WO1999050403A2 - Method and reagents for the treatment of diseases or conditions related to molecules involved in angiogenic responses - Google Patents

Method and reagents for the treatment of diseases or conditions related to molecules involved in angiogenic responses Download PDF

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WO1999050403A2
WO1999050403A2 PCT/US1999/006507 US9906507W WO9950403A2 WO 1999050403 A2 WO1999050403 A2 WO 1999050403A2 US 9906507 W US9906507 W US 9906507W WO 9950403 A2 WO9950403 A2 WO 9950403A2
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nucleic acid
acid molecule
enzymatic nucleic
rna
patient
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PCT/US1999/006507
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WO1999050403A9 (en
WO1999050403A3 (en
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Pamela A. Pavco
Elisabeth Roberts
Thale Jarvis
Claire Coeshott
James A. Mcswiggen
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Ribozyme Pharmaceuticals, Inc.
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Priority to EP99915032A priority Critical patent/EP1086212A2/en
Priority to JP2000541291A priority patent/JP2002509721A/en
Priority to CA002324421A priority patent/CA2324421A1/en
Priority to AU33647/99A priority patent/AU757789B2/en
Publication of WO1999050403A2 publication Critical patent/WO1999050403A2/en
Publication of WO1999050403A9 publication Critical patent/WO1999050403A9/en
Publication of WO1999050403A3 publication Critical patent/WO1999050403A3/en

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Definitions

  • This invention relates to methods and reagents for the treatment of diseases or conditions relating to the levels of expression of angiogenic factors and receptors involved in the regulation of angiogenesis .
  • the following is a discussion of relevant art, none of which is admitted to be prior art to the present invention.
  • yolk sac splanchnopleuric mesenchyme differentiates into vascular progenitor cells and then to blood island aggregates which are primitive blood cells surrounded by fused endothelial progenitors
  • angiogenesis vascular developmental step
  • vasculogenesis From the vessels formed during vasculogenesis, new blood vessels sprout, elongate and develop into capillary loop formations of endothelial cells. It is a highly complex event involving local basement membrane disruption, endothelial cell proliferation, migration and microvessel morphogenesis
  • Sprouting angiogenesis also originates from pre-existing blood vessels and consists of new blood vessels sprouting, elongating and developing into capillary loop formations of endothelial cells. It is a highly complex event involving disruption of extracellular matrix, endothelial cell proliferation, chemotaxic migration and microvessel morphogenesis (Rak, supra ) . Many factors regulating positive and negative control of angiogenesis have been reported demonstrating the sophistication of this process.
  • An example of an angiogenic factor is Vascular Endothelial Growth Factor receptor (VEGFr) which has been shown to be specific to endothelial cells and is discussed in Pavco et al . , Int. PCT Pub. No. WO 97/15662.
  • VEGFr Vascular Endothelial Growth Factor receptor
  • angiogenesis not only occurs in embyronic development, but can also occur throughout the lifespan of the organism during such events as wound healing, bone repair, inflammation, and female menstral cycles.
  • Local delivery of oxygen and nutrients and the removal of waste requires a complex system of blood vessels which has the ability to adapt as the tissue requirements changes.
  • Involvement of a large number of positive and negative factors in angiogenic regulation demonstrates the complexity of this process.
  • ARNT ryl Hydrocarbon Nuclear Transporter
  • HIF-l ⁇ forms heterodimers with several factors including HIF- ⁇ (Maxwell et al . , 1997, Proc . Na tl .
  • HIF-1 HIF-1 is believed to be regulate genes involved in the response to oxygen deprivation.
  • ARNT -/- embryonic stem cells fail to induce VEGF expression in response to hypoxia.
  • ARNT -/- mice are not viable beyond embryonic day 10.5.
  • VEGF knockout mice these embryos show defective angiogenesis of the yolk sac (Maltepe et al . , 1997, Na ture 386, 403- 407) .
  • Hepatoma cells containing an ARNT mutation that is functionally deficient in dimerizing with HIF-l ⁇ shows greatly reduced VEGF expression in response to hypoxia compared to normal cells (Wood et al . , 1996, J. Biol . Chem . 271, 15117-15123) .
  • Tumor xenografts derived from these cells show reduced vascularity and approximately 2- fold reduced tumor growth rates (Maxwell et al . , 1997, supra ) .
  • Tie-2 (also known as Tek) , is a tyrosine kinase protein receptor which consists of 1122 amino acids and is produced in endothelial (Merenmies et al . , 1997, Cell Growth & Differentia tion 8, 3-10) as well as early hematopoeitic cells (Maisonpierree et al . , 1993, Oncogene 8, 1631-1637). Tie-2 expression has been demonstrated in mice, rats and humans. The human gene is thought to be located on chromosome 9p21 (Dumont et al . , 1994, Genes & Developmen t 8, 1897-1909).
  • Tie-2 homozygous mutant endothelial cells were examined using anti-PECAM monoclonal antibody (Sato et al . , 1997, Na ture 376, 70- 74) . All of the homozygous mutants were dead within 10.5 days with obvious deformities in the head and heart present by day 9.5. In addition, large vessels were indistinguishable from small vessels and no capillary sprouts were seen in the brain. These observations suggested that Tie-2 plays an important role in angiogenesis rather than vasculogenesis. The earlier effects of Tie-2 mutant compared to the Tie-1 mutant indicates separate roles for the two RTK' s in angiogenesis .
  • angiopoietin 1 and 2 Ligands to Tie-2 have been discovered and named angiopoietin 1 and 2 (angl and 2) (Davis, S. et al . , 1993, Cell 87, 1161; Maisonpierre, P.C. et al . , 1997, Science, 277, 55-60) . Both factors consist of an NH 2 -terminal coiled-coil domain as well as a COOH-terminal fibrinogen- like domain.
  • Angl binds to Tie-2/Tek but not Tie-1 and stimulates angiogenesis through autophosphorylation.
  • Ang2 is a 496 amino acid polypeptide whose human and mouse homologs are 85% identical. Autophosphorylation caused by Angl binding to the Tie-2 receptor can be blocked with the addition of Ang2.
  • the Tie-2 receptor is unusual in that it utilizes both positive and negative control mechanisms.
  • Integrins are a family of cell adhesion and migration mediating proteins that are comprised of at least 15 alpha and 8 beta subunits that are expressed as a number of different ⁇ non-covalently bound heterodimers on cell surfaces (Varner, 1997, Regula tion of Angiogenesis, ed I.D Goldberg & E.M. Rosen, 361-390; Brooks, 1996, Eur J Cancer 14, 2423-2429) .
  • ECM extracellular matrix
  • Integrin production has been shown to be induced by a number a stimuli including intracellular pH increases, calcium concentration, inositol lipid synthesis, tyrosine phosphorylation of a focal contact associated tyrosine kinase, and activation of p34/cdc2 and cyclin A (Varner & Cheresh, 1996, Curr Op in Cell Biol 8,724-730).
  • ⁇ v ⁇ 3 a 160kDa protein is the most well characterized molecule of the integrin family and is believed to play a large role in angiogenesis (Varner, 1997, supra ) .
  • ⁇ v ⁇ 3 binds the largest number of ECM components of all known heterodimers indicating any cell with these molecules on the cell surface could adhere to or migrate on almost any of the ECM components (Varner, 1997, supra ) .
  • Antagonists to ⁇ v ⁇ 3 can inhibit angiogenesis in the chick chorioallentoic membrane (CAM) model and in SCID mice and even reduce the tumor volume.
  • CAM chick chorioallentoic membrane
  • Methionine Aminopeptidase (Arfin et al . , 1995, PNAS 92, 7714-7718 (Genbank Accession No. U29607) ; Sin, N. et . al . , 1997, PNAS 94, 6099-6103; Griffith et al . , 1997, Chem Biol . 4(6), 461-471); Transcription factor Ets- 1: (Iwasaka, C. et al . 1996. J. Cell Physiol . 169, 522-531; Chen, Z. et al .
  • Tumor necrosis factor receptor (TNFR1, Accession No. M11567)); Tumor necrosis factor receptor:
  • Interleukin-8 (IL-8) : (Elner et al., 1991, , Am J. Pathol. 139, 977-988; Strieter et al., 1992, Am. J. Pathol. 141, 1279-1284; Mukaida et al., 1989, J. Immunol. 143 (4), 1366-1371 (Genbank Accession No. M28130) ) ; Angiopoietin 1: (Davis, S. et al., 1996, Cell 87, 1161; Iwama, A. et al., 1993, Biochem Biophys . Res. Commun. 195, 301; Dumont, D.J.
  • Hepatocyte growth factor (Miyazawa et al . , 1991, Eur. J. Biochem . 197 (1), 15-22 (Genbank accession No. X57574); Proliferin: (Groskopf et al . , 1997, Endocrinology 138(7), 2835-2840; Jackson D, et al . , 1994, Science . 266(5190), 1581-1584; Volpert et al . , 1996 , Endocrinology 137(9): 3871-3876); Placental growth factor: (Kodama et al . , 1997, Eur J Gynaecol Oncol .
  • the invention features the use of enzymatic nucleic acid molecules and methods for their use to down regulate or inhibit the expression of angiogenic factors.
  • the enzymatic nucleic acids of the present invention are used as a treatment for indications relating to angiogenesis including but not limited to cancer, age related macular degeneration (ARMD) , diabetic retinopathy, inflammation, arthritis, psoriasis and the like.
  • the invention features enzymatic nucleic acid molecules that cleave RNAs encoding angiogenic selected from a group comprising: Tie-2, integrin subunit ⁇ 3, integrin subunit ⁇ 6, and aryl hydrocarbon nuclear transporter (ARNT) .
  • inhibit it is meant that the activity of the cleaved RNA is reduced below that observed in the absence of the nucleic acid.
  • inhibition with ribozymes preferably is below that level observed in the presence of an enzymatically inactive RNA molecule that is able to bind to the same site on the mRNA, but is unable to cleave that RNA.
  • angiogenic factors is meant a peptide molecule which is involved in a process or pathway necessary for the formation of novel blood vessels.
  • the invention features the use of enzymatic nucleic acids that cleave the RNAs encoded by angiogenic factors selected from a group comprising: Methionine Aminopeptidase; Ets-1 Transcription factor; integrins; platelet derived endothelial cell growth factor (PD-ECGF); PD-ECGF receptor; Transforming Growth factors (TGFs) ; Transforming growth factor receptor; Angiogenin; Endothelial cell stimulating angiogenesis factor (ESAF) ; Interleukin-8 (IL- 8); Angiopoietin 1 and 2; TIE-1; insulin-like growth factor (IGF-1); insulin-like growth factor receptor (IGF- lr) ; B61; B61 receptor (Eck) ; Protein kinase C; an SH2 domain (e.g.
  • Phospholipase c-g Phosphatidylinositol 3 kinase (PI-3), Ras GTPase activating protein (GAP); Oncogene adaptor protein Nek; Granulocyte Colony- Stimulating Factor; Hepatocyte growth factor; Proliferin; and Placental growth factor.
  • enzymatic nucleic acid it is meant a nucleic acid molecule capable of catalyzing reactions including, but not limited to, site-specific cleavage and/or ligation of other nucleic acid molecules, cleavage of peptide and amide bonds, and trans-splicing.
  • a molecule with endonuclease activity may have complementarity in a substrate binding region to a specified gene target, and also has an enzymatic activity that specifically cleaves RNA or DNA in that target. That is, the nucleic acid molecule with endonuclease activity is able to intramolecularly or intermolecularly cleave RNA or DNA and thereby inactivate a target RNA or DNA molecule.
  • the nucleic acids may be modified at the base, sugar, and/or phosphate groups.
  • the term enzymatic nucleic acid is used interchangeably with phrases such as ribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, catalytic oligonucleotides, nucleozyme, DNAzyme, RNA enzyme, endo- ribonuclease, endonuclease, minizyme, leadzyme, oligozyme or DNA enzyme.
  • nucleic acid molecules with enzymatic activity are not meant to be limiting and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it have a specific substrate binding site which is complementary to one or more of the target nucleic acid regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart a nucleic acid cleaving activity to the molecule (Cech et al., U.S. Patent No. 4,987,071; Cech et al., 1988, JAMA) .
  • enzyme portion or “catalytic domain” is meant that portion/region of the ribozyme essential for cleavage of a nucleic acid substrate (for example see Figure 1) .
  • substrate binding arm or “substrate binding domain” is meant that portion/region of a ribozyme which is complementary to (i.e., able to base-pair with) a portion of its substrate. Generally, such complementarity is 100%, but can be less if desired. For example, as few as 10 bases out of 14 may be base-paired. Such arms are shown generally in Figure 1. That is, these arms contain sequences within a ribozyme which are intended to bring ribozyme and target RNA together through complementary base-pairing interactions .
  • the ribozyme of the invention may have binding arms that are contiguous or noncontiguous and may be of varying lengths .
  • the length of the binding arm(s) are preferably greater than or equal to four nucleotides; specifically 12-100 nucleotides; more specifically 14-24 nucleotides long. If two binding arms are chosen, the design is such that the length of the binding arms are symmetrical (i.e., each of the binding arms is of the same length; e . g.
  • 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) .
  • DNAzyme is meant, an enzymatic nucleic acid molecule lacking a 2' -OH group.
  • the enzymatic nucleic acid molecule is formed in a hammerhead or hairpin motif, but may also be formed in the motif of a hepatitis ⁇ virus, group I intron, group II intron or RNase P RNA (in association with an RNA guide sequence) , Neurospora VS RNA or DNAzymes.
  • hammerhead motifs are described by Dreyfus, supra , Rossi et al . , 1992, AIDS Research and Human Retroviruses 8, 183; of hairpin motifs by Hampel et al . , EP0360257, Hampel and Tritz, 1989 Biochemistry 28, 4929, Feldstein et al .
  • RNA to Tie-2, integrin subunit ⁇ 3, integrin subunit ⁇ 6, or ARNT is meant to include those naturally occurring RNA molecules having homology (partial or complete) to Tie-2, integrin subunit ⁇ 3, integrin subunit ⁇ 6, or ARNT or encoding for proteins with similar function as Tie-2, integrin subunit ⁇ 3, integrin subunit ⁇ 6, or ARNT in various animals, including human, rodent, primate, rabbit and pig.
  • the equivalent RNA sequence also includes in addition to the coding region, regions such as 5' -untranslated region, 3' -untranslated region, introns, intron-exon junction and the like.
  • nucleotide sequence of two or more nucleic acid molecules is partially or completely identical .
  • complementarity is meant a nucleic acid molecules that can form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types (for example, Hoogsteen type) of base-paired interactions.
  • the invention provides a method for producing a class of enzymatic cleaving agents which exhibit a high degree of specificity for the RNA of a desired target.
  • the enzymatic nucleic acid molecule is preferably targeted to a highly conserved sequence region of a target RNAs encoding Tie-2, integrin subunit ⁇ 3, integrin subunit ⁇ 6, or ARNT proteins such that specific treatment of a disease or condition can be provided with either one or several enzymatic nucleic acids.
  • Such enzymatic nucleic acid molecules can be delivered exogenously to specific cells as required.
  • the ribozymes can be expressed from DNA/RNA vectors that are delivered to specific cells.
  • highly conserved sequence region is meant a nucleotide sequence of one or more regions in a nucleic acid molecule does not vary significantly from one generation to the other or from one biological system to the other.
  • Such ribozymes are useful for the prevention of the diseases and conditions including cancer, diabetic retinopathy, macular degeneration, neovascular glaucoma, myopic degeneration, arthritis, psoriasis, verruca vulgaris, angiofibroma of tuberous sclerosis, pot-wine stains, Sturge Weber syndrome, Kippel-Trenaunay-Weber syndrome, Osler-Weber-Rendu syndrome and any other diseases or conditions that are related to the levels of Tie-2, integrin subunit ⁇ 3, integrin subunit ⁇ 6, or ARNT activity in a cell or tissue.
  • diseases and conditions including cancer, diabetic retinopathy, macular degeneration, neovascular glaucoma, myopic degeneration, arthritis, psoriasis, verruca vulgaris, angiofibroma of tuberous sclerosis, pot-wine stains, Sturge Weber syndrome, Kippel-Trenaunay-Weber syndrome, O
  • integrin subunit ⁇ 3, integrin subunit ⁇ 6, and/or ARNT RNAs By “related” is meant that the inhibition of Tie-2, integrin subunit ⁇ 3, integrin subunit ⁇ 6, and/or ARNT RNAs and thus reduction in the level respective protein activity will relieve to some extent the symptoms of the disease or condition.
  • the ribozymes have binding arms which are complementary to the target sequences in Tables III-X. Examples of such ribozymes are also shown in Tables III-X. Tables III and IV display target sequences and ribozymes for ARNT, Tables V and VI display target sequences and ribozymes for Tie-2, tables VII and VIII display target sequences and ribozymes for integrin subunit alpha 6, and tables IX and X display target sequences and ribozymes for integrin subunit beta 3. Examples of such ribozymes consist essentially of sequences defined in these Tables.
  • the active ribozyme contains an enzymatic center or core equivalent to those in the examples, and binding arms able to bind mRNA such that cleavage at the target site occurs. Other sequences may be present which do not interfere with such cleavage.
  • the invention features ribozymes that inhibit gene expression and/or cell proliferation.
  • These chemically or enzymatically synthesized RNA molecules contain substrate binding domains that bind to accessible regions of their target mRNAs.
  • the RNA molecules also contain domains that catalyze the cleavage of RNA.
  • the RNA molecules are preferably ribozymes of the hammerhead or hairpin motif.
  • the ribozymes are DNAzymes .
  • the ribozymes cleave the target mRNAs, preventing translation and protein accumulation. In the absence of the expression of the target gene, cell proliferation is inhibited.
  • Chemically synthesized RNA molecules also include RNA molecules assembled together from various fragments of RNA using a chemical or an enzymatic ligation method.
  • ribozymes are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells.
  • the nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex vivo, or in vivo through injection, infusion pump or stent, with or without their incorporation in biopolymers .
  • the ribozyme is administered to the site of Tie-2, integrin subunit ⁇ 3, integrin subunit ⁇ 6, or ARNT expression (e.g. tumor cells, endothelial cells) in an appropriate liposomal vehicle.
  • ribozymes that cleave target molecules and inhibit Tie-2, integrin subunit ⁇ 3, integrin subunit ⁇ 6, or ARNT activity are expressed from transcription units inserted into DNA or RNA vectors .
  • the recombinant vectors are preferably DNA plasmids or viral vectors. Ribozyme expressing viral vectors could be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus .
  • the recombinant vectors capable of expressing the ribozymes are delivered as described above, and persist in target cells.
  • viral vectors may be used that provide for transient expression of ribozymes.
  • ribozymes cleave the target RNA. Delivery of ribozyme expressing vectors could be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex- planted from the patient followed by reintroduction into the patient, or by any other means that would allow for introduction into the desired target cell (for a review see Couture and Stinchcomb, 1996, TIG. , 12, 510).
  • ribozymes that cleave target molecules and inhibit cell proliferation are expressed from transcription units inserted into DNA, RNA, or viral vectors.
  • the recombinant vectors capable of expressing the ribozymes are locally delivered as described above, and transiently persist in smooth muscle cells.
  • other mammalian cell vectors that direct the expression of RNA may be used for this purpose.
  • patient is meant an organism which is a donor or recipient of explanted cells or the cells themselves.
  • Patient also refers to an organism to which enzymatic nucleic acid molecules can be administered.
  • a patient is a mammal or mammalian cells. More preferably, a patient is a human or human cells.
  • vectors any nucleic acid- and/or viral- based technique used to deliver a desired nucleic acid.
  • ribozymes individually, or in combination or in conjunction with other drugs, can be used to treat diseases or conditions discussed above.
  • the patient may be treated, or other appropriate cells may be treated, as is evident to those skilled in the art.
  • the described ribozymes can be used in combination with other known treatments to treat conditions or diseases discussed above.
  • the described ribozymes could be used in combination with one or more known therapeutic agents to treat cancer.
  • the ribozymes have binding arms which are complementary to the sequences in the tables, shown as Seq. I.D. Nos. 394-786, 849-910, 1612- 2312, 2381-2448, 3588-4726, 4821-4914, 5702-6488, and 6569-6648.
  • Examples of such ribozymes are shown as Seq. I.D. Nos.1-393, 787-848, 911-1611, 2313-2380, 2449-3587, 4727-4820. 4915-5701, and 6489-6568.
  • Other sequences may be present which do not interfere with such cleavage.
  • Figure 1 shows the secondary structure model for seven different classes of enzymatic nucleic acid molecules. Arrow indicates the site of cleavage.
  • Group I Intron P1-P9.0 represent various stem-loop structures (Cech et al . , 1994, Na ture Struc . Bio . , 1, 273).
  • RNase P (M1RNA) EGS represents external guide sequence (Forster et al . , 1990, Science, 249, 783; Pace et al . , 1990, J. Biol . Chem . , 265, 3587).
  • Group II Intron 5'SS means 5' splice site; 3'SS means 3' -splice site; IBS means intron binding site; EBS means exon binding site (Pyle et al . , 1994, Biochemistry, 33, 2716) .
  • VS RNA I-VI are meant to indicate six stem-loop structures; shaded regions are meant to indicate tertiary interaction (Collins, International PCT Publication No. WO 96/19577) .
  • stems I-III are meant to indicate three stem-loop structures; stems I-III can be of any length and may be symmetrical or asymmetrical (Usman et al . , 1996, Curr . Op . Struct . Bio . , 1, 527) .
  • Helix 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 may be covalently linked by one or more bases (i.e., r is base) .
  • Helix 1, 4 or 5 may also be extended by 2 or more base pairs (e.g., 4 - 20 base pairs) to stabilize the ribozyme structure, and preferably is a protein binding site.
  • each N and N' independently is any normal or modified base and each dash represents a potential base-pairing interaction. These nucleotides may be modified at the sugar, base or phosphate. Complete base-pairing is not required in the helices, but is preferred.
  • Helix 1 and 4 can be of any size (i.e., o and p is each independently from 0 to any number, e.g., 20) as long as some base- pairing is maintained.
  • Helix 4 can be formed from two separate molecules, i.e., without a connecting loop.
  • the connecting loop when present may be a ribonucleotide with or without modifications to its base, sugar or phosphate.
  • "q" is ⁇ 2 bases.
  • the connecting loop can also be replaced with a non-nucleotide linker molecule.
  • H refers to bases A, U, or C.
  • Y refers to pyrimidine bases.
  • " refers to a covalent bond. (Burke et al . , 1996, Nucleic Acids & Mol . Biol . , 10, 129; Chowrira et al . , US Patent No. 5,631,359).
  • Figure 2 is a diagrammatic representation of a hammerhead ribozyme targeted against Tie-2 at position 1037.
  • enzymatic nucleic acids act by first binding to a target R ⁇ A. Such binding occurs through the target binding portion of an enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target R ⁇ A.
  • the enzymatic nucleic acid first recognizes and then binds a target R ⁇ A through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target R ⁇ A. Strategic cleavage of such a target R ⁇ A will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its R ⁇ A target, it is released from that R ⁇ A to search for another target and can repeatedly bind and cleave new targets.
  • ribozyme The enzymatic nature of a ribozyme is advantageous over other technologies, since the concentration of ribozyme necessary to affect a therapeutic treatment is lower. This advantage reflects the ability of the ribozyme to act enzymatically .
  • a single ribozyme molecule is able to cleave many molecules of target RNA.
  • the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base-pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage. Single mismatches, or base-substitutions, near the site of cleavage can be chosen to completely eliminate catalytic activity of a ribozyme.
  • Nucleic acid molecules having an endonuclease enzymatic activity are able to repeatedly cleave other separate RNA molecules in a nucleotide base sequence- specific manner. Such enzymatic nucleic acid molecules can be targeted to virtually any RNA transcript, and efficient cleavage achieved in vitro (Zaug et al . , 324, Na ture 429 1986 ; Uhlenbeck, 1987 Na ture 328, 596; Kim et al., 84 Proc . Na tl . Acad. Sci . USA 8788, 1987; Dreyfus, 1988, Einstein Quart . J. Bio . Med.
  • Ribozymes can be designed to cleave specific RNA targets within the background of cellular RNA. Such a cleavage event renders the RNA non-functional and abrogates protein expression from that RNA. In this manner, synthesis of a protein associated with a disease state can be selectively inhibited.
  • Ribozymes that cleave the specified sites in Tie-2, integrin subunit ⁇ 3, integrin subunit ⁇ 6, and aryl hydrocarbon nuclear transporter (ARNT) mRNAs represent a novel therapeutic approach to treat cancer, macular degeneration, diabetic retinopathy, inflammation, psoriasis and other diseases. Applicant indicates that ribozymes are able to inhibit the activity of Tie-2; integrin subunit ⁇ 3; integrin subunit ⁇ 6; and aryl hydrocarbon nuclear transporter (ARNT) and that the catalytic activity of the ribozymes is required for their inhibitory effect.
  • Targets for useful ribozymes can be determined as disclosed in Draper et al . , WO 93/23569; Sullivan et al . , WO 93/23057; Thompson et al . , WO 94/02595; Draper et al . , WO 95/04818; McSwiggen et al . , US Patent No. 5,525,468 and hereby incorporated by reference herein in totality. Rather than repeat the guidance provided in those documents here, below are provided specific examples of such methods, not limiting to those in the art. Ribozymes to such targets are designed as described in those applications and synthesized to be tested in vi tro and in vivo, as also described. Such ribozymes can also be optimized and delivered as described therein.
  • the sequence of human Tie-2, integrin subunit ⁇ 3, integrin subunit ⁇ 6, and aryl hydrocarbon nuclear transporter (ARNT) mRNAs were screened for optimal ribozyme target sites using a computer folding algorithm. Hammerhead or hairpin ribozyme cleavage sites were identified. These sites are shown in Tables III-X (All sequences are 5' to 3' in the tables)
  • the nucleotide base position is noted in the Tables as that site to be cleaved by the designated type of ribozyme.
  • the nucleotide base position is noted in the tables as that site to be cleaved by the designated type of ribozyme.
  • ribozymes were designed that could bind and were individually analyzed by computer folding (Jaeger et al . , 1989 Proc . Na tl . Acad . Sci . USA, 86, 7706) to assess whether the ribozyme sequences fold into the appropriate secondary structure. Those ribozymes with unfavorable intramolecular interactions between the binding arms and the catalytic core are eliminated from consideration. Varying binding arm lengths can be chosen to optimize activity. Generally, at least 5 bases on each arm are able to bind to, or otherwise interact with, the target RNA. Ribozymes of the hammerhead or hairpin motif were designed to anneal to various sites in the mRNA message. The binding arms are complementary to the target site sequences described above.
  • nucleic acid motifs e.g., antisense oligonucleotides, hammerhead or the hairpin ribozymes
  • small nucleic acid motifs e.g., antisense oligonucleotides, hammerhead or the hairpin ribozymes
  • the simple structure of these molecules increases the ability of the nucleic acid to invade targeted regions of the mRNA structure.
  • these nucleic acid molecules can also be expressed within cells from eukaryotic promoters (e.g., Izant and Weintraub, 1985 Science 229, 345; McGarry and Lindquist, 1986 Proc . Na tl . Acad. Sci .
  • nucleic Acids Res . 23, 2259 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 W093/23569, and Sullivan et al . , PCT WO94/02595, both hereby incorporated in their totality by reference herein; Ohkawa et al . , 1992 Nucleic Acids Symp .
  • the ribozymes were chemically synthesized.
  • the method of synthesis used follows the procedure for normal RNA synthesis as described in Usman et al . , 1987 J. Am . Chem . Soc , 109, 7845; Scaringe et al . , 1990 Nucleic Acids Res . , 18, 5433; and Wincott et al . , 1995 Nucleic Acids Res . 23, 2677-2684 and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'- end, and phosphoramidites at the 3 '-end.
  • small scale synthesis were conducted on a 394 Applied Biosystems, Inc.
  • Deprotection of the R ⁇ A was performed as follows.
  • the base-deprotected oligoribonucleotide was resuspended in anhydrous TEA-HF/NMP solution (250 ⁇ L of a solution of 1.5mL N-methylpyrrolidinone , 750 ⁇ L TEA and 1.0 mL TEA»3HF to provide a 1.4M HF concentration) and heated to 65°C for 1.5 h.
  • the resulting, fully deprotected, oligomer was quenched with 50 mM TEAB (9 mL) prior to anion exchange desalting.
  • the TEAB solution was loaded onto a Qiagen 500 ® anion exchange cartridge (Qiagen Inc.) that was prewashed with 50 mM TEAB (10 mL) . After washing the loaded cartridge with 50 mM TEAB (10 mL) , the R ⁇ A was eluted with 2 M TEAB (10 mL) and dried down to a white powder.
  • Inactive hammerhead ribozymes were synthesized by substituting a U for G5 and a U for A14 (numbering from Hertel, K. J., et al . , 1992, Nucleic Acids Res . , 20, 3252) . The average stepwise coupling yields were >98% (Wincott et al . , 1995 Nucleic Acids Res . 23, 2677-2684).
  • Hairpin ribozymes are synthesized in two parts and annealed to reconstruct the active ribozyme (Chowrira and Burke, 1992 Nucleic Acids Res . , 20, 2835-2840). Ribozymes are also synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol . 180, 51).
  • Ribozymes are modified to enhance stability and/or enhance catalytic activity by modification with nuclease resistant groups, for example, 2 '-amino, 2'-C-allyl, 2'- flouro, 2'-0-methyl, 2'-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992 TIBS 17, 34; Usman et al . , 1994 Nucleic Acids Symp . Ser . 31, 163; Burgin et al . , 1996 Biochemistry 6, 14090). Ribozymes were purified by gel electrophoresis using general methods or are purified by high pressure liquid chromatography (HPLC; See Stinchcomb et al . , International PCT Publication No. WO 95/23225, the totality of which is hereby incorporated herein by reference) and are resuspended in water.
  • nuclease resistant groups for example, 2 '-amino, 2'-C-allyl, 2'- flouro
  • sequences of the ribozymes that are chemically synthesized, useful in this study are shown in Tables III-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.
  • stem-loop II sequence of hammerhead ribozymes can be altered (substitution, deletion, and/or insertion) to contain any sequences provided a minimum of two base- paired stem structure can form.
  • stem-loop IV sequence of hairpin ribozymes can be altered (substitution, deletion, and/or insertion) to contain any sequence, provided a minimum of two base-paired stem structure can form.
  • sequences listed in Tables III-X may be formed of ribonucleotides or other nucleotides or non-nucleotides.
  • Such ribozymes (which have enzymatic activity) are equivalent to the ribozymes described specifically in the Tables.
  • Catalytic activity of the ribozymes described in the instant invention can be optimized as described by Draper et al., supra . The details will not be repeated here, but include altering the length of the ribozyme binding arms, or chemically synthesizing ribozymes with modifications (base, sugar and/or phosphate) that prevent their degradation by serum ribonucleases and/or enhance their enzymatic activity (see e.g., Eckstein et al . , International Publication No. WO 92/07065; Perrault et al . , 1990 Na ture 344, 565; Pieken et al . , 1991 Science 253, 314; Usman and Cedergren, 1992 Trends in Biochem .
  • Ribozymes are modified to enhance stability and/or enhance catalytic activity by modification with nuclease resistant groups, for example, 2 '-amino, 2'-C-allyl, 2'-flouro, 2'- O-methyl, 2'-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992 TIBS 17, 34; Usman et al . , 1994 Nucleic Acids Symp . Ser . 31, 163; Burgin et al .
  • 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 on all RNA ribozyme.
  • Therapeutic ribozymes delivered exogenously must optimally be stable within cells until translation of the target RNA has been inhibited long enough to reduce the levels of the undesirable protein. This period of time varies between hours to days depending upon the disease state.
  • ribozymes must be resistant to nucleases in order to function as effective intracellular therapeutic agents. Improvements in the chemical synthesis of RNA (Wincott et al . , 1995 Nucleic Acids Res . 23, 2677; incorporated by reference herein) have expanded the ability to modify ribozymes by introducing nucleotide modifications to enhance their nuclease stability as described above.
  • nucleotide as used herein is as recognized in the art to include natural bases (standard), and modified bases well known in the art. Such bases are generally located at the 1' position of a sugar moiety.
  • Nucleotide 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 .
  • base modifications that can be introduced into enzymatic nucleic acids without significantly effecting their catalytic activity include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5- methylcytidine) , 5-alkyluridines (e.g., ribothymidine) , 5- halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g.
  • modified bases in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at 1' position or their equivalents; such bases may be used within the catalytic core of the enzyme and/or in the substrate-binding regions .
  • unmodified nucleoside is meant one of the bases adenine, cytosine, guanine, uracil joined to the 1' carbon of b-D-ribo-furanose .
  • modified nucleoside any nucleotide base which contains a modification in the chemical structure of an unmodified nucleotide base, sugar and/or phosphate.
  • ribozyme structure can be made to enhance the utility of ribozymes. Such modifications will enhance shelf-life, half-life in vitro, stability, and ease of introduction of such ribozymes to the target site, e.g., to enhance penetration of cellular membranes, and confer the ability to recognize and bind to targeted cells.
  • Ribozymes 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 .
  • ribozymes may be directly delivered ex vivo to cells or tissues with or without the aforementioned vehicles.
  • the RNA/vehicle combination is locally delivered by direct injection or by use of a catheter, infusion pump or stent.
  • routes of delivery include, but are not limited to, intravascular, intramuscular, subcutaneous or joint injection, aerosol inhalation, oral (tablet or pill form) , topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. More detailed descriptions of ribozyme delivery and administration are provided in Sullivan et al . , supra and Draper et al . , PCT W093/23569 which have been incorporated by reference herein.
  • the molecules of the instant invention can be used as pharmaceutical agents.
  • Pharmaceutical agents prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state in a patient.
  • the negatively charged polynucleotides of the invention can be administered (e.g., RNA, DNA or protein) and introduced into a patient by any standard means, with or without stabilizers, buffers, and the like, to form a pharmaceutical composition.
  • RNA, DNA or protein e.g., RNA, DNA or protein
  • standard protocols for formation of liposomes can be followed.
  • the compositions of the present invention may also be formulated and used as tablets, capsules or elixirs for oral administration; suppositories for rectal administration; sterile solutions; suspensions for injectable administration; and the like.
  • the present invention also includes pharmaceutically acceptable formulations of the compounds described. These formulations include salts of the above compounds, e.g., acid addition salts, for example, salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid.
  • a pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, e.g., systemic administration, into a cell or patient, preferably a human. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, or by injection. Such forms should not prevent the composition or formulation to reach a target cell (i.e., a cell to which the negatively charged polymer is desired to be delivered to) .
  • pharmacological compositions injected into the blood stream should be soluble.
  • Other factors are known in the art, and include considerations such as toxicity and forms which prevent the composition or formulation from exerting its effect.
  • systemic administration is meant in vivo systemic absorption or accumulation of drugs in the blood stream followed by distribution throughout the entire body.
  • Administration routes which lead to systemic absorption include, without limitations: intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular.
  • Each of these administration routes expose the desired negatively charged polymers, e.g., nucleic acids, to an accessible diseased tissue.
  • the rate of entry of a drug into the circulation has been shown to be a function of molecular weight or size.
  • a liposome or other drug carrier comprising the compounds of the instant invention can potentially localize the drug, for example, in certain tissue types, such as the tissues of the reticular endothelial system (RES) .
  • RES reticular endothelial system
  • a liposome formulation which can facilitate the association of drug with the surface of cells, such as, lymphocytes and macrophages is also useful. This approach may provide enhanced delivery of the drug to target cells by taking advantage of the specificity of macrophage and lymphocyte immune recognition of abnormal cells, such as the cancer cells.
  • the invention also features the use of the a composition comprising surface-modified liposomes containing poly (ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes) .
  • PEG-modified, or long-circulating liposomes or stealth liposomes are examples of poly (ethylene glycol) lipids.
  • These formulations offer an method for increasing the accumulation of drugs in target tissues.
  • This class of drug carriers resists opsonization and elimination by the mononuclear phagocytic system (MPS or RES) , thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated drug (Lasic et al . Chem . Rev. 1995, 95, 2601-2627; Ishiwataet al . , Chem . Pharm . Bull . 1995, 43, 1005-1011).
  • liposomes have been shown to accumulate selectively in tumors, presumably by extravasation and capture in the neovascularized target tissues (Lasic et al . , Science 1995, 267, 1275-1276; Oku et al . ,1995, Biochim . Biophys . Acta , 1238, 86-90).
  • the long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA, particularly compared to conventional cationic liposomes which are known to accumulate in tissues of the MPS (Liu et al . , J. Biol . Chem . 1995, 42, 24864-24870; Choi et al . , International PCT Publication No.
  • WO 96/10391 Ansell et al . , International PCT Publication No. WO 96/10390; Holland et al . , International PCT Publication No. WO 96/10392; all of these 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. All of these references are incorporated by reference herein.
  • compositions prepared for storage or administration which include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent.
  • Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington ' s Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro edit. 1985) hereby incorporated by reference herein.
  • preservatives, stabilizers, dyes and flavoring agents may be provided.
  • Id. at 1449. include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid.
  • antioxidants and suspending agents may be used. Id.
  • a pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state.
  • the pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors which those skilled in the medical arts will recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients is administered dependent upon potency of the negatively charged polymer.
  • the enzymatic 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 . Na tl . Acad. Sci . USA 83, 399; Scanlon et al . , 1991, Proc . Na tl . Acad. Sci . USA, 88, 10591-5; Kashani-Sabet et al . , 1992 An tisense Res . Dev. , 2 , 3-15; Dropulic et al . , 1992 J.
  • eukaryotic promoters e.g., Izant and Weintraub, 1985 Science 229, 345; McGarry and Lindquist, 1986 Proc . Na tl . Acad. Sci . USA 83, 399; Scanlon et al .
  • nucleic acid can be expressed in eukaryotic cells from the appropriate DNA/RNA vector.
  • the activity of such nucleic acids can be augmented by their release from the primary transcript by a ribozyme (Draper et al . , PCT WO 93/23569, and Sullivan et al . , PCT WO 94/02595; Ohkawa et al . , 1992 Nucleic Acids Symp . Ser . , 27, 15-6; Taira et al . , 1991, Nucleic Acids Res . , 19, 5125-30; Ventura et al . , 1993 Nucleic Acids Res . , 21, 3249-55; Chowrira et al . , 1994 J. Biol . Chem . 269, 25856; all of the references are hereby incorporated in their totality by reference herein) .
  • enzymatic nucleic acid molecules that cleave target molecules are expressed from transcription units (see for example Couture et al . , 1996, TIG. , 12, 510) inserted into DNA or RNA vectors.
  • the recombinant vectors are preferably DNA plasmids or viral vectors. Ribozyme expressing viral vectors could be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus.
  • the recombinant vectors capable of expressing the ribozymes are delivered as described above, and persist in target cells.
  • viral vectors may be used that provide for transient expression of ribozymes.
  • Such vectors might be repeatedly administered as necessary.
  • the ribozymes cleave the target RNA.
  • the active ribozyme contains an enzymatic center or core equivalent to those in the examples, and binding arms able to bind target nucleic acid molecules such that cleavage at the target site occurs. Other sequences may be present which do not interfere with such cleavage.
  • Delivery of ribozyme expressing vectors could be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that would allow for introduction into the desired target cell (for a review see Couture et al . , 1996, TIG. , 12, 510) .
  • an expression vector comprising nucleic acid sequence encoding at least one of the nucleic acid catalyst of the instant invention is disclosed.
  • the nucleic acid sequence encoding the nucleic acid catalyst of the instant invention is operable linked in a manner which allows expression of that nucleic acid molecule.
  • the expression vector comprises: 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 gene encoding at least one of the nucleic acid catalyst of the instant invention; and wherein said gene is operably linked to said initiation region and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.
  • the vector may optionally include an open reading frame (ORF) for a protein operably linked on the 5' side or the 3 '-side of the gene encoding the nucleic acid catalyst of the invention; and/or an intron (intervening sequences).
  • ORF open reading frame
  • RNA polymerase I RNA polymerase I
  • polymerase II RNA polymerase II
  • poly III RNA polymerase III
  • Transcripts from pol II or pol III promoters will be expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type will depend on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby.
  • Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990 Proc. Natl. Acad. Sci.
  • transcription units such as the ones derived from genes encoding U6 small nuclear (snRNA) , transfer RNA (tRNA) and adenovirus VA RNA are useful in generating high concentrations of desired RNA molecules such as ribozymes in cells (Thompson et al., supra; Couture and Stinchcomb, 1996, supra; Noonberg et al., 1994, Nucleic Acid Res., 22, 2830; Noonberg et al., US Patent No. 5,624,803; Good et al., 1997, Gene Ther. 4, 45; Beigelman et al., International PCT Publication No. NO 96/18736; all of these publications are incorporated by reference herein.
  • 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).
  • viral DNA vectors such as adenovirus or adeno- associated virus vectors
  • viral RNA vectors such as retroviral or alphavirus vectors
  • the invention features an expression vector comprising nucleic acid sequence encoding at least one of the catalytic nucleic acid molecule 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 gene encoding at least one said nucleic acid molecule; and wherein said gene is operably linked to said initiation region and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.
  • the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an open reading frame; d) a gene encoding at least one said nucleic acid molecule, wherein said gene is operably linked to the 3 ' -end of said open reading frame; and wherein said gene is operably linked to said initiation region, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.
  • the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) a gene encoding at least one said nucleic acid molecule; and wherein said gene is operably linked to said initiation region, said intron and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.
  • the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) an open reading frame; e) a gene encoding at least one said nucleic acid molecule, wherein said gene is operably linked to the 3 ' -end of said open reading frame; and wherein said gene is operably linked to said initiation region, said intron, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.
  • the methods described herein represent a scheme by which ribozymes may be derived that cleave other RNA targets required for angiogenesis. Also provided is a description of how such ribozymes may be delivered to cells. The examples demonstrate that upon delivery, the ribozymes inhibit cell proliferation in culture and modulate gene expression in vivo . Moreover, significantly reduced inhibition is observed if mutated ribozymes that are catalytically inactive are applied to the cells. Thus, inhibition requires the catalytic activity of the ribozymes .
  • Ribozyme target sites were chosen by analyzing genomic sequences of Tie-2 (Ziegler et al . , 1993, Oncogene 8 (3) , 663-670 (Genbank sequence HUMTEKRPTK accession number: M69238) and prioritizing the sites on the basis of folding. Hammerhead ribozymes were designed that could bind each target (see Figure 1) and were individually analyzed by computer folding (Christoffersen et al . , 1994 J. Mol . Struc . Theochem , 311, 273; Jaeger et al .
  • Ribozymes of the hammerhead or hairpin motif were designed to anneal to various sites in the RNA message.
  • the binding arms are complementary to the target site sequences described above.
  • the ribozymes were chemically synthesized. The method of synthesis used followed the procedure for normal RNA synthesis as described in Usman et al., (1987 J. Am. Chem. Soc, 109, 7845), Scaringe et al., (1990 Nucleic Acids Res., 18, 5433) and Wincott et al . , supra, and made use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'- end, and phosphoramidites at the 3 '-end. The average stepwise coupling yields were >98%.
  • Inactive ribozymes were synthesized by substituting a U for G5 and a U for A14 (numbering from Hertel et al., 1992 Nucleic Acids Res., 20, 3252). Hairpin ribozymes were synthesized in two parts and annealed to reconstruct the active ribozyme (Chowrira and Burke, 1992 Nucleic Acids Res., 20, 2835-2840). Ribozymes were also synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol. 180, 51) .
  • Ribozymes were modified to enhance stability by modification with nuclease resistant groups, for example, 2 '-amino, 2'-C-allyl, 2'-flouro, 2'-0-methyl, 2 ' -H (for a review see Usman and Cedergren, 1992 TIBS 17, 34) . Ribozymes were purified by gel electrophoresis using general methods or were purified by high pressure liquid chromatography (HPLC; See Wincott et al . , supra; the totality of which is hereby incorporated herein by reference) and were resuspended in water. The sequences of the chemically synthesized ribozymes used in this study are shown below in Table V-VI .
  • Example 4 Ribozyme Cleavage of TIE-2 RNA Target in vi tro
  • Ribozymes targeted to the human Tie-2 RNA are designed and synthesized as described above. These ribozymes can be tested for cleavage activity in vi tro, for example using the following procedure. The target sequences and the nucleotide location within the Tie-2 mRNA are given in Table V.
  • Cleavage Reactions Full-length or partially full- length, internally-labeled target RNA for ribozyme cleavage assay is prepared by in vi tro transcription in the presence of [a- 32 p] CTP, passed over a G 50 Sephadex column by spin chromatography and used as substrate RNA without further purification.
  • substrates are 5-_32p_ enc * labeled using T4 polynucleotide kinase enzyme.
  • Assays are performed by pre-warming a 2X concentration of purified ribozyme in ribozyme cleavage buffer (50 mM Tris- HCl, pH 7.5 at 37°C, 10 mM MgCl 2 ) and the cleavage reaction was initiated by adding the 2X ribozyme mix to an equal volume of substrate RNA (maximum of 1-5 nM) that was also pre-warmed in cleavage buffer.
  • ribozyme cleavage buffer 50 mM Tris- HCl, pH 7.5 at 37°C, 10 mM MgCl 2
  • o assays are carried out for 1 hour at 37 C using a final concentration of either 40 nM or 1 mM ribozyme, i.e., ribozyme excess.
  • the reaction is quenched by the addition of an equal volume of 95% formamide, 20 mM EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol after which the o sample is heated to 95 C for 2 minutes, quick chilled and loaded onto a denaturing polyacrylamide gel.
  • Substrate RNA and the specific RNA cleavage products generated by ribozyme cleavage are visualized on an autoradiograph of the gel. The percentage of cleavage is determined by Phosphor Imager® quantitation of bands representing the intact substrate and the cleavage products.
  • the rate of tumor growth is believed to be a function of blood supplied and therefore a function of angiogenesis (Rak, Supra ; Blood & Zetter, 1990, Biochimica et Biophysica Acta 1032, 89-118) . Elevated levels of a number of these angiogenic factors including Tie-2; integrin subunit ⁇ 3; integrin subunit ⁇ 6; and aryl hydrocarbon nuclear transporter have been reported in a number of cancers. Thus, inhibition of expression of these angiogenic factors (for example using ribozymes) would potentially reduce that rate of growth of these tumors.
  • Ribozymes With their catalytic activity and increased site specificity (see above) , are likely to represent a potent and safe therapeutic molecule for the treatment of cancer. Tumor angiogenesis and other indications are discussed below.
  • Tumor angiogenesis Angiogenesis has been shown to be necessary for tumors to grow into pathological size (Folkman, 1971, PNAS 76, 5217-5221; Wellstein & Czubayko, 1996, Breast Cancer Res and Trea tment 38, 109-119) . In addition, it allows tumor cells to travel through the circulatory system during metastasis. Increased levels of gene expression of a number of angiogenic factors such as vascular endothelial growth factor (VEGF) have been reported in vascularized and edema-associated brain tumors (Berkman et al . , 1993 J. Clini . Invest . 91, 153).
  • VEGF vascular endothelial growth factor
  • Neovascularization has been shown to cause or exacerbate ocular diseases including but not limited to, macular degeneration, neovascular glaucoma, diabetic retinopathy, myopic degeneration, and trachoma (Norrby, 1997, APMIS 105, 417-437) .
  • Aiello et al . , 1994 New Engl . J. Med. 331, 1480 showed that the ocular fluid, of a majority of patients suffering from diabetic retinopathy and other retinal disorders, contains a high concentration of VEGF.
  • Miller et al . , 1994 Am . J. Pa thol . 145, 574 reported elevated levels of VEGF mRNA in patients suffering from retinal ischemia.
  • Rheumatoid arthritis Immunohistochemistry and in si tu hybridization studies on tissues from the joints of patients suffering from rheumatoid arthritis show an increased level of VEGF and its receptors (Fava et al . , 1994 J. Exp . Med. 180, 341). Additionally, Koch et al . , 1994 J. Immunol . 152, 4149, found that VEGF-specific antibodies were able to significantly reduce the mitogenic activity of synovial tissues from patients suffering from rheumatoid arthritis. These observations support a direct role for VEGF in rheumatoid arthritis. Other angiogenic factors including those of the present invention may also be involved in arthritis. Animal Models
  • Ribozymes directed against ARNT, Tie-2 or integrin subunit RNAs would be delivered in the disk as well, or dropwise to the eye over the time course of the experiment.
  • hypoxia has been shown to cause both increased expression of VEGF and neovascularization in the retina (Pierce et al . , 1995 Proc . Na tl . Acad. Sci . USA. 92: 905-909; Shweiki et al . , 1992 J. Clin . Invest . 91: 2235-2243).
  • Another animal model that addresses neovascularization involves Matrigel, an extract of basement membrane that becomes a solid gel when injected subcutaneously (Passaniti et al .
  • Matrigel When the Matrigel is supplemented with angiogenesis factors, vessels grow into the Matrigel over a period of 3 to 5 days and angiogenesis can be assessed. Again, ribozymes directed against ARNT, Tie-2 or integrin subunit RNAs would be delivered in the Matrigel.
  • corneal vessel formation following corneal injury (Burger et al . , 1985 Cornea 4: 35-41; Lepri, et al . , 1994 J. Ocular Pharmacol . 10: 273- 280; Ormerod et al . , 1990 Am . J. Pa thol . 137: 1243-1252) or intracorneal growth factor implant (Grant et al . , 1993 Diabetologia 36: 282-291; Pandey et al . 1995 supra ; Zieche et al . , 1992 Lab . Invest .
  • the cornea model is the most common and well characterized anti-angiogenic agent efficacy screening model.
  • This model involves an avascular tissue into which vessels are recruited by a stimulating agent (growth factor, thermal or alkalai burn, endotoxin) .
  • the corneal model would utilize the intrastromal corneal implantation of a Teflon pellet soaked in a angiogenic compound-Hydron solution to recruit blood vessels toward the pellet which can be quantitated using standard microscopic and image analysis techniques.
  • ribozymes are applied topically to the eye or bound within Hydron on the Teflon pellet itself.
  • This avascular cornea as well as the Matrigel provide for low background assays. While the corneal model has been performed extensively in the rabbit, studies in the rat have also been conducted.
  • the mouse model (Passaniti et al . , supra ) is a non- tissue model which utilizes Matrigel, an extract of basement membrane (Kleinman et al . , 1986) or Millipore® filter disk, which can be impregnated with growth factors and anti-angiogenic agents in a liquid form prior to injection. Upon subcutaneous administration at body temperature, the Matrigel or Millipore® filter disk forms a solid implant.
  • an angiogenic compound would be embedded in the Matrigel or Millipore® filter disk which would be used to recruit vessels within the matrix of the Matrigel or Millipore® filter disk that can be processed histologically for endothelial cell specific vWF (factor VIII antigen) immunohistochemistry, Trichrome-Masson stain, or hemoglobin content.
  • the Matrigel or Millipore® filter disk are avascular; however, it is not tissue.
  • ribozymes are administered within the matrix of the Matrigel or Millipore® filter disk to test their anti-angiogenic efficacy.
  • delivery issues in this model as with delivery of ribozymes by Hydron- coated Teflon pellets in the rat cornea model, may be less problematic due to the homogeneous presence of the ribozyme within the respective matrix.
  • VEGF vascular endothelial growth factor
  • ribozymes will target only VEGFr RNA.
  • the involvement of other nonspecific types of stimuli in the cornea and Matrigel models is not advantageous from the standpoint of understanding the pharmacologic mechanism by which the anti-VEGFr RNA ribozymes produce their effects.
  • the models will allow for testing the specificity of the anti-VEGFr RNA ribozymes by using either a- or bFGF as a pro-angiogenic factor. Vessel recruitment using FGF should not be affected in either model by anti-VEGFr RNA ribozymes.
  • Identifying a common animal model for systemic efficacy testing of ribozymes is an efficient way of screening ribozymes for systemic efficacy.
  • the Lewis lung carcinoma and B-16 murine melanoma models are well accepted models of primary and metastatic cancer and are used for initial screening of anti-cancer. These murine models are not dependent upon the use of immunodeficient mice, are relatively inexpensive, and minimize housing concerns. Both the Lewis lung and B-16 melanoma models involve subcutaneous implantation of approximately 10' tumor cells from metastatically aggressive tumor cell lines (Lewis lung lines 3LL or D122, LLC-LN7; B-16-BL6 melanoma) in C57BL/6J mice. Alternatively, the Lewis lung model can be produced by the surgical implantation of tumor spheres (approximately 0.8 mm in diameter) . Metastasis also may be modeled by injecting the tumor cells directly i.v..
  • systemic pharmacotherapy with a wide variety of agents usually begins 1-7 days following tumor implantation/inoculation with either continuous or multiple administration regimens.
  • Concurrent pharmacokinetic studies can be performed to determine whether sufficient tissue levels of ribozymes can be achieved for pharmacodynamic effect to be expected.
  • primary tumors and secondary lung metastases can be removed and subjected to a variety of in vi tro studies (i.e. target RNA reduction) .
  • ribozyme formulations including cationic lipid complexes which may be useful for inflammatory diseases (e . g. DIMRIE/DOPE, etc . ) and RES evading liposomes which may be used to enhance vascular exposure of the ribozymes, are of interest in cancer models due to their presumed biodistribution to the lung.
  • liposome formulations can be used for delivering ribozymes to sites of pathology linked to an angiogenic response.
  • Ribozymes of this invention may be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of Tie-2; integrin subunit ⁇ 3; integrin subunit ⁇ 6; and/or aryl hydrocarbon nuclear transporter 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 may map nucleotide changes which are important to RNA structure and function in vi tro, as well as in cells and tissues.
  • Cleavage of target RNAs with ribozymes may be used to inhibit gene expression and define the role (essentially) of specified gene products in the progression of disease. In this manner, other genetic targets may be defined as important mediators of the disease.
  • 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
  • ribozymes of this invention include detection of the presence of RNAs associated with Tie-2; integrin subunit ⁇ 3; integrin subunit ⁇ 6; and/or aryl hydrocarbon nuclear transporter related condition. Such RNA is detected by determining the presence of a cleavage product after treatment with a ribozyme using standard methodology.
  • ribozymes which can cleave only wild-type or mutant forms of the target RNA are used for the assay.
  • the first ribozyme is used to identify wild-type RNA present in the sample and the second ribozyme will be used to identify mutant RNA in the sample.
  • synthetic substrates of both wild-type and mutant RNA will be cleaved by both ribozymes to demonstrate the relative ribozyme efficiencies in the reactions and the absence of cleavage of the "non-targeted" RNA species.
  • the cleavage products from the synthetic substrates will also serve to generate size markers for the analysis of wild-type and mutant RNAs in the sample population.
  • each analysis will require two ribozymes, two substrates and one unknown sample which will be combined into six reactions.
  • the presence of cleavage products will be determined using an RNAse protection assay so that full-length and cleavage fragments of each RNA can be analyzed in one lane of a polyacrylamide gel. It is not absolutely required to quantify the results to gain insight into the expression of mutant RNAs and putative risk of the desired phenotypic changes in target cells.
  • the expression of mRNA whose protein product is implicated in the development of the phenotype i.e., Tie-2; integrin subunit ⁇ 3; integrin subunit 6; ARNT is adequate to establish risk.
  • RNA levels will be adequate and will decrease the cost of the initial diagnosis. Higher mutant form to wild-type ratios will be correlated with higher risk whether RNA levels are compared qualitatively or quantitatively. Additional Uses
  • sequence-specific enzymatic nucleic acid molecules of the instant invention might have many of the same applications for the study of RNA that DNA restriction endonucleases have for the study of DNA
  • RNAs of unknown sequence could be used to establish sequence relationships between two related RNAs, and large RNAs could be specifically cleaved to fragments of a size more useful for study.
  • the ability to engineer sequence specificity of the ribozyme is ideal for cleavage of RNAs of unknown sequence.
  • Reaction mechanism attack by the 3' -OH of guanosine to generate cleavage products with 3' -OH and 5'- guanosine .
  • the small (4-6 nt) binding site may make this ribozyme too non-specific for targeted RNA cleavage, however, the Tetrahymena group I intron has been used to repair a "defective" ⁇ -galactosidase message by the ligation of new ⁇ -galactosidase sequences onto the defective message [—] .
  • RNAse P RNA (Ml RNA) • Size: -290 to 400 nucleotides.
  • RNA portion of a ubiquitous ribonucleoprotein enzyme • RNA portion of a ubiquitous ribonucleoprotein enzyme .
  • Reaction mechanism possible attack by M 2 + -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.
  • 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.
  • Reaction mechanism attack by 2' -OH 5' to the scissile bond to generate cleavage products with 2', 3'- cyclic phosphate and 5' -OH ends.
  • Reaction mechanism attack by 2' -OH 5' to the scissile bond to generate cleavage products with 2',3'- cyclic phosphate and 5' -OH ends.
  • RNA RNA as the infectious agent.
  • Hepatitis Delta Virus (HDV) Ribozyme • Size: -60 nucleotides. • Trans cleavage of target RNAs demonstrated [—]

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Abstract

Nucleic acid molecule which modulate the synthesis, expression and/or stability of an mRNA encoding for angiogenic factors selected from aryl hydrocarbon nuclear transport (ARNT), intergrin subunit beta 3 (β3), integrin subunit alpha 6 (α6) and tie - 2 RNA. This invention further provides a treatment for indications related to angiogenesis using the nucleic acid molecules.

Description

DESCRIPTION
Method And Reagents For The Treatment Of
Diseases Or Conditions Related To
Molecules Involved In Angiogenie Responses
Background Of The Invention
This invention relates to methods and reagents for the treatment of diseases or conditions relating to the levels of expression of angiogenic factors and receptors involved in the regulation of angiogenesis . The following is a discussion of relevant art, none of which is admitted to be prior art to the present invention.
The formation of blood vessels in vertebrates can be described in two embyronic stages. During the first stage, known as vasculogenesis, yolk sac splanchnopleuric mesenchyme differentiates into vascular progenitor cells and then to blood island aggregates which are primitive blood cells surrounded by fused endothelial progenitors
(angioblasts) . These blood islands then fuse and go on to form a vascular plexus which supplies nutrients to the embryo (Merenmies et al . , 1997, Gell Growth & Development 8, 3-10) . The next vascular developmental step is known as angiogenesis. From the vessels formed during vasculogenesis, new blood vessels sprout, elongate and develop into capillary loop formations of endothelial cells. It is a highly complex event involving local basement membrane disruption, endothelial cell proliferation, migration and microvessel morphogenesis
(Rak et al . , 1995, Anti-Cancer Drugs 6, 3-18). Organs such as the brain and kidney are vascularized through the angiogenic process (Dumont et al . , 1995, Developmental Dynamics 203, 80-92) . Angiogenesis has been described to occur through two mechanisms, vascular sprouting and intussusception. Intussusception of pre-existing vessels occur after proliferation of endothelial cells producing a wide lumen. Through the utilization of transcapillary pillars or posts of extracellular matrix, the lumen is split to form two vessels (Risau, 1997, Na ture 386, 671-674) . Sprouting angiogenesis also originates from pre-existing blood vessels and consists of new blood vessels sprouting, elongating and developing into capillary loop formations of endothelial cells. It is a highly complex event involving disruption of extracellular matrix, endothelial cell proliferation, chemotaxic migration and microvessel morphogenesis (Rak, supra ) . Many factors regulating positive and negative control of angiogenesis have been reported demonstrating the sophistication of this process. An example of an angiogenic factor is Vascular Endothelial Growth Factor receptor (VEGFr) which has been shown to be specific to endothelial cells and is discussed in Pavco et al . , Int. PCT Pub. No. WO 97/15662.
Unlike vasculogenesis, angiogenesis not only occurs in embyronic development, but can also occur throughout the lifespan of the organism during such events as wound healing, bone repair, inflammation, and female menstral cycles. Local delivery of oxygen and nutrients and the removal of waste requires a complex system of blood vessels which has the ability to adapt as the tissue requirements changes. Involvement of a large number of positive and negative factors in angiogenic regulation demonstrates the complexity of this process. When the balance between upregulating factors and downregulating factors is disrupted in favor of increased angiogenesis, disease states have been known to occur.
Many factors have been identified which contribute to increased angiogenesis including: 1) ryl Hydrocarbon Nuclear Transporter (ARNT) : ARNT
(also known as HIF-lβ) forms heterodimers with several factors including HIF-α (Maxwell et al . , 1997, Proc . Na tl .
Acad. Sci . USA 94, 8104-8109) . When HIF-α and ARNT complex together, they form a complex called HIF-1. HIF -1 is believed to be regulate genes involved in the response to oxygen deprivation. ARNT -/- embryonic stem cells fail to induce VEGF expression in response to hypoxia. ARNT -/- mice are not viable beyond embryonic day 10.5. Like VEGF knockout mice, these embryos show defective angiogenesis of the yolk sac (Maltepe et al . , 1997, Na ture 386, 403- 407) .
Hepatoma cells containing an ARNT mutation that is functionally deficient in dimerizing with HIF-lα shows greatly reduced VEGF expression in response to hypoxia compared to normal cells (Wood et al . , 1996, J. Biol . Chem . 271, 15117-15123) . Tumor xenografts derived from these cells show reduced vascularity and approximately 2- fold reduced tumor growth rates (Maxwell et al . , 1997, supra ) .
2) Tie-2 : Tie-2 (also known as Tek) , is a tyrosine kinase protein receptor which consists of 1122 amino acids and is produced in endothelial (Merenmies et al . , 1997, Cell Growth & Differentia tion 8, 3-10) as well as early hematopoeitic cells (Maisonpierree et al . , 1993, Oncogene 8, 1631-1637). Tie-2 expression has been demonstrated in mice, rats and humans. The human gene is thought to be located on chromosome 9p21 (Dumont et al . , 1994, Genes & Developmen t 8, 1897-1909). Tie-2 homozygous mutant endothelial cells were examined using anti-PECAM monoclonal antibody (Sato et al . , 1997, Na ture 376, 70- 74) . All of the homozygous mutants were dead within 10.5 days with obvious deformities in the head and heart present by day 9.5. In addition, large vessels were indistinguishable from small vessels and no capillary sprouts were seen in the brain. These observations suggested that Tie-2 plays an important role in angiogenesis rather than vasculogenesis. The earlier effects of Tie-2 mutant compared to the Tie-1 mutant indicates separate roles for the two RTK' s in angiogenesis .
Ligands to Tie-2 have been discovered and named angiopoietin 1 and 2 (angl and 2) (Davis, S. et al . , 1993, Cell 87, 1161; Maisonpierre, P.C. et al . , 1997, Science, 277, 55-60) . Both factors consist of an NH2-terminal coiled-coil domain as well as a COOH-terminal fibrinogen- like domain. Angl binds to Tie-2/Tek but not Tie-1 and stimulates angiogenesis through autophosphorylation. Ang2 is a 496 amino acid polypeptide whose human and mouse homologs are 85% identical. Autophosphorylation caused by Angl binding to the Tie-2 receptor can be blocked with the addition of Ang2. The Tie-2 receptor is unusual in that it utilizes both positive and negative control mechanisms.
3) Integrins : Integrins are a family of cell adhesion and migration mediating proteins that are comprised of at least 15 alpha and 8 beta subunits that are expressed as a number of different αβ non-covalently bound heterodimers on cell surfaces (Varner, 1997, Regula tion of Angiogenesis, ed I.D Goldberg & E.M. Rosen, 361-390; Brooks, 1996, Eur J Cancer 14, 2423-2429) . Each combination of integrin subunits is thought to have angiogenic capabilities, for example αββi has been implicated in capillary tube formation Additionally, distinct integrins allow for the attachment to many different extracellular matrix (ECM) components including fibronectin, vitronectin, laminin and collagen (Stromblad & Cheresh, 1996, Chemistry & Biochemistry 3, 881-885) . Integrin production has been shown to be induced by a number a stimuli including intracellular pH increases, calcium concentration, inositol lipid synthesis, tyrosine phosphorylation of a focal contact associated tyrosine kinase, and activation of p34/cdc2 and cyclin A (Varner & Cheresh, 1996, Curr Op in Cell Biol 8,724-730). αvβ3 a 160kDa protein is the most well characterized molecule of the integrin family and is believed to play a large role in angiogenesis (Varner, 1997, supra ) . αvβ3 binds the largest number of ECM components of all known heterodimers indicating any cell with these molecules on the cell surface could adhere to or migrate on almost any of the ECM components (Varner, 1997, supra ) . When vascular endothelial cells are in their quiescent state very little o_vβ3 is expressed, but is highly upregulated in several pathological conditions including neoplasms. Antagonists to αvβ3 can inhibit angiogenesis in the chick chorioallentoic membrane (CAM) model and in SCID mice and even reduce the tumor volume. When antibodies are administered for Avβ3, apoptosis is observed in the proliferating vascular vessels. This has led to suggestions that αvβ3 provides a survival signal for vascular cells allowing for continued proliferation (Stromblad & Cheresh, 1996, supra; Varner, 1997 supra ) .
Other angiogenic targets are included and their characteristics are defined in the following references, all of which are incorporated herein by reference in their entirety: Methionine Aminopeptidase : (Arfin et al . , 1995, PNAS 92, 7714-7718 (Genbank Accession No. U29607) ; Sin, N. et . al . , 1997, PNAS 94, 6099-6103; Griffith et al . , 1997, Chem Biol . 4(6), 461-471); Transcription factor Ets- 1: (Iwasaka, C. et al . 1996. J. Cell Physiol . 169, 522-531; Chen, Z. et al . ,1997, Cancer Res . 57, 2013-2019; Hultgardh-Nilsson A, et al . , 1996, Circ Res . 78(4), 589- 595; Reddy et al . , 1988, Oncogene Res . 3 (3), 239-246 (Genbank accession No. X14798)); Platelet-derived endothelial cell growth factor and its receptor (PD-ECGF & PD-ECGFr) : (Furukawa, T. et al . , 1992, Nature 356, 668; Moghaddam, A. et al., 1995, Proc. Natl. Acad. Sci.; Clark, R.A.F. et al. ,1996, Am J. Pathol. 148, 1407; Hoshina, T.M., et al., 1995, Int. J. Cancer 64, 79-82; Nakanishi, A.K., et al., 1992, J. Biol. Chem 267, 20311-20316; Finnis et al., unpublished (Genbank accession No. M63193) ; Transforming Growth factors (TGFs) : (Schreiber et al., 1986, Science 232, 1250; Maione, T.E. and Sharpe, R.J.,1990, Trends Pharm. Sci., 11, 457-461; Noma et al., 1991, Growth Factors 4 (4), 247-255; Sukurai (unpublished) (Genbank accession No. AB009356) transforming growth factor receptor: (Miyazono, K.,1996, Nippon Yakurigaku Zasshu 107, 133-140; Mahooti-Brooks . et al., 1996, J. Clin. Invest. 97, 1436-1446; Lopez-Casillas et al., 1991, Cell 67 (4), 797-805; Lopez-Casillas et al., 1991, Cell 67 (4), 785-795 (Genbank Accession No. L07594); Angiogenin: (Fett et al., 1985, Biochemistry 24, 5480-5486; Bicknell & Vallee, 1988, PNAS 85, 5961-5965; Vallee & Riordan, 1988 , Adv. Exp. Med. Biol 234, 41-53; Shapiro & Vallee, 1987, PNAS 84, 2238-2241; Shapiro et al., 1986, Bioc emis ry 25, 3527-3532; Olson et al., 1994, Cancer Res. 54, 4576-4579;
Kurachi et al., 1985, Biochemistry 24, 5494-5499; Kurachi et al., 1985, Biochemistry 24 (20), 5494-5499 (Genbank
Accession No. M11567)); Tumor necrosis factor receptor:
(Naismith et al., 1995,. J. Inflamm 47, 1-7; Loetscher et al., 1990, Cell 61, 351-359; Himmler et al., 1990, DNA
Cell Biol. 9, 705-715 (Genbank Accession No. M63121
M75861); Endothelial cell stimulating angiogenesis factor
(ESAF) : (Brown & Weiss, 1988,. Ann. Rheum. Dis., 47, 881-
885); Interleukin-8 (IL-8) : (Elner et al., 1991, , Am J. Pathol. 139, 977-988; Strieter et al., 1992, Am. J. Pathol. 141, 1279-1284; Mukaida et al., 1989, J. Immunol. 143 (4), 1366-1371 (Genbank Accession No. M28130) ) ; Angiopoietin 1: (Davis, S. et al., 1996, Cell 87, 1161; Iwama, A. et al., 1993, Biochem Biophys . Res. Commun. 195, 301; Dumont, D.J. et al., 1995, Genes Dev 8, 1897; Sato, T.N. et al., 1995, Nature 376, 70; Suri, C. et al., 1996) Cell 87, 1171 (Genbank Accession No. U83508)); Angiopoietin 2: (Maisonpierre, et al., 1997, Science, 277, 55-60; Hanahan, 1997, Science 277, 48-50; Genbank Accession No. AF004327 (unpublished) ); Insulin-like growth factor (IGF- 1): (Warren, R.S. et al. ,1996, J. Biol. Chem. 271, 29483- 29488; Grant et.al., 1993 , Diabetologia 36, 282-291; Nicosia et al., 1994, Am. J. Pathol. 145, 1023-1029; Steenbergh et al., Biochem. Biophys . Res. Commun. 175, 507-514 (Genbank Accession: X57025); Insulin-like growth factor receptor (IGF-lr) : (Ullrich et al., 1986, EMBO J. 5, 2503-2512 (Genbank Accession No. X04434 M24599) ; B61 : (Pandey, A. et al., 1995, Science 268, 567-569; Holzman et al., 1990, Mol. Cell. Biol. 10, 5830-5838 (Genbank Accession No. M57730 M37476); B61 receptor (Eck) : (Pandey, A. et al., 1995, Science 268, 567-569; Lindberg & Hunter, 1990, Mol. Cell. Biol. 10 (12), 6316-6324 (Genbank Accession No. M59371 M36395); Protein kinase C: (Morris et al., 1988, Cell Physiol. 23, C318-C322; Oikawa, T. et al., 1992, J. Antibiot. 45, 1155-1160; Finkenzeller . et al., 1992, Cancer Res. 52, 4821-4823; Kubo et al., 1987, FEBS Lett. 223 (1), 138-142 (Genbank Accession No. X06318 M27545); ); SH2 domain (Guo, D. et al., 1995, J. Biol. Chem 270, 6729-6733) a. Phospholipase c-g:(Guo, D. at al . , 1995, J. Biol. Chem 270, 6729-6733; Rhee, S.G. et al . (1992) J. Biol. Chem 267, 12393-12396; Burgess et al . , 1990, Mol. Cell. Biol. 10, 4770-4777 (Genbank Accession No. M34667)) b. Phosphatidylinositol 3 kinase (PI-3): (Downs, CP. et al., 1991, Cell Signalling 3, 501-513; Genbank accession No. Z29090; Genbank accession No. Z46973) c. Ras GTPase activating protein (GAP) : (Trahey, M. et al., 1987, Science 238, 542-545; Guo, D. et al., 1995, J. Biol. Chem 270, 6729-6733; Trahey et al., 1988, Science 242, 1697-1700 (Genbank accession No. M23612)) d. Oncogene adaptor protein Nek: (Park & Rhee, 1992, Mol . Cell . Biol . 12, 5816-5823; Johnson, 1990, Nucleic Acids Res . 18 (4), 1048 (Genbank accession No. X17576) ) ; Granulocyte Colony-Stimulating Factor: (Devlin et al . , 1987, J. Leukoc . Biol . 41, 302-306 (Genbank accession No. M17706) ) ; Hepatocyte growth factor: (Miyazawa et al . , 1991, Eur. J. Biochem . 197 (1), 15-22 (Genbank accession No. X57574); Proliferin: (Groskopf et al . , 1997, Endocrinology 138(7), 2835-2840; Jackson D, et al . , 1994, Science . 266(5190), 1581-1584; Volpert et al . , 1996 , Endocrinology 137(9): 3871-3876); Placental growth factor: (Kodama et al . , 1997, Eur J Gynaecol Oncol . ; 18(6), 508- 510; Ziche et al . , 1997, Lab Invest . 76(4), 517-531; Relf et al . , 1997, Cancer Res . 57(5), 963-969; Genbank accession No. Y09268)
Summary Of The Invention
The invention features the use of enzymatic nucleic acid molecules and methods for their use to down regulate or inhibit the expression of angiogenic factors. Specifically, the enzymatic nucleic acids of the present invention are used as a treatment for indications relating to angiogenesis including but not limited to cancer, age related macular degeneration (ARMD) , diabetic retinopathy, inflammation, arthritis, psoriasis and the like. In a preferred embodiment, the invention features enzymatic nucleic acid molecules that cleave RNAs encoding angiogenic selected from a group comprising: Tie-2, integrin subunit β3, integrin subunit α6, and aryl hydrocarbon nuclear transporter (ARNT) . By "inhibit" it is meant that the activity of the cleaved RNA is reduced below that observed in the absence of the nucleic acid. In one embodiment, inhibition with ribozymes preferably is below that level observed in the presence of an enzymatically inactive RNA molecule that is able to bind to the same site on the mRNA, but is unable to cleave that RNA.
By "angiogenic factors" is meant a peptide molecule which is involved in a process or pathway necessary for the formation of novel blood vessels.
In another preferred embodiment, the invention features the use of enzymatic nucleic acids that cleave the RNAs encoded by angiogenic factors selected from a group comprising: Methionine Aminopeptidase; Ets-1 Transcription factor; integrins; platelet derived endothelial cell growth factor (PD-ECGF); PD-ECGF receptor; Transforming Growth factors (TGFs) ; Transforming growth factor receptor; Angiogenin; Endothelial cell stimulating angiogenesis factor (ESAF) ; Interleukin-8 (IL- 8); Angiopoietin 1 and 2; TIE-1; insulin-like growth factor (IGF-1); insulin-like growth factor receptor (IGF- lr) ; B61; B61 receptor (Eck) ; Protein kinase C; an SH2 domain (e.g. Phospholipase c-g, Phosphatidylinositol 3 kinase (PI-3), Ras GTPase activating protein (GAP); Oncogene adaptor protein Nek; Granulocyte Colony- Stimulating Factor; Hepatocyte growth factor; Proliferin; and Placental growth factor.
By "enzymatic nucleic acid" it is meant a nucleic acid molecule capable of catalyzing reactions including, but not limited to, site-specific cleavage and/or ligation of other nucleic acid molecules, cleavage of peptide and amide bonds, and trans-splicing. Such a molecule with endonuclease activity may have complementarity in a substrate binding region to a specified gene target, and also has an enzymatic activity that specifically cleaves RNA or DNA in that target. That is, the nucleic acid molecule with endonuclease activity is able to intramolecularly or intermolecularly cleave RNA or DNA and thereby inactivate a target RNA or DNA molecule. This complementarity functions to allow sufficient hybridization of the enzymatic RNA molecule to the target RNA or DNA to allow the cleavage to occur. 100% complementarity is preferred, but complementarity as low as 50-75% may also be useful in this invention. The nucleic acids may be modified at the base, sugar, and/or phosphate groups. The term enzymatic nucleic acid is used interchangeably with phrases such as ribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, catalytic oligonucleotides, nucleozyme, DNAzyme, RNA enzyme, endo- ribonuclease, endonuclease, minizyme, leadzyme, oligozyme or DNA enzyme. All of these terminologies describe nucleic acid molecules with enzymatic activity. The specific enzymatic nucleic acid molecules described in the instant application are not meant to be limiting and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it have a specific substrate binding site which is complementary to one or more of the target nucleic acid regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart a nucleic acid cleaving activity to the molecule (Cech et al., U.S. Patent No. 4,987,071; Cech et al., 1988, JAMA) .
By "enzymatic portion" or "catalytic domain" is meant that portion/region of the ribozyme essential for cleavage of a nucleic acid substrate (for example see Figure 1) .
By "substrate binding arm" or "substrate binding domain" is meant that portion/region of a ribozyme which is complementary to (i.e., able to base-pair with) a portion of its substrate. Generally, such complementarity is 100%, but can be less if desired. For example, as few as 10 bases out of 14 may be base-paired. Such arms are shown generally in Figure 1. That is, these arms contain sequences within a ribozyme which are intended to bring ribozyme and target RNA together through complementary base-pairing interactions . The ribozyme of the invention may have binding arms that are contiguous or noncontiguous and may be of varying lengths . The length of the binding arm(s) are preferably greater than or equal to four nucleotides; specifically 12-100 nucleotides; more specifically 14-24 nucleotides long. If two binding arms are chosen, the design is such that the length of the binding arms are symmetrical (i.e., each of the binding arms is of the same length; e . g. , five and five nucleotides, six and six nucleotides or seven and seven nucleotides long) or asymmetrical (i.e., the binding arms are of different length; e.g., six and three nucleotides; three and six nucleotides long; four and five nucleotides long; four and six nucleotides long; four and seven nucleotides long; and the like) .
By DNAzyme is meant, an enzymatic nucleic acid molecule lacking a 2' -OH group.
In one of the preferred embodiments, the enzymatic nucleic acid molecule is formed in a hammerhead or hairpin motif, but may also be formed in the motif of a hepatitis δ virus, group I intron, group II intron or RNase P RNA (in association with an RNA guide sequence) , Neurospora VS RNA or DNAzymes. Examples of such hammerhead motifs are described by Dreyfus, supra , Rossi et al . , 1992, AIDS Research and Human Retroviruses 8, 183; of hairpin motifs by Hampel et al . , EP0360257, Hampel and Tritz, 1989 Biochemistry 28, 4929, Feldstein et al . , 1989, Gene 82, 53, Haseloff and Gerlach, 1989, Gene, 82, 43, and Hampel et al . , 1990 Nucleic Acids Res . 18, 299; of the hepatitis d virus motif is described by Perrotta and Been, 1992 Biochemistry 31, 16; of the RNaseP motif by Guerrier- Takada et al . , 1983 Cell 35, 849; Forster and Altman, 1990, Science 249, 783; Li and Altman, 1996, Nucleic Acids Res . 24, 835; Neurospora VS RNA ribozyme motif is described by Collins (Saville and Collins, 1990 Cell 61, 685-696; Saville and Collins, 1991 Proc . Na tl . Acad. Sci . USA 88, 8826-8830; Collins and Olive, 1993 Biochemistry 32, 2795-2799; Guo and Collins, 1995, EMBO. J. 14, 363); Group II introns are described by Griffin et al . , 1995, Chem . Biol . 2, 761; Michels and Pyle, 1995, Biochemistry 34, 2965; Pyle et al . , International PCT Publication No. WO 96/22689; of the Group I intron by Cech et al . , U.S. Patent 4,987,071 and of DNAzymes by Usman et al . , International PCT Publication No. WO 95/11304; Chartrand et al . , 1995, NAR 23, 4092; Breaker et al . , 1995, Chem . Bio . 2 , 655; Santoro et al . , 1997, PNAS 94, 4262. 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).
By "equivalent" RNA to Tie-2, integrin subunit β3, integrin subunit α6, or ARNT is meant to include those naturally occurring RNA molecules having homology (partial or complete) to Tie-2, integrin subunit β3, integrin subunit α6, or ARNT or encoding for proteins with similar function as Tie-2, integrin subunit β3, integrin subunit α6, or ARNT in various animals, including human, rodent, primate, rabbit and pig. 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 "complementarity" is meant a nucleic acid molecules that can form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types (for example, Hoogsteen type) of base-paired interactions.
In a preferred embodiment the invention provides a method for producing a class of enzymatic cleaving agents which exhibit a high degree of specificity for the RNA of a desired target. The enzymatic nucleic acid molecule is preferably targeted to a highly conserved sequence region of a target RNAs encoding Tie-2, integrin subunit β3, integrin subunit α6, or ARNT proteins such that specific treatment of a disease or condition can be provided with either one or several enzymatic nucleic acids. Such enzymatic nucleic acid molecules can be delivered exogenously to specific cells as required. Alternatively, the ribozymes can be expressed from DNA/RNA vectors that are delivered to specific cells.
By "highly conserved sequence region" is meant a nucleotide sequence of one or more regions in a nucleic acid molecule does not vary significantly from one generation to the other or from one biological system to the other.
Such ribozymes are useful for the prevention of the diseases and conditions including cancer, diabetic retinopathy, macular degeneration, neovascular glaucoma, myopic degeneration, arthritis, psoriasis, verruca vulgaris, angiofibroma of tuberous sclerosis, pot-wine stains, Sturge Weber syndrome, Kippel-Trenaunay-Weber syndrome, Osler-Weber-Rendu syndrome and any other diseases or conditions that are related to the levels of Tie-2, integrin subunit β3, integrin subunit α6, or ARNT activity in a cell or tissue.
By "related" is meant that the inhibition of Tie-2, integrin subunit β3, integrin subunit α6, and/or ARNT RNAs and thus reduction in the level respective protein activity will relieve to some extent the symptoms of the disease or condition.
In preferred embodiments, the ribozymes have binding arms which are complementary to the target sequences in Tables III-X. Examples of such ribozymes are also shown in Tables III-X. Tables III and IV display target sequences and ribozymes for ARNT, Tables V and VI display target sequences and ribozymes for Tie-2, tables VII and VIII display target sequences and ribozymes for integrin subunit alpha 6, and tables IX and X display target sequences and ribozymes for integrin subunit beta 3. Examples of such ribozymes consist essentially of sequences defined in these Tables. By "consists essentially of" is meant that the active ribozyme contains an enzymatic center or core equivalent to those in the examples, and binding arms able to bind mRNA such that cleavage at the target site occurs. Other sequences may be present which do not interfere with such cleavage.
Thus, in a first aspect, the invention features ribozymes that inhibit gene expression and/or cell proliferation. These chemically or enzymatically synthesized RNA molecules contain substrate binding domains that bind to accessible regions of their target mRNAs. The RNA molecules also contain domains that catalyze the cleavage of RNA. The RNA molecules are preferably ribozymes of the hammerhead or hairpin motif. Alternatively, the ribozymes are DNAzymes . Upon binding, the ribozymes cleave the target mRNAs, preventing translation and protein accumulation. In the absence of the expression of the target gene, cell proliferation is inhibited. Chemically synthesized RNA molecules also include RNA molecules assembled together from various fragments of RNA using a chemical or an enzymatic ligation method.
In a preferred embodiment, ribozymes are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells. The nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex vivo, or in vivo through injection, infusion pump or stent, with or without their incorporation in biopolymers . In another preferred embodiment, the ribozyme is administered to the site of Tie-2, integrin subunit β3, integrin subunit α6, or ARNT expression (e.g. tumor cells, endothelial cells) in an appropriate liposomal vehicle.
In another aspect of the invention, ribozymes that cleave target molecules and inhibit Tie-2, integrin subunit β3, integrin subunit α6, or ARNT activity are expressed from transcription units inserted into DNA or RNA vectors . The recombinant vectors are preferably DNA plasmids or viral vectors. Ribozyme expressing viral vectors could be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus . Preferably, the recombinant vectors capable of expressing the ribozymes are delivered as described above, and persist in target cells. Alternatively, viral vectors may be used that provide for transient expression of ribozymes. Such vectors might be repeatedly administered as necessary. Once expressed, the ribozymes cleave the target RNA. Delivery of ribozyme expressing vectors could be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex- planted from the patient followed by reintroduction into the patient, or by any other means that would allow for introduction into the desired target cell (for a review see Couture and Stinchcomb, 1996, TIG. , 12, 510). In another aspect of the invention, ribozymes that cleave target molecules and inhibit cell proliferation are expressed from transcription units inserted into DNA, RNA, or viral vectors. Preferably, the recombinant vectors capable of expressing the ribozymes are locally delivered as described above, and transiently persist in smooth muscle cells. However, other mammalian cell vectors that direct the expression of RNA may be used for this purpose.
By "patient" is meant an organism which is a donor or recipient of explanted cells or the cells themselves. "Patient" also refers to an organism to which enzymatic nucleic acid molecules can be administered. Preferably, a patient is a mammal or mammalian cells. More preferably, a patient is a human or human cells.
By "vectors" is meant any nucleic acid- and/or viral- based technique used to deliver a desired nucleic acid.
These ribozymes, 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 Tie-2, integrin subunit β3, integrin subunit α6, or ARNT, the patient may be treated, or other appropriate cells may be treated, as is evident to those skilled in the art.
In a further embodiment, the described ribozymes can be used in combination with other known treatments to treat conditions or diseases discussed above. For example, the described ribozymes could be used in combination with one or more known therapeutic agents to treat cancer.
In preferred embodiments, the ribozymes have binding arms which are complementary to the sequences in the tables, shown as Seq. I.D. Nos. 394-786, 849-910, 1612- 2312, 2381-2448, 3588-4726, 4821-4914, 5702-6488, and 6569-6648. Examples of such ribozymes are shown as Seq. I.D. Nos.1-393, 787-848, 911-1611, 2313-2380, 2449-3587, 4727-4820. 4915-5701, and 6489-6568. Other sequences may be present which do not interfere with such cleavage.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.
Description Of The Preferred Embodiments
The drawings will first briefly be described. 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, Na ture Struc . Bio . , 1, 273). RNase P (M1RNA) : EGS represents external guide sequence (Forster et al . , 1990, Science, 249, 783; Pace et al . , 1990, J. Biol . Chem . , 265, 3587). Group II Intron: 5'SS means 5' splice site; 3'SS means 3' -splice site; IBS means intron binding site; EBS means exon binding site (Pyle et al . , 1994, Biochemistry, 33, 2716) . VS RNA: I-VI are meant to indicate six stem-loop structures; shaded regions are meant to indicate tertiary interaction (Collins, International PCT Publication No. WO 96/19577) . HDV Ribozyme: : I-IV are meant to indicate four stem-loop structures (Been et al . , US Patent No. 5,625,047). Hammerhead Ribozyme: : I-III are meant to indicate three stem-loop structures; stems I-III can be of any length and may be symmetrical or asymmetrical (Usman et al . , 1996, Curr . Op . Struct . Bio . , 1, 527) . Hairpin 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 may be covalently linked by one or more bases (i.e., r is base) . Helix 1, 4 or 5 may also be extended by 2 or more base pairs (e.g., 4 - 20 base pairs) to stabilize the ribozyme structure, and preferably is a protein binding site. 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 may be modified at the sugar, base or phosphate. Complete base-pairing is not required in the helices, but is preferred. Helix 1 and 4 can be of any size (i.e., o and p is each independently from 0 to any number, e.g., 20) as long as some base- pairing is maintained. Essential bases are shown as specific bases in the structure, but those in the art will recognize that one or more may be modified chemically (abasic, base, sugar and/or phosphate modifications) or replaced with another base without significant effect. Helix 4 can be formed from two separate molecules, i.e., without a connecting loop. The connecting loop when present may be a ribonucleotide with or without modifications to its base, sugar or phosphate. "q" is ≥ 2 bases. The connecting loop can also be replaced with a non-nucleotide linker molecule. H refers to bases A, U, or C. Y refers to pyrimidine bases. " " refers to a covalent bond. (Burke et al . , 1996, Nucleic Acids & Mol . Biol . , 10, 129; Chowrira et al . , US Patent No. 5,631,359).
Figure 2 is a diagrammatic representation of a hammerhead ribozyme targeted against Tie-2 at position 1037.
Enzymatic Nucleic Acid Molecules
Seven basic varieties of naturally-occurring enzymatic RNAs are known presently. In addition, several in vitro selection (evolution) strategies (Orgel, 1979, Proc . R . Soc . London , B 205, 435) have been used to evolve new nucleic acid catalysts capable of catalyzing cleavage and ligation of phosphodiester linkages (Joyce, 1989, Gene, 82, 83-87; Beaudry et al . , 1992, Science 257, 635- 641; Joyce, 1992, Scientific American 267, 90-97; Breaker et al . , 1994, TIBTECH 12, 268; Bartel et al . , 1993, Science 261:1411-1418; Szostak, 1993, TIBS 17, 89-93; Kumar et al . , 1995, FASEB J. , 9, 1183; Breaker, 1996, Curr . Op . Biotech . , 7, 442; Santoro et al . , 1997, Proc . Na tl . Acad. Sci . , 94, 4262; Tang et al . , 1997, #NA 3, 914; Νakamaye & Eckstein, 1994, supra ; Long & Uhlenbeck, 1994, supra; Ishizaka et al., 1995, supra ; Vaish et al . , 1997, Biochemistry 36, 6495; all of these are incorporated by reference herein) . Each can catalyze a series of reactions including the hydrolysis of phosphodiester bonds in trans (and thus can cleave other RΝA molecules) under physiological conditions. Table I summarizes some of the characteristics of some of these ribozymes. In general, enzymatic nucleic acids act by first binding to a target RΝA. Such binding occurs through the target binding portion of an enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RΝA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RΝA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RΝA. Strategic cleavage of such a target RΝA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RΝA target, it is released from that RΝA to search for another target and can repeatedly bind and cleave new targets.
The enzymatic nature of a ribozyme is advantageous over other technologies, since the concentration of ribozyme necessary to affect a therapeutic treatment is lower. This advantage reflects the ability of the ribozyme to act enzymatically . Thus, a single ribozyme molecule is able to cleave many molecules of target RNA. In addition, the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base-pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage. Single mismatches, or base-substitutions, near the site of cleavage can be chosen to completely eliminate catalytic activity of a ribozyme. Nucleic acid molecules having an endonuclease enzymatic activity are able to repeatedly cleave other separate RNA molecules in a nucleotide base sequence- specific manner. Such enzymatic nucleic acid molecules can be targeted to virtually any RNA transcript, and efficient cleavage achieved in vitro (Zaug et al . , 324, Na ture 429 1986 ; Uhlenbeck, 1987 Na ture 328, 596; Kim et al., 84 Proc . Na tl . Acad. Sci . USA 8788, 1987; Dreyfus, 1988, Einstein Quart . J. Bio . Med. , 6, 92; Haseloff and Gerlach, 334 Na ture 585, 1988; Cech, 260 JAMA 3030, 1988; and Jefferies et al . , 17 Nucleic Acids Research 1371, 1989; Santoro et al . , 1997 supra ) .
Because of their sequence-specificity, trans-cleaving ribozymes show promise as therapeutic agents for human disease (Usman & McSwiggen, 1995 Ann . Rep . Med. Chem . 30, 285-294; Christoffersen and Marr, 1995 J. Med. Chem . 38, 2023-2037) . Ribozymes can be designed to cleave specific RNA targets within the background of cellular RNA. Such a cleavage event renders the RNA non-functional and abrogates protein expression from that RNA. In this manner, synthesis of a protein associated with a disease state can be selectively inhibited.
Ribozymes that cleave the specified sites in Tie-2, integrin subunit β3, integrin subunit α6, and aryl hydrocarbon nuclear transporter (ARNT) mRNAs represent a novel therapeutic approach to treat cancer, macular degeneration, diabetic retinopathy, inflammation, psoriasis and other diseases. Applicant indicates that ribozymes are able to inhibit the activity of Tie-2; integrin subunit β3; integrin subunit α6; and aryl hydrocarbon nuclear transporter (ARNT) and that the catalytic activity of the ribozymes is required for their inhibitory effect. Those of ordinary skill in the art will find that it is clear from the examples described that other ribozymes that cleave Tie-2, integrin subunit β3, integrin subunit α6, and aryl hydrocarbon nuclear transporter (ARNT) mRNAs may be readily designed and are within the scope of the invention.
Target sites
Targets for useful ribozymes can be determined as disclosed in Draper et al . , WO 93/23569; Sullivan et al . , WO 93/23057; Thompson et al . , WO 94/02595; Draper et al . , WO 95/04818; McSwiggen et al . , US Patent No. 5,525,468 and hereby incorporated by reference herein in totality. Rather than repeat the guidance provided in those documents here, below are provided specific examples of such methods, not limiting to those in the art. Ribozymes to such targets are designed as described in those applications and synthesized to be tested in vi tro and in vivo, as also described. Such ribozymes can also be optimized and delivered as described therein.
The sequence of human Tie-2, integrin subunit β3, integrin subunit α6, and aryl hydrocarbon nuclear transporter (ARNT) mRNAs were screened for optimal ribozyme target sites using a computer folding algorithm. Hammerhead or hairpin ribozyme cleavage sites were identified. These sites are shown in Tables III-X (All sequences are 5' to 3' in the tables) The nucleotide base position is noted in the Tables as that site to be cleaved by the designated type of ribozyme. The nucleotide base position is noted in the tables as that site to be cleaved by the designated type of ribozyme.
Hammerhead or hairpin ribozymes were designed that could bind and were individually analyzed by computer folding (Jaeger et al . , 1989 Proc . Na tl . Acad . Sci . USA, 86, 7706) to assess whether the ribozyme sequences fold into the appropriate secondary structure. Those ribozymes with unfavorable intramolecular interactions between the binding arms and the catalytic core are eliminated from consideration. Varying binding arm lengths can be chosen to optimize activity. Generally, at least 5 bases on each arm are able to bind to, or otherwise interact with, the target RNA. Ribozymes of the hammerhead or hairpin motif were designed to anneal to various sites in the mRNA message. The binding arms are complementary to the target site sequences described above.
Ribozyme Synthesis
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 (e.g., antisense oligonucleotides, hammerhead or the hairpin ribozymes) are used for exogenous delivery. The simple structure of these molecules increases the ability of the nucleic acid to invade targeted regions of the mRNA structure. However, these nucleic acid molecules can also be expressed within cells from eukaryotic promoters (e.g., Izant and Weintraub, 1985 Science 229, 345; McGarry and Lindquist, 1986 Proc . Na tl . Acad. Sci . USA 83, 399; Sullenger Scanlon et al . , 1991, Proc . Na tl . Acad. Sci . USA, 88, 10591-5; Kashani-Sabet et al . , 1992 Antisense Res . Dev. , 2, 3-15; Dropulic et al . , 1992 J. Virol , 66, 1432-41; Weerasinghe et al . , 1991 J. Virol , 65, 5531-4; Ojwang et al . , 1992 Proc . Na tl . 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). 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 W093/23569, and Sullivan et al . , PCT WO94/02595, both hereby incorporated in their totality by reference herein; Ohkawa et al . , 1992 Nucleic Acids Symp .
Ser. , 27, 15-6; Taira et al . , 1991, Nucleic Acids Res . ,
19, 5125-30; Ventura et al . , 1993 Nucleic Acids Res . , 21,
3249-55; Chowrira et al . , 1994 J. Biol . Chem . 269, 25856).
The ribozymes were chemically synthesized. The method of synthesis used follows the procedure for normal RNA synthesis as described in Usman et al . , 1987 J. Am . Chem . Soc , 109, 7845; Scaringe et al . , 1990 Nucleic Acids Res . , 18, 5433; and Wincott et al . , 1995 Nucleic Acids Res . 23, 2677-2684 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 synthesis were conducted on a 394 Applied Biosystems, Inc. synthesizer using a modified 2.5 μmol scale protocol with a 5 min coupling step for alkylsilyl protected nucleotides and 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. A 6.5-fold excess (163 μL of 0.1 M = 16.3 μmol) of phosphoramidite and a 24- fold excess of S-ethyl tetrazole (238 μL of 0.25 M **= 59.5 μmol) relative to polymer-bound 5 ' -hydroxyl was used in each coupling cycle. Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, were 97.5-99%. Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer : detritylation solution was 2% TCA in methylene chloride (ABI); capping was performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); oxidation solution was 16.9 mM I2, 49 mM pyridine, 9% water in THF (Millipore) . B & J Synthesis Grade acetonitrile was used directly from the reagent bottle. S- Ethyl tetrazole solution (0.25 M in acetonitrile) was made up from the solid obtained from American International Chemical, Inc.
Deprotection of the RΝA was performed as follows. The polymer-bound oligoribonucleotide, trityl-off, was transferred from the synthesis column to a 4mL glass screw top vial and suspended in a solution of methylamine (MA) at 65 °C for 10 min. After cooling to -20 °C, the supernatant was removed from the polymer support. The support was washed three times with 1.0 mL of EtOH:MeCΝ :H2θ/3 : 1 : 1, vortexed and the supernatant was then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, were dried to a white powder .
The base-deprotected oligoribonucleotide was resuspended in anhydrous TEA-HF/NMP solution (250 μL of a solution of 1.5mL N-methylpyrrolidinone , 750 μL TEA and 1.0 mL TEA»3HF to provide a 1.4M HF concentration) and heated to 65°C for 1.5 h. The resulting, fully deprotected, oligomer was quenched with 50 mM TEAB (9 mL) prior to anion exchange desalting.
For anion exchange desalting of the deprotected oligomer, the TEAB solution was loaded onto a Qiagen 500® anion exchange cartridge (Qiagen Inc.) that was prewashed with 50 mM TEAB (10 mL) . After washing the loaded cartridge with 50 mM TEAB (10 mL) , the RΝA was eluted with 2 M TEAB (10 mL) and dried down to a white powder. Inactive hammerhead ribozymes were synthesized by substituting a U for G5 and a U for A14 (numbering from Hertel, K. J., et al . , 1992, Nucleic Acids Res . , 20, 3252) . The average stepwise coupling yields were >98% (Wincott et al . , 1995 Nucleic Acids Res . 23, 2677-2684).
Hairpin ribozymes are synthesized in two parts and annealed to reconstruct the active ribozyme (Chowrira and Burke, 1992 Nucleic Acids Res . , 20, 2835-2840). Ribozymes are also synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol . 180, 51).
Ribozymes are modified to enhance stability and/or enhance catalytic activity by modification with nuclease resistant groups, for example, 2 '-amino, 2'-C-allyl, 2'- flouro, 2'-0-methyl, 2'-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992 TIBS 17, 34; Usman et al . , 1994 Nucleic Acids Symp . Ser . 31, 163; Burgin et al . , 1996 Biochemistry 6, 14090). Ribozymes were purified by gel electrophoresis using general methods or are purified by high pressure liquid chromatography (HPLC; See Stinchcomb et al . , International PCT Publication No. WO 95/23225, the totality of which is hereby incorporated herein by reference) and are resuspended in water.
The sequences of the ribozymes that are chemically synthesized, useful in this study, are shown in Tables III-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. For example, stem-loop II sequence of hammerhead ribozymes can be altered (substitution, deletion, and/or insertion) to contain any sequences provided a minimum of two base- paired stem structure can form. Similarly, stem-loop IV sequence of hairpin ribozymes, can be altered (substitution, deletion, and/or insertion) to contain any sequence, provided a minimum of two base-paired stem structure can form. Preferably, no more than 200 bases are inserted at these locations. The sequences listed in Tables III-X may be formed of ribonucleotides or other nucleotides or non-nucleotides. Such ribozymes (which have enzymatic activity) are equivalent to the ribozymes described specifically in the Tables.
Optimizing Ribozyme Activity
Catalytic activity of the ribozymes described in the instant invention can be optimized as described by Draper et al., supra . The details will not be repeated here, but include altering the length of the ribozyme binding arms, or chemically synthesizing ribozymes with modifications (base, sugar and/or phosphate) that prevent their degradation by serum ribonucleases and/or enhance their enzymatic activity (see e.g., Eckstein et al . , International Publication No. WO 92/07065; Perrault et al . , 1990 Na ture 344, 565; Pieken et al . , 1991 Science 253, 314; Usman and Cedergren, 1992 Trends in Biochem . Sci . 17, 334; Usman et al . , International Publication No. WO 93/15187; and Rossi et al . , International Publication No. WO 91/03162; Sproat, US Patent No. 5,334,711; and Burgin et al . , supra ; all of these describe various chemical modifications that can be made to the base, phosphate and/or sugar moieties of enzymatic RNA molecules). Modifications which enhance their efficacy in cells, and removal of bases from stem loop structures to shorten RNA synthesis times and reduce chemical requirements are desired. (All these publications are hereby incorporated by reference herein) .
There are several examples in the art describing sugar, base and phosphate modifications that can be introduced into enzymatic nucleic acid molecules without significantly effecting catalysis and with significant enhancement in their nuclease stability and efficacy. Ribozymes are modified to enhance stability and/or enhance catalytic activity by modification with nuclease resistant groups, for example, 2 '-amino, 2'-C-allyl, 2'-flouro, 2'- O-methyl, 2'-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992 TIBS 17, 34; Usman et al . , 1994 Nucleic Acids Symp . Ser . 31, 163; Burgin et al . , 1996 Biochemistry 35, 14090) . Sugar modification of enzymatic nucleic acid molecules have been extensively described in the art (see Eckstein et al . , Interna tional Publica tion PCT No. WO 92/07065; Perrault et al . Na ture 1990, 344, 565-568; Pieken et al . Science 1991, 253, 314- 317; Usman and Cedergren, Trends in Biochem . Sci . 1992, 1 1, 334-339; Usman et al . Interna tional Publica tion PCT No. WO 93/15187; Sproat, US Pa tent No. 5,334,711 and Beigelman et al . , 1995 J. Biol . Chem . 270, 25702; all of the references are hereby incorporated in their totality by reference herein) . Such publications describe general methods and strategies to determine the location of incorporation of sugar, base and/or phosphate modifications and the like into ribozymes without inhibiting catalysis, and are incorporated by reference herein. In view of such teachings, similar modifications can be used as described herein to modify the nucleic acid catalysts of the instant invention.
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 on all RNA ribozyme.
Therapeutic ribozymes delivered exogenously must optimally be stable within cells until translation of the target RNA has been inhibited long enough to reduce the levels of the undesirable protein. This period of time varies between hours to days depending upon the disease state. Clearly, ribozymes must be resistant to nucleases in order to function as effective intracellular therapeutic agents. Improvements in the chemical synthesis of RNA (Wincott et al . , 1995 Nucleic Acids Res . 23, 2677; incorporated by reference herein) have expanded the ability to modify ribozymes by introducing nucleotide modifications to enhance their nuclease stability as described above.
By "nucleotide" as used herein is as recognized in the art to include natural bases (standard), and modified bases well known in the art. Such bases are generally located at the 1' position of a sugar moiety. Nucleotide 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; all hereby incorporated by reference herein) . There are several examples of modified nucleic acid bases known in the art and has recently been summarized by Limbach et al . , 1994, Nucleic Acids Res . 22, 2183. Some of the non-limiting examples of base modifications that can be introduced into enzymatic nucleic acids without significantly effecting their catalytic activity include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3-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) and others (Burgin et al . , 1996, Biochemistry, 35, 14090). By "modified bases" in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at 1' position or their equivalents; such bases may be used within the catalytic core of the enzyme and/or in the substrate-binding regions .
By "unmodified nucleoside" is meant one of the bases adenine, cytosine, guanine, uracil joined to the 1' carbon of b-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.
Various modifications to ribozyme structure can be made to enhance the utility of ribozymes. Such modifications will enhance shelf-life, half-life in vitro, stability, and ease of introduction of such ribozymes to the target site, e.g., to enhance penetration of cellular membranes, and confer the ability to recognize and bind to targeted cells.
Administration of Ribozymes
Sullivan et al . , PCT WO 94/02595, describes the general methods for delivery of enzymatic RNA molecules . Ribozymes 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, ribozymes may be directly delivered ex vivo to cells or tissues with or without the aforementioned vehicles. Alternatively, the RNA/vehicle combination is locally delivered by direct injection or by use of a catheter, infusion pump or stent. 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 ribozyme delivery and administration are provided in Sullivan et al . , supra and Draper et al . , PCT W093/23569 which have been incorporated by reference herein.
The molecules of the instant invention can be used as pharmaceutical agents. Pharmaceutical agents prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state in a patient.
The negatively charged polynucleotides of the invention can be administered (e.g., RNA, DNA or protein) and introduced into a patient by any standard means, with or without stabilizers, buffers, and the like, to form a pharmaceutical composition. When it is desired to use a liposome delivery mechanism, standard protocols for formation of liposomes can be followed. The compositions of the present invention may also be formulated and used as tablets, capsules or elixirs for oral administration; suppositories for rectal administration; sterile solutions; suspensions for injectable administration; and the like.
The present invention also includes pharmaceutically acceptable formulations of the compounds described. These formulations include salts of the above compounds, e.g., acid addition salts, for example, salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid. A pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, e.g., systemic administration, into a cell or patient, preferably a human. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, or by injection. Such forms should not prevent the composition or formulation to reach a target cell (i.e., a cell to which the negatively charged polymer is desired to be delivered to) . For example, pharmacological compositions injected into the blood stream should be soluble. Other factors are known in the art, and include considerations such as toxicity and forms which prevent the composition or formulation from exerting its effect. By "systemic administration" is meant in vivo systemic absorption or accumulation of drugs in the blood stream followed by distribution throughout the entire body. Administration routes which lead to systemic absorption include, without limitations: intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular. Each of these administration routes expose the desired negatively charged polymers, e.g., nucleic acids, to an accessible diseased tissue. The rate of entry of a drug into the circulation has been shown to be a function of molecular weight or size. The use of a liposome or other drug carrier comprising the compounds of the instant invention can potentially localize the drug, for example, in certain tissue types, such as the tissues of the reticular endothelial system (RES) . A liposome formulation which can facilitate the association of drug with the surface of cells, such as, lymphocytes and macrophages is also useful. This approach may provide enhanced delivery of the drug to target cells by taking advantage of the specificity of macrophage and lymphocyte immune recognition of abnormal cells, such as the cancer cells.
The invention also features the use of the a composition comprising surface-modified liposomes containing poly (ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes) . These formulations offer an method for increasing the accumulation of drugs in target tissues. This class of drug carriers resists opsonization and elimination by the mononuclear phagocytic system (MPS or RES) , thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated drug (Lasic et al . Chem . Rev. 1995, 95, 2601-2627; Ishiwataet al . , Chem . Pharm . Bull . 1995, 43, 1005-1011). Such liposomes have been shown to accumulate selectively in tumors, presumably by extravasation and capture in the neovascularized target tissues (Lasic et al . , Science 1995, 267, 1275-1276; Oku et al . ,1995, Biochim . Biophys . Acta , 1238, 86-90). The long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA, particularly compared to conventional cationic liposomes which are known to accumulate in tissues of the MPS (Liu et al . , J. Biol . Chem . 1995, 42, 24864-24870; Choi et al . , International PCT Publication No. WO 96/10391; Ansell et al . , International PCT Publication No. WO 96/10390; Holland et al . , International PCT Publication No. WO 96/10392; all of these 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. All of these references are incorporated by reference herein.
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 Remington ' s Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro edit. 1985) hereby incorporated by reference herein. For example, preservatives, stabilizers, dyes and flavoring agents may be provided. Id. at 1449. These include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. In addition, antioxidants and suspending agents may be used. Id.
A pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state. The pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors which those skilled in the medical arts will recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients is administered dependent upon potency of the negatively charged polymer.
Alternatively, the enzymatic 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 . Na tl . Acad. Sci . USA 83, 399; Scanlon et al . , 1991, Proc . Na tl . Acad. Sci . USA, 88, 10591-5; Kashani-Sabet et al . , 1992 An tisense Res . Dev. , 2 , 3-15; Dropulic et al . , 1992 J. Virol , 66, 1432-41; Weerasinghe et al . , 1991 J. Virol , 65, 5531-4; Ojwang et al . , 1992 Proc . Natl . Acad. Sci . USA 89, 10802-6; Chen et al . , 1992 Nucleic Acids Res . , 20, 4581-9; Sarver et al . , 1990 Science 247, 1222-1225; Thompson et al . , 1995 Nucleic Acids Res . 23, 2259; Good et al . , 1997, Gene Therapy, 4, 45; all of 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 Symp . Ser . , 27, 15-6; Taira et al . , 1991, Nucleic Acids Res . , 19, 5125-30; Ventura et al . , 1993 Nucleic Acids Res . , 21, 3249-55; Chowrira et al . , 1994 J. Biol . Chem . 269, 25856; all of the references are hereby incorporated in their totality by reference herein) .
In another aspect of the invention, enzymatic nucleic acid molecules that cleave target molecules are expressed from transcription units (see for example Couture et al . , 1996, TIG. , 12, 510) inserted into DNA or RNA vectors. The recombinant vectors are preferably DNA plasmids or viral vectors. Ribozyme expressing viral vectors could be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Preferably, the recombinant vectors capable of expressing the ribozymes are delivered as described above, and persist in target cells. Alternatively, viral vectors may be used that provide for transient expression of ribozymes. Such vectors might be repeatedly administered as necessary. Once expressed, the ribozymes cleave the target RNA. The active ribozyme contains an enzymatic center or core equivalent to those in the examples, and binding arms able to bind target nucleic acid molecules such that cleavage at the target site occurs. Other sequences may be present which do not interfere with such cleavage. Delivery of ribozyme expressing vectors could be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that would allow for introduction into the desired target cell (for a review see Couture et al . , 1996, TIG. , 12, 510) .
In one aspect the invention features, an expression vector comprising nucleic acid sequence encoding at least one of the nucleic acid catalyst of the instant invention is disclosed. The nucleic acid sequence encoding the nucleic acid catalyst of the instant invention is operable linked in a manner which allows expression of that nucleic acid molecule.
In another aspect the invention features, the expression vector comprises: 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 gene encoding at least one of the nucleic acid catalyst of the instant invention; and wherein said gene is operably linked to said initiation region and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule. The vector may optionally include an open reading frame (ORF) for a protein operably linked on the 5' side or the 3 '-side of the gene encoding the nucleic acid catalyst of the invention; and/or an intron (intervening sequences).
Transcription of the ribozyme 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 will be expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type will depend on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby. Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990 Proc. Natl. Acad. Sci. U S A, 87, 6743-7; Gao and Huang 1993 Nucleic Acids Res., 21, 2867-72; Lieber et al . , 1993 Methods Enzymol., 217, 47-66; Zhou et al . , 1990 Mol. Cell. Biol., 10, 4529-37). Several investigators have demonstrated that ribozymes expressed from such promoters can function in mammalian cells (e.g. Kashani-Sabet et al., 1992 Antisense Res. Dev. , 2, 3-15; Ojwang et al . , 1992 Proc. Natl. Acad. Sci. U S A, 89, 10802-6; Chen et al., 1992 Nucleic Acids Res., 20, 4581-9; Yu et al . , 1993 Proc. Natl. Acad. Sci. U S A, 90, 6340-4; L'Huillier et al., 1992 EMBO J. 11, 4411-8; Lisziewicz et al., 1993 Proc. Natl. Acad. Sci. U. S. A., 90, 8000-4; Thompson et al., 1995 Nucleic Acids Res. 23, 2259; Sullenger & Cech, 1993, Science, 262, 1566) . More specifically, transcription units such as the ones derived from genes encoding U6 small nuclear (snRNA) , transfer RNA (tRNA) and adenovirus VA RNA are useful in generating high concentrations of desired RNA molecules such as ribozymes in cells (Thompson et al., supra; Couture and Stinchcomb, 1996, supra; Noonberg et al., 1994, Nucleic Acid Res., 22, 2830; Noonberg et al., US Patent No. 5,624,803; Good et al., 1997, Gene Ther. 4, 45; Beigelman et al., International PCT Publication No. NO 96/18736; all of these publications are incorporated by reference herein. The above ribozyme transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno- associated virus vectors), or viral RNA vectors (such as retroviral or alphavirus vectors) (for a review see Couture and Stinchcomb, 1996, supra). In yet another aspect the invention features an expression vector comprising nucleic acid sequence encoding at least one of the catalytic nucleic acid molecule 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 gene encoding at least one said nucleic acid molecule; and wherein said gene 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 gene encoding at least one said nucleic acid molecule, wherein said gene is operably linked to the 3 ' -end of said open reading frame; and wherein said gene is operably linked to said initiation region, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule. In yet another embodiment the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) a gene encoding at least one said nucleic acid molecule; and wherein said gene is operably linked to said initiation region, said intron and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule. In another embodiment, the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) an open reading frame; e) a gene encoding at least one said nucleic acid molecule, wherein said gene is operably linked to the 3 ' -end of said open reading frame; and wherein said gene is operably linked to said initiation region, said intron, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.
Examples The following are non-limiting examples showing the selection, isolation, synthesis and activity of enzymatic nucleic acids of the instant invention.
The following examples demonstrate the selection of ribozymes that cleave Tie-2, integrin subunit b3, integrin subunit α6, and aryl hydrocarbon nuclear transporter
(ARNT) . The methods described herein represent a scheme by which ribozymes may be derived that cleave other RNA targets required for angiogenesis. Also provided is a description of how such ribozymes may be delivered to cells. The examples demonstrate that upon delivery, the ribozymes inhibit cell proliferation in culture and modulate gene expression in vivo . Moreover, significantly reduced inhibition is observed if mutated ribozymes that are catalytically inactive are applied to the cells. Thus, inhibition requires the catalytic activity of the ribozymes .
Example 1: Identification of Potential Ribozyme Cleavage Sites in TIE-2
The sequence of human Tie-2 was screened for accessible sites using a computer folding algorithm. Regions of the mRNA that did not form secondary folding structures and contained potential hammerhead and/or hairpin ribozyme cleavage sites were identified. The sequences of these cleavage sites are shown in tables V- VI. Example 2: Selection of Ribozyme Cleavage Sites in Human TIE-2 RNA
To test whether the sites predicted by the computer- based RNA folding algorithm corresponded to accessible sites in Tie-2 RNA, 20 hammerhead sites were selected for analysis. Ribozyme target sites were chosen by analyzing genomic sequences of Tie-2 (Ziegler et al . , 1993, Oncogene 8 (3) , 663-670 (Genbank sequence HUMTEKRPTK accession number: M69238) and prioritizing the sites on the basis of folding. Hammerhead ribozymes were designed that could bind each target (see Figure 1) and were individually analyzed by computer folding (Christoffersen et al . , 1994 J. Mol . Struc . Theochem , 311, 273; Jaeger et al . , 1989, Proc . Na tl . Acad. Sci . USA, 86, 7706) to assess whether the ribozyme sequences fold into the appropriate secondary structure. Those ribozymes with unfavorable intramolecular interactions between the binding arms and the catalytic core were eliminated from consideration. As noted below, varying binding arm lengths can be chosen to optimize activity. Generally, at least 5 bases on each arm are able to bind to, or otherwise interact with, the target RNA. An example of a ribozyme targeted to Tie-2 is shown in figure 2.
Example 3: Chemical Synthesis and Purification of Ribozymes for Efficient Cleavage of TIE-2 RNA
Ribozymes of the hammerhead or hairpin motif were designed to anneal to various sites in the RNA message. The binding arms are complementary to the target site sequences described above. The ribozymes were chemically synthesized. The method of synthesis used followed the procedure for normal RNA synthesis as described in Usman et al., (1987 J. Am. Chem. Soc, 109, 7845), Scaringe et al., (1990 Nucleic Acids Res., 18, 5433) and Wincott et al . , supra, and made use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'- end, and phosphoramidites at the 3 '-end. The average stepwise coupling yields were >98%.
Inactive ribozymes were synthesized by substituting a U for G5 and a U for A14 (numbering from Hertel et al., 1992 Nucleic Acids Res., 20, 3252). Hairpin ribozymes were synthesized in two parts and annealed to reconstruct the active ribozyme (Chowrira and Burke, 1992 Nucleic Acids Res., 20, 2835-2840). Ribozymes were also synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol. 180, 51) . Ribozymes were modified to enhance stability by modification with nuclease resistant groups, for example, 2 '-amino, 2'-C-allyl, 2'-flouro, 2'-0-methyl, 2 ' -H (for a review see Usman and Cedergren, 1992 TIBS 17, 34) . Ribozymes were purified by gel electrophoresis using general methods or were purified by high pressure liquid chromatography (HPLC; See Wincott et al . , supra; the totality of which is hereby incorporated herein by reference) and were resuspended in water. The sequences of the chemically synthesized ribozymes used in this study are shown below in Table V-VI .
Example 4: Ribozyme Cleavage of TIE-2 RNA Target in vi tro Ribozymes targeted to the human Tie-2 RNA are designed and synthesized as described above. These ribozymes can be tested for cleavage activity in vi tro, for example using the following procedure. The target sequences and the nucleotide location within the Tie-2 mRNA are given in Table V. Cleavage Reactions : Full-length or partially full- length, internally-labeled target RNA for ribozyme cleavage assay is prepared by in vi tro 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_enc * labeled using T4 polynucleotide kinase enzyme. Assays are performed by pre-warming a 2X concentration of purified ribozyme in ribozyme cleavage buffer (50 mM Tris- HCl, pH 7.5 at 37°C, 10 mM MgCl2) and the cleavage reaction was initiated by adding the 2X ribozyme mix to an equal volume of substrate RNA (maximum of 1-5 nM) that was also pre-warmed in cleavage buffer. As an initial screen, o assays are carried out for 1 hour at 37 C using a final concentration of either 40 nM or 1 mM ribozyme, i.e., ribozyme excess. The reaction is quenched by the addition of an equal volume of 95% formamide, 20 mM EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol after which the o sample is heated to 95 C for 2 minutes, quick chilled and loaded onto a denaturing polyacrylamide gel. Substrate RNA and the specific RNA cleavage products generated by ribozyme cleavage are visualized on an autoradiograph of the gel. The percentage of cleavage is determined by Phosphor Imager® quantitation of bands representing the intact substrate and the cleavage products.
Use of Ribozymes Targeting TIE-2
The rate of tumor growth is believed to be a function of blood supplied and therefore a function of angiogenesis (Rak, Supra ; Blood & Zetter, 1990, Biochimica et Biophysica Acta 1032, 89-118) . Elevated levels of a number of these angiogenic factors including Tie-2; integrin subunit β3; integrin subunit α6; and aryl hydrocarbon nuclear transporter have been reported in a number of cancers. Thus, inhibition of expression of these angiogenic factors (for example using ribozymes) would potentially reduce that rate of growth of these tumors. The use of ribozymes would be desirable over such therapies as chemotherapeutics since, chemotherapeutic compounds such as doxorubicin because of its highly specific inhibition and reduction of the likelihood for side effects. Ribozymes, with their catalytic activity and increased site specificity (see above) , are likely to represent a potent and safe therapeutic molecule for the treatment of cancer. Tumor angiogenesis and other indications are discussed below.
Indications
1) Tumor angiogenesis: Angiogenesis has been shown to be necessary for tumors to grow into pathological size (Folkman, 1971, PNAS 76, 5217-5221; Wellstein & Czubayko, 1996, Breast Cancer Res and Trea tment 38, 109-119) . In addition, it allows tumor cells to travel through the circulatory system during metastasis. Increased levels of gene expression of a number of angiogenic factors such as vascular endothelial growth factor (VEGF) have been reported in vascularized and edema-associated brain tumors (Berkman et al . , 1993 J. Clini . Invest . 91, 153). A more direct demostration of the role of VEGF in tumor angiogenesis was demonstrated by Jim Kim et al . , 1993 Na ture 362,841 wherein, monoclonal antibodies against VEGF were successfully used to inhibit the growth of rhabdomyosarcoma, glioblastoma multiforme cells in nude mice. Similarly, expression of a dominant negative mutated form of the flt-1 VEGF receptor inhibits vascularization induced by human glioblastoma cells in nude mice (Millauer et al . , 1994, Na ture 367, 576).
2) Ocular diseases: Neovascularization has been shown to cause or exacerbate ocular diseases including but not limited to, macular degeneration, neovascular glaucoma, diabetic retinopathy, myopic degeneration, and trachoma (Norrby, 1997, APMIS 105, 417-437) . Aiello et al . , 1994 New Engl . J. Med. 331, 1480, showed that the ocular fluid, of a majority of patients suffering from diabetic retinopathy and other retinal disorders, contains a high concentration of VEGF. Miller et al . , 1994 Am . J. Pa thol . 145, 574, reported elevated levels of VEGF mRNA in patients suffering from retinal ischemia. These observations support a direct role for VEGF in ocular diseases. Other factors including those that stimulate VEGF synthesis may also contribute to these indications.
3) Dermatological Disorders: Many indications have been identified which may by angiogenesis dependent including but not limited to psoriasis, verruca vulgaris, angiofibroma of tuberous sclerosis, pot-wine stains, Sturge Weber syndrome, Kippel-Trenaunay-Weber syndrome, and Osler-Weber-Rendu syndrome (Norrby, supra ) . Intradermal injection of the angiogenic factor b-FGF demonstrated angiogenesis in nude mice (Weckbecker et al . , 1992, Angiogenesis : Key principles-Science-Technology- Medicine, ed R. Steiner) Detmar et al . , 1994 J. Exp . Med. 180, 1141 reported that VEGF and its receptors were over-expressed in psoriatic skin and psoriatic dermal microvessels, suggesting that VEGF plays a significant role in psoriasis.
4) Rheumatoid arthritis: Immunohistochemistry and in si tu hybridization studies on tissues from the joints of patients suffering from rheumatoid arthritis show an increased level of VEGF and its receptors (Fava et al . , 1994 J. Exp . Med. 180, 341). Additionally, Koch et al . , 1994 J. Immunol . 152, 4149, found that VEGF-specific antibodies were able to significantly reduce the mitogenic activity of synovial tissues from patients suffering from rheumatoid arthritis. These observations support a direct role for VEGF in rheumatoid arthritis. Other angiogenic factors including those of the present invention may also be involved in arthritis. Animal Models
There are several animal models in which the anti- angiogenesis effect of nucleic acids of the present invention, such as ribozymes, directed against ARNT RNAs can be tested. Typically a corneal model has been used to study angiogenesis in rat and rabbit since recruitment of vessels can easily be followed in this normally avascular tissue (Pandey et al . , 1995 Science 268: 567- 569) . In these models, a small Teflon or Hydron disk pretreated with an angiogenic compound is inserted into a pocket surgically created in the cornea. Angiogenesis is monitored 3 to 5 days later. Ribozymes directed against ARNT, Tie-2 or integrin subunit RNAs would be delivered in the disk as well, or dropwise to the eye over the time course of the experiment. In another eye model, hypoxia has been shown to cause both increased expression of VEGF and neovascularization in the retina (Pierce et al . , 1995 Proc . Na tl . Acad. Sci . USA. 92: 905-909; Shweiki et al . , 1992 J. Clin . Invest . 91: 2235-2243). Another animal model that addresses neovascularization involves Matrigel, an extract of basement membrane that becomes a solid gel when injected subcutaneously (Passaniti et al . , 1992 Lab . Invest . 67: 519-528). When the Matrigel is supplemented with angiogenesis factors, vessels grow into the Matrigel over a period of 3 to 5 days and angiogenesis can be assessed. Again, ribozymes directed against ARNT, Tie-2 or integrin subunit RNAs would be delivered in the Matrigel.
Several animal models exist for screening of anti- angiogenic agents. These include corneal vessel formation following corneal injury (Burger et al . , 1985 Cornea 4: 35-41; Lepri, et al . , 1994 J. Ocular Pharmacol . 10: 273- 280; Ormerod et al . , 1990 Am . J. Pa thol . 137: 1243-1252) or intracorneal growth factor implant (Grant et al . , 1993 Diabetologia 36: 282-291; Pandey et al . 1995 supra ; Zieche et al . , 1992 Lab . Invest . 67: 711-715), vessel growth into Matrigel matrix containing growth factors (Passaniti et al . , 1992 supra ) , female reproductive organ neovascularization following hormonal manipulation (Shweiki et al . , 1993 Clin . Invest . 91: 2235-2243), several models involving inhibition of tumor growth in highly vascularized solid tumors (O'Reilly et al . , 1994 Cell 79: 315-328; Senger et al . , 1993 Cancer and Metas . Rev. 12: 303-324; Takahasi et al . , 1994 Cancer Res . 54: 4233-4237; Kim et al . , 1993 supra ) , and transient hypoxia- induced neovascularization in the mouse retina (Pierce et al . , 1995 Proc . Na tl . Acad . Sci . USA. 92: 905-909).
The cornea model, described in Pandey et al . supra , is the most common and well characterized anti-angiogenic agent efficacy screening model. This model involves an avascular tissue into which vessels are recruited by a stimulating agent (growth factor, thermal or alkalai burn, endotoxin) . The corneal model would utilize the intrastromal corneal implantation of a Teflon pellet soaked in a angiogenic compound-Hydron solution to recruit blood vessels toward the pellet which can be quantitated using standard microscopic and image analysis techniques. To evaluate their anti-angiogenic efficacy, ribozymes are applied topically to the eye or bound within Hydron on the Teflon pellet itself. This avascular cornea as well as the Matrigel (see below) provide for low background assays. While the corneal model has been performed extensively in the rabbit, studies in the rat have also been conducted. The mouse model (Passaniti et al . , supra ) is a non- tissue model which utilizes Matrigel, an extract of basement membrane (Kleinman et al . , 1986) or Millipore® filter disk, which can be impregnated with growth factors and anti-angiogenic agents in a liquid form prior to injection. Upon subcutaneous administration at body temperature, the Matrigel or Millipore® filter disk forms a solid implant. An angiogenic compound would be embedded in the Matrigel or Millipore® filter disk which would be used to recruit vessels within the matrix of the Matrigel or Millipore® filter disk that can be processed histologically for endothelial cell specific vWF (factor VIII antigen) immunohistochemistry, Trichrome-Masson stain, or hemoglobin content. Like the cornea, the Matrigel or Millipore® filter disk are avascular; however, it is not tissue. In the Matrigel or Millipore® filter disk model, ribozymes are administered within the matrix of the Matrigel or Millipore® filter disk to test their anti-angiogenic efficacy. Thus, delivery issues in this model, as with delivery of ribozymes by Hydron- coated Teflon pellets in the rat cornea model, may be less problematic due to the homogeneous presence of the ribozyme within the respective matrix.
These models offer a distinct advantage over several other angiogenic models listed previously. The ability to use VEGF as a pro-angiogenic stimulus in both models is highly desirable since ribozymes will target only VEGFr RNA. In other words, the involvement of other nonspecific types of stimuli in the cornea and Matrigel models is not advantageous from the standpoint of understanding the pharmacologic mechanism by which the anti-VEGFr RNA ribozymes produce their effects. In addition, the models will allow for testing the specificity of the anti-VEGFr RNA ribozymes by using either a- or bFGF as a pro-angiogenic factor. Vessel recruitment using FGF should not be affected in either model by anti-VEGFr RNA ribozymes. Other models of angiogenesis including vessel formation in the female reproductive system using hormonal manipulation (Shweiki et al . , 1993 supra ) ; a variety of vascular solid tumor models which involve indirect correltations with angiogenesis (O'Reilly et al . , 1994 supra ; Senger et al . , 1993 supra ; Takahasi et al . , 1994 supra ; Kim et al., 1993 supra) ; and retinal neovascularization following transient hypoxia (Pierce et al . , 1995 supra ) were not selected for efficacy screening due to their non-specific nature, although there is a correlation between VEGF and angiogenesis in these models .
Other model systems to study tumor angiogenesis is reviewed by Folkman, 1985 Adv. Cancer . Res . . 43, 175.
Use of murine models
For a typical systemic study involving 10 mice (20 g each) per dose group, 5 doses (1, 3, 10, 30 and 100 mg/kg daily over 14 days continuous administration) , approximately 400 mg of ribozyme, formulated in saline would be used. A similar study in young adult rats (200 g) would require over 4 g. Parallel pharmacokinetic studies may involve the use of similar quantities of ribozymes further justifying the use of murine models.
Ribozymes and Lewis lung carcinoma and B-16 melanoma murine models
Identifying a common animal model for systemic efficacy testing of ribozymes is an efficient way of screening ribozymes for systemic efficacy.
The Lewis lung carcinoma and B-16 murine melanoma models are well accepted models of primary and metastatic cancer and are used for initial screening of anti-cancer. These murine models are not dependent upon the use of immunodeficient mice, are relatively inexpensive, and minimize housing concerns. Both the Lewis lung and B-16 melanoma models involve subcutaneous implantation of approximately 10' tumor cells from metastatically aggressive tumor cell lines (Lewis lung lines 3LL or D122, LLC-LN7; B-16-BL6 melanoma) in C57BL/6J mice. Alternatively, the Lewis lung model can be produced by the surgical implantation of tumor spheres (approximately 0.8 mm in diameter) . Metastasis also may be modeled by injecting the tumor cells directly i.v.. In the Lewis lung model, microscopic metastases can be observed approximately 14 days following implantation with quantifiable macroscopic metastatic tumors developing within 21-25 days. The B-16 melanoma exhibits a similar time course with tumor neovascularization beginning 4 days following implantation. Since both primary and metastatic tumors exist in these models after 21-25 days in the same animal, multiple measurements can be taken as indices of efficacy. Primary tumor volume and growth latency as well as the number of micro- and macroscopic metastatic lung foci or number of animals exhibiting metastases can be quantitated. The percent increase in lifespan can also be measured. Thus, these models would provide suitable primary efficacy assays for screening systemically administered ribozymes/ribozyme formulations. In the Lewis lung and B-16 melanoma models, systemic pharmacotherapy with a wide variety of agents usually begins 1-7 days following tumor implantation/inoculation with either continuous or multiple administration regimens. Concurrent pharmacokinetic studies can be performed to determine whether sufficient tissue levels of ribozymes can be achieved for pharmacodynamic effect to be expected. Furthermore, primary tumors and secondary lung metastases can be removed and subjected to a variety of in vi tro studies (i.e. target RNA reduction) .
Delivery of ribozymes and ribozyme formulations in the Lewis lung model
Several ribozyme formulations, including cationic lipid complexes which may be useful for inflammatory diseases (e . g. DIMRIE/DOPE, etc . ) and RES evading liposomes which may be used to enhance vascular exposure of the ribozymes, are of interest in cancer models due to their presumed biodistribution to the lung. Thus, liposome formulations can be used for delivering ribozymes to sites of pathology linked to an angiogenic response.
Diagnostic uses
Ribozymes of this invention may be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of Tie-2; integrin subunit β3; integrin subunit α6; and/or aryl hydrocarbon nuclear transporter 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 may map nucleotide changes which are important to RNA structure and function in vi tro, as well as in cells and tissues. Cleavage of target RNAs with ribozymes may be used to inhibit gene expression and define the role (essentially) of specified gene products in the progression of disease. In this manner, other genetic targets may be defined as important mediators of the disease. These experiments will lead to better treatment of the disease progression by affording the possibility of combinational therapies (e.g., multiple 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 in vitro uses of ribozymes of this invention are well known in the art, and include detection of the presence of RNAs associated with Tie-2; integrin subunit β3; integrin subunit α6; and/or aryl hydrocarbon nuclear transporter 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 will be used to identify mutant RNA in the sample. As reaction controls, synthetic substrates of both wild-type and mutant RNA will be cleaved by both ribozymes to demonstrate the relative ribozyme efficiencies in the reactions and the absence of cleavage of the "non-targeted" RNA species. The cleavage products from the synthetic substrates will also serve to generate size markers for the analysis of wild-type and mutant RNAs in the sample population. Thus each analysis will require two ribozymes, two substrates and one unknown sample which will be combined into six reactions. The presence of cleavage products will be determined using an RNAse protection assay so that full-length and cleavage fragments of each RNA can be analyzed in one lane of a polyacrylamide gel. It is not absolutely required to quantify the results to gain insight into the expression of mutant RNAs and putative risk of the desired phenotypic changes in target cells. The expression of mRNA whose protein product is implicated in the development of the phenotype (i.e., Tie-2; integrin subunit β3; integrin subunit 6; ARNT) is adequate to establish risk. If probes of comparable specific activity are used for both transcripts, then a qualitative comparison of RNA levels will be adequate and will decrease the cost of the initial diagnosis. Higher mutant form to wild-type ratios will be correlated with higher risk whether RNA levels are compared qualitatively or quantitatively. Additional Uses
Potential usefulness of sequence-specific enzymatic nucleic acid molecules of the instant invention might have many of the same applications for the study of RNA that DNA restriction endonucleases have for the study of DNA
(Nathans et al . , 1975 Ann . Rev. Biochem . 44:273). For example, the pattern of restriction fragments could be used to establish sequence relationships between two related RNAs, and large RNAs could be specifically cleaved to fragments of a size more useful for study. The ability to engineer sequence specificity of the ribozyme is ideal for cleavage of RNAs of unknown sequence.
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 maintainance of the active structure . • Over 300 known members of this class. Found as an intervening sequence in Tetrahymena thermophila rRNA, fungal mitochondria, chloroplasts, phage T4, blue-green algae, and others.
• Major structural features largely established through phylogenetic comparisons, mutagenesis, and biochemical studies [-,-] .
• Complete kinetic framework established for one ribozyme [-,-,-,-] .
- Michel, Francois; esthof, Eric. Slippery substrates. Nat. Struct. Biol. (1994), 1(1), 5-7.
- 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 Studies of ribozyme folding and substrate docking underway [-,-,-].
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 Apparent 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 folding of a group I ribozyme. Biochemistry (1995), 34(19), 6504-12.
- Zarrinkar, Patrick 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. • Chemical modification investigation of important residues well established [—,—] .
• The small (4-6 nt) binding site may make this ribozyme too non-specific for targeted RNA cleavage, however, the Tetrahymena group I intron has been used to repair a "defective" β-galactosidase message by the ligation of new β-galactosidase sequences onto the defective message [—] .
RNAse P RNA (Ml RNA) • Size: -290 to 400 nucleotides.
• RNA portion of a ubiquitous ribonucleoprotein enzyme .
• Cleaves tRNA precursors to form mature tRNA [—] .
• Reaction mechanism: possible attack by M 2 + -OH to generate cleavage products with 3' -OH and 5' -phosphate .
— 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.
— Strobel, Scott A.; Cech, Thomas R.. Exocyclic Amine of the Conserved G.cntdot.U Pair at the Cleavage Site of the Tetrahymena Ribozyme Contributes to 5 '-Splice Site Selection and Transition State Stabilization. Biochemistry (1996), 35(4), 1201-11.
— Sullenger, Bruce A. ; Cech, Thomas R.. Ribozyme-mediated repair of defective mRNA by targeted trans-splicing. Nature (London) (1994), 371(6498), 619-22.
^ Robertson, H.D.; Altman, S . ; Smith, J.D. J. Biol. Chem., 247,
5243-5251 (1972) . • 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
• Important phosphate and 2' OH contacts recently identified i^,11]
Group II Introns
• Size: >1000 nucleotides.
• Trans cleavage of target RNAs recently demonstrated [— ,— ] .
— Forster, Anthony C; Altman, Sidney. External guide sequences for an RNA enzyme. Science (Washington, D. C, 1883-) (1990), 249(4970), 783-6.
— Yuan, Y.; Hwang, E. S.; Altman, S. Targeted cleavage of mRNA by human RNase P. Proc. Natl. Acad. Sci. USA (1992) 89, 8006-10.
— Harris, Michael E.; Pace, Norman R.. Identification of phosphates involved in catalysis by the ribozyme RNase P RNA. RNA
(1995), 1(2), 210-18.
— Pan, Tao; Loria, Andrew; Zhong, Kun. Probing of tertiary interactions in RNA: 2 ' -hydroxyl-base contacts between the RNase P RNA and pre-tRNA. Proc. Natl. Acad. Sci. U. S. A. (1995), 92(26), 12510- 14.
— 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. • Sequence requirements not fully determined.
• Reaction mechanism: 2' -OH of an internal adenosine generates cleavage products with 3' -OH and a "lariat" RNA containing a 3' -5' and a 2' -5' branch point. • Only natural ribozyme with demonstrated participation in DNA cleavage [—,—] in addition to RNA cleavage and ligation.
• Major structural features largely established through phylogenetic comparisons [—] . • Important 2' OH contacts beginning to be identified [—]
— 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 Structure/Function Relationships. Biochemistry (1995), 34(9), 2965-77.
— Zimmerly, Steven; Guo, Huatao; Eskes, Robert; Yang, Jian; Perlman, Philip S.; La bowitz, 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.
— 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.
— Michel, Francois; Ferat, Jean Luc. Structure and activities of group II introns. Annu. Rev. Biochem. (1995), 64, 435-61.
— 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. • Kinetic framework under development [—]
Neurospora VS RNA
• Size: -144 nucleotides.
• Trans cleavage of hairpin target RNAs recently demonstrated [— ] .
• 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 generate cleavage products with 2', 3'- cyclic phosphate and 5' -OH ends.
— 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.
— Guo, Hans C. T.; Collins, 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. • 14 known members of this class. Found in a number of plant pathogens (virusoids) that use RNA as the infectious agent.
• Essential structural features largely defined, including 2 crystal structures [—,—]
• Minimal ligation activity demonstrated (for engineering through in vitro selection) [— ]
• Complete kinetic framework established for two or more ribozymes [— ] . • Chemical modification investigation of important residues well established [— ] .
Hairpin Ribozyme
• Size: -50 nucleotides.
— 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.
— McKay, Structure and function of the hammerhead ribozyme: an unfinished story. RNA, (1996), 2, 395-403.
— Long, D., Uhlenbeck, 0., Hertel, K. Ligation with hammerhead ribozymes. US Patent No. 5,633,133.
— Hertel, K.J., Herschlag, D., Uhlenbeck, 0. 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.
— Beigelman, L., et al., Chemical modifications of hammerhead ribozymes. J. Biol. Chem., (1995) 270, 25702-25708. • Requires the target sequence GUC immediately 3' of the cleavage site.
• Binds 4-6 nucleotides at the 5 '-side of the cleavage site and a variable number to the 3 '-side of the cleavage site.
Reaction mechanism: attack by 2' -OH 5' to the scissile bond to generate cleavage products with 2',3'- cyclic phosphate and 5' -OH ends.
• 3 known members of this class. Found in three plant pathogen (satellite RNAs of the tobacco ringspot virus, arabis mosaic virus and chicory yellow mottle virus) which uses RNA as the infectious agent.
• Essential structural features largely defined r31 32 33 34-,
L I I i J
— 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.
— 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.
— 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.
— 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. • Ligation activity (in addition to cleavage activity) makes ribozyme amenable to engineering through in vi tro selection [— 35-]
Complete kinetic framework established for one ribozyme [—] .
Chemical modification investigation of important residues begun [—,—]
Hepatitis Delta Virus (HDV) Ribozyme • Size: -60 nucleotides. • Trans cleavage of target RNAs demonstrated [—]
— Berzal-Herranz, Alfredo; Joseph, Simpson; Burke, John M.. In vitro selection of active hairpin ribozymes by sequential RNA- catalyzed cleavage and ligation reactions. Genes Dev. (1992), 6(1), 129-34.
— Hegg, Lisa A.; Fedor, Martha J.. Kinetics and Thermodynamics of Intermolecular Catalysis by Hairpin Ribozymes. Biochemistry (1995), 34(48), 15813-28.
— Grasby, Jane A.; Mersmann, Karin; Singh, Mohinder; Gait, Michael J.. Purine Functional Groups in Essential Residues of the Hairpin Ribozyme Required for Catalytic Cleavage of RNA. Biochemistry (1995), 34(12), 4068-76.
— Schmidt, Sabine; Beigelman, Leonid; 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.
— 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. • Binding sites and structural requirements not fully determined, although no sequences 5 ' of cleavage site are required. Folded ribozyme contains a pseudoknot structure [— ] . • Reaction mechanism: attack by 2' -OH 5' to the scissile bond to generate cleavage products with 2', 3'- cyclic phosphate and 5' -OH ends.
• Only 2 known members of this class. Found in human HDV. • Circular form of HDV is active and shows increased nuclease stability [—]
— 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.
— Puttaraju, M. ; Perrotta, Anne T.; Been, Michael D.. A circular trans-acting hepatitis delta virus ribozyme. Nucleic Acids Res. (1993), 21(18), 4253-8.
Table I I : 2 . 5 μmol RNA Synthesi s Cycle
Reagent Equ¬ivalents Amount Time*
Phosphoramidites 6.5 163 μL 2.5
5-Ethyl Tetrazole 23.8 238 μL 2.5
Acetic Anhydride 100 233 μL 5 sec
W-Methyl Imidazole 186 233 μL 5 sec
TCA 83.2 1.73 mL 21 sec
Iodine 8.0 1.18 mL 45 sec
Acetonitrile NA 6.67 mL NA
* Wait time does not include contact time during delivery.
TABLE III: HAMMERHEAD RIBOZYME AND SITE SEQUENCES FOR ARNT
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Figure imgf000099_0001
oε
9Z
02
91
oτ
Figure imgf000100_0001
86
_.0S90/66Sn/_L3d £0fr0S/66 OM
Figure imgf000101_0001
Figure imgf000102_0001
oε
92
02
91
01
Figure imgf000103_0001
τoτ
__0S90/66Sf_/_LOd £0tOS/66 OM oε
92
02
91
01
Figure imgf000104_0001
201
_.0S90/66Sπ/XOd £0tOS/66 OM
Figure imgf000105_0001
oε
92
02
91
oτ
Figure imgf000106_0001
froτ
Z.0S90/66Sn/XDd eθfrOS/66 OM
Figure imgf000107_0001
Figure imgf000108_0001
oε
92
02
91
01
Figure imgf000109_0001
LOT
_.0S90/66Sn/_LDd eθtOS/66 OM
Figure imgf000110_0001
Figure imgf000111_0001
Figure imgf000112_0001
oε
92
02
91
01
Figure imgf000113_0001
τxτ
Z.0S90/66Sn/13d εθfrOS/66 OM
Figure imgf000114_0001
oε
92
02
91
01
Figure imgf000115_0001
εττ
__0S90/66Sπ/lOd εθtOS/66 OM oε
92
02
91
oτ
Figure imgf000116_0001
frττ
__0S90/66Sπ/X3d εθ.>OS/66 OM
Figure imgf000117_0001
Figure imgf000118_0001
Figure imgf000119_0001
oε
92
02
91
01
Figure imgf000120_0001
8iτ
_.0S90/66Sn/IDd εθt-OS/66 OM
Figure imgf000121_0001
oε
92
02
91
01
Figure imgf000122_0001
021
_.0S90/66Sπ/IOd εθfrOS/66 OM oε
92
02
91
01
Figure imgf000123_0001
121
_.os9o/66snyj-Od εθfrOS/66 OM oε
92
02
91
01
Figure imgf000124_0001
221
__0S90/66Sn/_LDd εθfrOS/66 OM oε
92
02
91
01
Figure imgf000125_0001
i_OS90/66Sn/IDd εθtOS/66 OM
Figure imgf000126_0001
Figure imgf000127_0001
Figure imgf000128_0001
Figure imgf000129_0001
Figure imgf000130_0001
oε
92
02
91
oτ
Figure imgf000131_0001
621
_.0S90/66Sri/JL3d £0t>0S/66 OM
Figure imgf000132_0001
oε
92
02
91
oτ
Figure imgf000133_0001
τετ
Z.0S90/66SΩ/13d εθfrOS/66 OM oε
92
02
91
oτ
Figure imgf000134_0001
2ετ
Z.0S90/66Sn/_L3d εθt-OS/66 OM oε
92
02
91
oτ
Figure imgf000135_0001
εετ
_L0S90/66Sπ/lDd εθt-OS/66 OM oε
92
02
91
oτ
Figure imgf000136_0001
_.0S90/66Sn/lOd εθtOS/66 OM
Figure imgf000137_0001
TABLE VI : HAIRPIN RIZOZYMES AND TRARGET SITES FOR TIE-2
Figure imgf000138_0001
Figure imgf000139_0001
Figure imgf000140_0001
Figure imgf000141_0001
Figure imgf000142_0001
TABLE VII: HAMMERHEAD RIBOZYME AND TARGET SITE SEQUENCES FOR INTEGRIN
ALPHA 6 SUBUNIT
Figure imgf000143_0001
Figure imgf000144_0001
Figure imgf000145_0001
oε
92
02
91
oτ
Figure imgf000146_0001
τ
L0S90/66Sn/JLDd εθt-OS/66 OM oε
92
02
91
01
Figure imgf000147_0001
9
,0S90/66Sf_/J_Dd εβtOS/66 OM oε
92
02
51
01
Figure imgf000148_0001
9E-I
_.0S90/66Sα/13d εθtOS/66 OM
Figure imgf000149_0001
oε
92
02
91
01
Figure imgf000150_0001
8 l
__0S90/66Sπ/IDd εθfrOS/66 OM
Figure imgf000151_0001
oε
92
02
91
oτ
Figure imgf000152_0001
091
_10S90/66Sn/_LOd εθfrOS/66 OM oε
92
02
91
01
Figure imgf000153_0001
191
__0S90/66S.T/_L3d εθfrOS/66 OM
Figure imgf000154_0001
oε
92
02
91
01
Figure imgf000155_0001
εgτ
_.0S90/66Sfl/_LDd εθtOS/66 OM oε
92
02
91
01
Figure imgf000156_0001
frsτ
Z.0S90/66SriΛLDd εθt>OS/66 OM oε
92
02
91
01
Figure imgf000157_0001
991
Z,0S90/66Sπ/I3d εθfrOS/66 OM
Figure imgf000158_0001
oε
92
02
9T
01
Figure imgf000159_0001
Z.9I
_.0S90/66Sn/JL3d £0t>0S/66 OM
Figure imgf000160_0001
Figure imgf000161_0001
Figure imgf000162_0001
oε
92
02
91
01
Figure imgf000163_0001
191
_.0S90/66Sn/iDd εθfOS/66 OM oε
92
02
91
01
Figure imgf000164_0001
291
__0S90/66Sn_LDd εθfrOS/66 OM oε
92
02
91
01
Figure imgf000165_0001
__0S90/66SΩ/_LDd εθfrOS/66 OM
Figure imgf000166_0001
oε
92
02
91
01
Figure imgf000167_0001
991
__0S90/66Sf_/IDd εθt-OS/66 OM oε
92
02
91
01
Figure imgf000168_0001
99 1
Z.0S90/66Sfl/_L3d εθfrOS/66 OM oε
92
02
91
01
Figure imgf000169_0001
L9 \
i-OS90/66SflΛLDd εθfrOS/66 OM
Figure imgf000170_0001
Figure imgf000171_0001
oε
92
02
91
01
Figure imgf000172_0001
0Z.I
_.0S90/66Sfl/lDd εθtOS/66 OM
Figure imgf000173_0001
oε
52
02
91
01
Figure imgf000174_0001
Z Ll
__0S90/66Sn/_LOd εθt-OS/66 OM
Figure imgf000175_0001
oε
92
02
91
01
Figure imgf000176_0001
LI
_.0S90/66SnyiDd εθtOS/66 OM oε
92
02
51
01
Figure imgf000177_0001
5 /.I
_.0S90/66Sn/_L3d εθtOS/66 OM
Figure imgf000178_0001
oε
52
02
51
oτ
Figure imgf000179_0001
LLT
__0S90/66SΩ/lDd εθfrOS/66 OM
Figure imgf000180_0001
oε
52
02
51
01
Figure imgf000181_0001
6LI
,0S90/66SOIOd εθfOS/66 OM
Figure imgf000182_0001
oε
52
02
51
01
Figure imgf000183_0001
181
_.0S90/66SflΛLOd εθfrOS/66 OM
Figure imgf000184_0001
oε
92
02
91
oτ
Figure imgf000185_0001
εδi
_.0S90/66Sfl/lOd εθtOS/66 OM oε
92
02
51
01
Figure imgf000186_0001
8I
_.0S90/66Sn/13d εθtOS/66 OM
Figure imgf000187_0001
Figure imgf000188_0001
oε
52
02
51
01
Figure imgf000189_0001
L81
_.0S90/66Sn/-LDd εθtOS/66 OM oε
52
02
51
01
Figure imgf000190_0001
_.0S90/66Sn/IOd εθfrOS/66 OM oε
52
02
51
01
Figure imgf000191_0001
_.0S90/66Sπ/13d εθfOS/66 OM oε
52
02
91
01
Figure imgf000192_0001
061
_.0S90/66Sfl/_LDd εθt-OS/66 OM oε
52
02
51
01
Figure imgf000193_0001
161
Z.0S90/66SflΛLDd εθfrOS/66 OM
Figure imgf000194_0001
oε
52
02
51
oτ
Figure imgf000195_0001
_.0S90/66Sfl/IOd εβtOS/66 OM
Figure imgf000196_0001
Figure imgf000197_0001
Figure imgf000198_0001
Figure imgf000199_0001
oε
52
02
51
01
Figure imgf000200_0001
861
_.0S90/66Sn/_LOd εθtOS/66 OM oε
52
02
sτ
oτ
Figure imgf000201_0001
661
j_OS90/66Sn/_LDd εθt-OS/66 OM oε
52
02
51
oτ
Figure imgf000202_0001
002
_.0S90/66Sn/JLDd εθtOS/66 OM
Figure imgf000203_0001
oε
52
02
51
01
Figure imgf000204_0001
202
Z.0S90/66Sr_yJL3d εθfrOS/66 OM
Figure imgf000205_0001
Figure imgf000206_0001
oε
52
02
91
01
Figure imgf000207_0001
902
.0S90/66Sπ/lDd εθtOS/66 OM
Figure imgf000208_0001
Figure imgf000209_0001
oε
92
02
91
01
Figure imgf000210_0001
802
Z.0S90/66Sfl/13d εθfrOS/66 OM oε
92
02
91
oτ
Figure imgf000211_0001
602
_-0S90/66Sn/_L3d εθtOS/66 OM
Figure imgf000212_0001
Figure imgf000213_0001
Figure imgf000214_0001
TABLE VIII : HAIRPIN RIBOZYME AND TARGET SEQUENCES FOR INTEGRIN ALPHA 6 SUBUNIT
Figure imgf000215_0001
Figure imgf000216_0001
Figure imgf000217_0001
Figure imgf000219_0001
Figure imgf000220_0001
Figure imgf000221_0001
Figure imgf000222_0001
Figure imgf000223_0001
Figure imgf000224_0001
TABLE IX: HAMMERHEAD RIBOZYME AND TARGET SEQUENCES FOR INTEGRIN SUBUNIT BETA 3
Figure imgf000225_0001
Figure imgf000226_0001
Figure imgf000227_0001
Figure imgf000228_0001
Figure imgf000229_0001
oε
92
02
91
01
Figure imgf000230_0001
822
_-0S90/66Sπ/IDd εθfrOS/66 OM oε
92
02
91
01
Figure imgf000231_0001
622
_.0S90/66Sπ/XDd εθfrOS/66 OM
Figure imgf000232_0001
Figure imgf000233_0001
Figure imgf000234_0001
oε
92
02
91
01
Figure imgf000235_0001
εε2
_.0S90/66SfTΛLDd εθt>OS/66 OM
Figure imgf000236_0001
Figure imgf000237_0001
oε
92
02
91
01
Figure imgf000238_0001
9ε2
Z,0S90/66Sn/.LDd εθtOS/66 OM
Figure imgf000239_0001
Figure imgf000240_0001
oε
92
02
91
01
Figure imgf000241_0001
6ε2
Z.0S90/66Sπ/13d εθt-OS/66 OM
Figure imgf000242_0001
oε
92
02
91
01
Figure imgf000243_0001
T 2
_.0S90/66Sπ/lDd εθt>OS/66 OM
Figure imgf000244_0001
Figure imgf000245_0001
oε
92
02
91
01
Figure imgf000246_0001
fvZ
Z.0S90/66Sn/lDd εθfrOS/66 OM
Figure imgf000247_0001
oε
92
02
91
01
Figure imgf000248_0001
9^2
_.0S90/66Sπ/IOd εθt-OS/66 OM
Figure imgf000249_0001
Figure imgf000250_0001
Figure imgf000251_0001
Figure imgf000252_0001
oε
92
02
91
01
Figure imgf000253_0001
192
__0S90/66SflΛLDd εθtOS/66 OM
Figure imgf000254_0001
Figure imgf000255_0001
Figure imgf000256_0001
Figure imgf000257_0001
oε
92
02
91
01
Figure imgf000258_0001
952
Z.0S90/66Sn/LDd εθtOS/66 OM oε
52
02
51
oτ
Figure imgf000259_0001
Z.52
__0S90/66Sn/_LOd εθt-OS/66 OM oε
52
02
51
oτ
Figure imgf000260_0001
852
__0S90/66Sf_/±Dd εθfrOS/66 OM
Figure imgf000261_0001
oε
52
02
sτ
oτ
Figure imgf000262_0001
092
_.0S90/66Si /_lDd εθfrOS/66 OM
Figure imgf000263_0001
Figure imgf000264_0001
Figure imgf000265_0001
oε
52
02
51
01
Figure imgf000266_0001
92
_.0S90/66Sπ/IOd εθtOS/66 OM
Figure imgf000267_0001
Figure imgf000268_0001
oε
52
02
51
01
Figure imgf000269_0001
Z.9 2
_.0S90/66SriΛLDd εθtOS/66 OM
Figure imgf000270_0001
Figure imgf000271_0001
Figure imgf000272_0001
Figure imgf000273_0001
Figure imgf000274_0001
TABLE X : HAIRPIN RIBOZYME AND TARGET SEQUENCES FOR INTEGRIN SUBUNIT BETA 3
Figure imgf000275_0001
Figure imgf000276_0001
Figure imgf000277_0001
Figure imgf000278_0001
Figure imgf000279_0001
Figure imgf000280_0001

Claims

Claims
1. An enzymatic nucleic acid molecule with RNA cleaving activity, wherein said enzymatic nucleic acid molecule specifically cleaves RNA encoded by an aryl hydrocarbon nuclear transporter (ARNT) gene.
2. An enzymatic nucleic acid molecule with RNA cleaving activity, wherein said enzymatic nucleic acid molecules specifically cleaves RNA encoded by an integrin subunit beta 3 (╬▓3) gene.
3. An enzymatic nucleic acid molecule with RNA cleaving activity, wherein said enzymatic nucleic acid molecules cleaves RNA encoded by a integrin subunit alpha 6 (╬▒6) gene.
4. An enzymatic nucleic acid molecule with RNA cleaving activity, wherein said enzymatic nucleic acid molecules cleaves RNA encoded by a Tie-2 gene.
5. The enzymatic nucleic acid molecule of any of claims 1-4, wherein said enzymatic nucleic acid molecule is in a hammerhead configuration.
6. The enzymatic nucleic acid molecule of claim 5, wherein said enzymatic nucleic acid molecule comprises a stem II region of length greater than or equal to 2 base pairs .
7. The enzymatic nucleic acid molecule of any of claims 1-4, wherein said enzymatic nucleic acid molecule is in a hairpin configuration.
8. The enzymatic nucleic acid molecule of any of claims 1-4, wherein said enzymatic nucleic acid is in a hepatitis delta virus, group I intron, group II intron, VS nucleic acid or RNase P nucleic acid configuration.
9. The enzymatic nucleic acid molecule of any of claims 1-4, wherein said enzymatic nucleic molecule is a DNAzyme .
10. The enzymatic nucleic acid of claim 7, wherein said enzymatic nucleic acid molecule comprises a stem II region of length between three and seven base-pairs.
11. The enzymatic nucleic acid molecule of any of claims 1-4, wherein said enzymatic nucleic acid molecule comprises between 12 and 100 bases complementary to said RNA.
12. The enzymatic nucleic acid molecule of any of claims 1-4, wherein said enzymatic nucleic acid molecule comprises between 14 and 24 bases complementary to said mRNA.
13. The enzymatic nucleic acid molecule of claim 5 wherein said enzymatic nucleic acid molecule consists essentially of any sequence defined as Seq. I.D. Nos 1- 393, 911-1611, 2449-3587, and 4915-5701.
14 The enzymatic nucleic acid molecule of claim 7, wherein said enzymatic nucleic acid molecule consists essentially of any sequence defined as Seq. ID 787-848, 2313-2380, 4727-4820 and 6489-6568.
15. A mammalian cell including an enzymatic nucleic acid molecule of any of claims 1-4.
16. The mammalian cell of claim 15, wherein said mammalian cell is a human cell.
17. An expression vector comprising nucleic acid sequence encoding at least one enzymatic nucleic acid molecule of any of claims 1-4 in a manner which allows expression of that enzymatic nucleic acid molecule.
18. A mammalian cell including an expression vector of claim 17.
19. The mammalian cell of claim 18, wherein said mammalian cell is a human cell.
20. A method for treatment of cancer, diabetic retinopathy, age related macular degeneration (ARMD) , inflammation, and arthritis comprising the step of administering to a patient an enzymatic nucleic acid molecule of any of claims 1-4.
21. A method for treatment of cancer comprising the step of administering to a patient, an expression vector of claim 17.
22. A method for the treatment of cancer, diabetic retinopathy, age related macular degeneration (ARMD) , inflammation, and arthritis comprising the step of administering to a patient an expression vector of claim 17.
23. A method for treatment of cancer comprising the steps of: a) isolating cells from a patient; b) administering to said cells an enzymatic nucleic acid molecule of any of claims 1-4; and c) introducing said cells back into said patient.
24. A pharmaceutical composition comprising the enzymatic nucleic acid molecule of any of claims 1-4.
25. A method of treatment of a patient having a condition associated with an elevated level of aryl hydrocarbon nuclear transporter (ARNT) , comprising the step of administration to said patient an enzymatic nucleic acid molecule of claim 1.
26. A method of treatment of a patient having a condition associated with the level of Tie-2 comprising the step of administration to said patient an enzymatic nucleic acid molecule of claim 2.
27. A method of treatment of a patient having a condition associated with the level of integrin subunit alpha 6, comprising the step of administration to said patient an enzymatic nucleic acid molecule of claim 3.
28. A method of treatment of a patient having a condition associated with the level of integrin subunit beta 3 comprising the step of administration to said patient an enzymatic nucleic acid molecule of claim 4.
29. A method of treatment of a patient having a condition associated with the level of aryl hydrocarbon nuclear transporter (ARNT), comprising the steps of: (a) contacting cells of said patient with an enzymatic nucleic acid molecule of claim 1; and (b) administering to said patient one or more additional drugs.
30. A method of treatment of a patient having a condition associated with the level of Tie-2, comprising the steps of: (a) contacting cells of said patient with an enzymatic nucleic acid molecule of claim 2; and (b) administering to said patient one or more additional drugs .
31. A method of treatment of a patient having a condition associated with the level of integrin subunit alpha 6, comprising the steps of: (a) contacting cells of said patient with an enzymatic nucleic acid molecule of claim 3; and (b) administering to said patient one or more additional drugs.
32. A method of treatment of a patient having a condition associated with the level of integrin subunit beta 3, comprising the steps of: (a) contacting cells of said patient with an enzymatic nucleic acid molecule of claim 4; and (b) administering to said patient one or more additional drugs.
33. The enzymatic nucleic acid molecule of claim 5, wherein said enzymatic nucleic acid molecule comprises at least five ribose residues, phosphorothioate linkages in at least three of the 51 terminal nucleotides, a 2'-C- allyl modification at position No. 4 of said nucleic acid, at least ten 2 ' -O-methyl modifications, and a 3'- end modification.
34. The enzymatic nucleic acid of claim 33, wherein said enzymatic nucleic acid comprises a 3 ' -3 ' linked inverted ribose moiety at said 31 end.
35. The enzymatic nucleic acid molecule of claim 5, wherein said enzymatic nucleic acid molecule comprises at least five ribose residues; phosphorothioate linkages at least three of the 5 ' terminal nucleotides 2 ' -amino modification at position No. 4 and/or at position No. 7 of said enzymatic nucleic acid molecule; at least ten 2 ' -0- methyl modifications; and a 3'- end modification.
36. The enzymatic nucleic acid molecule of claim 5, wherein said enzymatic nucleic acid molecule comprises at least five ribose residues; phosphorothioate linkages at least three of the 5' terminal nucleotides, abasic substitution at position No. 4 and/or at position No. 7 of said enzymatic nucleic acid molecule; at least ten 2'-0- methyl modifications; comprises a 3 ' -end modification.
37. The enzymatic nucleic acid molecule of claim 5, wherein said enzymatic nucleic acid molecule comprises of at least five ribose residues; phosphorothioate linkages at least three of the 51 terminal nucleotides; a 6-methyl uridine substitution at position No. 4 and/or at position No. 7 of said enzymatic nucleic acid molecule; at least ten 2 ' -O-methyl modifications; and comprises a 3' end modification.
38. A method for modulating expression of ARNT gene in a mammalian cell comprising the step of administering to said cell an enzymatic nucleic acid molecule of claim 1.
39. A method for modulating expression of integrin subunit beta 3 in a mammalian cell comprising the step of administering to said cell an enzymatic nucleic acid molecule of claim 2.
40. A method for modulating expression of integrin subunit alpha 6 in a mammalian cell comprising the step of administering to said cell an enzymatic nucleic acid molecule of claim 3.
41. A method for modulating expression of Tie-2 in a mammalian cell comprising the step of administering to said cell an enzymatic nucleic acid molecule of claim 4.
42. A method of cleaving an ARNT RNA molecule comprising the step of, contacting the enzymatic nucleic acid molecule of claim 1 with said ARNT RNA molecule under conditions suitable for the cleavage of said ARNT RNA molecule .
43. A method of cleaving a integrin subunit beta 3 RNA molecule comprising the step of, contacting the enzymatic nucleic acid molecule of claim 2 with said integrin subunit beta 3 RNA molecule under conditions suitable for the cleavage of said integrin subunit beta 3 RNA molecule.
44. A method of cleaving a integrin subunit alpha 6 RNA molecule comprising the step of, contacting the enzymatic nucleic acid molecule of claim 3 with said integrin subunit alpha 6 RNA molecule under conditions suitable for the cleavage of said integrin subunit alpha 6 RNA molecule.
45. A method of cleaving a Tie-2 RNA molecule comprising the step of, contacting the enzymatic nucleic acid molecule of claim 4 with said Tie-2 RNA molecule under conditions suitable for the cleavage of said Tie-2 RNA molecule.
46. The method of any of claims 42-45, wherein said cleavage is carried out in the presence of a divalent cation.
47. The method of claim 46, wherein said divalent cation is g2+.
48. The enzymatic nucleic acid molecule of any of claims 1-4, wherein said enzymatic nucleic acid molecule is chemically synthesized.
49. The expression vector of claim 17, wherein said expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) a gene encoding at least one said nucleic acid molecule; and wherein said gene 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.
50. The expression vector of claim 17, wherein said expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an open reading frame; d) a gene encoding at least one said nucleic acid molecule, wherein said gene is operably linked to the 3'- end of said open reading frame; and wherein said gene is operably linked to said initiation region, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.
51. The expression vector of claim 17, wherein said expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) a gene encoding at least one said nucleic acid molecule; and wherein said gene is operably linked to said initiation region, said intron and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.
52. The expression vector of claim 18, wherein said vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) an open reading frame; e) a gene encoding at least one said nucleic acid molecule, wherein said gene is operably linked to the 3'- end of said open reading frame; and wherein said gene 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.
53. The enzymatic nucleic acid molecule of claim 1, wherein said enzymatic nucleic acid comprises sequences that are complementary to any of sequences defined as Seq ID Nos 394-786 and 849-910.
54. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid comprises sequences that are complementary to any of sequences defined as Seq ID Nos 5702-6488 and 6569-6648.
55. The enzymatic nucleic acid molecule of claim 3, wherein said enzymatic nucleic acid comprises sequences that are complementary to any of sequences defined as Seq ID Nos 3588-4726 and 4821-4914.
56. The enzymatic nucleic acid molecule of claim 4, wherein said enzymatic nucleic acid comprises sequences that are complementary to any of sequences defined as Seq ID Nos 1612-2312 and 2381-2448.
57. The enzymatic nucleic acid molecule of any of claims 1-4, wherein said enzymatic nucleic acid comprises at least one 2' -sugar modification.
58. The enzymatic nucleic acid molecule of any of claims 1-4, wherein said enzymatic nucleic acid comprises at least one nucleic acid base modification.
59. The enzymatic nucleic acid molecule of any of claims 1-4, wherein said enzymatic nucleic acid comprises at least one phosphorothioate modification.
PCT/US1999/006507 1998-03-27 1999-03-24 Method and reagents for the treatment of diseases or conditions related to molecules involved in angiogenic responses WO1999050403A2 (en)

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WO2001030362A2 (en) * 1999-10-26 2001-05-03 Immusol Incorporated Ribozyme therapy for the treatment of proliferative skin and eye diseases
EP1189918A1 (en) * 1999-06-25 2002-03-27 Isis Pharmaceuticals, Inc. Antisense modulation of integrin beta 3 expression
US7618947B2 (en) * 2004-08-25 2009-11-17 Isis Pharmaceuticals, Inc. Modulation of HIF-1 beta expression
EP1414964B1 (en) * 2001-08-01 2010-03-24 University Of Bristol Vegf isoform
US7723293B2 (en) 1998-10-28 2010-05-25 Cornell Research Foundation, Inc. Methods for increasing capillary density and maintaining viability of microvascular cardiac endothelial cells using trk receptor ligands
WO2015179660A3 (en) * 2014-05-22 2016-01-14 Baylor College Of Medicine Epigenetic engineering to study the role of aberrant dna methylation in disease
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WO2022204714A1 (en) * 2021-03-24 2022-09-29 Kansas State University Research Foundation Zinc-based physionanocomposites and methods of use thereof

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