WO1999020753A2 - Formation de liaison peptidique utilisant des catalyseurs d'acide nucleique - Google Patents

Formation de liaison peptidique utilisant des catalyseurs d'acide nucleique Download PDF

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WO1999020753A2
WO1999020753A2 PCT/US1998/021401 US9821401W WO9920753A2 WO 1999020753 A2 WO1999020753 A2 WO 1999020753A2 US 9821401 W US9821401 W US 9821401W WO 9920753 A2 WO9920753 A2 WO 9920753A2
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nucleic acid
acid catalyst
rna
cell
gene
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PCT/US1998/021401
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WO1999020753A3 (fr
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Thomas R. Cech
Biliang Zhang
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Ribozyme Pharmaceuticals, Inc.
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Priority to AU11870/99A priority Critical patent/AU1187099A/en
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Publication of WO1999020753A3 publication Critical patent/WO1999020753A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
    • C12N2310/124—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes based on group I or II introns

Definitions

  • This invention relates to nucleic acid molecules with catalytic activity and derivatives thereof.
  • RNA In the early evolutionary pathway of life on Earth, RNA is thought to have had dual roles as genetic material and reaction catalyst (Joyce, 1996, Current Biology, 6, 965- 967) . While DNA has replaced RNA as the main vehicle of genetic information, the catalytic capability of nucleic acids can still be demonstrated in several enzymatic nucleic acid molecules, an example of which is ribozymes (catalytic RNA) .
  • Enzymatic nucleic acid molecules are nucleic acid molecules capable of catalyzing one or more of a variety of reactions, including the ability to repeatedly cleave other separate nucleic acid molecules in a nucleotide base sequence-specific manner.
  • Such enzymatic nucleic acid molecules can be used, for example, to target virtually any RNA transcript (Zaug et al . , 324, Na ture 429 1986, Cech, 260 JAMA 3030, 1988; and Jefferies et al . f 17 Nucleic Acids Research 1371, 1989) .
  • nucleic acid catalyts can be employed to perform other reactions .
  • One such reaction may occur naturally during protein synthesis. After disruption of much but not all of the protein portion of ribosomes using proteinase K, SDS and phenol extraction, the large ribosomal subunit can still carry out peptidyl transfer (Noller, H. F. et al . 1992
  • the resulting ribonucleoside-terminated product has nearly identical electrophoretic mobility compared with the amide-terminated product of the amide cleavage reaction when analyzed in a denaturing polyacrylamide gel at pH 8.3.
  • the two products can be separated by polyacrylamide gel electrophoresis at pH 6.5, revealing that the second pathway dominates over amide bond cleavage.
  • the rate of RNA catalyzed amide cleavage is about 50-fold slower than we reported thus representing only about 10 2 ⁇ fold rate acceleration when compared with the uncatalyzed reaction.”
  • the references cited above are distinct from the presently claimed invention since they do not disclose and/or contemplate the catalytic nucleic acid molecules of the instant invention.
  • nucleic acid catalysts novel nucleic acid molecules with catalytic activity (nucleic acid catalysts), which are particularly useful for the formation and/or cleavage of amide bonds.
  • the nucleic acid catalysts of the instant invention are distinct from other nucleic acid catalysts known in the art.
  • the nucleic acid catalysts of the instant invention do not share sequence homology with other known ribozymes.
  • nucleic acid catalysts of the instant invention are capable of catalyzing an intermolecular amide bond formation and/or cleavage between two derivatived amino acids or peptides .
  • the invention features nucleic acid catalysts which are capable of catalyzing a reaction between two modified amino acids. The reaction causes formation of an amide bond between the two amino acids in a peptide .
  • the invention features a nucleic acid catalyst capable of cleaving an amide bond within a peptide molecule.
  • cleavage is meant disruption of the amide bond between and amine and a carboxyl group.
  • Cleavage activity can be used interchangeably with protease, amidase, peptidase, endopeptidase, hydrolysis or amide bond cleaving activity.
  • formation is meant the formation of an amide bond between a carboxyl group of one amino acid and an amine group of another amino acid. Bond formation can be used interchangeably with peptidyl transferase activity.
  • amino acid any molecule which contains both a carboxylic acid group and an amino group.
  • peptide is meant a molecule comprising 2 or more amino acids; preferably 2—100, specifically 100-500, 500- 1000 or greater than 1000.
  • the nucleic acid catalyst cleaves a peptide involved in a disease, infection or indication including but not limited to ligands (e.g. vascular endothelial growth factor (VEGF) ; acidic and basic fibroblast growth factors, etc.); an human immunodeficiency virus (HIV) protein such as tat, rev; protein receptors
  • ligands e.g. vascular endothelial growth factor (VEGF) ; acidic and basic fibroblast growth factors, etc.
  • HIV human immunodeficiency virus
  • VEGF receptor e.g. VEGF receptor, Tie-1, Tie-2
  • metalloproteases e.g. stromelysin, collagenase
  • kinases mutant raf protein
  • G- proteins mutant ras protein
  • phosphatases histones; oxidases; dehydrogenases; aminases; mutases; synthetases; isomerases; deaminases; esterases; transferases; oxygenases; replicases; structural proteins (e.g. collagen, fibronectin, laminin, etc.); peptide hormones (e.g. insulin, lactin, thrombin etc.); hydroxylases; dehydratases; carboxylases; reductases; opsins; peroxidases; phosphorylases; and polymerases .
  • metalloproteases e.g. stromelysin, collagenase
  • kinases mutant
  • the nucleic acid of the invention is linked to a chemical moiety where the chemical moiety comprises chemical structures including but not limited to hydrocarbon chains, disulfide bonds, ether bonds, ester bonds, carboxy groups, polyethylene glycol (PEG), phosphorothioates, epoxides, sulfides, thioethers, and the like.
  • the linker can be a combination of one or more different bonds.
  • Such chemical moiety is linked via a linker to the catalysts.
  • linker can be defined as any moiety which connects the chemical moiety with the nucleic acid catalyst.
  • hydrocarbon is meant a moiety which contains both carbon and hydrogen atoms .
  • disulfide bond is meant a covalent bond between two sulfur atoms.
  • ether bond is meant an oxygen atom bonded to two carbon atoms which may or may not be further bonded to additional atoms .
  • ester bond is meant two chemical structures joined with a carboxylic acid derivative.
  • thioether is meant a sulfur atom bonded to two carbon atoms which may or may not be the same.
  • phosphorothioate is meant a phosphate group in which an oxygen has been replaced by a sulfur atom.
  • the enzymatic nucleic acid includes one or more stretches of RNA, which provide the enzymatic activity of the molecule, linked to the non- nucleotide moiety.
  • RNA is meant a molecule comprising at least one ribonucleotide residue .
  • the invention features ribozymes that inhibit cellular function (s) or structure (s) .
  • These chemically or enzymatically synthesized nucleic acid catalysts could contain substrate binding domains that bind to accessible regions of specific target peptide molecules.
  • the nucleic acid molecules also contain domains that catalyze the cleavage of a target. Upon binding, the enzymatic nucleic acid molecules cleave the target molecules, preventing for example, protein function.
  • the invention features ribozymes that repair damaged or mutated sequences.
  • ribozymes that repair damaged or mutated sequences.
  • These chemically or enzymatically synthesized nucleic acid molecules could cleave off a mutated sequence which can then be juxtaposed with a correct sequence.
  • the protein function could be repaired and normal function could resume.
  • a ribozyme capable of this activity could be identified using appropriate selection pressure.
  • an expression vector comprising nucleic acid sequence encoding at least one of the nucleic acid catalysts of the instant invention.
  • 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.
  • patient is meant an organism which is a donor or recipient of explanted cells or the cells themselves.
  • tient 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.
  • the invention features a method of synthesis of enzymatic nucleic acid molecules of instant invention which follows the procedure for normal chemical synthesis of RNA 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 di ethoxytrityl at the 5 '-end, and phosphoramidites at the 3'-end.
  • common nucleic acid protecting and coupling groups such as di ethoxytrityl at the 5 '-end, and phosphoramidites at the 3'-end.
  • enhanced enzymatic activity is meant to include activity measured in cells and/or in vivo where the activity is a reflection of both catalytic activity and ribozyme stability.
  • the product of these properties is increased or not significantly (less that 10 fold) decreased in vivo compared to an all RNA ribozyme.
  • 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.
  • 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.
  • Figure 1 is a schematic representation of a non- limiting in vitro selection strategy used to evolve nucleic acid catalysts .
  • Figure 2 is a comparison of the peptide bond formation by a ribosome (left) and by a ribozyme (right) .
  • the standard linker (wavy vertical line) joining GMPS and Phe was CH 2 C (0)NHCH 2 CH 2 SSCH 2 CH 2 NH.
  • One experiment employed a shortened linker, SCH 2 CH 2 NH, attached directly to the 5' G rather than through the thiophosphate (GMPS).
  • Figure 3 is a schematic representation of the reaction between 5' -Phe-SS-RNA and AMP-Met-Bio.
  • Figure 4 Activity of the selected RNA pool and transcripts from individual clones from generation 18.
  • a Polyacrylamide gel showing shift of a portion of the radiolabeled RNA after incubation with AMP-Met-Bio and subsequently with streptavidin .
  • Control reactions for pool 9 RNA lane 9c, no streptavidin; lane 9c', RNA treated with DTT prior to streptavidin incubation.
  • b The distribution of the observed rate constants of 75 cloned RNAs . RNAs with no detectable activity ( ⁇ 10 ⁇ 6 min -1 ) are indicated by squares with diagonal line.
  • Figure 5 shows the sequence of clone 25 and the nucleotide differences compared to clones 08, 14, 45, and 60.
  • Figure 6 Formation of biotinylated dipeptide catalyzed by clone 25 RNA.
  • a Reaction kinetics of clone 25 RNA at 25 °C . Best-fit values of m and k cat were obtained by fitting the data to the Michaelis-Menten equation by KaleidoGraph program.
  • b The standard reaction contained 0.4 mM AMP-Met- Bio substrate and 4 ⁇ M clone 25 5' -Phe-SS-RNA in 300 mM KC1 and 200 mM Mg 2+ .
  • Figure 7 Validation of the peptide-bond formed by catalysis of the clone 25 RNA by HPLC-ESI/MS analysis of the dipeptide product.
  • N- biotinylamidocaproyl-L-methioninyl-L-phenylalanine-2' - pyridyldithioethyl amide Bio-Met-Phe-SS-Py
  • N-biotinylamidocaproyl-methionyl-N- hydroxyl-succinimide ester with L-phenylalanine-2' -pyridyldithioethyl amide (made by reacting t-Boc-L-phenylalanine-N- hydroxyl succinimide ester with pyridyldithioethylamine) .
  • Figure 8 demonstrates amino acid specificity of peptide bond formation catalyzed by clone 25.
  • Figure 9 Schematically demonstrates both the trans- (a) and cis- (b) amide bond cleavage and/or ligation reactions catalyzed by ribozymes.
  • the invention provides nucleic acid catalysts which can cleave and/or ligate amino acid molecules.
  • the enzymatic nucleic acid molecule is preferably targeted to a highly conserved sequence region of a target such that specific diagnosis and/or treatment of a disease or condition in a variety of biological systems can be provided with a single enzymatic nucleic acid.
  • Such enzymatic nucleic acid molecules can be delivered exogenously to specific cells as required.
  • the size of the molecule (less than 300 nucleotides) allows the cost of treatment to be reduced compared to larger molecules.
  • nucleic acid catalyst is meant a nucleic acid molecule capable of catalyzing (increasing the velocity and/or rate of) a variety of reactions including the ability to repeatedly cleave or ligate amino acids or peptides in an amino acid sequence-specific manner.
  • a molecule with protease/ligation activity may have affinity to a specified peptide sequence, and also has an enzymatic activity that specifically cleaves and/or ligates amino acids and peptides in that target. That is, the nucleic acid molecule with protease activity is able to intramolecularly or intermolecularly cleave amide bonds and thereby inactivate a target peptide.
  • the nucleic acid molecule could modify a peptide sequence by cleaving the amide bond and reforming it by attaching a different sequence.
  • 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, nucleozyme, DNAzyme, RNA enzyme, endoribonuclease, endonuclease, minizyme, leadzyme, oligozyme or DNA enzyme. All of these terminologies describe nucleic acid molecules with enzymatic activity.
  • nucleic acid molecule as used herein is meant a molecule comprising nucleotides.
  • the nucleic acid can be composed of modified or unmodified nucleotides or non- nucleotides or various mixtures and combinations thereof.
  • oligonucleotide as used herein, is meant a molecule comprising two or more nucleotides .
  • enzymatic nucleic acid molecules described in the instant application are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it have preference to one or more of the target regions, and that it have sequences within or surrounding that target site which impart a peptidyl cleaving/ligating activity to the molecule.
  • enzymatic portion is meant that part of the ribozyme essential for cleavage or ligation of a peptide.
  • Ribozyme synthesis was carried out using the methods well known in the art. Small scale synthesis were conducted on a 394 Applied Biosystems, Inc. synthesizer using a modified 2.5 ⁇ mol scale protocol with a 5 min coupling step for alkylsilyl protected nucleotides and 2.5 min coupling step for 2' -O-methylated nucleotides. Table I outlines the amounts, and the contact times, of the reagents used in the synthesis cycle.
  • 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 12, 49 mM pyridine, 9% water in THF (Millipore) .
  • B & J Synthesis Grade acetonitrile is used directly from the reagent bottle.
  • S-Ethyl tetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc.
  • deprotection of the chemically synthesized nucleic acid catalysts of the invention is performed as follows.
  • the polymer-bound oligoribonucleotide, trityl-off, is 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 is removed from the polymer support.
  • the support is washed three times with 1.0 mL of EtOH :MeCN : H20/3 : 1 : 1, vortexed and the supernatant is then added to the first supernatant.
  • the combined supernatants, containing the oligoribonucleotide are dried to a white powder.
  • the base-deprotected oligoribonucleotide is resuspended in anhydrous TEA-HF/NMP solution (250 ⁇ L of a solution of 1.5mL AZmethylpyrrolidinone, 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 is quenched with 50 mM TEAB (9 mL) prior to anion exchange desalting .
  • the TEAB solution is loaded on to a Qiagen 500® anion exchange cartridge (Qiagen Inc.) that is prewashed with 50 mM TEAB (10 mL) .
  • the RNA is eluted with 2 M TEAB (10 mL) and dried down to a white powder.
  • the average stepwise coupling yields are generally >98% (Wincott et al . , 1995 Nucleic Acids Res . 23, 2677-2684) .
  • Ribozymes of the instant invention are also synthesized from DNA templates using an RNA polymerase, such as that encoded by bacteriophage T7 (Milligan and Uhlenbeck, 1989, Methods Enzymol . 180, 51) . Ribozymes are purified by gel electrophoresis using general methods or are purified by high pressure liquid chromatography (HPLC; See Wincott et al . , supra ) the totality of which is hereby incorporated herein by reference) .
  • HPLC high pressure liquid chromatography
  • 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, 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.
  • ribozymes delivered exogenously, must remain 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 to enhance their nuclease stability.
  • the term "nucleotide” is used as recognized in the art to include natural bases, and modified bases well known in the art.
  • a nucleotide generally comprises a base, sugar and a phosphate group.
  • the nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (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) .
  • modified nucleic acid bases known in the art and has recently been summarized by Limbach et al . , 1994, Nucleic Acids Res . 22, 2183.
  • base modifications that can be introduced into enzymatic nucleic acids without significantly effecting their catalytic activity include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyluracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 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.
  • the invention features modified ribozymes having a base substitution selected from pyridin- 4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6- trimethoxy benzene, 3-methyluracil, dihydrouracil, naphthyl, 6-methyl-uracil and aminophenyl.
  • 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 Biochemis try 35, 14090).
  • Sugar modification of enzymatic nucleic acid molecules have been extensively described in the art (see Eckstein et al . , In terna tional Publi ca tion PCT No. WO
  • a trans-acting ribozyme which can ligate and/or cleave amide bonds could be evolved which would not require the attachment of an amino acid for proper function.
  • a ribozyme could be selected which can recognize specific sequences of a peptide and preferentially ligate and/or cleave at that site.
  • the selection site could be a mutation of a protein such that only mutated copies of proteins are cleaved and made inactive. While one example of a starting nucleic acid is provide above, those in the art will recognize that different pools of nucleic acid molecules can be used and screened as described herein or by equivalent methodology.
  • this invention is not limited to the motif decribed herein, but can be readily expanded, with the knowledge provided herein, to other motifs. Such molecules are readily optimized by technologies described in the art cited herein. Similarly, smaller catalysts can be designed based on the motifs described herein, specificity is selected as described herein.
  • 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.
  • 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 composi tion 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.
  • systemic administration 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) .
  • 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 a composition comprising surface-modified liposomes containing polyethylene 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) .
  • 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 .
  • 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. These include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid.
  • antioxidants and suspending agents may be used.
  • a pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state.
  • the pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors which those skilled in the medical arts will recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients is administered dependent upon potency of the negatively charged polymer.
  • the invention provides enzymatic nucleic acid molecules that can be delivered exogenously to specific cells as required.
  • the enzymatic nucleic acid molecules are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to smooth muscle cells.
  • the RNA or RNA complexes can be locally administered to relevant tissues through the use of a catheter, infusion pump or stent, with or without their incorporation in biopolymers .
  • other enzymatic nucleic acid molecules that cleave target nucleic acid may be derived and used as described above. Specific examples of nucleic acid catalysts of the instant invention are provided below in the Tables and figures .
  • 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. Natl . Acad. Sci . USA 83, 399; 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.
  • 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 mRNA.
  • 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 catalysts 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
  • 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 or viral RNA polymerase promoters are also used, providing that the prokaryotic or viral RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990 Proc .
  • 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.
  • transcription units such as the ones derived from genes encoding U6 small nuclear (snRNA), transfer RNA (tRNA) and adenovirus VA RNA are useful in generating high concentrations of desired RNA molecules such as ribozymes in cells (Thompson et al., supra; Couture and Stinchcomb, 1996, supra; Noonberg et al., 1994, Nucleic Acid Res., 22, 2830; Noonberg et al., US Patent No. 5,624,803; Good et al., 1997, Gene Ther. 4, 45; Beigelman et al., International PCT Publication No. WO 96/18736; all of these publications are incorporated by reference herein.
  • transcription units suitable for expression of ribozymes of the instant invention are shown in Figure 15.
  • 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).
  • the invention features an expression vector comprising nucleic acid sequence encoding at least one of the catalytic nucleic acid molecules of the invention, in a manner which allows expression of that nucleic acid molecule.
  • the expression vector comprises in one embodiment; a) a transcription initiation region; b) a transcription termination region; c) a 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.
  • Applicant employed an in vi tro selection strategy (figure 1) to comprehensively test whether a ribozyme could be employed to catalyze the peptidyl transferase reaction similar to the ribosome (figure 2) .
  • the process on the ribosome involves two binding sites, the P (peptidyl) and A (aminoacyl) sites.
  • the 5'-Phe-SS- RNA (functioning similarly to aminoacyl-tRNA in the A site of the ribosome) provides the amino group that can attack the aminoacyl carbonyl carbon of AMP-Met-Bio (mimicking the fMet-tRNA or peptidyl-tRNA in the P site of the ribosome) to form a peptide bond (thick line) .
  • Example 1 Oligonucleotide and aminoacyl substrate synthesis .
  • the oligodeoxynucleotides PM1 : 5 ' AGCGAATTCTAATACGACTCACTATAGGGAGAGCCATACCTGAC-3' ; PM2 : 5 ' -CACGGATCCTGACGACTGAC-3 ' ; N70DNA, 5' -GGGAGAGCCATACCTGAC-N-7 0 -CAGGTTACGCATCC-3' ; and N72DNA, 5'- CACGGATCCTGACGACTGAC-N 72 -GGATGCGTAACCTG-3' were synthesized on an Applied Biosystems DNA Synthesizer Model 394.
  • a mixture of phosphoramidites in a ratio (3 : 3 : 2 : 2 A : C : G : T) was used to synthesize the random positions (N) .
  • AMP-Met-Bio was synthesized by esterification of AMP with N-biotinylamidocaproyl-L-methionine (Azhayev, A. V. et al.,1977, Nucleic Acids Res . 4, 2223-2234).
  • the other AMP- aminoacyl-Bio compounds were similarly synthesized from the corresponding N-biotinylamidocaproyl-L-amino acids.
  • the full length 222-mer dsDNA was synthesized by PCR- amplification with N70DNA (120 ⁇ g) and N72DNA(120 ⁇ g) as templates.
  • PCR with primers PM1 and PM2 was run in a total volume of 39.2 mL in 196 tubes, each containing 200 ⁇ L of 10 mM Tris-HCl, pH 8.3; 50 mM KC1; 0.01% (W/V) gelatin; 0.05% Tween 20; 0.2 mM dNTPs; 1.0 ⁇ M primers and 3.0 mM MgCl 2 , in 10 cycles (94 °C, 1 min; 52 °C, 1 min; 72 °C, 2 min) .
  • the initial RNA pool was prepared from PCR- amplified DNA (800 ⁇ g) by in vi tro transcription in a 20 mL of reaction mixture [40 mM Tris-HCl, pH 7.5; 10 mM DTT; 4 mM spermidine; 0.05% Triton X-100; 12 mM MgCl 2 ; 1 mM each ATP, CTP, UTP; 8 mM GMPS; 0.4 mM GTP; 100 ⁇ Ci [a-32P]-ATP and T7 RNA polymerase (supplied by A.
  • GMPS 5'- deoxy-5' -thioguanosine-5' -monophosphate
  • the 5'-GMPS-RNA (50 ⁇ M) was reacted with N-bromoacetyl-N' -phenylalanyl-cystamine (10 mM) in chemical linking buffer (20 mM HEPES, pH 7.7; 150 mM NaCl and 1 mM EDTA) for 1 hour at room temperature and overnight at 4 °C giving a yield of 40-60%. After phenol extraction as above, the 5' -Phe-SS-RNA pellet was collected by precipitation with ethanol and used for the selection.
  • Example 3 Selection.
  • reaction buffer 50 mM HEPES, pH 7.4; 300 mM KC1 and 50 mM MgCl 2
  • the pellet was redissolved in 1.0 mL of binding buffer (1.0 M NaCl; 20 mM HEPES, pH 7.4; 5 M EDTA) and transferred to a tube containing 1 L (5 L for 1st cycle selection) of 50% streptavidin agarose gel slurry (prewashed twice with binding buffer) .
  • the slurry mixture was mixed gently for 40-60 min by a roller, loaded on a column, and then washed with 20 mL of binding buffer, 20 mL of water, 20 mL of 7.0 M urea, and 20 mL of water.
  • the bound RNA was eluted with 3.0 mL of 100 mM DTT in binding buffer.
  • the eluted RNA was precipitated with 100 ⁇ g of glycogen and 2.5 volumes of ethanol.
  • the eluted RNA was converted to cDNA by reverse transcription.
  • a 20 ⁇ L annealing mixture (40 mM Tris-HCl, pH 8.3; 8.0 mM DTT; 48 mM NaCl; 5 pmol 3' -primer) was heated for 90 s at 90 °C, and slowly cooled to room temperature.
  • the cDNA was amplified by PCR in a 100 ⁇ L reaction (50 ⁇ l of RT- reaction mix; 2.4 mM MgCl 2 ; 23 mM Tris-HCl, pH 8.3; 12.5 mM KC1; 0.2 mM dNTPs; 1.0 ⁇ M primers and 5 U of Taq DNA polymerase) and purified as described above, transcribed, and linked with phenylalanine for the next selection. In subsequent selection cycles, 1-2 ⁇ M 5' -Phe-SS-RNA and 200- 400 ⁇ M AMP-Met-Bio were incubated for 1-5 h, with other conditions the same as described above.
  • Selection cycle 10 was different from the others in that it was done with 1 ⁇ M 5' -Phe-SS-RNA and 1 mM AMP-Met-Bio for 5 h.
  • the enriched RNA pool showed a band-shift upon gel electrophoresis that represented a streptavidin-RNA complex (Fig. 4a).
  • the 19th generation cDNA molecules were cloned. PCR- amplified DNAs from 75 plasmids were used as templates for run-off transcription by T7 RNA polymerase. Kinetic analysis of the individual RNA species revealed that 9 of 75 had better activity than the final RNA pool, which 18 clones were inactive (Fig. 4b). Sequence analysis (24 clones) showed that the most active RNAs comprised at least two sequence classes. The sequence of one of these classes, which included clones 8, 14, 25, 45, and 60 ( figure 5) . The deletion of ⁇ 20 nucleotides from either the 5'- or the 3'- end or from both ends of clone 25 RNA decreased dramatically its catalytic activity (data not shown) . Therefore, both the 5' and 3' constant regions of this ribozyme are necessary for formation of the active structure.
  • the rate enhancement of the peptide bond formation catalyzed by clone 25 RNA is about 1 x 10 6 .
  • the reaction of clone 25 RNA required Mg 2+ and reached a maximal rate when [Mg 2+ ] ⁇ 100 mM.
  • Mg 2+ may be required for proper RNA folding and may also be involved directly in the chemical step of peptide bond formation.
  • Divalent metal ions also are required for peptidyl transfer to puromycin by the ribosomeJVladen, B. E. et al. 1968 Eur . J. Biochem . 6, 309-316)_ Controls were run which contained 0.4 mM AMP-Met-Bio substrate and 4 ⁇ M clone 25 5' -Phe-SS-RNA in 300 mM KCl and 200 mM Mg 2+ ( Figure 6b). After 2.5 h at 25 °C, aliquots were removed from the reaction mixture for streptavidin gel-shift assays.
  • Polyacrylamide gel showing shift of a portion of the radiolabeled RNA after incubation with AMP-Met-Bio and subsequently with streptavidin is shown in figure 4a.
  • the selection reactions were performed at 25 ° C with 4 ⁇ M 5'- Phe-SS-RNA and 8 mM AMP-Met-Bio substrate for 20 h except for selection cycle 10, which was done with 1 ⁇ M 5' -Phe-SS- RNA and 1 mM AMP-Met-Bio for 5 h.
  • Control reactions for pool 9 RNA were performed and are as follows: lane 9c, no streptavidin; lane 9c', RNA treated with DTT prior to streptavidin incubation.
  • RNAs with no detectable activity are indicated by squares with diagonal line .
  • linker provides specific contacts with the RNA that are required for proper positioning of the 5 ' -amino group of 5'- Phe-SS-RNA in the active site of the ribozyme. Because inhibition by the free linker requires it to compete with the linker attached intramolecularly to the ribozyme, the interaction energy may be much greater than would be indicated by an apparent K & of 5-10 mM.
  • these novel nucleic acid catalysts can be used as a laboratory reagent. Deletion analysis of target peptide sequences using ribozymes would give insight into structure- function relationship of the target peptides. Alternatively, various peptide molecules can be joined together to form chimeric peptides, or to introduce novel domains or structures on these peptides . Using these ribozymes, novel proteins can be designed and produced whose function is tailored to the needs of the researcher.
  • DNA binding domain of a transcription factor can be linked to the activation domain from a different protein using the ribozymes of the present invention to generate novel proteins; this protein may be useful in the study of, for example, the mechanism of transcription in a mammalian system.
  • Ribozymes could also be used as a diagnostic tool to detect pathogens or mutant proteins in a biological sample. Ribozymes can be designed to cleave or ligate mutant proteins or proteins specific to a particular pathogen (e.g. HIV, clymidia, etc) within a sample. Positive identification of cleavage or ligation products would then be an indicator of the pathogen' s presence within the sample. Additionally, a tagged peptide sequence which is comprised of an epitope tag (e.g. hemagglutinin, Myc) , fluorescent tag (e.g. green fluorescent protein, fluorocein, rhodamine) , or a Hise tag could be ligated onto the desired target to allow for detection.
  • an epitope tag e.g. hemagglutinin, Myc
  • fluorescent tag e.g. green fluorescent protein, fluorocein, rhodamine
  • Hise tag e.g. green fluorescent protein, fluorocein,
  • the cleavage products could be quantitated to yield information such as viral load. Knowing the viral load of the patient can be invaluable in determining appropriate therapeutic approaches. This would reduce the number of effective treatments that a patient receives and also lower the cost of treatment.
  • nucleic acid catalysts can be utilized as part of an industrial synthesis scheme for protein splicing or adding a tag to one end of a protein via peptide bond formation.
  • the tag may consist of a number of different molecules including epitopes, fluorescent molecules (e.g. fluorocein, rhodamine), lipids, vitamins, peptides, nucleic acids, nucleotides, amino acids, antibiotics, carbohydrates, antibodies and the like. Use of these molecules may reduce the amount of chemical by-products generated through traditional manufacturing methods .
  • nucleic acid catalysts hold an advantage over protein enzymes which perform similar functions in terms of enzymatic activity and half-lives. If modifications are introduced into the nucleic acid catalyst to stabilize the molecule, they should continue to catalyze the peptidyl transferases reaction longer than proteins which are quickly oxidized and made inactive. Additionally, reaction conditions (e.g. pH, salinity, temperature) may exist which prohibit the use of protein enzymes but allow ribozymes with peptide bond ligating and/or cleavage activity to execute its function. Furthermore, through in vi tro selection, ribozymes can be identified which are highly specific to various peptide sequences and offer researchers flexibility to cleave at almost any specific site within a target peptide molecule . Other embodiments are within the following claims.

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Abstract

La présente invention concerne des catalyseurs d'acide nucléique avec de nouveaux motifs structuraux ayant une activité catalytique, les méthodes de synthèse, et leur utilisation.
PCT/US1998/021401 1997-10-21 1998-10-09 Formation de liaison peptidique utilisant des catalyseurs d'acide nucleique WO1999020753A2 (fr)

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US5595873A (en) * 1994-05-13 1997-01-21 The Scripps Research Institute T. thermophila group I introns that cleave amide bonds
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001066721A2 (fr) * 2000-03-06 2001-09-13 Ribozyme Pharmaceuticals, Inc. Molecules detectrices a acides nucleiques
WO2001066721A3 (fr) * 2000-03-06 2002-07-25 Ribozyme Pharm Inc Molecules detectrices a acides nucleiques

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