'-OH) group within its ovcm nucleic acid sequence for activity.

By "zinzyme" motif is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described in Figure 4. Zinzymes possess endonuclease activity to cleave RNA substrates having a cleavage triplet including but not limited to YG/Y, where Y is uridine or cytidine, and G is guanosine and / represents the cleavage site. Zinzymes can be chemically modified to increase nuclease stability through substitutions as are generally shown in Figure 4, including substituting 2'-O-methyl guanosine nucleotides for guanosine nucleotides. In addition, differing nucleotide andlor non-nucleotide linkers can be used to substitute the 5'-gaaa-2' loop shown in the figure. Zinzymes represent a non limiting example of an enzymatic nucleic acid molecule that does not require a ribonucleotide (2'-OH) group within its own nucleic acid sequence for activity.
By 'DNAzyme' is meant, an enzymatic nucleic acid molecule that does not require the presence of a 2'-OH group fox its activity. In particular embodiments the enzymatic nucleic acid molecule can have an attached linkers) or other attached or associated groups, moieties, or chains containing one or more nucleotides with 2'-OH groups.
DNAzymes can be synthesized chemically or expressed endogenously in vivo, by means of a single stranded DNA vector or equivalent thereof. An example of a DNAzyme is shown in Figure 5 and is generally reviewed in Usman et al., International PCT
Publication No. WO
95/11304; Chartrand et al., 1995, NAR 23, 4092; Breaker et al., 1995, Chem.
Bio. 2, 655;
Santoro et al., 1997, PNAS 94, 4262; Breaker, 1999, Natu~°e Biotechnology, 17, 422-423;
and Santoro et. al., 2000, J. Am. Cl2em. Soc., 122, 2433-39. Additional DNAzyme motifs can be selected for using techniques similar to those described in these references, and hence, are within the scope of the present invention.
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) that is sufficient to the intended purpose (e.g., cleavage of target RNA by an enzyme).
By "equivalent" RNA to GRID is meant to include those naturally occurring RNA
molecules having homology (partial or complete) to GRID proteins or encoding for proteins with similar function as GRID 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.
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 Scierace 261, 1004 and Woolf et al., US
patent No. 5,849,902). Typically, antisense molecules are complementary to a target sequence along a single contiguous sequence of the antisense molecule.
However, in certain embodiments, an antisense molecule can bind to substrate such that the substrate molecule forms a loop, and/or an antisense molecule can bind such that the antisense molecule forms a loop. Thus, the antisense molecule can be complementary to two (or even more) non-contiguous substrate sequences or two (or even more) non-contiguous sequence portions of an antisense molecule can 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, Antiserase N. A. Drug Dev., 7, 151, Crooke, 2000, Methods Enzyrraol., 313, 3-45; Croolce, 1998, Biotech. Genet. Eng. Rev., 15, 121-157, Crooke, 1997, Ad. Pharrnacol., 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. The antisense oligonucleotides can comprise one or more RNAse H activating region, which is capable of activating RNAse H cleavage of a target RNA. Antisense DNA can be synthesized chemically or expressed .via the use of a single stranded DNA expression vector or equivalent thereof.
By "RNase H activating region" is meant a region (generally greater than or equal to 4-25 nucleotides in length, preferably from 5-11 nucleotides in length) of a nucleic acid molecule capable of binding to a target RNA to form a non-covalent complex that is recognized by cellular RNase H enzyme (see for example Arrow et al., US
5,849,902;
Arrow et al., US 5,989,912). The RNase H enzyme binds to the nucleic acid molecule-target RNA complex and cleaves the target RNA sequence. The RNase H activating region comprises, for example, phosphodiester, phosphorothioate (preferably at least four of the nucleotides are phosphorothiote substitutions; morepreferably, 4-11 of the nucleotides are phosphorothiote substitutions); phosphorodithioate, 5'-thiophosphate, or methylphosphonate backbone chemistry or a combination thereof. In addition to one or more backbone chemistries described above, the RNase H activating region can also comprise a variety of sugar chemistries. For example, the RNase H activating region can comprise deoxyribose, arabino, fluoroarabino or a combination thereof, nucleotide sugar chemistry. Those skilled in the art will recognize that the foregoing are non-limiting examples and that any combination of phosphate, sugar and base chemistry of a nucleic acid that supports the activity of RNase H enzyme is within the scope of the definition of the RNase H activating region and the instant invention.
By "2-SA antisense chimera" is meant an antisense oligonucleotide containing a 5'-phosphorylated 2'-5'-linked adenylate residue. These chimeras bind to target RNA in a sequence-specific manner and activate a cellular 2-SA-dependent ribonuclease which, in turn, cleaves the target RNA (Torrence et al., 1993 P>"oc. Natl. Acad. Sci.
USA 90, 1300;
Silverman et al., 2000, Methods Enzyrnol., 313, 522-533; Player and Torrence, 1998, Plzas~rnacol. They., 78, 55-113).
By "triplex forming oligonucleotides" 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 Pt~oc. Natl. Acad. Sci. USA 89, 504; Fox, 2000, Cum°. Med. Chenz., 7, 17-37; Praseuth et. al., 2000, Biochim. Biophys.
Acta, 1489, 181-206).
By "gene" it is meant a nucleic acid that encodes RNA, for example, nucleic acid sequences including but not limited to structural genes encoding a polypeptide.
"Complementarity" refers to the ability of a nucleic acid to form hydrogen bonds) with another RNA sequence by either traditional Watson-Cxick 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 taxget or complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., enzymatic nucleic acid cleavage, antisense or triple helix inhibition. Determination of binding free energies fox nucleic acid molecules is well known in the art (see, e.g., Turner et al., 1987, CSH Syrnp.
Quant. Biol. LII pp.123-133; Frier et al., 1986, PYOG. Nat. Acad. Sci. USA
83:9373-9377;
Turner et al., 1987, J. Am. Chezn. 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-Criclc 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.
By "RNA" is meant a molecule comprising at least one ribonucleotide residue.
By "ribonucleotide" or "2'-OH" is meant a nucleotide with a hydroxyl group at the 2' position of a (3-D-ribo-furanose moiety.
By "decoy RNA" is meant a RNA molecule that mimics the natural binding domain for a ligand. The decoy RNA therefore competes with natural binding target for the binding of a specific ligand. For example, it has been shown that over-expression of HIV trans-activation response (TAR) RNA can act as a "decoy" and efficiently binds HIV
tat protein, thereby preventing it from binding to TAR sequences encoded in the HIV RNA
(Sullenger et al., 1990, Cell, 63, 601-608). This is but a specific example and those in the art will recognize that other embodiments can be readily generated using techniques generally known in the art.
Several varieties of naturally occurring 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 GRID-specific RNAs represent a novel therapeutic approach to treat a variety of pathologic indications, including but not limited to tissue/graft rejection and leukemia.

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 can 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, 5 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 Biochenaistry 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 &

10 McSwiggen, US. Patent No. 5,631,359. The hepatitis delta virus motif is described by Perrotta and Been, 1992 Biochemistry 31, 16. The RNase P motif is described by Guerrier-Takada et al., 1983 Cell 35, 849; Forster and 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, Chena. 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, Claem. 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
99116871.
Additional motifs include the Aptazyme (Breaker et al., WO 98/43993), Amberzyme (Class I motif; Figure 3; Beigelman et al., International PCT publication No.
WO
99/55857) and Zinzyme (Beigelman et al., International PCT publication No. WO
99/55857), all these references are incorporated by reference herein in their totalities, including drawings and can also be used in the present invention. 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 of the instant invention can be between 13 and 100 nucleotides in length. Exemplary enzymatic nucleic acid molecules of the invention are shown in Tables III-VIII and X.
For example, enzymatic nucleic acid molecules of the invention are preferably between 15 and 50 nucleotides in length, more preferably between 25 and 40 nucleotides in length, e.g., 34, 36, or 38 nucleotides in length (for example see Jarvis et al., 1996, J. Biol.
ClZem., 271, 29107-29112). Exemplary DNAzymes of the invention are preferably between 15 and 40 nucleotides in length, more preferably between 25 and 35 nucleotides in length, e.g., 29, 30, 31, or 32 nucleotides in length (see for example Santoro et al., 1998, Bioche~raistry, 37, 13330-13342; Chartrand et al., 1995, Nucleic Acids Research, 23, 4092-4096 and Cairns et al., 2000, Aratisense & Nucleic Acid Drug Dev., 10, 323-332). Exemplary antisense molecules of the invention are preferably between 15 and 75 nucleotides in length, more preferably between 20 and 35 nucleotides in length, e.g., 25, 26, 27, or 28 nucleotides in length (see for example Woolf et al., 1992, PNAS., 89, 7305-7309; Milner et al., 1997, Nature Bioteclanology, 15, 537-541). Exemplary triplex forming oligonucleotide molecules of the invention are preferably between 10 and 40 nucleotides in length, more preferably between 12 and 25 nucleotides in length, e.g., 18, 19, 20, or 21 nucleotides in length (see for example Maher et al., 1990, Bioclae~aistry, 29, 8820-8826;
Strobel and Dervan, 1990, Science, 249, 73-75). Those skilled in the art will recognize that all that is required is for the nucleic acid molecule to be of length and conformation sufficient and suitable for the nucleic acid molecule to catalyze a reaction contemplated herein. The length of the nucleic acid molecules of the instant invention are not limiting within the general limits stated.
Preferably, a nucleic acid molecule that down regulates the replication of GRID or GRID-like gene comprises between 12 and 100 bases complementary to a GRID or GRID-lilce RNA. Even more preferably, a nucleic acid molecule ~ that down regulates the replication of GRID or GRID-like gene comprises between 14 and 24 bases complementary to a GRID or GRID-lilce RNA.
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 GRID or GRID
lilce proteins such that specific treatment of a disease or condition can be provided with either one or several nucleic acid molecules of the invention. Such nucleic acid molecules can be delivered exogenously to specific tissue or cellular targets as required.
Alternatively, the nucleic acid molecules (e.g., ribozymes and antisense) can be expressed from DNA andlor RNA vectors that are delivered to target cells.

In a preferred embodiment, the invention features the use of nucleic acid-based inhibitors of the invention to specifically target genes that share homology with the GRID
gene. For example, the invention describes the use of nucleic acid-based inhibitors to target the Grb2 (GenBank accession No. NM 002086) and GRAP (GenBank accession No.
h1M-006613) genes.
As used in herein "cell" is used in its usual biological sense and does not refer to an entire multicellular organism. The cell can be present in an organism which includes humans 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 can be prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., mammalian or plant cell).
By "GRID proteins" is meant, a protein or a mutant protein derivative thereof, comprising an adapter-protein type of association to the activated CD28 co-stimulatory receptor, and to other signaling proteins including but not limited to SLP-76 and LAT.
By "highly conserved sequence region" is meant a nucleotide sequence of one or more regions in a target gene that does not vary significantly from one generation to the other or from one biological system to the other.
The nucleic acid-based inhibitors of GRID expression are useful for the prevention and/or treatment of diseases and conditions that are related to or will respond to the levels of GRID in a cell or tissue, alone or in combination with other therapies. For example, the nucleic acid-based inhibitors of GRID expressions are useful for the prevention and/or treatment of tissue/graft rejection and cancer, such as leukemia, among other conditions.
By "related" is meant that the reduction of GRID expression (specifically GRID
gene) RNA levels and thus reduction in the level of the respective protein will relieve, to some extent, the symptoms of the disease or condition.
In a preferred embodiment, the invention features the use of nucleic acid-based inhibitors of the invention to specifically target regions of GRID gene that are not homologous to Grb2 gene. Specifically, the invention describes the use of nucleic acid-based inhibitors to target sequences that are unique to GRID gene.
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 using well-lrnown methods described herein and generally lrnown in the art. 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 III to X. Examples of such enzymatic nucleic acid molecules also are shown in Tables III to VIII and X. Examples of such enzymatic nucleic acid molecules consist essentially of sequences defined in these Tables.
In yet another embodiment, the invention features antisense nucleic acid molecules and 2-SA chimera including sequences complementary to the substrate sequences shown in Tables III to X. Such nucleic acid molecules can include sequences as shown for the binding arms of the enzymatic nucleic acid molecules in Tables III to VIII and X and sequences shown as GeneBlocTM sequences in Table X. 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 are complementary to a target sequence along a single contiguous sequence of the antisense molecule.
However, in certain embodiments, an antisense molecule can bind to substrate such that the substrate molecule forms a loop, andlor an antisense molecule can bind such that the antisense molecule forms a loop. Thus, the antisense molecule can be complementary to two (or even more) non-contiguous substrate sequences or two (or even more) non-contiguous sequence portions of an antisense molecule can be complementary to a target sequence or both.
By "consists essentially of 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 RNA
such that cleavage at the target site occurs. Other sequences can be present which do not interfere with such cleavage. Thus, a core region can, for example, include one or more loop, stem-loop structure, or linker which does not prevent enzymatic activity. Thus, the underlined regions in the sequences in Tables III and IV can be such a loop, stem-loop, nucleotide linker, and/or non-nucleotide linker and can be represented generally as sequence "X". For example, a core sequence for a hammerhead enzymatic nucleic acid can comprise a conserved sequence, such as 5'-CUGAUGAG-3' and 5'-CGAA-3' connected by a sequence X, where X is 5'-GCCGUUAGGC-3' (SEQ ID NO 2236) or any other stem II
region known in the art or a nucleotide and/or non-nucleotide linker.
Similarly, for other nucleic acid molecules of the instant invention, such as Inozyme, G-cleaver, amberzyme, zinzyme, DNAzyme, antisense, 2-SA antisense, triplex forming nucleic acid, and decoy nucleic acids, other sequences or non-nucleotide linkers may be present that do not interfere with the function of the nucleic acid molecule.

Sequence X can be a linker of >_ 2 nucleotides in length, preferably 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 26, 30, where the nucleotides can preferably be internally base-paired to form a stem of preferably >_ 2 base pairs. Alternatively or in addition, sequence X
can be a non-nucleotide linker. In yet another embodiment, the nucleotide linker X can be a nucleic acid aptamer, such as an ATP aptamer, HIV Rev aptamer (RRE), HIV Tat aptamer (TAR) and others (for a review see Gold et al., 1995, Anhu. Rev. Biochem., 64, 763; and Szostalc &
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 a nucleic acid sequence capable of interacting with a ligand. The ligand can be any natural or a synthetic molecule, including but not limited to a resin, metabolites, nucleosides, nucleotides, drugs, toxins, transition state analogs, peptides, lipids, proteins, amino acids, nucleic acid molecules, hormones, carbohydrates, receptors, cells, viruses, bacteria and others.
In yet another embodiment, the non-nucleotide linker X is as defined herein.
The term "non-nucleotide linker" as used herein include either abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, or polyhydrocarbon compounds.
Specific examples include those described by Seela and Kaiser, Nucleic Acids Res. 1990, 18:6353 and Nucleic Acids Res. 1987, 15:3113; Cload and Schepartz, J. Aru.
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 Bioclaemist~y 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., 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

and Ferentz and Verdine, J. Arn. Chem. Soc. 1991, 113:4000, all hereby incorporated by reference herein. The term "non-nucleotide" further refers to any group or compound which can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions and allows the remaining bases to exhibit their enzymatic activity. The group or compound can be abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine. 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.
In another aspect of the invention, ribozymes or antisense molecules that interact with target RNA molecules and inhibit GRID activity (e.g., inhibit GRID gene) 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 can 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 can be used that provide for transient expression of 5 ribozymes or antisense. Such vectors can be repeatedly administered as necessary. Once expressed, the ribozymes or antisense bind to the target RNA and inhibit its function or expression. Delivery of ribozyme or antisense expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that 10 would allow for introduction into the desired target cell. Antisense DNA
can be expressed endogenously via the use of a single stranded DNA intracellular expression vector.
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 15 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.
By "enhanced enzymatic activity" is meant to include activity measured in cells and/or in vivo where the activity is a reflection of both the catalytic activity and the stability of the nucleic acid molecules of the invention. In this invention, the product of these properties can be increased in vivo compared to an all RNA enzymatic nucleic acid or all DNA enzyme. In some cases, the individual catalytic activity or stability of the nucleic acid molecule can be decreased (i.e., less than ten-fold), but the overall activity of the nucleic acid molecule is enhanced in vivo.
The nucleic acid molecules of the instant invention, individually, or in combination or in conjunction with other drugs, can be used to treat diseases or conditions discussed above. For example, to treat a disease or condition associated with the levels of GRID, the patient can be treated, or other appropriate cells can 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 can be used in combination with one or more known therapeutic agents to treat tissue/graft rejection, leukemia and/or other disease states or conditions which respond to the modulation of GRID
expression.
In another preferred embodiment, the invention features nucleic acid-based inhibitors (e.g., enzymatic nucleic acid molecules (ribozymes), antisense nucleic acids, antisense chimeras, triplex DNA, antisense nucleic acids containing RNA
cleaving chemical groups) and methods for their use to down regulate or inhibit the expression of genes (e.g., GRID) related to the progression and/or maintenance of tissue/graft rejection, leukemia and/or other disease states or conditions which respond to the modulation of GRID expression.
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 can independently be targeted to the same or different sites.
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.
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 First the drawings will be described briefly.
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, ~'cietace, 249, 783; Pace et al., 1990, J. Biol. Chena., 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 96119577). HDV Ribozyme: : I-IV are meant to indicate four stem-loop structures (Been et al., US Patent No. 5,625,047). Hammerhead Ribozyme:
I-III are meant to indicate three stem-loop structures; stems I-III can be of any length and can be symmetrical or asymmetrical (Unman et al., 1996, Cur. Op.
Sty°uct. Bio., 1, 527).
Hairpin Ribozyme: 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 (preferably 3 - 20 bases, i.e., m is from 1 - 20 or more). Helix 2 and helix 5 can be covalently linked by one or more bases (i.e., r is >_ 1 base). Helix 1, 4 or 5 can 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-pairing interaction. These nucleotides can 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 can 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 can 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 (Usman et al., 1996, GuYY. Op.
Stf°uct. Bio., l, 527); NCH Rz represents the NCH ribozyme motif (Ludwig & Sproat, International PCT
Publication No. WO 98/58058); G-Cleaver, represents G-cleaver ribozyme motif (Kore et al., 1998, Nucleic Acids Reseaf~ch 26, 4116-4120). N or n, represent independently a nucleotide which can 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-ally!
modification, but those skilled in the art will recognize that this position can be modified with other modifications well !mown 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
99155857, incorporated by reference herein; also referred to as Class I
Motif). The Amberzyme motif is a class of enzymatic nucleic molecules that do not require the presence of a ribonucleotide (2'-OH) group for its activity.
Figure 4 shows an example of the Zinzyme A ribozyme motif that is chemically stabilized (Beigelman et al., International PCT publication No. WO 99/55857, incorporated by reference herein; also referred to as Class A or Class II
Motif). The Zinzyme motif is a class of enzymatic nucleic molecules that do not require the presence of a ribonucleotide (2'-OH) group for its activity.
Figure 5 shows an example of a DNAzyme motif described by Santoro et al., 1997, PNAS, 94, 4262.
Figure 6 shows a graph of optimization of GeneBloc concentration. A
fluoresceinated randomized antisense GeneBloc (fGB) was used as a marker for uptake using a axed concentration of lipid. Cells were either untreated (A) or treated continuously for 24hrs with 10-200nM antisense GeneBloc (B-F). Following treatment, cells were analyzed by flow cytometry. Gate M1 represents either untransfected cells or cells refractory to transfection. Gate M2 represents the transfected cells.
Figure 7 shows a bar graph of a primary screen of twelve GRID GeneBlocs.
Taqman mRNA assay was used to quantify the level of GRID transcript in Jurkat cells treated continuously for 24 hours with 100nM antisense GeneBloc and S.O~.gml-' cationic lipid.
For comparison, all data was normalized to the level of (3-actin. Error bars represent the standard error of the mean of triplicate points.
Figure 8 shows a graph demonstrating that flow cytometric sorting of transfected cells improves antisense GeneBloc mediated inhibition of GRID mRNA expression.
Jurlcat cells were treated continuously for 24 and 72 hours with GB 14540 (75nM) or control GeneBloc GBC3.3 (75nM) spiked with 25nM fluorescent randomized GeneBloc (A) to facilitate the identiftcation of transfected cells. After transfection, the 10% most and least fluorescent cells (gates M2 and M1 respectively) were sorted on a FACStar Plus. Post-sort low transfecting (B) and high transfecting (C) fractions were re-analyzed fox purity.
Histograms A-D are representative of results obtained in all experiments and were taken from cells treated for 72 hours. The GRID mRNA content of all samples was quantified by Taqman RNA assay and normalized to the (3-actin content. For the purposes of inter-experiment comparison, all GB 14540 values were also normalized to the appropriate , control GBC3.3 value. (D) Normalized GRID mRNA levels in pre-sort samples;
(E) Normalized GRID mRNA levels in the post-sort low transfecting fraction; (F) Normalized GRID mRNA levels in the post-sort high transfecting fraction. Error bars represent the range of duplicate points.
Figure 9 shows a graph representing the phenotypic analysis of antisense GeneBloc treated Jurlcat cells following activation with anti-CD3 and anti-CD28 anti-sera. Jurkat cells were treated continuously for 72 hours with the anti-GRID reagent GB
14540 (A, C) and the mismatch control reagent GB 17477 (B, D), activated for 22 hours (C, D) and stained for the surface activation marker CD69. Unactivated samples are shown in (A, B).
Mechanism of action of Nucleic Acid Molecules of the Invention Antisense: Antisense molecules can be modified or unmodified RNA, DNA, or mixed polymer oligonucleotides which primarily function by specifically binding to matching sequences resulting in inhibition of peptide synthesis (Wu-Pong, Nov 1994, BioPharrn, 20-33). The antisense oligonucleotide binds to target RNA by Watson Criclc base-pairing and blocks gene expression by preventing ribosomal translation of the bound sequences either by steric blocking or by activating RNase H enzyme. Antisense molecules can also alter protein synthesis by interfering with RNA processing or transport from the nucleus into the cytoplasm (Mukhopadhyay & Roth, 1996, Crit. Rev. in Oncogenesis 7, 151-190).

In addition, binding of single stranded DNA to RNA can result in nuclease degradation of the heteroduplex (Wu-Pong, supra; Crooke, supr°a). To date, the only backbone modified DNA chemistry known to act as substrates for RNase H are phosphorothioates, phosphorodithioates, and borontrifluoridates. Recently it has been 5 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
98113526;
10 Thompson et al., International PCT Publication No. WO 99/54459; Hartmann et al., USSN
60/101,174 which was filed on September 21, 1998) all of these are incorporated by reference herein in their entirety.
In addition, antisense deoxyoligoribonucleotides can be used to taxget RNA by means of DNA-RNA interactions, thereby activating RNase H, which digests the target 15 RNA in the duplex. Antisense DNA can be expressed endogenously in vivo via the use of a single stranded DNA intracellular expression vector or equivalents and variations thereof.
Triplex Formint~~ Oli~onucleotides (TFO): Single stranded DNA can be designed to bind to genomic DNA in a sequence specific manner. TFOs are comprised of pyrimidine-rich oligonucleotides which bind DNA helices through Hoogsteen Base-pairing (Wu-Pong, 20 supra). The resulting triple helix composed of the DNA sense,.DNA
antisense, and TFO
disrupts RNA synthesis by RNA polymerase. The TFO mechanism can result in gene expression or cell death since binding may be irreversible (Mulchopadhyay &
Roth, supra).
2-SA Antisense Chimera: The 2-SA 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-SA synthetase and RNase L, are required for RNA cleavage. The 2-SA synthetases require double stranded RNA to form 2'-5' oligoadenylates (2-SA). 2-SA then acts as an allosteric effector for utilizing RNase L
which has the ability to cleave single stranded RNA. The ability to form 2-SA
structures with double stranded RNA makes this system particularly useful for inhibition of viral replication.
(2'-5') oligoadenylate structures can be covalently linked to antisense molecules to form chimeric oligonucleotides capable of RNA cleavage (Torrence, supra).
These molecules putatively bind and activate a 2-SA dependent RNase, the oligonucleotide/enzyme complex then binds to a target RNA molecule which can then be cleaved by the RNase enzyme.
Enzymatic Nucleic Acid: Several varieties of naturally occurring 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, Scieface 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 &
Uhlenbeclc, 1994, supra; Ishizalca 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 tr°ans (and thus can cleave other RNA molecules) under physiological conditions.
Nucleic acid molecules of this invention can block to some extent GRID protein expression and can be used to treat disease or diagnose disease associated with levels of GRID.
The enzymatic nature of an enzymatic nucleic acid has significant advantages, such as the concentration of enzymatic nucleic acid necessary to affect a therapeutic treatment is lower. This advantage reflects the ability of the enzymatic nucleic acid to act enzymatically. Thus, a single enzymatic nucleic acid molecule is able to cleave many molecules of target RNA. In addition, the enzymatic nucleic acid 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 an enzymatic nucleic acid molecule.
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. LISA
8788, 1987;
Dreyfus, 1988, Eitasteita Quart. J. Bio. Med., 6, 92; Iiaseloff and Gerlach, 334 Nature 585, 1988; Cech, 260 JAMA 3030, 1988; and Jefferies et al., 17 Nucleic Acids Researcla 1371, 1989; Santoro et al., 1997 supra).
Because of their sequence specificity, traps-cleaving enzymatic nucleic acid molecules show promise as therapeutic agents for human disease (iJsman &
McSwiggen, 1995 Atan. Rep. Med. Ghena. 30, 285-294; Christoffersen and Marr, 1995 J. Med.
Chena.
38, 2023-2037). Enzymatic nucleic acid molecules 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 GeneBloc reagents, which are essentially nucleic acid molecules (e.g., ribozymes, antisense) capable of down-regulating gene expression.
GeneBlocs are modified oligonucleotides, including ribozymes and modified antisense oligonucleotides, that bind to and target specific mRNA molecules.
Because GeneBlocs can be designed to target any specific mRNA, their potential applications are quite broad. Traditional antisense approaches have often relied heavily on the use of phosphorothioate modifications to enhance stability in biological samples, leading to a myriad of specificity problems stemming from non-specific protein binding and general cytotoxicity (Stein, 1995, Nature Medicine, 1, 1119). In contrast, GeneBlocs contain a number of modifications that confer nuclease resistance while making minimal use of phosphorothioate linkages, which reduces toxicity, increases binding affinity, and minimizes non-specific effects compared with traditional antisense oligonucleotides.
Similar reagents have recently been utilized successfully in various cell culture systems (Vassar, et al., 1999, Science, 286, 735) and in vivo (Jarvis et al., manuscript in preparation). In addition, novel cationic lipids can be utilized to enhance cellular uptake in the presence of serum. Since ribozymes and antisense oligonucleotides regulate gene expression at the RNA level, the ability to maintain a steady-state dose of GeneBloc over several days is important for target protein and phenotypic analysis. The advances in resistance to nuclease degradation and prolonged activity in vitro have supported the use of GeneBlocs in target validation applications.
Target 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.

All of these publications are hereby incorporated by reference herein in their totality.
Other examples include the following PCT applications, which concern inactivation of expression of disease-related genes: WO 95/23225, WO 95/13380, WO 94/02595, all of which are incorporated by reference herein. Rather than repeat the guidance provided in those documents here, specific examples of such methods are provided herein, 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 sequences of human GRID RNAs were screened for optimal enzymatic nucleic acid and antisense target sites using a computer-folding algorithm. Antisense, hammerhead, DNAzyme, NCH, amberzyme, zinzyme, or G-Cleaver. ribozyme binding/cleavage sites were identified. These sites are shown in Tables III to VIII and X (all sequences are 5' to 3' in the tables; underlined regions can be any sequence or linker X as previously defined herein, 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. 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 are also useful to test efficacy of action of the enzymatic nucleic acid molecule and/or antisense prior to testing in humans.
Antisense, hammerhead, DNAzyme, NCH, amberzyme, zinzyme or G-Cleaver ribozyme binding/cleavage sites were identified. The nucleic acid molecules were individually analyzed by computer folding (Jaeger et al., 1989 Proc. Natl.
Acad. Sci. USA, 86, 7706) to assess whether the sequences fold into the appropriate secondary structure.
Those nucleic acid molecules with unfavorable intramolecular interactions, such as between the binding arms and the catalytic core, were eliminated from consideration.
Varying binding arm lengths can be chosen to optimize activity.
Antisense, hammerhead, DNAzyme, NCH, amberzyme, zinzyme or G-Cleaver ribozyme binding/cleavage sites were identified and were designed to anneal to various sites in the RNA target. The binding arms are complementary to the target site sequences described above. The nucleic acid molecules were chemically synthesized. The method of synthesis used follows the procedure for normal DNA/RNA synthesis as described below and in Usman et al., 1987 J. Ana. 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 be similarly synthesized.
Oligonucleotides (e.g.; antisense GeneBlocs) are synthesized using protocols known in the art as described in Caruthers et al., 1992, Methods in Enzyfnology 211, 3-19, Thompson et al., W ternational PCT Publication No. WO 99154459, 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 pmol 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 IT
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 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 ~L of 0.25 M =
15 ~cmol) can be used in each coupling cycle of 2'-O-methyl residues relative to polymer-bound 5'-hydroxyl. A 22-fold excess (40 ~,L of 0.11 M = 4.4 ~.mol) of deoxy phosphoramidite and a 70-fold excess of S-ethyl tetrazole (40 ~L of 0.25 M = 10 ~.mol) 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; 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 (PERSEPTIVET~. 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 5 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 10 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, axe 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.
Arn. Chern.
15 Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res., 18, 5433;
Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684 and Wincott et al., 1997, Methods Mol. Bio., 74, 59, and makes use of common.nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end. In a non-limiting example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 p,mol 20 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 25 ~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 ~mol) can be used in each coupling cycle of 2'-O-methyl residues relative to polymer-bound 5'-hydroxyl. A 66-fold excess (120 ~,L of 0.11 M =
13.2 ~,mol) of allcylsilyl (ribo) protected phosphoramidite and a 150-fold excess of S-ethyl tetrazole (120 ~.L of 0.25 M = 30 wmol) 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; detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); oxidation solution is 16.9 mM I2, 49 mM pyridine, 9% water in THF (PERSEPTIVETM). Burdick & Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc. 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 ~L of a solution of 1.5 mL N-methylpyrrolidinone, 750 ~L TEA and 1 mL TEA~3HF
to provide a 1.4 M HF concentration) and heated to 65 °C. After 1.5 h, the oligomer is quenched with 1.5 M 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 15 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 examples 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 Iigation (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, Biocofzjugate Claena. 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 Syynp. See. 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 III to X. 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 III to X can be formed of ribonucleotides or other nucleotides or non-nucleotides. Such ribozymes with enzymatic activity are equivalent to the ribozymes described specifically in the Tables.
Optimizing Activity of the nucleic acid molecule of the invention.
Chemically synthesizing nucleic acid molecules with modifications (base, sugar and/or phosphate) that prevent their degradation by serum ribonucleases can increase their potency (see e.g., Eclcstein et al., International Publication No. WO
92/07065; Perrault et al., 1990 Natuf°e 344, 565; Pieken et al., 1991, Science 253, 314;
Usman and Cedergren, 1992, Trends ih BioclZeyn. 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 described herein). All these references are incorporated by reference herein. Modifications which enhance their efficacy in cells, and removal of bases from nucleic acid molecules to shorten oligonucleotide synthesis times and reduce chemical requirements are preferably desired.

There are several examples in the art describing sugar, base and phosphate modifications that can be introduced into nucleic acid molecules with significant enhancement in their nuclease stability and efficacy. For example, oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-O-methyl, 2'-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992, TIBS. 17, 34;
Usman et al., 1994, Nucleic Acids Synap. Ser. 31, 163; Burgin et al., 1996, Biochemistry , 35, 14090). Sugar modifications of nucleic acid molecules have been extensively described in the art (see Eckstein et al., InteYnatiorzal 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, Treads in Biochem. Sci. , 1992, 17, 334-339; Usman et al.
Internrational Publication PCT No. WO 93/15187; Sproat, US Patent No.
5,334,711 and Beigelman et al., 1995, J. Biol. Claem., 270, 25702; Beigelman et al., International PCT
publication No. WO 97/26270; Beigelman et al., US Patent No. 5,716,824; Usman et al., US patent No. 5,627,053; Woolf et al., International PCT Publication No. WO
98/13526;
Thompson et al., USSN 60/082,404 which was filed on April 20, 1998; Karpeislcy et al., 1998, Tet>"alaedr~on Lett., 39, 1131; Earnshaw and Gait, 1998, Biopolyrner°s (Nucleic acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Arrnu. Rev. Biochern., 67, 99-134; and Burlina et al., 1997, Bioorg. Med. Claem., 5, 1999-2010; all of the references are hereby incorporated by reference herein in their totalities). 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. In view of such teachings, similar modifications can be used as described herein to modify the nucleic acid molecules of the instant invention.
While chemical modification of oligonucleotide internucleotide linkages with phosphorothioate, phosphorothioate, and/or 5'-methylphosphonate linkages improves stability, too many of these modifications may cause some toxicity. Therefore, when designing nucleic acid molecules the amount of these 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.
Use of the nucleic acid-based molecules of the invention can lead to improved 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 can 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 should preferably 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. The nucleic acid molecules should be resistant to nucleases in order to function as effective intracellular therapeutic agents when delivered exogenously.
Improvements in the chemical synthesis of nucleic acid molecules described in the instant invention and in the art (see, e.g., Wincott et al., 1995, Nucleic Acids Res., 23:2677;
Carruthers, et al., 1992, Methods in Enzymology, 211:3-19, each 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.
In yet another preferred embodiment, nucleic acid catalysts having chemical modifications which maintain or enhance enzymatic activity are provided. Such nucleic acid is also generally more resistant to nucleases than unmodified nucleic acid. 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 5' 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 can help in delivery and/or localization within a cell. The cap can be present at the 5'-terminus (5'-cap) or at the 3'-terminus (3'-cap) or can be present on both termini. In non-limiting examples, the 5'-cap is selected from the group consisting of inverted abasic residue (moiety), 4',5'-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide, 4'-thin 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).
Suitable 3'-caps include 4',5'-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4'-thio nucleotide, carbocyclic nucleotide; 5'-amino-alkyl phosphate; 1,3-5 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;
tlaYeo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; 3,4-dihydroxybutyl nucleotide;
3,5-dihydroxypentyl nucleotide, 5'-5'-inverted nucleotide moiety; 5'-5'-inverted abasic 10 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).
15 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, 20 uracil or thymine.
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 can be substituted or unsubstituted. When substituted the substituted 25 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 30 allcenyl group can 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 has 1 to 12 carbons. More preferably it is a lower allcynyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkynyl group can 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 can also include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups. An "aryl" group refers to an aromatic group which has at least one ring having a conjugated ~ electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which can 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 "allcylaryl"
group refers to an alkyl group (as described above) covalently joined to an aryl group (as described above).
Carbocyclic aryl groups are groups wherein the ring atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted. Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted. An "amide" refers to an -C(O)-NH-R, where R is either alkyl, aryl, alkylaryl or hydrogen. An "ester" refers to an -C(O)-OR', where R is either alkyl, aryl, allcylaryl or hydrogen.
By "nucleotide" is meant a heterocyclic nitrogenous base in N-glycosidic linkage with a phosphorylated sugar. Nucleotides are recognized in the art to include natural bases (standard), and modified bases well known in the art. Such bases are generally located at the 1' position of a nucleotide sugar moiety. Nucleotides generally comprise a base, sugar and a phosphate group. The nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and other; see for example, Usman and McSwiggen, supra; Eckstein et al., International PCT
Publication No.
WO 92/07065; Usman et al., International PCT Publication No. WO 93/15187;
Uhlman &
Peyman, supra all are hereby incorporated by reference herein). 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 chemically modified and other natural nucleic acid bases that can be introduced into nucleic acids include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, quesosine, 2-thiouridine, 4-thiouridine, wybutosine, wybutoxosine, 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 5'-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, beta-D-galactosylqueosine, 1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine, methylcytidine, 2-methyladenosine, 2-methylguanosine, N6-methyladenosine, 7-methylguanosine, 5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylcarbonylmethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine, beta-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine, threonine derivatives 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 can 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.
By "nucleoside" is meant a heterocyclic nitrogenous base in N-glycosidic linkage with a sugar. Nucleosides are recognized in the art to include natural bases (standard), and modified bases well known in the art. Such bases are generally located at the 1' position of a nucleoside sugar moiety. Nucleosides generally comprise a base and sugar group. The nucleosides can be unmodified or modified at the sugar, and/or base moiety, (also referred to interchangeably as nucleoside analogs, modified nucleosides, non-natural nucleosides, non-standard nucleosides and other; see for example, Usman and McSwiggen, supra;
Eclcstein 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 chemically modified and other natural nucleic acid bases that can be introduced into nucleic acids include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-allcyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, quesosine, 2-thiouridine, 4-thiouridine, wybutosine, wybutoxosine, 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 5'-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, beta-D-galactosylqueosine, 1-methyladenosine, methylinosine, 2,2-dimethylguanosine, 3-methylcytidine, 2-methyladenosine, 2-methylguanosine, N6-methyladenosine, 7-methylguanosine, 5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylcarbonylmethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine, beta-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine, threonine derivatives and others (Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra).
By "modified bases" in this aspect is meant nucleoside bases other than adenine, guanine, cytosine and uracil at 1' position or their equivalents; such bases can 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.
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 PropeYties, in Modey~n Synthetic Methods, VCH, 331-417, and Mesmaeker et al., 1994, Novel Backbone Replacements fos~ Oligohucleotides, in Carbohydrate Modificatiofas iya Antisehse ReseaYCh, 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" is meant one of the bases adenine, cytosine, guanine, thymine, uracil joined to the 1' carbon of (3-D-ribo-furanose.
By "modified nucleoside" 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 can be modified or unmodified.
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, respectively, which are both incorporated by reference herein in their entireties.
Various modifications to nucleic acid (e.g., antisense and ribozyme) structure can be made to enhance the utility of these molecules. For example, modifications can enhance shelf life, half life in vita°o, 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 can 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) andlor other chemical or biological molecules). The treatment of patients with nucleic acid molecules can also include combinations of different types of nucleic acid molecules.
Therapies can be devised which include a mixture of ribozymes (including different ribozyrne 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, Treyads Gell Bio., 2, 139; and Delivery Strategies for Ahtisefase Oligonucleotide Tlaerapeutics, ed. Alchtar, 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 can be utilized for the delivery of virtually any nucleic acid molecule. Nucleic acid molecules can 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 can be directly delivered ex vivo to cells or tissues with or without the aforementioned vehicles. Alternatively, the nucleic acid/vehicle combination can be locally delivered by direct injection or by use of a catheter, infusion pump or stmt. 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 have been incorporated by reference herein.
The molecules of the instant invention can be used as pharmaceutical agents.
Pharmaceutical agents prevent, inhibit the occurrence, or treat (i.e., 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 5 liposomes can be followed as described in the art. The compositions of the present invention can also be formulated and used as tablets, capsules or elixirs for oral administration; suppositories for rectal administration; sterile solutions;
suspensions for injectable administration; and other compositions known in the art.
The present invention also includes pharmaceutically acceptable formulations of the 10 compounds described. These formulations include salts of the above compounds, e.g., acid addition salts, including salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid.
A pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, e.g., systemic administration, into a cell 15 ox 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 20 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 that lead to systemic absorption include, without limitations:
25 intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular. Each of these administration routes exposes the desired negatively charged polymers, e.g., nucleic acids, to an accessible diseased tissue. The rate of entry of a drug into the circulation has been shown to be a function of molecular weight or size. The use of a liposome or other drug carrier comprising the compounds of the instant invention can 30 potentially localize the drug, for example, in certain tissue types, such as the tissues of the reticular endothelial system (RES). A liposome formulation that can facilitate the association of drug with the surface of cells, such as, lymphocytes and macrophages is also useful. This approach can provide enhanced delivery of the drug to target cells by taking advantage of the specificity of macrophage and lymphocyte immune recognition of 35 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.
Non-limiting 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 as those made of polybutylcyanoacrylate, which can deliver drugs across the blood brain barrier and can alter neuronal uptake mechanisms (Pj°og Neuropsychopharnzacol Biol Psyclaiat~-y, 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. PharnZ. Sci., 87, 1308-1315; Tyler et al., 1999, FEBS Lett., 421, 280-284;
Pardridge et al., 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv.
Dr~ugDelivery 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 Iiposomes 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. Chern. Rev. 1995, 95, 2601-2627; Ishiwata et al., Cl2em. Pharm. Bull.
1995, 43, 1005-1011). All incorporated by reference herein. Such liposomes have been shown to accumulate selectively in tumors, presumably by extravasation and capture in the neovascularized target tissues (Lasic et al., Scierace 1995, 267, 1275-1276;
Oku et a1.,1995, Biochim. Biophys. Acta, 1238, 86-90). All incorporated by reference herein.
The long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA
and RNA, particularly compared to conventional cationic liposomes which are lrnown to accumulate in tissues of the MPS (Liu et al., J. Biol. Claem. 1995, 42, 24864-24870; Choi et al., International PCT Publication No. WO 96110391; Ansell et al., International PCT
Publication No. WO 96/10390; Holland et al., International PCT Publication No.
WO
96/10392; all of which are incorporated by reference herein). Long-circulating liposomes are also likely to protect drugs from nuclease degradation to a greater extent compared to cationic liposomes, based on their ability to avoid accumulation in metabolically aggressive MPS tissues such as the liver and spleen.
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 carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Renzington'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 can 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 mglkg 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 can 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, Ps°oc. Natl. Acad. Sci. USA, 88, 10591-5; Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Dropulic et al., 1992, J. Tirol., 66, 1432-41;
Weerasinghe et al., 1991, J. ViYOI., 65, 5531-4; Ojwang et al., 1992, Pf~oc. 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 the references are hereby incorporated in their totality by reference herein). 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 Syrnp. 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.
Chern., 269, 25856; all of these references are hereby incorporated in their totalities 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 can 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 can be used that provide for transient expression of nucleic acid molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the nucleic acid molecule binds to the target mRNA. Delivery of nucleic acid molecule expressing vectors can be systemic, such as by intravenous or infra-muscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that 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 disclosed in the instant invention. The nucleic acid sequence encoding the nucleic acid molecule of the instant invention is operable linked in a manner Which allows expression of that nucleic acid molecule.
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 can 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 III), Transcripts from pol II or pol III promoters are expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type depends on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby.
Prokaryotic RNA
polymerase promoters also can be used, providing that the prokaryotic RNA
polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990, Pnoc. Natl.
Acad. Sci. U S A, 87, 6743-7; Gao and Huang 1993, Nucleic Acids Res.., 21, 2867-72;
Lieber et al., 1993, Metlzods Erazyznol., 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, Pz°oc. Natl.. Acad. Sci. U S A, 90, 6340-4; L'Huillier et al., 1992, EMBO J., 1 l, 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; and 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., sup>"a; 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; and 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 a nucleic acid sequence encoding at least one of the nucleic acid molecules of the invention, in a manner which allows expression of that nucleic acid molecule. The expression vector comprises in one embodiment; a) a transcription initiation region; b) a transcription termination region; c) a nucleic acid sequence encoding at least one said nucleic acid molecule; and wherein said sequence is operably linked to said initiation region and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.
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 5 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 10 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 15 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 20 and activity of nucleic acids of the instant invention.
The following examples demonstrate the selection and design of Antisense, hammerhead, DNAzyme, NCH, Amberzyme, Zinzyme, or G-Cleaver enzymatic nucleic acid molecules and binding/cleavage sites within GRID RNA.
Nucleic acid inhibition of GRID target RNA
25 The use of GeneBlocs to modulate the activity of GRID, a putative component of co-stimulatory signaling in T cells, is herein described. An array of GeneBlocs were designed and screened for their ability to reduce GRID mRNA levels whilst leaving transcripts from the closely related genes Grb2 and GRAD unaffected. A series of experiments were conducted to optimize delivery of GeneBlocs to the Jurlcat T cell line. Using these 30 conditions, applicant has demonstrated the efficacy of these reagents at both the mRNA
and protein level. Anti-CD3/CD28 triggering of Jurkat cells pre-treated with the anti-GRID
GeneBloc results in an impairment of CD69 up-regulation consistent with an important role for GRID in transducing the co-stimulatory signal.

Example 1: Identification of Potential Targ-et Sites in Human GRID RNA
The sequence of human GRID were screened for accessible sites using a computer-folding algorithm. Regions of the RNA were identified that do not form secondary folding structures. These regions contain potential ribozyme andlor antisense binding/cleavage sites. The sequences of these binding/cleavage sites are shown in Tables III-X.
Example 2: Selection of Enzymatic Nucleic Acid Cleavage Sites in Human GRID
RNA
Enzymatic nucleic acid target sites are chosen by analyzing sequences of Human GR>I7 (for example, GenBank accession numbers: AJ011736 and Y18051) and prioritizing the sites on the basis of folding. Enzymatic nucleic acids are designed that bind each target and are individually analyzed by computer folding (Christoffersen et al., 1994 J. Mol.
Struc. Tlaeochena, 311, 273; Jaeger et al., 1989, Proc. Natl. Acad. Sci. USA, 86, 7706) to assess whether the enzymatic nucleic acid sequences fold into the appropriate secondary structure. Those enzymatic nucleic acids with unfavorable intramolecular interactions between the binding arms and the catalytic core are eliminated from consideration. As noted below, varying binding arm lengths can be chosen to optimize activity.
Generally, at least 5 bases on each arm are able to bind to, or otherwise interact with, the target RNA.
Example 3: Chemical Synthesis and Purification of Enzymatic nucleic acids and Antisense for Efficient Cleavage and/or blocking of GRID RNA
Enzymatic nucleic acids and antisense constructs are designed to anneal to various sites in the RNA message. The binding arms of the enzymatic nucleic acids are complementary to the target site sequences described above, while the antisense constructs are fully complimentary to the target site sequences described above. The enzymatic nucleic acids and antisense constructs were chemically synthesized. The method of synthesis used followed the procedure for normal RNA or DNA synthesis as described above and in Usman et al., (1987 J. Arn. Chern. 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 typically >98%.
Enzymatic nucleic acids and antisense constructs also can be synthesized from DNA
templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzyrnol. 180, 51). Enzymatic nucleic acid and antisense constructs are purified by gel electrophoresis using general methods or are purified by high pressure liquid chromatography (HPLC; see Wincott et al., supra; the totality of which is hereby incorporated herein by reference) and are resuspended in water. The sequences of the chemically synthesized enzymatic nucleic acid and antisense constructs used in this study are shown below in Table III-X.
Example 4: Enzymatic nucleic acid Cleavage of GRID RNA Target in vitro Enzymatic nucleic acids targeted to the human GRID RNA are designed and synthesized as described above. These enzymatic nucleic acids can be tested for cleavage activity in vitro, for example, using the following procedure. The target sequences and the nucleotide location within the GRID RNA are given in Tables III-X.
Cleavage Reactions: Full-length or partially full-length, internally-labeled target RNA for enzymatic nucleic acid cleavage assay is prepared by ita 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 lcinase enzyme. Assays are performed by pre-wanning a 2X concentration of purified enzymatic nucleic acid in enzymatic nucleic acid 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 enzymatic nucleic acid 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., enzymatic nucleic acid 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 enzymatic nucleic acid 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: Nucleic acid inhibition of GRID i~ vivo Antisense nucleic acid molecules (GeneBlocs) targeted to the human GRID RNA
are designed and synthesized as described above. These nucleic acid molecules can be tested for cleavage activity in vivo, for example, using the following procedure. The target sequences and the nucleotide location within the GRID RNA are given in Tables III-X.
GRID shares 60.3% and 57.3% homology at the nucleotide level with the closely related adapter proteins Grb2 and GRAP. In order to discriminate between human GRID
and other Grb2 family members, twelve GeneBlocs (see Methods for details) targeting human GRID (GenBank accession number Y18051) were designed, each containing a minimum of six mismatches versus human Grb2 (M96995) and human GRAD (U52518).
In order to determine the optimal site for GeneBloc binding and inhibition of the target mRNA, the efficacy of the GeneBlocs was tested on Jurkat cells. A Taqman RNA
assay was used to quantify the level of GRID transcript in cells treated continuously for 24hrs.
The efficacy of the twelve GeneBlocs, normalized to the levels of a house-keeping gene ((3-actin), is shown in Figure 7. The GeneBloc targeting site 152 (GeneBloc 14540) was the most efficacious, reducing GRID mRNA levels by up to 55% when compared with a randomized control GeneBloc (GBC3.3). To confirm that these effects were target specific, a four base-pair mismatch GeneBloc (GB 17477) was synthesized. GRID mRNA
expression was unaffected in cells treated with the mismatch control GeneBloc compared to untreated cells.
Efficacy of the anti-GRID GeneBloc (GB 14540) in Jurkat cells From the primary screen (Figure 7), the optimal GeneBloc, GB 14540, suppressed GRID mRNA levels by up to 55%. However, this represents the inhibition in a bulk population of cells, some of which are refractory to transfection (see Figure 6D-F). To investigate the correlation between dose and efficacy, GB 14540 was spiked with 25% fGB.
Based on mixture experiments with active GeneBlocs in other systems, it was not expected that the presence of the fluorescent GeneBloc would interfere With anti-GRID
activity of GB 14540. Thus, the most highly fluorescent cells represent the population of cells transfected with the highest concentration of active GeneBloc ('high transfecting'), whilst the cells that appear to be refractory to transfection should contain a significantly lower concentration active GeneBloc ('low transfecting').
Following transfection of a GB14540:fGB mixture, the high transfecting cells (Figure 8A, Gate M2, the 10% most fluorescent cells) and the low transfecting cells (Figure 8A, Gate Ml, the 10% least fluorescent cells) were purified by FACS
sorting. Re-analysis of the sorted cell populations confirmed greater than 95% purity (Figure 8B-C).
Taqman RNA analysis of the treated cells pre- and post-sort (Figure 8D-F) shows that although GB 14540 inhibition of GRID mRNA expression in an unsorted population is variable between experiments (0-30°l0, Figure 8D), the level of inhibition is significantly increased to 45-63% in the 'high transfecting' fraction (Figure 8F). In contrast, GRID
mRNA levels in the 'low transfecting' fraction was similar to that of cells treated with control GBC3.3 (Figure 8E). These data suggest that the degree of GRID mRNA
inhibition is dependent on the dose of GeneBloc delivered to the cells.

To identify the optimal time-point for inhibition of GRID mRNA levels, samples were sorted as described above at 24 and 72 hours following continuous transfection.
Analysis of pre- and post-sort samples at these time-points revealed that in pre-sort samples, inhibition of GRID transcript occurred within 24 hours and did not significantly increase throughout the time-course of the experiment (Figure 8D). In the 'high transfecting' fractions, reduction of GRID transcript was ~45% at 24 hours and increased only fractionally at the 72 hour time-point (50-65%, Figure 8F). This suggests that GB 14540 reduced GRID mRNA levels rapidly following transfection and that inhibition was sustained in the continued presence of GB 14540.
Analysis of GRID protein levels in GB 14540 treated cells To determine whether the reduction in GRID transcript levels was associated with a loss of GRID protein, the level of GRID protein in cells treated continuously with active GeneBloc reagent GB 14540 and the mismatch control GB 17477 was assessed. When delivered continuously for 72 hours, GB 14540 caused a substantial reduction in GRID
protein levels as determined by the intensity of the GRID specific band whilst at earlier time-points (24 and 48 hrs) no reduction in protein was observed. Cells treated with the mismatch control GB 17477 showed GRID levels comparable to the untreated sample.
Cells treated continuously with GB 14540 for periods up to 144 hours showed no further reduction in GRID protein levels, suggesting that the effect of the GeneBloc was maximal and sustained from 72 hours onwards. Whilst the effects of the anti-GRID
GeneBloc on mRNA levels are seen at 24 hours, the reduction in GRID protein is delayed a further 48 hours indicating that GRID protein may have a relatively long half life.
The GeneBlocs were designed to target and discriminate GRID from the closely related adapter proteins Grb2 and GRAP. GB 14540 contains 6 and 7 mismatches respectively when aligned with the human Gxb2 and GRAD sequences. Due to the presence of these mismatches, GB 14540 was not expected to inhibit Grb2 mRNA
expression. The Western blots used for the GRID assay were stripped and re-probed using an anti-Grb2 antibody. No difference in Grb2 protein levels was observed between the untreated sample and cells treated with either GB 14540 or the mismatch control reagent GB
17477, confirming that the GB 14540 was specific for GRID.
Phenotypic effects of the anti-GRID GeneBloc on T cell activation GRID is a novel member of the Grb2 family of adapter proteins. A role for GRID
in T cell signaling has been postulated due to its association with known T cell signaling proteins [Law, 1999 #3296][Asada, 1999 #3243][Liu, 1999 #3245] and more recently the T cell co-stimulatory receptor CD28 following activation by cross-linking antibodies (Ellis et al.). To further elucidate the role of GRID in T cell co-stimulatory pathways, applicant studied the expression of early surface activation marker CD69 (Jung et al., 1988, Cellulaf°
Immuzzology, 117, 352, Lanier et al., 1988, .I. Exp. Med., 167, 1572) following activation of 5 Jurkat cells treated with GB 14540 and GB 17477. Jurkat cells were activated by cross-linking anti-CD3 and anti-CD28 monoclonal antibodies using a sub-maximal stimulus to increase the sensitivity of the assay. In cells treated with the mismatch control GeneBloc, GB 17477, 5.7% stained CD69 positive following activation compared with 0.7%

positive in unactivated cells (Figure 9D vs. 9B). In cells treated with the anti-GRID
10 reagent GB 14540, there was a marked reduction in the proportion of activated cells, with only 1.3% staining positive for CD69 (Figure 9C). Expression of CD69 in the unactivated sample remained unaltered at 0.6% (Figure 9A). As the activation stimulus was increased, the relative difference between the cells treated with GB 14540 and GB 17477 decreased even though the proportion of cells staining positive for CD69 increased. This can be 15 attributed to the combination of residual GRID protein and supra-maximal activation stimulus. The latter component is particularly relevant to T cell activation since the dependency on co-stimulation is reduced as the strength of the CD3 signal increases (Geppert and Lipslcy, 1988, J. Clin. Invest., 81, 1497, Geppert and Lipslcy, 1987, Journal oflmmunology, 138, 1660).
20 Taken together, these data suggest that the phenotypic effects described above can be attributed to GRID and not the closely related adapter protein Grb2. The inhibitory effects of GB 14540 on CD69 expression support a role for GRID in T cell co-stimulatory signaling.
Example 6: Delivery of GeneBloc reagents to Jurkat cells 25 As in many mammalian cell culture systems (Marcusson et al., 1998, Nuc.
Acids, Res. 26, 2016), a cationic lipid was found to be necessary to facilitate cellular uptake of oligonucleotide. In preliminary experiments using a fluoresceinated randomized GeneBloc as a marker for uptake, a lipid concentration of 2.5-5.0 q,gml-' was found to be optimal.
Although some cells are readily transfected by the GeneBloc, a sub-population of cells 30 remained refractory to transfection (see Gate M2 vs. M1 in Figures 6D-6F).
In order to minimize the refractory population, the concentration of GeneBloc was varied between 10-200nM. Transfection frequencies of up to 75% (as determined by fraction of cells in Gate M2) were observed in the 50-100nM range of GeneBloc concentration. At lower concentrations (10-25nM), the transfection frequency dropped off very steeply whilst at 35 higher concentrations, no further enhancement of transfection was observed.
Cationic lipids however are not essential for the use of oligonucleotides in vivo (see McGraw et al., 1997, A~zti-Cancer Ds°ug Design, 12, 315-326; Henry et al., 1997, Ahti-Cafacer Drug Design, 12, 409-420).
Example 7: Flow Cytometry Cultures were harvested, washed once and re-suspended in PBS containing 2%
FCS.
Cells were stained with a human anti-CD69 PE-conjugated antibody (Caltag) using an IgG2a PE-conjugate as an isotype control (Becton Dickinson). Cells were analyzed on a Becton Dickinson FACScan using CellQuest software. Cells were sorted on the basis of fluorescence in the FL1 channel using a Becton Diclcinson FACStar Plus. In order to compare the efficiency of GeneBloc uptake using different transfection conditions, a coefficient of transfection was calculated by multiplying the proportion of control GeneBloc (as a fraction of total GeneBloc) and the transfection frequency.
Example 8: Protein Studies Actively growing Jurkat cells (0.1-1.0 x 106) were harvested, washed once in PBS
and re-suspended in 25p.1 PBS. Cells were lysed by the addition of an equal volume of ice-cold 2x RIPA buffer (2% NP40, 1.0% sodium deoxycholate, 0.2% SDS in PBS with 2x protease and phosphatase inhibitors). Following a 30 minute incubation on ice, cell debris was removed by centrifugation and the supernatant denatured at 100°C
for 5 minutes following the addition of an equal volume of 2x SDS protein sample buffer.
Prior to separation by SDS-PAGE electrophoresis, protein content was normalized using a Coomassie~ Plus-200 protein assay reagent (Pierce). For Western blotting, SDS-PAGE
gels were transferred to PVDF membrane (Millipore). Antisera specific for GRID
(rabbit polyclonal courtesy of Claire Ashman, GlaxoWellcome), p85 sub-unit of PI-3-kinase (#06-195, Upstate Biotechnology) and Grb2 (sc-255, Santa Cruz) were used as primary antibodies with an anti-rabbit HRP conjugate as the secondary antibody. Bound antibody was visualized using the SuperSignal~ West Dura chemiluminescent reagent. For re-probing, chemiluminescent substrate and bound antibody were removed with TBST
(TBS
+ 0.5% Tween-20) and ImmunoPure~ IgG Elution Buffer (Pierce) respectively.
Example 9: Cell Culture Human Jurlcat cell lines E6.1 and J6 were maintained at 37°C in 5% COZ
in flasks in RPMI 1641 (+ 25mM HEPES) supplemented with 10% fetal calf serum and glutamine.
Cells were passaged at a density of 1 x 106 cells ml-'. GeneBlocs were delivered to the cells using a modified centrifugation-based transfection protocol (Verma et al., 1998, BioTeclaniques, 25, 46). Cells were grown to a density of 1 x 106 cells m1', harvested by centrifugation and re-suspended in fresh media at 0.75 x 106 cells ml-'.
GeneBloc at lOX

final concentration and cationic lipid (25p,gml-') at lOX anal concentration were prepared separately in RPMI media (no FCS or glutamine), mixed 1:1 and incubated at 37°C for 30 minutes. 1.6m1 aliquots of the cell suspension was dispensed into a 6-well tissue-culture treated plate and 0.4m1 of the GeneBloc:lipid mixture added drop-wise. The GeneBloc:lipid solution was evenly distributed by gentle agitation. Following centrifugation at 1000rpm for 60 minutes at room temperature, the 6-well plates were incubated for 24-72 hours at 37°C.
Example 10: Real-time quantitative PCR (Taqman) Human GRID oligonucleotide Taqman probe 6FAM-(5'-ACTCCAGTTTCCCAAATGGTTTCACGAA-3') (SEQ ID NO 2237) -TAMRA and human actin Taqman probe JOE-(5'-TCGAGCACGGCATCGTCACCAA-3') (SEQ ID
NO 2238) -TAMRA were purchased from PE Applied Biosystems. GRID primers (forward, 5'-AGGATATGTGCCCAAGAATTTCATA-3') (SEQ ID NO 2239) and reverse, (5'-TGCCTGGTGTCGAGAGAGG-3') (SEQ ID NO 2240) and actin primers (forward, 5'-GCATGGGTCAGAAGGATTCCTAT-3') (SEQ ID NO 2241) and reverse, (5'-TGTAGAAGGTGTGGTGCCAGATT-3') (SEQ ID NO 2242) were purchased from Life Technologies. The Taqman probes were labeled with a reporter dye (FAM or JOE) at the 5' termini and a quencher dye (TAMRA) at their 3' termini. A combination RT-PCR
and Taqman PCR was performed for each sample in triplicate on an ABI PRISM

Sequence Detection System using the following program: 48°C for 30 minutes, 95°C for 10 minutes and then 40 cycles of 95°C for 15 seconds and 60°C
fox 1 minute. The reaction was performed in a total volume of 40p.1 with each tube containing 10U RNase inhibitor (Promega), 1.25U Amplitaq Gold (PE Biosystems), 100nM of the GRID and Actin primers, 100nM GRID FAM Taqman probe, 100nM Actin JOE Taqman probe and 10U
MuLV reverse transcriptase. PCR Buffer (PE Biosystems #4304441) and dNTPs (PE
Biosystems #N808-0261) were added according to the manufacturer's guidelines.
A
standard curve was generated using serially diluted purified RNA (300, 100, 33 and 1 lng) prepared from untreated Jurkat cells.
Example 11: RNA isolation Total RNA was isolated from Jurkat J6 or Jurkat E6.1 cells using the 96-well RNeasy lcit (Qiagen) and a minor modification of their protocol. 901 of RLT buffer was added to each sample, followed by an equal volume of 70% ethanol. Samples were mixed and transferred to a RNeasy-96-plate. A vacuum was applied for 15-60sec until the wells were dry. 80,1 of lx DNase solution was added (40mM Tris-HCl pH 7.5, lOmM MgCl2, lOmM
CaClz, lOmM NaCl, 1.2U/~,l RNase-free DNase I). Following incubation at room temperature for 15 minutes, lml of Buffer RW1 was added and incubated for a further 5 minutes. The buffer was removed by applying a vacuum. The wells were washed once in lml of RPE. A second lml aliquot of Buffer RPE was added and the RNeasy-96-plate centrifuged at 6000 rpm for 10 minutes. The RNA was eluted by the addition of 100m1 of RNase-free water. Following incubation at room temperature for 1 minute, the RNA was recovered by centrifugation at 6000rpm for 4 minutes and stored at -70°C.
Indications Particular conditions and disease states that can be associated with GRID
expression modulation include, but are not limited to. tissue/graft rejection and cancer, such as leukemia.
The present body of knowledge in GRID research indicates the need for methods to assay GRID activity and for compounds that can regulate GRID expression for research, diagnostic, and therapeutic use.
Radiation, chemotherapeutic treatments, and Cyclosporin are non-limiting examples of compounds and/or methods that can be combined with or used in conjunction with the nucleic acid molecules (e.g. ribozymes and antisense molecules) of the instant invention.
Those slcilled in the art will recognize that other drug compounds and therapies can be similarly be readily combined with the nucleic acid molecules of the instant invention (e.g.
ribozymes and antisense molecules) are hence within the scope of the instant invention.
Diagnostic uses The nucleic acid molecules of this invention (e.g., ribozyrnes) can be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of GRID RNA in a cell. The close relationship between ribozyme activity and the structure of the target RNA allows the detection of mutations in any region of the molecule which alters the base-pairing and three-dimensional structure of the target RNA.
By using multiple ribozymes described in this invention, one can map nucleotide changes which are important to RNA structure and function in vitro, as well as in cells and tissues.
Cleavage of target RNAs with ribozymes can be used to inhibit gene expression and define the role (essentially) of specified gene products in the progression of disease. In this manner, other genetic targets can be defined as important mediators of the disease. These experiments can lead to better treatment of the disease progression by affording the possibility of combinational therapies (e.g., multiple ribozymes targeted to different genes, ribozymes coupled with known small molecule inhibitors, or intermittent treatment with combinations of ribozymes and/or other chemical or biological molecules).
Other ira vitro uses of ribozymes of this invention include detection of the presence of mRNAs associated with GRID-related condition. Such RNA is detected by determining the presence of a cleavage product after treatment with a ribozyme using standard methodology.
In a specific example, ribozymes which can cleave only wild-type or mutant forms of the target RNA are used for the assay. The first ribozyme is used to identify wild-type RNA present in the sample and the second ribozyme is used to identify mutant RNA in the sample. As reaction controls, synthetic substrates of both wild-type and mutant RNA are cleaved by both ribozymes to demonstrate the relative ribozyme efficiencies in the reactions and the absence of cleavage of the "non-targeted" RNA species. The cleavage products from the synthetic substrates also serve to generate size markers for the analysis of wild-type and mutant RNAs in the sample population. Thus, each analysis can require two ribozymes, two substrates and one unknown sample, which are combined into six reactions. The presence of cleavage products is determined using an RNAse protection assay so that full-length and cleavage fragments of each RNA can be analyzed in one lane of a polyacrylamide gel. It is not absolutely required to quantify the results to gain insight into the expression of mutant RNAs and putative risk of the desired phenotypic changes in target cells. The expression of mRNA whose protein product is implicated in the development of the phenotype (i.e., GRID) is adequate to establish rislc. If probes of comparable specific activity are used for both transcripts, then a qualitative comparison of RNA levels is adequate and will decrease the cost of the initial diagnosis.
Higher mutant form to wild-type ratios are correlated with higher risk whether RNA levels are compared qualitatively or quantitatively.
Additional Uses Potential usefulness of sequence-specific enzymatic nucleic acid molecules of the instant invention 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 Aran. Rev.
Biochena. 44:273). For example, the pattern of restriction fragments can be used to establish sequence relationships between two related RNAs, and large RNAs can 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 eulcaryotic systems including plant, or mammalian cells.
All patents and publications mentioned in the specification are indicative of the levels of skill of those slcilled 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 5 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 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 10 modifications can 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 15 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 20 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 25 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 Marlcush group or other group.
30 Other embodiments are within the following claims.

TABLE I
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 maintenance of the active structure.
~ Over 300 known members of this class. Found as an intervening sequence in TetrahynZesia therrnophila rRNA, fungal mitochondria, chloroplasts, phage T4, blue-green algae, and others.
~ Major structural features largely established through phylogenetic comparisons, mutagenesis, and biochemical studies [;ii].
~ Complete kinetic framework established for one ribozyme (ii ~x ~ ~~i~
~ Studies of ribozyme folding and substrate docking underway (~i ~~ixi,iX].
~ Chemical modification investigation of important residues well established (~,Xi].
~ The small (4-6 nt) binding site may make this ribozyme too non-specific for targeted RNA cleavage, however, the Tetrahymena group 1 intron has been used to repair a "defective" beta-galactosidase message by the ligation of new beta-galactosidase sequences onto the defective message RNAse P RNA (M1 RNA) ~ Size: 290 to 400 nucleotides.
~ RNA portion of a ubiquitous ribonucleoprotein enzyme.
~ Cleaves tRNA precursors to form mature tRNA [Xiii]
~ Reaction mechanism: possible attack by M2+-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~ ~XVii~
Group II Introns ~ Size: >1000 nucleotides.
~ Trans cleavage of target RNAs recently demonstrated [X~ii ~XiX~.
~ 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' br anch point.
~ Only natural ribozyme with demonstrated participation in DNA
cleavage [X ;xXi] in addition to RNA cleavage and ligation.
~ Major structural features largely established through phylogenetic comparisons [Xxii].
~ Important 2' OH contacts beginning to be identified [XXiii]
~ Kinetic framework under development [XXiV]

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.
~ Only 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 genes ate 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.
~ Essenfiial structural features largely defined, including 2 crystal structures [xxv ~XX~ii' ~ Minimal ligation activity demonstrated (for engineering through irc vitro selection) [XX~iii]
~ Complete kinetic framework established for two or more ribozymes ~ 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 vixus, arabis mosaic virus and chicory yellow mottle virus) which uses RNA as the infectious agent.
~ Essential structural features largely defined (XXXi XXxii XXXiii XXXi~]
~ Ligation activity (in addition to cleavage activity) makes ribozyme amenable to engineering through in vitro selection (XXX~~
~ Complete kinetic framework established for one ribozyme (XXX~i]
~ Chemical modification investigation of important residues begun (xxxvii xxxviii,.
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 (xl~.
~ 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.

XiiCircular form of HDV isXlii active and shows increased nuclease stability [xlill~
' . Michel, Francois; Westhof, Eric. Slippery substrates. Nat. Struct. Biol.
(1994),1(1), 5-7.
ii . Lisacek, Frederique; Diaz, Yolande; Michel, Francois. Automatic identification of group I intron cores in genomic DNA sequences. J. Mol. Biol. (1994), 235(4),1206-17.
"' . Herschlag, Daniel; Cech, Thomas R.. Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme.1. Kinetic description of the reaction of an RNA
substrate complementary to the active site. Biochemistry (1990), 29(44),10159-71.
'° . Herschlag, Daniel; Cech, Thomas R.. Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 2. Kinetic description of the reaction of an RNA
substrate that forms a mismatch at the active site. Biochemistry (1990), 29(44),10172-80.
Knitt, Deborah S.; Herschlag, Daniel. pH Dependencies of the Tetrahymena Ribozyme Reveal an Unconventional Origin of an Appaxent pKa. Biochemistry (1996), 35(5), 1560-70.
°' . Bevilacqua, Philip C.; Sugimoto, Naoki; Turner, Douglas H.. A
mechanistic framework for the second step of splicing catalyzed by the Tetrahymena ribozyme.
Biochemistry (1996), 35(2), 648-58.
°" . Li, Yi; Bevilacqua, Philip C.; Mathews, David; Turner, Douglas H..
Thermodynamic and activation parameters for binding of a pyrene-labeled substrate by the Tetrahymena ribozyme: docking is not diffusion-controlled and is driven by a favorable entropy change.
Biochemistry (1995), 34(44),14394-9.
°"' . Banerjee, Aloke Raj; Turner, Douglas H.. The time dependence of chemical modification reveals slow steps in the foldW g of a group I ribozyme.
Biochemistry (1995), 34(19), 6504-12.
'x . Zarrinkar, Patxick P.; Williamson, James R.. The P9.1-P9.2 peripheral extension helps guide folding of the Tetrahymena ribozyme. Nucleic Acids Res. (1996), 24(5), 854-8.
X . Strobel, Scott A.; Cech, Thomas R.. Minor groove recognition of the conserved G.cntdot.U pair at the Tetrahymena ribozyme reaction site. Science (Washington, D. C.) (1995), 267(5198), 675-9.
X' . Strobel, Scott A.; Cech, Thomas R.. Exocyclic Amine of the Conserved G.cntdot.U
Paix at the Cleavage Site of the Tetrahymena Ribozyme Contributes to 5'-Splice Site Selection and Transition State Stabilization. Biochemistry (1996), 35(4),1201-11.
x". Sullenger, Bruce A.; Cech, Thomas R.. Ribozyme-mediated repair of defective mRNA by targeted traps-splicing. Nature (London) (1994), 371(6498), 619-22.
X"'. Robertson, H.D.; Altman, S.; Smith, J.D. J. Biol. Chem., 247, 5243-5251 (1972).
X'°. Forster, Anthony C.; Altman, Sidney. External guide sequences for an RNA enzyme.
Science (Washington, D. C.,1883-) (1990), 249(4970), 783-6.
x°. yuan, Y.; Hwang, E. S.; Altman, S. Targeted cleavage of mRNA by human RNase P.
Proc. Natl. Acad. Sci. USA (1992) 89, 8006-10.
x°' . Harris, Michael E.; Pace, Norman R.. Identification of phosphates involved in catalysis by the ribozyme RNase P RNA. RNA (1995),1(2), 210-18.
X°" . Pan, Tao; Loria, Andrew; Zhong, Kun. Probing of tertiary interactions in RNA: 2'-hydroxyl-base contacts between the RNase P RNA and pxe-tRNA. Proc. Natl. Acad.
Sci. U. S.
A. (1995), 92(26),12510-14.
X°"' . Pyle, Anna Marie; Green, Justin B.. Building a Kinetic Framework for Group II Intron Ribozyme Activity: Quantitation of Interdomain Binding and Reaction Rate.
Biochemistry (1994), 33(9), 2716-25.

XtX . Michels, William J. Jr.; Pyle, Anna Marie. Conversion of a Group II
Intron into a New Multiple-Turnover Ribozyme that Selectively Cleaves Oligonucleotides:
Elucidation of Reaction Mechanism and Strucfure/Function Relationships. Biochemistry (1995), 34(9), 2965-77.
xX . Zimmerly, Steven; Guo, Huatao; Eskes, Robert; Yang, Jian; Perlman, Philip S.;
Lambowitz, Alan M.. A group II intron RNA is a catalytic component of a DNA
endonuclease involved in intron mobility. Cell (Cambridge, Mass.) (1995), 83(4), 529-38.
XXa . Griffin, Edmund A., Jr.; Qin, Zhifeng; Michels, Williams J., Jr.; Pyle, Anna Marie.
Group II intron ribozymes that cleave DNA and RNA linkages with similar efficiency, and lack contacts with substrate 2'-hydroxyl groups. Chem. Biol. (1995), 2(11), 761-70.
xxsa . Michel, Francois; Ferat, Jean Luc. Structure and activities of group II
introns. Annu.
Rev. Biochem. (1995), 64, 435-61.
x%"' . Abramovitz, Dana L.; Friedman, Richard A.; Pyle, Anna Marie. Catalytic role of 2'-hydroxyl groups within a group II intron active site. Science (Washington, D.
C.) (1996), 271 (5254),1410-13.
Xxa . Daniels, Danette L.; Michels, William J., Jr.; Pyle, Anna Marie. Two competing pathways for self-splicing by group II introns: a quantitative analysis of in vitro reaction rates and products. J. Mol. Biol. (1996), 256(1), 31-49.
XX° . Guo, Hans C. T.; Collies, Richard A.. Efficient trans-cleavage of a stem-loop RNA
substrate by a ribozyme derived from Neurospora VS RNA. EMBO J. (1995),14(2), 368-76.
xX°i , Scott, W.G., Finch, J.T., Aaron,K. The crystal structure of an all RNA hammerhead ribozyme:Aproposed mechanism for RNA catalytic cleavage. Cell, (1995), 81, 991-1002.
Xx°" . McICay, Structure and function of the hammerhead ribozyme: an unfinished story.
RNA, (1996), 2, 395-403.
Xx°"' . Long, D., Uhlenbeck, O., Hertel, K. Ligation with hammerhead ribozymes. US Patent No. 5,633,133.
xXiX . Hertel, K.J., Herschlag, D., Uhlenbeck, O. A kinetic and thermodynamic framework for the hammerhead ribozyme reaction. Biochemistry, (1994) 33, 3374-3385.Beigelman, L., et al., Chemical modifications of hammerhead ribozymes. J. Biol. Chem., (1995) 270, 25702-25708.
xxx . Beigelman, L., et al., Chemical modifications of hammerhead ribozymes.
J. Biol.
Chem., (1995) 270, 25702-25708.
XxXt , Hampel, Arnold; Tritz, Richard; Hicks, Margaret; Cruz, Phillip.
'Hairpin' catalytic RNA model: evidence for helixes and sequence requirement for substrate RNA.
Nucleic Acids Res. {1990),18(2), 299-304.
xXxa , Chowrira, Bharat M.; Berzal-Herranz, Alfredo; Burke, John M.. Novel guanosine requirement for catalysis by the hairpin ribozyme. Nature (London) (1991), 354(6351), 320-2.
Xxx"' . Berzal-Herranz, Alfredo; Joseph, Simpson; Chowrira, Bharat M.;
Butcher, Samuel E.;
Burke, John M.. Essential nucleotide sequences and secondary structure elements of the hairpin ribozyme. EMBO J. (1993),12(6), 2567-73.
XxX» , Joseph, Simpson; Berzal-Herranz, Alfredo; Chowrira, Bharat M.; Butcher, Samuel E..
Substrate selection rules for the hairpin ribozyme determined by in vitro selection, mutation, and analysis of mismatched substrates. Genes Dev. (1993), 7(1),130-8.
xxx° , Berzal-Herranz, Alfredo; Joseph, Simpson; Burke, John M.. In vitro selection of active hairpW ribozymes by sequential RNA-catalyzed cleavage and ligation reactions.
Genes Dev. (1992), 6(1),129-34.
xxx~' . Hegg, Lisa A.; Fedor, Martha J.. Kinetics and Thermodynamics of Intermolecular Catalysis by Hairpin Ribozymes. Biochemistry (1995), 34(48),15813-28.
xXx°" . Grasby, Jane A.; Mersmann, Karin; Singh, Mohinder; Gait, Michael J.. Purine Functional Groups in Essential Residues of the Hairpin Ribozyme Required fox Catalytic Cleavage of RNA. Biochemistry (1995), 34(12), 4068-76.
xXX°'.' , Schmidt, Sabine; Beigelman, Leorud; Karpeisky, Alexander;
Usman, Nassim;
Sorensen, Ulrik S,; Gait, Michael J.. Base and sugar requirements for RNA
cleavage of essential nucleoside residues in internal loop B of the hairpin ribozyme:
implications for secondary structure. Nucleic Acids Res. (1996), 24(4), 573-81.
XxxiX . Perrotta, Anne T.; Been, Michael D.. Cleavage of oligoribonucleotides by a ribozyme derived from the hepatitis .delta. virus RNA sequence. Biochemistry (1992), 31(1),16-21.
%t . Perrotta, Anne T.; Been, Michael D.. A pseudoknot-like structure required for efficient self-cleavage of hepatitis delta virus RNA. Nature (London) (1991), 350(6317), 434-6.
xn Xn~
x<<" . Puttaraju, M.; Perrotta, Arule T.; Been, Michael D.. A circular trans-acting hepatitis delta virus ribozyme. Nucleic Acids Res. (1993), 21(18), 4253-8.

Table II;
A. 2.5 umnl Synthesis Cycle Agl 39d Instrument Reagent EquivalentsAmount Wait Time* Wait Time* Wait Time*RNA
DNA 2'-O-methyl Phosphoramidites6.5 163 NL 45 sec 2.5 min 7.5 min S-Ethyl 23.8 238 NL 45 sec 2.5 min 7.5 min Tetrazole Acetic 100 233 NL 5 sec 5 sec 5 sec Anhydride N-Methyl 186 233 NL 5 sec 5 sec 5 sec Imidazole TCA 176 2.3 mL 21 sec 21 sec 21 sec Iodine 11.2 1.7 mL 45 sec 45 sec 45 sec Beaucage 12.9 645 NL 100 sec 300 sec 300 sec AcetonitrileNA 6.67 NA NA NA
mL

B. 0.2 umol Synthesis Cycle ABI 394 Instrument Reagent EquivalentsAmount Wait Time* Wait Time* Wait Time*RNA
DNA 2'-O-methyl Phosphoramidites15 31 NL 45 sec 233 sec 465 sec S-Ethyl 38.7 31 NL 45 sec 233 min 465 sec Tetrazole Acetic 655 124 NL 5 sec 5 sec 5 sec Anhydride N-Methyl 1245 124 IIL 5 sec 5 sec 5 sec Imidazole TCA 700 732 [~L 10 sec 10 sec 10 sec Iodine 20.6 244 NL 15 sec 15 sec 15 sec Beaucage 7.7 232 NL 100 sec 300 sec 300 sec AcetonitrileNA 2.64 NA NA NA
mL

C. 0.2 umol Synthesis Cycle 96 well Instrument Reagent Equivalents:DNAIAmount: DNA/2'-O-Wait Time* Wait Time*Wait Time*
2'-O-methyIIRibomethyl/Ribo DNA 2'-O Ribo methyl Phosphoramidites22/33/66 40/60/120 60 sec 180 sec 360sec NL

S-Ethyl 70/105/21040/60/120 60 sec 180 min 360 sec Tetrazole NL

Acetic 265/265/26550150/50 NL 10 sec 10 sec 10 sec Anhydride N-Methyl 5021502/50250/50/50 NL 10 sec 10 sec 10 sec Imidazole TCA 23S/475/475250/500/500 15 sec 15 sec 15 sec NL

Iodine 6.8/6,.8/6.880/80/80 NL 30 sec 30 sec 30 sec Beaucage 34/51/51 80/1201120 100 sec 200 sec 200 sec AcetonitrileNA 1150/1150/1150NA NA NA
uL

Wait time does not include contact time during delivery.

Table III: Human GRID Hammerhead Ribozyme and Substrate Sequence Pos Substrate Seq Ribozyme Seq ID ID

21' UAAUGGAUC UGUAAACU3 AGUUUACACUGAUGAGGCCGUUAGGCCGAA AUCCAUUA908 CUGAUGAG

CUGAUGAG

41' ACCCUCUUU CAGAGUGG8 CCACUCUGCUGAUGAGGCCGUUAGGCCGAA AAGAGGGU913 99 CAGUAACUC UGAUGCUUl5 AAGCAUCACUGAUGAGGCCGUUAGGCCGAA AGUUACUG920 112 GCUUGAAUU UGUUCUCCl7 GGAGAACACUGAUGAGGCCGUUAGGCCGAA AUUCAAGC922 119 UUUGUUCUC CCUUCUUG2l CAAGAAGGCUGAUGAGGCCGUUAGGCCGAA AGAACAAA926 CUGAUGAG

CUGAUGAG

CUGAUGAG

CUGAUGAG

CUGAUGAG

CUGAUGAG

Sl5 AUGACGUUC AACACUUC100 GAAGUGUUCUGAUGAGGCCGUUAGGCCGAA AACGUCAU1005 CUGAUGAG

CUGAUGAG

CUGAUGAG

Input Sequence = HSA011736. Cut Site = UH/.
Stem Length = 8 . Core Sequence = CUGAUGAG GCCGUUAGGC CGAA
HSA011736 (Homo sapiens mRNA for growth factor receptor binding protein (GRBLG) ; 1303 bp) Underlined region can be any X sequence or linker as defined herein.

Table IV: Human GRID NCH Ribozyme and Substrate Sequence Pos Substrate Se Riboz Se ID me ID

39 GCACCCUCU UUCAGAGU187 ACUCUGAA GCCGUUAGGCCGA~ IAGGGUGC1092 CUGAUGAG

43 CCUCUUUCA GAGUGGUA188 UACCACUCCUGAUGAGGCCGUUAGGCCGA1~' IAAAGAGG1093 173 ACAUAAACU CAAUCUCU214 AGAGAUUG~ GCCGUUAGGCCGAA IUUUAUGU1119 CUGAUGAG

, CUGAUGAG

CUGAUGAG

AAUUCCAU

CUGAUGAG

743 AAGAAAUCC GACCUUCG341 CGAAGGUCCUGAUGAGGCCGUUAGGCCGAA IAUUUCUU124'6 CUGAUGAG

L ~CCCCCACA GCAGCGAU391 AUCGCUGCCUGAUGAGGCCGUUAGGCCGAA IUGGGGGC1296 CUGAUGAG

CUGAUGAG

CUGAUGAG

CUGAUGAG

CUGAUGAG

I

Input Sequence = HSA011736. Cut Site = CH/.
Stem Length = 8 . Core Sequence = CUGAUGAG GCCGUUAGGC CGAA
HSA011736 (Homo Sapiens mRNA for growth factor receptor binding protein (GRBLG); 1303 bp) Underlined region can be any X sequence or linker as defined herein.
I = Inosine Table V: Human GRID G-cleaver Ribozyme and Substrate Sequence Pos Substrate Se Riboz me Se ID ID

AAGUUUAC

AAGCAUCA

AAGAAGGG

AAACUUGG

AACUGAGC

AAAACAUC

906 GGAACGCCG AGGAGGCA528 UGCCUCCUUGAUGGCAUGCACUAUGCGCG'GGCGUUCC1433 AAGGCUGC

AAAGUCAU

1073AGGAUGACG AGCUGGGG542. CCCCAGCUUGAUGGCAUGCACUAUGCGCGGUCAUCCU1447 Input Sequence = HSA011736. Cut Site = YG/M or UG/U.
Stem Length = 8. Core Sequence = UGAUG GCAUGCACUAUGC GCG
HSA011736 (Homo Sapiens mRNA for growth factor receptor binding protein (GRBLG); 1303 bp) Table VI: Human GRID Zlnzyme and Substrate Sequence Pos Substrate Se Zinz me Se ID ID

UUAAUGGA GCCGAAAGGCGAGUCAAGGUCU

UAAACUUG

CACCCUCU AAGUUUAC

UGGUACAU

UACAUGGA

CACAAAGU

G

CAGUAACU

UAACUCUG

CUUGAAUU

UUCUCCCU GCCGAAAGGCGAGUCAAGGUCU
AAAUUCAA

CCAGAAAG AAGAAGGG

UGUCAAAG

UCAAAGCC

G

G

201 AGCUUCACG 562 UGCUGUAA GUGAAGCU1477.
UUACAGCA GCCGAAAGGCGAGUCAAGGUCU

CAUGGAAG

CUGUUGCC

UUGCCAAG GCCGAAAGGCGAGUCAAGGUCU

CCAAGUUU AACAGCUU

UUUGAUUU

CUUCAGGU

UGAGGAUG

CUUUCACA

UUUUGAAG

G

UGGUUUAA

UUUAAGGC GCCGAAAGGCGAGUCAAGGUCU

CGGAGCUU

CUUGGGAG

CCAGGAAG

344 AAGGAUAUG 576 CUUGGGCAGCCGAAAGGCGAGUCAAGGUCUAUAUCCUU7.487 UGCCCAAG

CCCAAGAA

UUUCCCAA

UUUCACGA

CCUCUCUC

CAGAGAAC

CAAGGAGG

UUGGCUUC GCCGAAAGGCGAGUCAAGGUCU

CUUCUUCA

CCAGCCAG

CCAGAGCU

CUCCCCAG

UCAGGCAU

CAUGAGGA

UUCAACAC GCCGAAAGGCGAGUCAAGGUCU

UCAUGCGA

CGAGACAA

UAAUUACU

UGGACUGA

G GCCGAAAGGCGAGUCAAGGUCU

G

UAGACUAC

G

UCACCGGG

CAACAGCC

CCUGGACC

UCCCAGGG

CCCACACC

UGGGGCUG

CUGUGGGA

UGGGAGAA

G

UCGGAUCA

CAGCAGCA

CAGCACCA

CACCAGCA

CACCAGCC

CCACAGCC

CCUCCGCA

CAAUAUGC

CCCCAGCG

CGCCCCAG

CCCCAGCA

CAGCUGCA

CUGCAGCA

CAGCAGCC

CAGCCCCC

CCCCCACA

CAGCGAUA

CGAUAUCU

CAGCACCA

CACCACCA

CCGAGGAG

CAGCCUUG

CCUUGACA

CAUUGUGG

UGGCACCG AAUGCCCA

CACCGGCU

CUUGGGCA

CAGUGAAA

UGAAAUGA

CGGCCCUC

CCCUCAUG

CAUCGGAG

UGCAGCUC

CAGCUCCA

CUCCAGGC

CGGCAGGG

CAGGGCGA

CGAGUGCG

UGCGGUGG

CGGUGGGC

I UGGGCCCG

CCCGGGCG

CGCUGUAU

CUGUAUGA

UAUGACUU

CCCUGGAG

CUGGGGUU

UUCCACAG GCCGAAAGGCGAGUCAAGGUCU

CGGGGAGG

UGGUGGAG

UGGAGGUC

UCCUGGAU

CUCCAACC

UGGACCGG

CCGCCUGC

CCUGCACA

CACAACAA

G

CCUCUUCC

CCAACUAC

UGGCACCC

CACCCAUG

G

UCUGGAGC AAAAAGCU

CUGCCCAC

CCCACAAG

CAAGGAAA

I CUGGACUC
I

Input Sequence = HSA011736. Cut Site = G/Y
Stem Length = 8 . Core Sequence = GCcgaaagGCGaGuCaaGGuCu HSA011736 (Homo sapiens mRNA for growth factor receptor binding protein (GRBLG); 1303 bp) Table VII: Human GRID DNAzyme and Substrate Sequence Pos Substrate Se DNAz me Se ID
ID

UUAAUGGA GGCTAGCTACAACGA

UGGAUCUG

UCUGUAAA

UAAACUUG

A

CACCCUCU AAGTTTAC

CCCUCUUU

UGGUACAU

UACAUGGA

CAUGGAAG

UGGAAGAC

CAGCACAA

CACAAAGU

65 AAGACAGCA 7.91 CCACTTTGGGCTAGCTACAACGAGCTGTCTT1600 CAAAGUGG

G

UCCAUACU

UACUCUGA

CUCUGAAA

UGCAGUAA

CAGUAACU

UAACUCUG

CUCUGAUG

UGCUUGAA

CUUGAAUU

UUUGUUCU GGCTAGCTACAACGA

UUCUCCCU AAATTCAA

CCAGAAAG AAGAAGGG

UUCUAAUA GGCTAGCTACAACGA

UAACUCGG

CUCGGUGU

UGUCAAAG

UCAAAGCC

G

CAUAAACU

UAAACUCA

A

UCUCUUCU

G

CGUUACAG

UUACAGCA GGCTAGCTACAACGA

CAGCAUGG AACGTGAA

CAUGGAAG

UGGAAGCU

CUGUUGCC

UUGCCAAG

CCAAGUUU AACAGCTT

G GGCTAGCTACAACGA

UUUCACUG GGCTAGCTACAACGA

CUGCUUCA

CUUCAGGU

GGCTAGCTACAACGA

G

GGCTAGCTACAACGA

GGCTAGCTACAACGA

A

AAGTTCTC

GGCTAGCTACAACGA

UGCGAGAC

CGAGACAA

CAACAAGG

CAAGGGUA

UAAUUACU

UUACUUUC GGCTAGCTACAACGA

CUUUCUGU AATTACCC

UGGACUGA

CUGAGAAG

G

UCCCUAAA

UAAGCUGG

G

UAGACUAC

CUACUACA

CUACAGGA

CAGGACAA

CAAAUUCC

UUCCAUCU GGCTAGCTACAACGA

UCUCCAGA

CAGAAGCA

CAGAUCUU

UCUUCCUU

CAGAACCC

CCCGAGAA

CCAGGGUC

UCACCGGG

CCGGGGCA

CAACAGCC

CAGCCUGG

CCUGGACC

CCGGAGGU

UCCCAGGG

CCCACACC

CACCUCAG

CCUCAGUG

UGGGGCUG

CUGUGGGA

UGGGAGAA

A

CCUUCGAU

UGAACCGG

CCGGAAGC

CUGUCGGA

UCGGAUCA

UCACCCCC

CCCCCCGA

CCCUUCCC

CAGCAGCA

CAGCACCA

CACCAGCA

CCAGCACC

CACCAGCC

CCAGCCAC

CCACAGCC

CAGCCUCC

CCUCCGCA

CAAUAUGC

UAUGCCCC

UGCCCCAG

CCCCAGCG

CGCCCCAG

CCCCAGCA

CAGCUGCA

CUGCAGCA

CAGCAGCC

CAGCCCCC

CCCCCACA

CAGCAGCG

CAGCGAUA

CGAUAUCU

UAUCUGCA

UCUGCAGC

CAGCACCA

CACCACCA

CCACCAUU

CCAUUUCC

UUUCCACC GGCTAGCTACAACGA

CCAGGAAC

CGCCGAGG

CCGAGGAG

CAGCCUUG

CCUUGACA

CAUAAAUG

UAAAUGAU

A

UGGGCAUU

CAUUGUGG

UUGUGGCA GGCTAGCTACAACGA

UGGCACCG

CACCGGCU

CCGGCUUG

CUUGGGCA

CAGUGAAA

UGAAAUGA

A

UGCGGCCC

CGGCCCUC

CCCUCAUG

UGCAUCGG

CAUCGGAG

UCGGAGAC

CACACAGA

CACAGACC

CAGACCCA

CCCAGUGC

UGCAGCUC

CAGCUCCA

1068 CCUGGAGGA UGACGAGC721 GCTCGTCAGGCTAGCTACAACGACCTCCAGG187.3 GGCTAGCTACAACGA

A

G

Input Sequence = HSA011736. Cut Site = R/Y
Stem Length = 8 . Core Sequence = GGCTAGCTACAACGA
HSA011736 (Homo Sapiens mRNA for growth factor receptor binding protein (GRBLG); 1303 bp) M<HLf1lDr OD01O r1N M~HLn10r N01O o-1N MV~l7710r aD01Or1N
~

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L7 U' La U C9 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
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C7 U C7 L5 C7 L7 L7 C7 U' C7 C7 L7 U' C7 C7 C7 C7 L7 C7 C7 C7 C7 Lh U' C7 C7 L7 C7 C7 C7 C7 L7 C7 Ch C7 C7 U U L7 C7 C7 C7 L7 U C7 L7 L7 L7 U L7 U' U C7 C7 C7 C7 L7 C7 C7 C7 L7 C7 C7 ',~ U' ~ C7 U "~' U' L7 p C7 U r.C '',.~ U '',.~ D "a C7 U p p ''..~' ''.~
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Claims (57)

94 What is claimed is:

1. A nucleic acid molecule which down regulates expression of a Grb2-related with Insert Domain (GRID) gene.

2. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule is used to treat conditions selected from the group consisting of tissue/graft rejection and leukemia.

3. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule is an enzymatic nucleic acid molecule having at least one binding arm.

4. The nucleic acid molecule of claim 3, wherein one or more binding arms of the enzymatic nucleic acid molecule comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOS. 1-905 and 2256-2279.

5. The nucleic acid molecule of claim 3, wherein the enzymatic nucleic acid molecule comprises a sequence selected from the group consisting of SEQ ID NOS. 906-2199 and 2280-2304.

6. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule is an antisense nucleic acid molecule.

7. The nucleic acid molecule of claim 6, wherein said antisense nucleic acid molecule comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOS. 1-905, 2200-2211 and 2256-2279

8. The nucleic acid molecule of claim 6, wherein said antisense nucleic acid molecule comprises a sequence selected from the group consisting of SEQ ID NOS. 2212-2235.

9. The nucleic acid molecule of claim 3, wherein said enzymatic nucleic acid molecule is in a hammerhead (HH) motif.

10. The nucleic acid molecule of claim 3, wherein said enzymatic nucleic acid molecule is in a hairpin, hepatitis Delta virus, group I intron, VS nucleic acid, amberzyme, zinzyme or RNAse P nucleic acid motif.

11. The nucleic acid molecule of claim 3, wherein said enzymatic nucleic acid molecule is in an Inozyme motif.

12. The nucleic acid molecule of claim 3, wherein said enzymatic nucleic acid molecule is in a G-cleaver motif.

13. The nucleic acid molecule of claim 3, wherein said enzymatic nucleic acid molecule is a DNAzyme.

14. The nucleic acid molecule of claim 3, wherein said enzymatic nucleic acid molecule comprises between 12 and 100 bases complementary to the RNA of a GRID gene.

15. The nucleic acid molecule of claim 3, wherein said enzymatic nucleic acid molecule comprises between 14 and 24 bases complementary to the RNA of a GRID gene.

16. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule is chemically synthesized.

17. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises at least one 2'-sugar modification.

18. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises at least one nucleic acid base modification.

19. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises at least one phosphate backbone modification.

20. A mammalian cell including the nucleic acid molecule of claim 1.

21. The mammalian cell of claim 20, wherein said mammalian cell is a human cell.

22. A method of reducing GRID activity in a cell comprising the step of contacting said cell with the nucleic acid molecule of claim 1 under conditions suitable for said reduction of GRID activity.

23. A method of treatment of a patient having a condition associated with the level of GRID, comprising contacting cells of said patient with the nucleic acid molecule of claim 1, under conditions suitable for said treatment.

24. The method of claim 23 further comprising the use of one or more therapies under conditions suitable for said treatment.

25. A method of cleaving RNA of a GRID gene comprising the step of contacting the nucleic acid molecule of claim 1 with said RNA under conditions suitable for the cleavage of said RNA.

26. The method of claim 25, wherein said cleavage is carried out in the presence of a divalent cation.

27. The method of claim 26, wherein said divalent cation is Mg2+.

28. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises a cap structure at the 5'-end, the 3'-end or both the 5'-end and the 3'-end.

29. The nucleic acid molecule of claim 9, wherein one or more binding arms of the hammerhead motif comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOS. 1-179 and 2256-2260.

30. The nucleic acid molecule of claim 11, wherein one or more binding arms of the NCH
motif comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOS. 180-492 and 2261-2265.

31. The nucleic acid molecule of claim 12, wherein one or more binding arms of the G-cleaver motif comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOS. 493-657.

32. The nucleic acid molecule of claim 13, wherein one or more binding arms of the DNAzyme comprises a sequence complementary to a sequence selected from the group consisting of substrate sequences shown in Table VII.

33. The nucleic acid molecule of claim 10, wherein one or more binding arms of the zinzyme comprises a sequence complementary to a sequence selected from the group consisting of substrate sequences shown in Table VI.

34. The nucleic acid molecule of claim 10, wherein one or more binding arms of the amberzyme comprises a sequence complementary to a sequence selected from the group consisting of substrate sequences shown in Table VIII.

35. An expression vector comprising a nucleic acid sequence encoding at least one nucleic acid molecule of claim 1 in a manner which allows expression of the nucleic acid molecule.

36. A mammalian cell including the expression vector of claim 35.

37. The mammalian cell of claim 36, wherein said mammalian cell is a human cell.

38. The expression vector of claim 35, wherein said nucleic acid molecule is an enzymatic nucleic acid molecule.

39. The expression vector of claim 35, wherein said expression vector further comprises a sequence for an antisense nucleic acid molecule complementary to the RNA of a GRID
gene.

40. The expression vector of claim 35, wherein said expression vector comprises a sequence encoding two or more of said nucleic acid molecules, which may be the same or different.

41. The expression vector of claim 40, wherein said expression vector comprises a nucleic acid sequence encoding an antisense nucleic acid molecule complementary to the RNA of a GRID gene.

42. The expression vector of claim 40, wherein said expression vector comprises a nucleic acid sequence encoding an enzymatic nucleic acid molecule complementary to the RNA
of a GRID gene.

43. A method for treatment of tissue/graft rejection comprising the step of administering to a patient the nucleic acid molecule of claim 1 under conditions suitable for said treatment.

44. A method for treatment of leukemia comprising the step of administering to a patient the nucleic acid molecule of claim 1 under conditions suitable for said treatment.

45. An enzymatic nucleic acid molecule which cleaves RNA derived from a GRID
gene.

46. The enzymatic nucleic acid molecule of claim 45, wherein said enzymatic nucleic acid molecule is selected from the group consisting of Hammerhead, Hairpin, Inozyme, G-cleaver, DNAzyme, Amberzyme and Zinzyme.

47. The method of any of claims 43 or 44, wherein said method further comprises administering to said patient one or more other therapies.

48. The method of claim 47, wherein said other therapies are therapies selected from the group consisting of radiation, chemotherapy, and cyclosporin treatment.

49. The nucleic acid molecule of claim 7, wherein said nucleic acid molecule comprises at least five ribose residues, at least ten 2'-O-methyl modifications, and a 3'-end modification.

50. The nucleic acid molecule of claim 49, wherein said nucleic acid molecule further comprises a phosphorothioate core with a 3' and a 5' -end modification.

51. The nucleic acid molecule of any of claims 49 and 50, wherein said 3' and/or 5'- end modification is 3'-3' inverted abasic moiety.

52. The nucleic acid molecule of claim 3, wherein said nucleic acid molecule comprises at least five ribose residues, at least ten 2'-O-methyl modifications, and a 3'-end modification.

53. The nucleic acid molecule of claim 52, wherein said nucleic acid molecule further comprises phosphorothioate linkages on at least three of the 5' terminal nucleotides.

54. The nucleic acid molecule of claim 52, wherein said 3'- end modification is 3'-3' inverted abasic moiety.

55. The enzymatic nucleic acid molecule of claim 13, wherein said DNAzyme comprises at least ten 2'-O-methyl modifications and a 3'-end modification.

56. The enzymatic nucleic acid molecule of claim 55, wherein said DNAzyme further comprises phosphorothioate linkages on at least three of the 5' terminal nucleotides.

57. The enzymatic nucleic acid molecule of claim 55, wherein said 3'- end modification is 3'-3' inverted abasic moiety.