WO2000004141A2 - Use of nucleic acid molecules as antiviral agents - Google Patents
Use of nucleic acid molecules as antiviral agents Download PDFInfo
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- WO2000004141A2 WO2000004141A2 PCT/US1999/016253 US9916253W WO0004141A2 WO 2000004141 A2 WO2000004141 A2 WO 2000004141A2 US 9916253 W US9916253 W US 9916253W WO 0004141 A2 WO0004141 A2 WO 0004141A2
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- 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
- C12N15/1131—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 against viruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/13—Decoys
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/31—Chemical structure of the backbone
- C12N2310/317—Chemical structure of the backbone with an inverted bond, e.g. a cap structure
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/33—Chemical structure of the base
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/33—Chemical structure of the base
- C12N2310/332—Abasic residue
Definitions
- Viruses are obligate parasites that depend upon the infected host for many of the basic processes needed for a successful infection. Because viruses depend on the enzymatic and synthetic functions of the host cell, it is very difficult to treat viral infections without affecting cellular processes. Given that several viral diseases are at pandemic proportions, including influenza, AIDS, and hepatitis, the design of effective virus-specific drugs is increasingly important.
- Aziduovir which is utilized more readily by the reverse transcriptase of the human immunodeficiency virus (HIV) than by host cellular polymerases.
- HAV human immunodeficiency virus
- Aziduovir and other viral inhibitors were generally discovered through an intensive and costly drug screening program.
- RNA-dependent RNA polymerase is a complex composed of viral and cellular proteins that directs viral RNA synthesis from infecting RNA templates. Many viral RdRp proteins have been sequenced and analyzed. However, a comprehensive mechanism describing RNA synthesis is lacking. Consequently, general knowledge of RdRp is significantly less than that of other RNA and DNA polymerases.
- RdRp RNA-dependent RNA polymerase
- antisense molecules and ribozymes have been successfully utilized to prevent the translation of mRNA into protein.
- oligonucleotides have been found which are able to bind directly to protein and thereby reduce or eliminate its function.
- RNA binding oligonucleotides are through a process known as in vitro selection. From a random pool of RNA molecules, sequences are identified which are capable of binding to specific proteins though cycles of affinity selection. For example, Tuerk et al . , 1992, Proc . Na tl . Acad. Sci . USA 89, 6988-6992 describes the use of the selection procedure to identify a ligand capable of binding to HIV reverse transcriptase and inhibiting cDNA synthesis. Other nucleic acid ligands (aptamers) have also been described that bind to proteins with high affinity. However, all these RNA aptamers are highly structured with complex secondary and tertiary structures .
- the invention provides nucleic acid molecules that are useful as specific inhibitors of the viral polymerase.
- the present invention provides an oligonucleo- tide of at least four nucleotides, wherein the oligonucleo- tide comprises a viral nucleic acid sequence which includes the viral initiation nucleotide.
- the oligo- nucleotide includes viral promoter and initiation sequences.
- the invention features a linear single stranded nucleic acid molecule capable of binding to a viral polymerase thereby inhibiting the polymerase activity and as a result viral replication is inhibited.
- nucleic acid molecules may include preferably initiation sequences, or other sequences necessary for the initiation of, for example, RNA transcription by a viral RNA polymerase.
- the nucleic acid molecule preferably includes the initiation nucleotide and at least the first, and more preferably the first two nucleotides which are 3' of the initiation nucleotide in the viral sequence.
- nucleic acid molecule is meant a macromolecule comprised of at least one nucleotide.
- the nucleic acid molecule of the invention may be DNA, RNA or a mixture thereof.
- the nucleic acid molecule is comprised of DNA. More specifically, the nucleic acid molecule of the invention comprises nucleotides with modifications in the base, sugar or phosphate groups.
- nucleic acid molecule of the invention comprise at least about four to at least about fifty, more preferably at least about forty, and even more preferably at least about twenty to at least about thirty five, specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 contiguous nucleotides, and that the nucleic acid molecules preferably have identity to the wild-type viral sequence from which they are derived.
- the nucleic acid molecule may optionally also include cap structures at the 5' or 3' end.
- 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. Nucleotides generally comprise a base, sugar and a phosphate group. The nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and other; see for example, Usman and McSwiggen, supra; Eckstein et al., International PCT Publication No.
- base modifications that can be introduced into nucleic acids include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6- trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl ⁇ , 5-alkylcytidines (e.g., 5-methylcytidine) , 5-alkyluridines (e.g., ribothymidine) , 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g.
- modified bases in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at 1' position or their equivalents; such bases may be used at any position, for example, within the catalytic core of an enzymatic nucleic acid molecule and/or in the substrate-binding regions of the nucleic acid molecule.
- unmodified nucleotide is meant a nucleotide with one of the bases adenine, cytosine, guanine, thymine, uracil joined to the 1' carbon of ⁇ -D-ribo-furanose.
- modified nucleotide is meant a nucleotide which contains a modification in the chemical structure of an unmodified nucleotide base, sugar and/or phosphate.
- cap structure is meant chemical modifications which have been incorporated at the terminus of the oligonucleotide. These terminal modifications protect the nucleic acid molecule from exonuclease degradation, and may help in delivery and/or localization within a cell.
- oligonucleotide as used herein, is meant a molecule comprising two or more nucleotides.
- the nucleic acid molecule of the invention a plurality thereof, or in combination or in conjunction with other drugs, can be used to treat diseases or conditions discussed below are useful to prepare an antiviral composition.
- the antiviral compositions of the invention are particularly useful in methods to inhibit the infection or replication of viruses which are able to use the host's cellular machinery to generate viral polymerases.
- viruses are (+) single strand RNA viruses, including the alpha virus super-family, which includes bacterial, plant and animal viruses.
- viruses which may be inhibited by the nucleic acid molecules of the present invention include but are not limited to hepatitis C virus, Hepatitis B, Hepatitis A, HIV,
- the method comprises the administration to a cell having, or suspected of having, a viral infection, an effective amount of at least one nucleic acid molecule of the invention, wherein the nucleic acid molecule comprises the initiation nucleotide for the virus.
- an effective amount is meant, the minimum quantity of the nucleic acid molecule necessary to induce a reduction in the viral load carried in an cell, tissue or whole organism.
- the nucleic acid molecule includes modifications selected from a group comprising 2'-0-alkyl (e.g. 2'-0-allyl; Sproat et al., supra); 2' -O-alkylthioalkyl (e.g. 2' -O-methylthiomethyl; Karpeisky et al., 1998, Nucleosides & Nucleotides 16, 955- 958); L-nucleotides (Tazawa et al., 1970, Biochemistry 3499; Ashley, 1992, J. Am. Chem. Soc.
- the 5' cap structure is selected from a group comprising inverted abasic residue,. ', 5 ' -methylene nucleotide; 1- (beta-D- erythrofuranosyl) nucleotide, 4 ' -thio nucleotide, carbocyclic nucleotide; 1, 5-anhydrohexitol nucleotide; L- nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3 ' , 4 ' -seco nucleotide; acyclic 3, 4-dihydroxybutyl nucleotide; acyclic 3, 5-dihydroxypentyl nucleotide, 3 ' —3 ' — inverted nucleotide moiety; 3 ' -3
- the 3' cap structure is selected from a group comprising, 4 ',5'- methylene nucleotide; 1- (beta-D-erythrofuranosyl) nucleo- tide; 4 ' -thio nucleotide, carbocyclic nucleotide; 5 ' -amino- alkyl phosphate; 1, 3-diamino-2-propyl phosphate, 3- aminopropyl phosphate; 6-aminohexyl phosphate; 1,2- aminododecyl phosphate; hydroxypropyl phosphate; 1,5- anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; threo- pentofuranosyl nucleotide; acyclic 3 ' , 4 ' -seco nucleotide; 3, 4-dihydroxybutyl nucleotide
- the nucleic acid molecule of the present invention is conjugated with another moiety including but not limited to abasic nucleotides, polyether, polyamine, polyamides, peptides, carbohydrates, lipid, or polyhydrocarbon compounds. Those skilled in the art will recognize that these molecules may be linked to one or more of any nucleotides comprising the nucleic acid molecule at several positions on the sugar, base or phosphate group.
- the nucleic acid molecules that bind to viral polymerases 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. Such vectors might be repeatedly administered as necessary.
- the nucleic acid molecules can bind specifically to viral polymerase.
- nucleic acid molecule 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).
- nucleic acid molecules that bind to viral RNA polymerases and inhibit viral replication are expressed from transcription units inserted into DNA, RNA, or viral vectors.
- 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.
- the present invention may be superior to other known antiviral agents because it requires only the nucleic acid sequence (s) for initiation of viral nucleic acid synthesis.
- the sequence is readily prepared, does not require complex manipulation by molecular biology techniques, and only a minimal screening regime is necessary.
- the present oligonucleotides may be modified to increase their efficacy at lower concentrations, e.g., modified so as to contain nucleotide analogs or circularized, for RNAs, to provide agents that are more stable in vivo so as to, for example, have increased resistance to degradation in serum.
- the chemistry of the nucleic acid molecule may also be altered to increase the binding affinity of said nucleic acid molecule.
- nucleic acid molecules may be effective in reducing the effective amount of nucleic acid molecules.
- at least one therapeutic can be designed for every virus.
- these inhibitors can also be used to disrupt other steps in viral replication, e.g., translation, protein and nucleic acid modification, which may be tightly linked to the initiation of RNA synthesis.
- Figure 1 demonstrates the ability of BMV RdRp to accurately initiate RNA synthesis from RNA or DNA pro- scripts.
- (Top) Proscript -20/13 complementary to the viral (+) -strand RNA3 from positions 1222 to 1252, contains the WT BMV subgenomic promoter directing the synthesis of a 13-nt product and serves as the WT control.
- the initiation nucleotide is denoted by an arrow with the sequence of the RdRp product shown above.
- Schematics of various constructs tested are listed and to the right are the lane numbers showing the amount of RNA synthesis relative to that from the WT control in the corresponding autoradiograph.
- RNA sequences are denoted by bold capital letters while DNA sequences are in lower-case letters.
- RNA substitutions in both RNA and DNA constructs are shown below each sequence.
- the ⁇ -lg proscripts lack the 3' terminal guanylate at position -1 relative to the initiation site.
- (Bottom) Autoradiographs of BMV RdRp reaction products. The amount of RNA synthesis from 25 nM of proscript -20/13 WT is shown in lanes 1 and 10. T7 generated markers containing the expected sequence of the RdRp products were used to determine the sizes of the accurately initiated 13- and 14- nt BMV RdRp products. The 14-nt product is due to a 1-nt nontemplated addition by RdRp.
- RNA synthesis and accurate initiation from proscript d(- 20/13) were verified by the treatments indicated above the gel in lanes 3-5 and lanes 6-9, respectively.
- the amount of RdRp product from 125 nM of RNA or DNA templates with a penultimate initiation site is shown in lanes 11-21.
- the treatments shown above lanes 12-14 and 19-21 demonstrate the initiation requirements from r(-l/13) and d(-l/13), respectively.
- the treatments indicated above lanes 16-18 verify RNA synthesis from d(-l/13).
- Lane ⁇ represents the products of a control reaction with no added template while Std lanes represent products with no additional treatments.
- Figure 2 displays ribose moieties which facilitate RNA synthesis by RdRp.
- the sequence of the -20/13 WT proscript is shown with the initiation site marked by an arrow.
- the sequences of hybrid proscripts, containing both ribose and deoxyribose residues, are listed below.
- RNA sequences are denoted by bold capital letters while DNA sequences are in lower-case letters.
- Proscripts containing substitutions of the 2' -OH at position -11 relative to the initiation site were constructed to determine how this functional group interacts with the BMV RdRp.
- the lane number containing the RdRp product generated from each proscript in the autoradiograph below is shown to the right.
- Figure 3 displays the role of ribose 2'-OHs in stable interaction with RdRp.
- the sequence of the -20/15 WT proscript, directing synthesis of a 15-nt product from the initiating cytidylate (arrow) is shown.
- RNA construct containing a WT subgenomic promoter sequence.
- the -20/-1 proscript contains the WT subgenomic promoter from position -20 to -1 relative the initiation site and serves as a negative control.
- the concentration of competitor needed to reduce synthesis from 25 nM of the -20/15 proscript by 50% ( 150) are listed to the right.
- Bottom Determination of 150 values for RNA and DNA subgenomic promoters.
- the amount of 15-nt product generated from the -20/15 RNA proscript was measured and plotted as a function of the concentration of each competitor.
- the identities of the competitors are shown to the right of the graph. Data points represent the mean of three independent standard deviations shown.
- FIG. 4 Minimal DNA proscripts can inhibit viral RNA synthesis in vitro.
- the d(- 1/13) Rev proscript serves as a negative control.
- the names and 150 values are listed to the sides. (Bottom) Determination of 150 values for the DNA inhibitors.
- the amount of 15-nt product generated from the -20/15 RNA proscript was measured in the presence of increasing amounts of DNA templates.
- the 150 value was quantitated as the concentration of inhibitor needed to reduce the 15-nt product from 25 nM of -20/15 proscript by 50%.
- the identities of the inhibitors are shown to the right of the graph. Data points represent the mean of three independent experiments with deviations shown.
- Figure 5 displays functional moieties in the subgenomic promoter required for initiation of RNA synthesis.
- A Predicted functional groups required for interaction with the BMV RdRp. The sequence shown is proscript -20/13 containing the WT BMV subgenomic promoter directing synthesis of a 13-nt product and serves as the WT control. The subgenomic initiation site is denoted by an arrow with the sequence of the RdRp product shown above.
- the base moieties predicted to interact with the RdRp by previous mutational studies (Siegel et al., 1997, Proc. Natl. Acad. Sci. 94, 11238-11243) are indicated below the four nucleotides essential for RNA synthesis.
- the bands denoted by asterisks represent terminal transferase labeling of the input template.
- the structures of the particular base for each position and of the nucleosides in the case of the guanylate at positions -17 and -11, are shown with arrows indicating defined changes in the functional groups mediated by the insertion of various base analogs. Numbers in parenthesis indicate the lane in the autoradiograph containing the RdRp reaction products from proscripts containing the indicated base analog.
- Lane WT represents the products directed by the -20/13 proscript while lane ⁇ represents the products of a control reaction with no added template.
- the reaction products were separated by denaturing PAGE and visualized by autoradiography with their sizes denoted on the side.
- the predominant RdRp product was 14-nucleotides due to the non-templated addition of one residue at the 3' end of the RNA product (Siegel et al., supra) .
- Accurate initiation was verified by described enzymatic manipulations (Siegel et al., supra) and comparison to T7 generated size markers. Values listed below the gels represent the percent activity from promoters containing each base analog compared to that from the WT promoter sequence.
- Figure 6 displays the template requirements for stable interaction with RdRp.
- A Schematic of proscripts containing 5' truncations of the template. The initiation site is denoted with an arrow. Proscripts containing the +1 c/g mutation (circled) are unable to direct RNA synthesis. The names and 150 values for each construct are listed to the sides.
- B Determination of 150 values. The amount of activity from the -20/15 proscript directing synthesis of a 15-nt product was measured in the presence of increasing amounts (up to 10-fold molar excess, 250 nM) of competitor templates. The 150 value was determined as the concentration of competitor needed to reduce the 15-nt product from 25 nM of the -20/15 proscript by 50%.
- FIG. 7 demonstrates the recognition of the subgenomic initiation site.
- the sequence of the WT -20/13 proscript is shown with the initiation site denoted.
- the structure of the base analogs incorporated at positions -1, +1, and +2 are listed below with the names of the various proscripts shown on the left.
- the lanes containing the RdRp reaction products from the proscripts containing the base analogs are indicated to the right.
- Lane ⁇ represents the products of a control reaction with no added template. The reaction products were separated by denaturing PAGE and visualized by autoradiography with their sizes denoted on the side. Values listed below the gels represent the percent activity from promoters containing each base analog compared to that from the WT promoter sequence.
- Figure 8 displays a model for the interaction between the BMV RdRp and the subgenomic promoter elements needed to initiate RNA synthesis.
- Essential nucleotides are boxed with the key features putatively required for hydrogen bond formation with amino acid residues in RdRp shown above.
- the 2' -hydroxyl at position -11 also contributes to RNA synthesis.
- Recognition of the initiating nucleotide may occur by the rGTP primer bound by the RdRp.
- the oval represents a low resolution structure of the RdRp complex.
- Figure 9 demonstrates a schematic representation of a nucleic acid molecule inhibiting viral replication.
- Figure 10. 150 values of selected oligonucleotides.
- Figure 11. Graph of 150 values of DNA inhibitors.
- the reaction mixture contained 25 nM of the -20/15 WT template RNA with 10 ⁇ l BMV RdRp in a 40 ⁇ l volume containing 20 mM sodium glutamate (pH 8.2), 4 mM MgC12, 12.5 mM DTT, 0.5% (vol/vol) Triton X-100, 2 mM MnC12, 200 ⁇ M ATP and UTP, 500 ⁇ M GTP, and 250 nM [ ⁇ -32P] CTP.
- RdRp products from reactions containing DNA inhibitors were quantified and their relative activities were plotted against the amount of inhibitor present. 150 values were determined by calculating the amount of inhibitor need to reduce synthesis by 50% of the control reaction.
- nucleic acid Molecules Synthesis of Nucleic acid Molecules Synthesis of nucleic acids greater than 100 nucleotides in length is difficult using automated methods, and the therapeutic cost of such molecules is prohibitive.
- 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 RNA structure.
- the molecules of the instant invention were chemically synthesized. Oligodeoxy- ribonucleotides were synthesized using standard protocols as described in Caruthers et al., 1992, Methods in Enzymology 211,3-19, and is incorporated by reference.
- RNA including certain enzymatic nucleic acid molecules follows the procedure as described in Usman et al., 1987 J. Am. Chem. Soc, 109, 7845; Scaringe et al . , 1990 Nucleic Acids Res., 18, 5433; and Wincott et al., 1995 Nucleic Acids Res. 23, 2677-2684 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.
- RNA Deprotection of the RNA was performed as follows.
- MA methylamine
- the base-deprotected oligoribonucleotide was resus- pended in anhydrous TEA ⁇ F/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 pre-washed with 50 mM TEAB (10 mL) . After washing the loaded cartridge with 50 mM TEAB (10 mL) , the RNA was eluted with 2 M TEAB
- RNA may be modified to enhance stability with nuclease resistant groups, for example, 2 '-amino, 2'-C-allyl, 2'- flouro, 2'-0-methyl, 2'-H, nucleotide base modifications
- nuclease resistant groups for example, 2 '-amino, 2'-C-allyl, 2'- flouro, 2'-0-methyl, 2'-H, nucleotide base modifications
- sequences are representative only of many more such sequences where the chemical composition of the nucleotides may be altered.
- a "cap” structure may be added to the 5' or 3' end to increase stability.
- nucleic Acid Molecules Methods for the delivery of nucleic acid molecules is described in Akhtar et al . , 1992, Trends Cell Bio., 2, 139; and Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995 which are both incorporated herein by reference.
- Sullivan et al., PCT WO 94/02595 further describes the general methods for delivery of enzymatic RNA molecules . These protocols may be utilized for the delivery of virtually any nucleic acid molecule.
- Nucleic acid molecules may be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres .
- nucleic acid molecules may be directly delivered ex vivo to cells or tissues with or without the aforementioned vehicles.
- the nucleic acid/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 nucleic acid 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) . 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 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 PEG-modified, 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; Ishiwata et 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. These 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.
- nucleic acid molecules that cleave target molecules are expressed from transcription units (for example of ribozymes, see Couture et al., 1996, TIG., 12, 510) inserted into DNA or RNA vectors.
- the recombinant vectors are preferably DNA plasmids or viral vectors.
- Nucleic acid molecule 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 nucleic acid molecules are delivered as described above, and persist in target cells.
- viral vectors may be used that provide for transient expression of nucleic acid molecules. Such vectors might be repeatedly administered as necessary. Once expressed, the nucleic acid molecules bind to target viral polymerases. Delivery of nucleic acid molecule 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 molecules of the instant invention is disclosed.
- the nucleic acid sequence encoding the nucleic acid molecule of the instant invention is operable linked in a manner, which allows expression of that nucleic acid molecule.
- 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.
- a transcription initiation region e.g., eukaryotic pol I, II or III initiation region
- 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 molecule of the invention; and/or an intron (intervening sequences) .
- ORF open reading frame
- intron intervening sequences
- 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 also 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.
- RNA 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 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.
- Brome mosaic virus is a useful model to define the steps in RNA synthesis for (+) -strand RNA virus.
- BMV has three genomic RNAs, designated RNAl, 2, and 3 and a subgenomic RNA4. These RNAs encode four proteins: the helicase-like la (109 kDA) , the polymerase-like 2a (96 kDa) , the movement protein 3a (34 kDa) , and the capsid protein (20 kDa) .
- Each BMV RNA contains a highly conserved 3 ' region which folds into a tRNA-like structure that is required to direct the synthesis of (-) -strand RNA.
- the (-) -strand RNA serves as template and provides cis-acting sequences for genomic (+) -strand and subgenomic RNA synthesis
- the BMV RNA replication enzyme is a complex localized in the endoplasmic reticulum. It contains the BMV-encoded 1 a and 2a proteins and yet unidentified host proteins. Membrane-associated replicase can be solubilized with nonionic detergents and still retain the ability to direct synthesis of (-) -strand RNAs or subgenomic (+) -strand RNA from exogenously added genomic RNAs or (-) strand BMV RNA3, respectively.
- RNA-dependent RNA polymerase can utilize (+) -strand RNAs of less than 160 nucleotides containing the conserved tRNA-like sequence to direct BMV specific RNA synthesis in vitro.
- BMV is a positive strand RNA virus and is the type member of the bromovirus group of plant viruses in the alphavirus-like superfamily of positivesense RNA viruses.
- Monocistronic RNAl and RNA2 encode proteins la (containing putative methyltransferase and helicase domains) and 2a (containing polymerase-like domains) , respectively.
- RNA3 encodes the 3a movement protein and the coat protein, whose translation is directed by the subgenomic RNA4 (0.88 kb) .
- Synthesis of subgenomic RNA4 is by internal initiation from a (-) -strand copy of RNA3.
- the BMV RdRp complex is integrated into plant membranes but can be solubilized with high-concentration-salt and nonionic detergents, such as Triton X-100.
- the BMV RdRp has been highly enriched and can specifically synthesize minus- strand RNA from input plus-strand RNA templates in a sequence-specific manner.
- the BMV RdRp can also synthesize subgenomic plus-strand RNA 4 by initiating with a guanylate residue internally within the minus-strand of RNA 3.
- the synthesis of minus-strand RNA initiates from the conserved 3' ends of plus-strand BMV RNAs. This conserved region can fold into a tRNA-like structure.
- BMV RNA synthesis is amenable to biochemical studies because the viral RdRp can use exogenously added templates containing BMV promoter sequences. Accurate initiation of
- (-) -strand RNA synthesis from input (+) -strand templates has been demonstrated.
- steps in (-) -strand RNA synthesis have been defined, including initiation, primer- induced RNA synthesis, the synthesis of abortive initiation products of up to 8 nts accumulating at a 10-fold molar excess to full-length RNA (Sun et al . , 1996) and the transition of the RdRp from initiation to elongation (Sun & Kao, 1997a, 1997b) .
- the mechanism of subgenomic RNA synthesis has not been carefully studied.
- Short regions of (-) -strand RNA3 have been employed to refine previous characterizations of the subgenomic promoter and determine how the RdRp recognizes the promoter (Adkins et al . , 1997; Siegel et al . , 1997). As described hereinbelow, the mechanism of subgenomic (+) -strand RNA synthesis, including initiation and termination, is discerned and compared to (-) -strand synthesis.
- DdRp DNA-dependent RNA polymerases
- DNA-dependent RNA synthesis has been divided into a number of biochemically distinct steps: binding of the DdRp to the promoter, formation of a transcriptionally active open complex, synthesis of the first phosphodiester bond, abortive RNA synthesis, promoter clearance, processive elongation and termination.
- the progression of these steps is accompanied by increases in the affinity of the interaction between the polymerase and the template, with commitment of the polymerase to the template taking place during/soon after the first translocation step.
- the committed polymerase is thought to remain stably associated with the template even though additional nucleotides needed for elongation may be lacking in the reaction) .
- RNA-dependent RNA polymerases RdRp
- the genomic (+) -strand RNA serves as a template for synthesis of (-) -strand RNA which, in turn, serves as a template for synthesis of additional copies of genomic (+) -strand RNA and, in many viruses, (+) -strand subgenomic RNAs.
- RNA synthesis by RdRp may provide the foundation for studies of RNA repair and recombination and allow a comparison to RNA synthesis by DdRp.
- Results from previous characterization of in vi tro RNA synthesis by the BMV RdRp defined several steps, including: (1) initiation of RNA synthesis at the penultimate cytidylate at the 3' end of BMV (+) -strand templates (Miller et al . , 1985, Na ture (London) , 31S, 68-70; Kao & Sun, 1996, J. Virol . , X, 6826-6830), (2) abortive oligoribonucleotide synthesis (Sun et al .
- RNA synthesis by RdRp appears to mirror those seen in transcription by DdRps, including the release of abortive initiation products and the progression to elongation after the synthesis of nascent RNAs of 8 to 10 nt. This is perhaps not surprising since the catalytic subunits of all polymerases share common structural and functional motifs. Despite the overall similarities in RNA synthesis by DdRps and RdRp, several differences should be mentioned.
- RdRp usually initiates RNA synthesis from the ends of RNA templates rather than exclusively from a promoter within a DNA molecule as does DdRp (Miller et al . , 1986, J. Mol . Riol . 18 7, 537-546; Ishihama and Nagata, 1988; Kao and Sun, 1996, , J. Virol . , X, 6826-6830).
- RdRp appears to dissociate from the template during the abortive initiation step (Sun and Kao, 1997), whereas the T7 RNA polymerase remains more stably bound to supercoiled DNA, although the stability of the T7 RNA polymerase-DNA interaction is highly dependent on the structure of the template (Diaz et al . , 1996, Biochemistry 35, 10837-10843) .
- stability of the DdRp ternary complex is maintained primarily by RNA- protein and DNA-protein interactions, and not by RNA-DNA interactions (Altmann et al . , 1994, Proc Na tl Acad Sci U S A. 91, 3784-3788) .
- RdRp For RdRp, it is possible that for some viruses, an intermediate of (-) -strand RNA synthesis is a double-stranded hybrid composed of the nascent and template RNAs (Baltimore, 1968; Takeda et al . , 1986; Bienz et al . , 1992; de Graaffet al . , 1995). If true, then the duplex may contribute to the stability of RdRp ternary complex.
- a 33 nucleotide prescript (-20/13) was constructed which contains the WT promoter sequence 20 nucleotide 3 ' of the subgenomic initiation start site in (- ) -strand RNA3.
- Chemical synthesis of the prescripts containing base analogs were performed on a ABI 394 automated DNA synthesizer (ABI, Foster city, CA) using conventional phosphoramidite elongation cycles according to Wincott et. al., 1995. Nucleic Acids Res, 8, 1421.
- This prescript directs the synthesis of a 13-nt product, the first 11 nt of which are BMV sequence followed by two guanylates added by T7 RNA polymerase to allow labeling of RdRp products with [ ⁇ -32P]CTP.
- the BMV sequence within proscript -20/13 is complementary to the viral (+)- strand RNA3 from positions 1,222 to 1,252 and serves as the WT control.
- subgenomic synthesis was relatively unaffected by replacement of nucleotides from -2 to the initiation site, +1.
- Previous work has demonstrated that the identity of the initiating cytidylate for the subgenomic RNA4 must be maintained.
- the three nucleotide transversion in the -2+1 proscript places a cytidylate at position -1 and it is conceivable that the BMV RdRp is able to utilize this nucleotide for initiation of RNA synthesis directing a product of the same size as the WT proscript by either facilitating the use of a nontemplated guanylate (instead of the templated cytidylate at the +1 position) or bypassing the +1 position completely.
- Proscript +1C/G specifically replaces the initiating cytidylate with a guanylate while leaving all other positions as WT sequence.
- Proscript - 1/+1 G inserts a guanylate into the initiation site and moves the WT +1 cytidylate into the +2 position.
- This proscript encodes a 14 nt product (15 nt with nontemplated addition) if the inserted guanylate at the +1 position is recognized by the BMV RdRp.
- the RdRp products from proscripts -2+1, - 1/+1 G. and WT -20/13 were all initiated with GTP as judged by the reactions lacking GTP and by RNase Tl treatment and are the same size (13 and 14 nt) as T7 size markers. However, no product was synthesized from the +1 G/G proscript. Thus, a cytidylate residue is required as the initiating nucleotide, confirming with earlier observations. It appears that the BMV RdRp can recognize a cytidylate at either one nucleotide 3' or 5' proximal to the original initiation site.
- Positions - 17, - 14, - 13, - 11, - 10, and -5 were important for synthesis because transversions significantly decreased RNA synthesis by the BMV RdRp.
- proscript -20/13 competed efficiently for synthesis because it also contains a WT subgenomic promoter. Synthesis from - 20/15 was reduced 50% when -20/13 was present in an equimolar amount. In contrast, proscript - 17 G/C was not an effective competitor, decreasing synthesis from -20/ 15 by only 15% when present at an equimolar ratio. The - 17 G/U proscript inhibited synthesis from -20/15 at a reduced level (35%) from that observed with -20/13. This level of inhibition is consistent with the result that synthesis from this mutant template was diminished but not abolished. The result from this experiment is consistent with the idea that position - 17, and most likely the other key nucleotides identified by mutagenesis, is contacted by the BMV RdRp.
- Alphaviral Subgenomic Promoters A comparison of the BMV subgenomic promoter with those of other members of the alphavirus-like super-family, infecting both plants and animals, reveals a striking conservation of the four nucleotides critical for synthesis by the BMV RdRp. In addition, all of these promoters contain a pyrimidine as the initiating nucleotide (uridylate in animal viruses and either a cytidylate or uridylate in plant viruses) with a highly conserved adenylate at the +2 position. This similarity implies that RdRps from members of the alphavirus-like superfamily recognize subgenomic promoters by a conserved mechanism (see for example Tables 1-3) .
- a proscript containing the subgenomic promoter from SFV was constructed and tested its ability to be recognized by the BMV RdRp. So that the RNA product can be visually distinguished from the 13-nt product generated from the BMV proscript, the SFV proscript was designed to direct synthesis of an 1 1-nt product, the first 9 nt of which are WTSFV sequence followed by two guanylates added by T7 RNA polymerase to allow labeling of RdRp products with [ ⁇ -32P] CTP.
- the SFV proscript directs the BMV RdRp to synthesize a product which is dependent upon ATP, used only as the initiating nucleotide for subgenomic synthesis from this template.
- the amount of synthesis was only 0.25% of the synthesis from an equimolar amount of WT - 20/13 but significantly above background. This result demonstrates a heterologous interaction between an RdRp from a plant-infecting virus with an RNA template containing the subgenomic promoter from an animal-infecting virus.
- RdRp Activity Assays BMV RdRp was prepared from infected barley essentially as described by Sun et al. (1996) . RdRp preparations used in abortive initiation studies were passed through an additional PD10 (Pharmacia) gel filtration column to remove NTPs and other low molecular weight contaminants.
- Standard RdRp activity assays consisted of 43 I1L reactions containing 20 mM sodium glutamate (pH 8.2), 4 mM MgCI2, 12 mM dithiothreitol, 0.5% (v/v) Triton X-100, 2 mM MnC12, 200 ⁇ M ATP, 500 ⁇ M GTP, 200 , ⁇ M UTP, 242 nM [a-32P]CTP (400 Ci/mmol, 10 mCi/mL, Amersham) , equal moles (generally 1.0 pmol) template RNA, and 5- 10 ⁇ L RdRp. Reactions were incubated 90 minutes at 30 °C unless indicated otherwise. Reaction products were extracted with phenol/chloroform (1 : 1, v/v) and precipitated with three volumes of ethanol and 10 ⁇ g glycogen following standard protocols (Sambrook et al., 1989) .
- Denaturing loading buffer was added to Sl- treated products prior to analysis by denaturing polyacrylamide gel electrophoresis on 5% acrylamide gels while native loading buffer (5% (v/v) glycerol, 0.04% (w/v) bromophenol blue, 0.04% (w/v xylene cyanol) was added to S 1 -treated products prior to analysis by non-denaturing electrophoresis on 1% agarose gels. All gels were exposed to film at -80°C and the amount of label incorporated into newly synthesized RNAs was determined with a phosphorimager (Molecular Dynamics) .
- PCR was used to synthesize cDNA copies of either (-) -strand BMV RNA3 encompassing the subgenomic promoter or (+) -strand BMV RNA3 or RNAl encompassing the (-) -strand promoter. Pairs of synthetic oligonucleotides, one of which contained a T7 promoter, were used in PCR reactions with cDNA clones of BMV RNA3 (pB3TP8) or RNAl (pBlTP3) , respectively (Janda et al., 1987) .
- PCR products were purified as described above (Sambrook et al., 1989) and used as templates for in vitro transcription.
- the T7 DdRp was used for all transcription reactions (Ampliscribe, Epicentre) (Table 4). Synthesis of the B3-198 template has been previously described (Sun & Kao, 1997a) .
- Molecular weight markers of 8 and 13 nts were synthesized by the protocol of Milligan et al.
- transcripts Prior to RdRp assays, transcripts were purified by anion exchange chromatography on Qiagen tip-20 columns using the manufacturer's protocol. All RdRp templates contain two non-viral guanylates at the 5' end incorporated during initiation of T7 polymerase transcription on the cDNA templates. Concentration of RdRp templates was determined by toluidine blue staining following denaturing PAGE and/or by using a spectrophotometer as previously described (Adkins et al., 1997) .
- Mutation of the +3 uridylate to a cytidylate or guanylate reduced the ability of the template to direct RNA synthesis to 64% or 58%, respectively, of the wild-type level. Mutation of the +4 adenylate to a cytidylate had no adverse effect on its ability to direct RNA synthesis. Mutation of the +4 adenylate to a guanylate reduced the ability of the template to direct RNA synthesis to 57% of the wild-type level. Thus, the effect of template sequence on RNA synthesis 5 appears to decrease as the distance from the initiation nucleotide increases.
- Each template directed synthesis of a 242 nt (-) -strand RNAl product containing a change of the +2 cytidylate to an adenylate or uridylate.
- Synthesis from B 1 242 +2 C/A or B 1 -242 +2 C/U was compared with synthesis from a second template (B3-198), which directed synthesis of a 198 nt (-)- strand RNA3 product, present at the same molar concentration in the same reaction.
- a change of the +2 cytidylate to a uridylate reduced synthesis to 31 % (similar to that previously observed by Sun et al .
- Primer GpU is complementary to the initiation sequence for subgenomic RNA synthesis while GpG is complementary to the initiation sequence for (-) -strand RNA synthesis. GDP is expected to serve as a primer for both subgenomic and (-) -strand RNA synthesis
- Control reactions contained 200 ⁇ M GTP and equimolar mixtures of templates for subgenomic (up/45) and genomic (- ) -strand (B3-198) synthesis and resulted in synthesis of approximately equal molar amounts of subgenomic and (-)- strand products.
- GTP was reduced to 4 ⁇ M
- synthesis of both subgenomic and (-) -strand products decreased to 1.5% of that observed at 200 ⁇ M GTP.
- the addition of GpG to reactions to final concentrations of 250-1250 ⁇ M stimulated (-) strand synthesis from 7 to 10-fold over the basal level while subgenomic RNA synthesis remained unchanged.
- [ ⁇ -32P]ATP was used as a label in these experiments due to the lack of cytidylates in the expected product prior to position +14 and the inefficient labeling observed with UTP (noted above) . Greater synthesis of full-length products was consistently observed with [ ⁇ - 32P]CTP than with [ ⁇ -32P]ATP, perhaps due to ATP hydrolysis by the BMV 1 a helicase-like protein, a component of RdRp.
- oligonucleotides were observed during synthesis from proscript 12/26 (containing an 8 nt polyuridylate tract and directing synthesis of a 26 nt subgenomic product) and also during synthesis of full-length subgenomic RNA from (-) -strand RNA3.
- the oligonucleotides were 6, 7, and 9 nts in size by comparison to the T7 DdRp- generated RNAs of the sequences 5' GUAUUA 3', 5' GUAUUAA 3' and 5 ' GUAUUAAUA 3 ' .
- the RdRp-produced oligonucleotides of 6, 7 and 9 nt were in 12, 7 and 3-fold molar excess, respectively, to the full-length 26 nt product and in 20, 8 and 7-fold molar excess, respectively, to the full-length subgenomic RNA as determined by phosphorimager quantitation. Other sizes of oligonucleotides were present but observed less reproducibly.
- RNAs While some endogenous BMV RNA was present in the RdRp preparation and directed synthesis of high molecular weight products in the absence of added template, no oligonucleotides were synthesized unless a (-) -strand RNA3 template was added. The oligonucleotide products were judged to be correctly initiated based on the following lines of evidence. Labeling of both oligonucleotides and full-length products was significantly reduced or eliminated when GTP, the initiating nucleotide, was omitted from the reactions. Correctly initiated RNAs do not contain a cytidylate until position +14. Thus, abortive products should lack cytidylates.
- RNA and elongated products were detectable when the ATP concentration was increased to 30 ⁇ M indicating that the synthesis of oligonucleotides is an innate property of the BMV RdRp and not due simply to limiting substrates.
- MnC12 increases synthesis of full-length products by 8-28% and the synthesis of the 9 nt RNA by 95- 127%.
- Addition of MnC12 to more than 2 mM reduced synthesis of both oligonucleotides and elongated products although elongated products were more sensitive at lower MnC12 concentrations.
- Example 3 Inhibition of RNA Synthesis Using a Competitor Nucleic Acid Molecule To determine whether short RNAs corresponding to the initiation sequence can inhibit RNA synthesis, an in vitro assay was employed (Table 4) . The reaction was performed with a tester template and increasing concentrations of the 20 competitor RNA. The results were then plotted as the percentage of activity of the tester template in the presence of increasing concentrations of inhibitors. This plot yielded thVe inhibitor concentration needed to reduce synthesis by 50% (I50)
- a competitor RNA of 33 nts which contained the wild-type promoter and initiation sequence reduced the synthesis from the tester template (wt-15) .
- the concentration for the wt-13 RNA needed to reduce wt-15 synthesis to 50% (I50) was 20 nM.
- an RNA of 23 nt containing the promoter and an additional three nucleotides was employed. This RNA had an I 50 of 80 nM.
- the control, an RNA which did not contain an initiation nucleotide (-20/-1) had an I 50 much greater than 250 nM. Moreover, no significant inhibition was observed with this template even when it was present at 250 nM.
- RdRp's ability to recognize and accurately initiate RNA synthesis from a DNA version of the subgenomic promoter was tested.
- a 33-nt proscript (designated 20/13) was constructed which contains the WT promoter sequence directing the synthesis of a 13-nt product, the first 11-nts of which are BMV sequence followed by two guanylates which allow labeling of RdRp products with [a-32 P] CTP (12) .
- Standard assays consisted of 25 nM of proscript RNA (unless stated otherwise) with 10 ⁇ l of RdRp in a 40 ⁇ l reaction containing 20 mM sodium glutamate (pH 8.2), 4 mM MgC12, 12.5 mM dithiothreitol, 0.5% (v/v) Triton X-100, 2 mM MnC12, 200 ⁇ M ATP and UTP, 500 ⁇ M GTP, and 250 nM [a-32 PjCTP (Amersham) .
- the predominant RdRp product was 14-nts due to the nontemplated addition of one residue, a phenomenon common to many polymerases (Fig. 1, lanes 1 & 10) .
- the all DNA proscript designated d(-20/13), inserted deoxyriboses in every position while still containing otherwise WT subgenomic promoter and template sequences.
- This construct was able to direct RNA synthesis by RdRp (Fig. 1, lanes 2-8); however, the predominant product was now 13-nts rather than 14-nts as it was with the RNA template. This change may reflect the need for 2'-OHs in the template in order to efficiently add the nontemplated nucleotide.
- Product synthesis was resistant to inhibitors of DNA-dependent RNA polymerases, such as actinomycin D and rifampicin. Pretreating the d (-20/13) proscript with DNase I abolished product synthesis.
- RNA synthesis from the d(-l/13) template was verified as above; treatment with DNase I degraded the DNA template and abolished RNA synthesis while the product was resistant to DNase I, but degraded by RNase A (Fig. 1, lanes 16-18) .
- the RdRp from healthy tomato leaves has also been observed to initiate RNA synthesis from the end of a DNA template, but this initiation did not occur in a sequence-specific manner nor were the requirements for initiation fully characterized (Schiebel et al . , 1993, J Biol Chem 268, 11858) .
- RNA synthesis from the d(-l/13) template was verified as above; treatment with DNase I degraded the DNA template and abolished RNA synthesis while the product was resistant to DNase I, but degraded by RNase A (Fig. 1, lanes 16-18) .
- the RdRp from healthy tomato leaves has also been observed to initiate RNA synthesis from the end of a DNA template, but this initiation did not occur in a sequence-specific manner nor were the requirements for initiation fully characterized (Schiebel et al . , supra) .
- Hybrid proscripts containing both ribose and deoxyribose residues, were generated to determine the locations of residues that facilitate RNA synthesis by RdRp (Fig. 2) .
- Hybrid HI containing riboses only in the subgenomic promoter and the +1 and +2 positions, directed an increased amount of RNA synthesis (20%) relative to the d(- 20/13) proscript (6%) .
- synthesis was still below that obtained from the -20/13 WT proscript (Fig. 2, lanel vs. 2), indicating a preference for ribose residues in the template portion of the proscript.
- H3 which contains deoxyriboses at every position except those at -17, -14, - 13, -11, +1, and +2, directed a similar amount of RNA synthesis as that from the HI proscript (Fig. 2, lane 2 vs. 4) .
- Previously (11) applicant had found that the placement of a deoxyguanosine at position -17 in an otherwise RNA proscript had no effect on RNA synthesis, but a deoxyguanosine at position -11 reduced synthesis by over half relative to the -20/13 WT control (Fig.
- the -11 sugar containing a 2'-OH (ribose), 2'-OCH3 and 2'-F were chosen since these substitutions should be able to form the C3' endo conformation (16).
- 2'-NH2 which forms the C3' endo conformation at a reduced frequency.
- Potential H- bond acceptors include: 2' -OH, 2'-NH2, 2'-OCH3 and 2'-F while the 2'OH and 2'-NH2 can also act as hydrogen donors.
- the amount of RNA synthesis from the proscripts containing these replacements were determined (Fig. 2, lanes 8-11) .
- Proscript -20/13 WT reduced the level of 15-nt synthesis by half when present in the same molar ratio as the -20/15 proscript, generating an 150 of 25 nM (Fig. 3).
- the ability of d(- 20/13) to be bound by RdRp was only mildly affected, having an 150 value of 90 nM (Fig. 3) .
- This 3- to 4-fold reduction in 150 value was surprising given that d (-20/13) was reduced in the ability to direct RNA synthesis by over 15-fold relative to that from -20/13 WT proscript.
- DNA constructs of minimal lengths could be used as potential inhibitors of viral synthesis.
- DNA inhibitors containing a WT initiation sequence beginning at position -1 with increasing truncations at their 5' ends, were tested in template competition assays. As expected, all constructs were found to direct 13-, 8-, or 6-nt RNA products. All of these constructs also effectively reduced synthesis from the RNA -20/15 proscript in a manner dependent on the length of the 5' sequence (Fig. 4) . As a negative control, the d(-l/13) Rev proscript which does not contain the WT initiation sequence was not able to inhibit synthesis over the range of inhibitor tested (500-fold molar excess) .
- the sequence-specific reduction of viral RNA synthesis in vitro by relatively stable DNA inhibitors should allow the rational design of viral therapeutics.
- RdRp has the ability to recognize and initiate accurate RNA synthesis from either an internal or terminal initiation site on a DNA template.
- the functional and binding data from chemically synthesized proscripts suggest that the 2' -OH at position - 11 may be involved in hydrogen bonding with RdRp during the initiation of RNA synthesis, either directly or through a water molecule.
- ribonucleotides in the template portion of the proscript may be needed to direct WT levels of RNA synthesis, perhaps by stabilizing the conformational change in the polymerase as it translocates out of the initiation stage (17) .
- PCR Polymerase chain reaction
- pairs of primers one of which contained a T7 promoter, allowed proscript RNAs to be generated using T7 RNA polymerase (Ampliscribe, Epicentre) as described previously (5) .
- Transcription reactions which incorporate inosines were preformed with 2 mM of the primer GpG which allows initiation to take place and ITP in place of GTP in the reaction.
- RNAs were purified with Qiagen columns (Chatsworth, CA) using the manufacturer's protocol to remove NTPs and proteins remaining from the T7 transcription reaction. RNAs were visually inspected by denaturing polyacrylamide gel electrophoresis (PAGE) and quantified by UV absorbance. Synthesis of 2' -O-TBDMSi-3' -O-phosphoramidites of purine riboside (Liu et al . , 1997, J. Mol . Biol . 267, 163- 171), 2-amino purine riboside (Konforti et al . , 1998, J. Mol.
- Standard assays consisted of 25 nM of template RNA (unless stated otherwise) with 10 ⁇ l of RdRp in a 40 ⁇ l reaction containing 20 mM sodium glutamate (pH 8.2), 4 mM MgC12, 12.5 mM dithiothreitol, 0.5% (v/v) Triton X-100, 2 mM MnC12, 200 ⁇ M ATP and UTP, 500 ⁇ M GTP, and 250 nM [ ⁇ -32 P]CTP (Amersham) .
- Nucleotides located at positions -17, -14, -13, and -11 relative to the subgenomic initiation site (+1) were required for RNA synthesis and predictions were made regarding the functional moieties mediating this effect (Fig. 5A) (Siegel et al . , supra ) .
- RNAs containing base analogs at each of the four critical positions were made to determine the importance of these functional groups (Fig. 5) .
- a 33-nt proscript (designated - 20/13) was constructed which contains the WT promoter sequence 20-nts 3' of the subgenomic initiation start site in (-) -strand RNA3.
- This proscript directs the synthesis of a 13-nt product, the first 11-nts of which are BMV sequence followed by two guanylates which allow labeling of RdRp products with [ -32 P] CTP (Fig. 5B and Fig 5C, lane WT) .
- Applicant first examined the recognition of the guanylate at position -17 (Fig. 5B) .
- the C6 keto (and possibly the Nl) group was predicted to interact with the BMV RdRp.
- a proscript RNA containing the base analog 2- aminopurine which removes the C6 keto group and forms a double bond with the Nl moiety decreased RNA synthesis to background levels (Fig. 5B, lane 1) .
- the mutational analysis did not implicate roles for the C2 amine and the N7 i ine (Siegel et al . , supra) .
- the more limited role of the C2 amine in interaction with RdRp can be surmised by the severity of the 2-aminopurine substitution which indicates that the C6 keto and possibly the Nl groups are the determining moieties.
- As negative control a change of the -17 ribose to a deoxyribose was unaffected in RdRp recognition (Fig. 5B, lane 2) .
- RNA synthesis was not reduced to background levels, consistent with the prediction that the N7 imine was also important.
- the N7 deaza base analog was only available in a deoxyribose form; therefore, a control RNA with deoxyguanosine at position -11 was first tested.
- a deoxyribose at position -11 reduced RNA synthesis to 46% of wildtype (Fig. 5B, lane 5) , implicating that the RNA backbone does mediate some aspect of subgenomic promoter recognition.
- Removal of both the N7 imine and the 2' -OH further reduced synthesis to 25% of wildtype, indicating recognition of the predicted N7 imine (Fig 5B, lane 6) . Again RNA synthesis was not abolished since this base analog retained the C6 keto group.
- a likely candidate for RdRp recognition of the adenylate at position -14 is the exocyclic C6 amine group.
- a purine riboside analog (Liu et al . , supra ) was substituted for the adenylate at this position.
- An RNA with this change retained synthesis at 60% of wildtype, suggesting some other features of the adenylate are important for recognition by RdRp (Fig 5C, lane 1) .
- the -13 cytidylate was predicted to interact with RdRp by the exocyclic C4 amine group.
- a proscript containing the base analog pyrimidine-2-one (12) which specifically removes this functional group reduced the level of RNA synthesis to 7% (Fig. 5C, lane 2) .
- RdRp contacts within the subgenomic promoter A template competition assay was used to test whether various promoter mutations affected the ability of the RNA to interact with the BMV RdRp (Table 6) .
- the amount of synthesis from a WT promoter directing the production of a 15-nt product (proscript -20/15) was determined in the absence and presence of various competitor templates. If a mutant proscript used as a competitor had lost its ability to be recognized by RdRp, then its presence in a reaction should not adversely affect the amount of synthesis from the WT -20/15 proscript.
- the WT proscript -20/13 (generating a 13-nt product) was tested for its ability to inhibit synthesis of the 15-nt product from -20/15.
- this proscript was present in the same molar amount as proscript -20/15, the level of 15-nt synthesis was reduced by half. Mutations at positions -14, -13, and -11 which abolished the ability to direct synthesis did not inhibit the BMV RdRp from productively interacting with a WT promoter even when present in molar excess (Table 6) ; the same result as was obtained with mutations at position -17 (Siegal et al., supra).
- Minimal template needed for RdRp to interact with the subgenomic promoter In order to delineate the length of template required for stable interaction with RdRp, proscripts containing template truncations were constructed and tested for their ability to inhibit synthesis of the 15- nt product from proscript -20/15. The concentration of the competitor proscript required to reduce the activity from - 20/15 by 50% was termed the 150 value. This refinement of the template competition assay allowed us to compare various proscripts' ability to interact with RdRp. Better competitors (able to interact more strongly with RdRp and, therefore, reduce synthesis from proscript -20/15) will have lower 150 values.
- truncated proscripts all contained WT promoter sequences but varied in the length of their templates from 13-, 5-, 3-, 2-, 1-, and 0-nts (Fig. 6A) .
- these truncated proscripts all contained a mutated initiation site (+lc/g) .
- Comparison of the 150 value for proscripts -20/13 +lc/g and -20/13 WT revealed a 5-fold difference in the ability to inhibit synthesis of the 15-nt product (Fig. 6B) , consistent with the results observed in Table 6.
- the nucleic acid molecules of the invention may be utilized to prevent viral replication by inhibiting RNA transcription. This is particularly effective when the virus uses its own specific viral polymerase rather than the host cell's mechanism for RNA synthesis.
- Brome Mosaic Virus uses RNA dependent RNA polymerase to generate viral RNA. Because the polymerase is specific to the virus it may be inhibited without effecting the normal cellular mechanism of the host cell. This reduces the potential for side effects and allows for a virus specific therapeutic. Other RNA viruses may be inhibited using this technology if the virus utilizes a viral polymerase to transcribe RNA. The concept of inhibition using nucleic acid molecules is shown in figure 9.
- Figure 9A demonstrates normal initiation of RNA transcription while figures 9B and 9C demonstrate the effects of the nucleic acid molecule inhibitor.
- the nucleic acid molecules of the present invention may also be useful diagnostic applications, ie., by using the molecules for the detection of viral polymerases.
- One measure of viral burden may be the quantity of viral polymerase expressed within relevant cell types or patients.
- a diagnostic kit may be designed wherein the binding of the nucleic acid molecule and viral polymerase obtained from a patient, releases a fluorimetric or colorimetric signal. This signal may be detected and quantified to determine the level of viral load within a patient.
- Promoter Location initiates internally using a promoter distinct from template while (-) -strand synthesis initiates near end of template using a promoter contained within template
- Primer use Primers used when (-) -strand synthesis
- Termination Sequence-independent Table 2 Comparison of initiation sequences for genomic ( + ) -strand, subgenomic and genomic ( - ) -strand RNA synthesis in the alphavirus-like superfamily .
Abstract
Description
Claims
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AU53169/99A AU5316999A (en) | 1998-07-20 | 1999-07-15 | Use of nucleic acid molecules as antiviral agents |
EP99938756A EP1098969A2 (en) | 1998-07-20 | 1999-07-15 | Use of nucleic acid molecules as antiviral agents |
JP2000560239A JP2002520051A (en) | 1998-07-20 | 1999-07-15 | Use of nucleic acid molecules as antiviral agents |
CA002334161A CA2334161A1 (en) | 1998-07-20 | 1999-07-15 | Use of nucleic acid molecules as antiviral agents |
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US60/093,489 | 1998-10-26 |
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JP (1) | JP2002520051A (en) |
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2005500055A (en) * | 2001-08-07 | 2005-01-06 | ベーリンガー インゲルハイム (カナダ) リミテッド | Competitive binding assay identifying HCV polymerase inhibitors |
WO2007119889A1 (en) | 2006-04-18 | 2007-10-25 | Japan Tobacco Inc. | Novel piperazine compound, and use thereof as hcv polymerase inhibitor |
US7659263B2 (en) | 2004-11-12 | 2010-02-09 | Japan Tobacco Inc. | Thienopyrrole compound and use thereof as HCV polymerase inhibitor |
EP2206715A1 (en) | 2004-02-24 | 2010-07-14 | Japan Tobacco, Inc. | Fused heterotetracyclic compounds and use thereof as hcv polymerase inhibitor |
US7977331B1 (en) | 2004-02-24 | 2011-07-12 | Japan Tobacco Inc. | Tetracyclic fused heterocyclic compound and use thereof as HCV polymerase inhibitor |
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WO1997010328A2 (en) * | 1995-07-13 | 1997-03-20 | Ribozyme Pharmaceuticals, Inc. | Compositions and method for modulation of gene expression in plants |
WO1997026270A2 (en) * | 1996-01-16 | 1997-07-24 | Ribozyme Pharmaceuticals, Inc. | Synthesis of methoxy nucleosides and enzymatic nucleic acid molecules |
WO1998003668A1 (en) * | 1996-07-22 | 1998-01-29 | Zeneca Limited | Virus resistance in plants |
WO1999049031A1 (en) * | 1998-03-26 | 1999-09-30 | Advanced Research And Technology Institute, Inc. | Use of nucleic acid molecules as antiviral agents |
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1999
- 1999-07-15 AU AU53169/99A patent/AU5316999A/en not_active Abandoned
- 1999-07-15 CA CA002334161A patent/CA2334161A1/en not_active Abandoned
- 1999-07-15 WO PCT/US1999/016253 patent/WO2000004141A2/en not_active Application Discontinuation
- 1999-07-15 EP EP99938756A patent/EP1098969A2/en not_active Withdrawn
- 1999-07-15 JP JP2000560239A patent/JP2002520051A/en active Pending
Patent Citations (4)
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WO1997010328A2 (en) * | 1995-07-13 | 1997-03-20 | Ribozyme Pharmaceuticals, Inc. | Compositions and method for modulation of gene expression in plants |
WO1997026270A2 (en) * | 1996-01-16 | 1997-07-24 | Ribozyme Pharmaceuticals, Inc. | Synthesis of methoxy nucleosides and enzymatic nucleic acid molecules |
WO1998003668A1 (en) * | 1996-07-22 | 1998-01-29 | Zeneca Limited | Virus resistance in plants |
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ADKINS S ET AL: "Minimal templates directing accurate initiation of subgenomic RNA synthesis in vitro by the brome mosaic virus RNA-dependent RNA polymerase." RNA, (1997 JUN) 3 (6) 634-47., XP002110077 cited in the application * |
ADKINS S ET AL: "Subgenomic RNA promoters dictate the mode of recognition by bromoviral RNA-dependent RNA polymerases." VIROLOGY, (1998 DEC 5) 252 (1) 1-8., XP002110079 * |
HUNTLEY C C ET AL: "MINUS SENSE TRANSCRIPTS OF BROME MOSAIC VIRUS RNA-3 INTERCISTRONIC REGION INTERFERE WITH VIRAL REPLICATION" VIROLOGY, vol. 192, 1993, pages 290-297, XP002047298 ISSN: 0042-6822 * |
SIEGEL, R. ET AL.: "Moieties in an RNA promoter specifically recognized by a viral RNA-dependent RNA polymerase" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA., vol. 95, September 1998 (1998-09), pages 11613-11618, XP002110078 ISSN: 0027-8424 * |
SIEGEL, R. ET AL.: "Sequence-specific recognition of a subgenomic RNA promoter by a viral RNA polymerase" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA., vol. 94, October 1997 (1997-10), pages 11238-11243, XP002110076 ISSN: 0027-8424 cited in the application * |
ZACCOMER B ET AL: "TRANSGENIC PLANTS THAT EXPRESS GENES INCLUDING THE 3' UNTRANSLATED REGION OF THE TURNIP YELLOW MOSAIC VIRUS (TYMV) GENOME ARE PARTIALLY PROTECTED AGAINST TYMV INFECTION" GENE, vol. 136, 1 January 1993 (1993-01-01), pages 87-94, XP002047299 ISSN: 0378-1119 * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2005500055A (en) * | 2001-08-07 | 2005-01-06 | ベーリンガー インゲルハイム (カナダ) リミテッド | Competitive binding assay identifying HCV polymerase inhibitors |
EP2206715A1 (en) | 2004-02-24 | 2010-07-14 | Japan Tobacco, Inc. | Fused heterotetracyclic compounds and use thereof as hcv polymerase inhibitor |
US7977331B1 (en) | 2004-02-24 | 2011-07-12 | Japan Tobacco Inc. | Tetracyclic fused heterocyclic compound and use thereof as HCV polymerase inhibitor |
US7659263B2 (en) | 2004-11-12 | 2010-02-09 | Japan Tobacco Inc. | Thienopyrrole compound and use thereof as HCV polymerase inhibitor |
WO2007119889A1 (en) | 2006-04-18 | 2007-10-25 | Japan Tobacco Inc. | Novel piperazine compound, and use thereof as hcv polymerase inhibitor |
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AU5316999A (en) | 2000-02-07 |
JP2002520051A (en) | 2002-07-09 |
EP1098969A2 (en) | 2001-05-16 |
WO2000004141A3 (en) | 2000-04-27 |
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