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WO1999050403A2 - Procede et reactifs pour le traitement de maladies ou d'affections associees a des molecules impliquees dans les reactions angiogeniques - Google Patents

Procede et reactifs pour le traitement de maladies ou d'affections associees a des molecules impliquees dans les reactions angiogeniques 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 (fr
WO1999050403A3 (fr
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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 CA002324421A priority Critical patent/CA2324421A1/fr
Priority to EP99915032A priority patent/EP1086212A2/fr
Priority to AU33647/99A priority patent/AU757789B2/en
Priority to JP2000541291A priority patent/JP2002509721A/ja
Publication of WO1999050403A2 publication Critical patent/WO1999050403A2/fr
Publication of WO1999050403A9 publication Critical patent/WO1999050403A9/fr
Publication of WO1999050403A3 publication Critical patent/WO1999050403A3/fr

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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

L'invention se rapporte à une molécule d'acide nucléique qui module la synthèse, l'expression et/ou la stabilité d'un ARNm codant pour des facteurs angiogéniques sélectionnés parmi un transporteur nucléaire aryle-hydrocarbure (ARNT), une sous-unité intégrine bêta 3 (β3), une sous-unité intégrine alpha 6 (α6) et de l'ARN du récepteur 'tie-2'. L'invention se rapporte également à un traitement des affections associées à l'angiogenèse faisant usage de ces molécules d'acide nucléique.
PCT/US1999/006507 1998-03-27 1999-03-24 Procede et reactifs pour le traitement de maladies ou d'affections associees a des molecules impliquees dans les reactions angiogeniques WO1999050403A2 (fr)

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Cited By (7)

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WO2001030362A3 (fr) * 1999-10-26 2002-01-17 Immusol Inc Therapie ribozymique destinee au traitement de maladies proliferantes de la peau et des yeux
EP1189918A4 (fr) * 1999-06-25 2004-12-01 Isis Pharmaceuticals Inc Modulation antisens de l'expression d'integrine beta 3
US7618947B2 (en) * 2004-08-25 2009-11-17 Isis Pharmaceuticals, Inc. Modulation of HIF-1 beta expression
EP1414964B1 (fr) * 2001-08-01 2010-03-24 University Of Bristol Isoforme de facteur de croissance
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 (fr) * 2014-05-22 2016-01-14 Baylor College Of Medicine Ingénierie épigénétique pour étudier le rôle de la méthylation de l'adn aberrant dans une maladie
WO2022204714A1 (fr) * 2021-03-24 2022-09-29 Kansas State University Research Foundation Composites de physio-nanocomposites à base de zinc et leurs méthodes d'utilisation

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US5378822A (en) * 1993-04-08 1995-01-03 Wisconsin Alumni Research Nucleic acids encoding murine and human Ah receptors
FR2733913B1 (fr) * 1995-05-09 1997-08-01 Sanofi Sa Sequence d'adn a titre de medicament, et compositions pharmaceutiques en contenant
US6346398B1 (en) * 1995-10-26 2002-02-12 Ribozyme Pharmaceuticals, Inc. Method and reagent for the treatment of diseases or conditions related to levels of vascular endothelial growth factor receptor

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
US8853163B2 (en) 1998-10-28 2014-10-07 Cornell Research Foundation, Inc. Methods for increasing vascular density and maintaining viability of microvascular endothelial cells using trk receptor ligands
EP1189918A4 (fr) * 1999-06-25 2004-12-01 Isis Pharmaceuticals Inc Modulation antisens de l'expression d'integrine beta 3
WO2001030362A3 (fr) * 1999-10-26 2002-01-17 Immusol Inc Therapie ribozymique destinee au traitement de maladies proliferantes de la peau et des yeux
EP1414964B1 (fr) * 2001-08-01 2010-03-24 University Of Bristol Isoforme de facteur de croissance
US7820178B2 (en) 2001-08-01 2010-10-26 University of Brisol VEGF isoforms and their use as anti-angiogenic, anti-vasodilatory, anti-permeability and anti-proliferative agents
US8933211B2 (en) 2001-08-01 2015-01-13 University Of Bristol Growth factor isoform
US7618947B2 (en) * 2004-08-25 2009-11-17 Isis Pharmaceuticals, Inc. Modulation of HIF-1 beta expression
US7799764B2 (en) 2004-08-25 2010-09-21 Isis Pharmaceuticals, Inc. Modulation of HIF1-beta expression
WO2015179660A3 (fr) * 2014-05-22 2016-01-14 Baylor College Of Medicine Ingénierie épigénétique pour étudier le rôle de la méthylation de l'adn aberrant dans une maladie
US10470446B2 (en) 2014-05-22 2019-11-12 Baylor College Of Medicine Engineered cell comprising a recombinant pro-methylation cis-element construct that resides in a regulatory region of a target gene
WO2022204714A1 (fr) * 2021-03-24 2022-09-29 Kansas State University Research Foundation Composites de physio-nanocomposites à base de zinc et leurs méthodes d'utilisation

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