OLIGONUCLEOTIDES WITH ANTI-RESPIRATORY SYNCYTIAL VIRUS ACTIVITY

Abstract: Disclosed are oligonucleotides which hybridize to a portion of respiratory syncytial virus (RSV) genomic RNA under physiological conditions, and, in doing so, inhibit viral replication. Also disclosed are pharmaceutical compositions and methods useful for inhibiting and treating RSV infection and including at least one or two of these oligonucleotides, or at least one of these oligonucleotides and ribavirin.

Description: OLIGONUC EOTIDES WITH ANTI-RESPIRATORY SYNCYTIAL VIRUS ACTIVITY BACKGROUND OF THE INVENTION This invention relates to respiratory syncytial virus infection. More particularly, this invention relates to oligonucleotides complementary to portions of the genome of the virus which, when hybridized to it, inhibit viral replication. Respiratory syncytial virus (RSV) is a major cause of pneumonia, bronchiolitis, and other respiratory diseases in infants, children, and even adults (Hruska et al . (1980) Antimicrob. Ag. Chemother. 17 : 770-775; Kim et al . (1973) Am. J. Epidem. 98:216-225; Mclntosh et al . (1985) Virol. 1985:1285- 1304) . Mortality resulting from RSV infection has been estimated as 0.5% in infected children (Report to the Medical Research Council (1978) Br. Med. J. 2:796-798) , but this may increase significantly in immunocompromised infants (Hall et al. (1981) Pediatr. 15:613) or those with congenital heart disease (MacDonald et al . (1982) N. Engl. J. Med. 307 :397-400) . RSV is a member of the Paramyxoviridae family and comprises the genus Pneumovirus . It is an enveloped virus approximately 150 to 300 nm in size, so named because virus replication leads to fusion of neighboring cells into large multinucleated syncytia (Chanock et al . (1957) Am. J. Hyg. 66:281-290) . The single-stranded RNA genome codes for ten virus-specific proteins. This negative stranded genome is contained in a helical nucleocapsid surrounded by a lipid envelope bearing two glycoproteins, one of which is the fusion protein which facilitates entry of virus into the cell by fusing host and viral membranes . Upon entry, portions of the RSV genomic RNA are transcribed and translated, yielding viral proteins that mediate subsequent steps in virus reproduction. The next step is replication of the genome, which provides additional templates for transcription and further genome replication, amplifying the populations of virus-specific macromolecules within the cell, and supplying progeny genomes in large amounts for incorporation into new virus particles . In genome replication, the entire base sequence of the negative-strand RNA template must be conserved as a single entity. Therefore, the sequences that are not expressed as mRNA during transcriptive RNA synthesis must be incorporated into the positive-strand RNA (antigenome) that serves as an intermediate template for the synthesis of progeny negative-strand genomes. This requires that the RNA synthesizing machinery enter an antitermination mode, ignoring all of the signals at gene boundaries and at the boundary between the leader RNA template and various viral genes . At present, the only clinically recognized treatment for RSV infection is the broad-spectrum antiviral drug ribavirin (1-β-D-ribofuranosyl- 1, 2, 4-triazole-3-carb-oxamide) , a synthetic nucleoside resembling guanosine. It has been proposed that this drug functions by decreasing the intracellular concentration of GTP due to competitive inhibition of IMP dehydrogenase and two virus-specific actions, by inhibiting the function of virus-coded RNA polymerases necessary to prime and elongate viral mRNAs (reviewed in Gilbert et al . (1986) Antimicrob. Ag. Chemother. 30:201- 205) . When administered in aerosol form, ribavirin has been reported to reduce the severity of illness and the amount of virus shed (Hall et al. (1983) N. Eng. J. Med. 308:1443-1447) . However, there is as yet no evidence that it decreases the duration of hospitalization or diminishes the need for supportive therapies. Studies in experimental animals suggest that it may be possible to use immunotherapy for the prevention or treatment of serious RSV lower respiratory tract disease of infants and young children (Prince et al . (1985) Virus Res. 3:193-206; Prince et al . (1987) J. Virol. 61:1851-1854; Mclntosh et al . , "Respiratory Syncytial Virus" in Virology (2d ed.) Raven Press, New York (1990) pp.1045-1072) . However, the unusual age distribution of RSV bronchiolitis and pneumonia constitutes a major obstacle to the development of an effective vaccine for prevention of serious disease during very early infancy. If a vaccine is to have a major impact on RSV infection, it must be capable of stimulating effective resistance by the second month of life, since the peak incidence of disease occurs at this time (Parrott et al . (1973) Am. J. Epidemiol. 98:289-290) . Unfortunately in this age group immunosuppression by maternally derived antibodies and immunologic immaturity act to decrease vaccine efficacy (Murphy et al . (1986) J. Clin. Microbiol. 23:1009-1014; Murphy (1988) J. Virol. 62:3907-3910) . Vaccine safety and reactogenicity, especially in the case of live vaccines, may also pose special problems in this group. Hence, alternative antiviral strategies are needed. Recently, new chemotherapeutic agents have been developed which are capable of modulating cellular and foreign gene expression. These agents, called antisense oligonucleotides, bind to a target single-stranded nucleic acid molecules according to the Watson-Crick or the Hoogsteen rule of base pairing, and in doing so, disrupt the function of the target by one of several mechanisms : by preventing the binding of factors required for normal transcription, splicing, or translation; by triggering the enzymatic destruction of mRNA by RNase H, or by destroying the target via reactive groups attached directly to the antisense oligonucleotide. Antisense oligodeoxynucleotides have been designed to specifically inhibit the expression of HIV-1, influenza, and other viruses (see, e.g., Agrawal et al . , U.S. Patent No. 5,194,428; Pederson et al . , U.S. Patent Nos . 5,149,797; Agr awa 1 (1992) Trends in Biotechnology 10:152-158; Agrawal et al . in Gene Regulation: Biology of Antisense RNA and DNA (Erickson and Izant, eds . ) Raven Press Ltd., New York (1992) pp. 273-283) ; Matsukura et al . in Prospects for Antisense Nucleic Acid Therapy of Cancer and AIDS, Wiley-Liss, Inc. (1992) pp. 159-178; and Agrawal (1991) in Prospects for Antisense Nucleic Acid Therapy or Cancer and AIDS , (Wickstrom, ed.) Liss, New York, pp. 145-148) . A need remains for the development of oligonucleotides that are capable of inhibiting the replication or propagation of respiratory syncytial virus and whose uses are accompanied by low or no cellular toxicity. SUMMARY OF THE INVENTION The present invention provides synthetic oligonucleotides which hybridize under normal physiological conditions to a portion of respiratory syncytial virus genomic RNA (vRNA) . As used herein, the term "synthetic oligonucleotide" includes chemically synthesized polymers of three or up to 100 and preferably from about 12 to about 50 ribonucleotide and/or deoxyribonucleotide monomers connected together or linked by at least one 5' to 3' internucleotide linkage. In some aspects of the invention, the oligonucleotides are modified. The term "modified oligonucleotide" is used herein as an oligonucleotide in which at least two of its nucleotides are covalently linked via a synthetic linkage, i.e., a linkage other than a phosphodiester between the 5' end of one nucleotide and the 3' end of another nucleotide in which the 5' nucleotide phosphate has been replaced with any number of chemical groups. Preferable synthetic linkages include alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, phosphoramidates, phosphate esters, carbamates, carbonates, phosphate triesters, acetamidate, and carboxymethyl esters . In one preferred embodiment of the invention, the oligonucleotide comprises at least one phosphorothioate linkage. The term "modified oligonucleotide" also encompasses oligonucleotides with a modified base and/or sugar. For example, a 3', 5' -substituted oligonucleotide is a modified oligonucleotide having a sugar which, at both its 3' and 5' positions is attached to a chemical group other than a hydroxyl group (at its 3' position) and other than a phosphate group (at its 5' position) . A modified oligonucleotide may also be a capped species. In addition, unoxidized or partially oxidized oligonucleotides having a substitution in one nonbridging oxygen per nucleotide in the molecule are also considered to be modified oligonucleotides. Also considered as modified oligonucleotides are oligonucleotides having nuclease resistance-conferring bulky substituents at their 3' and/or 5' end(s) and/or various other structural modifications not found in vivo without human intervention are also considered herein as modified. The oligonucleotides of the invention are complementary to a portion of the vRNA and hybridize to the vRNA under normal physiological conditions existing within a cell harboring the vRNA. Such conditions include pH, temperature, and ionic conditions. The portion of the vRNA to which the oligonucleotides of the invention are complementary includes any region of the genome. This region is at least 6, and preferably 12 to 60 nucleotides in length. In some preferred embodiments, the oligonucleotides are complementary to 15 to 25 nucleotides of the RSV vRNA. In some embodiments, the region is at least a portion of the leader, NS1, NS2, and P RSV genes such as nucleotides (starting from the 3' end of vRNA) 1-20 (leader region) , 31-50 or 41-60 (NSl/leader) , and 590-609 (NS2/P) . Oligonucleotides complementary to these regions have the nucleotides sequences set forth herein as SEQ ID N0:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively. In some aspects, the invention provides an oligonucleotide which has antiviral activity against respiratory syncytial virus effected by hybridization with a portion of the vRNA. In others, a pharmaceutical composition including at least one of the oligonucleotides of the invention, and in some embodiments, at least two different oligonucleotides of the invention, and a pharmaceutically acceptable carrier are provided. In yet other embodiments, the composition further includes ribavirin. The pharmaceutical composition is used in a method of inhibiting, preventing, and/or reducing RSV replication in a cell. In this method, a therapeutic amount of the pharmaceutical composition is administered to the cell which is to be protected from infection or treated for an existing infection. The oligonucleotide (or oligonucleotides) in the pharmaceutical composition enters the cell, wherein it (they) hybridize (s) to RSV vRNA, thereby inhibiting RSV replication. Ribavirin in ribavirin-containing compositions inhibits replication of vRNA in the cell in three possible ways: it causes a decrease in the intracellular concentration of GTP; it inhibits 5' -cap formation of mRNAs; and/or it inhibits the function of virus-coded RNA polymerases . The pharmaceutical composition is also utilized in a method of treating respiratory syncytial virus infection wherein the composition is administered to an infected mammal or cell . BRIEF DESCRIPTION OF THE DRAWINGS The foregoing and other objects of the present invention, the various features thereof, as well a:- the invention itself may be more fully understock from the following description, when read together with the accompanying drawings in which: FIG. 1 is a diagrammatic representation of the genomic map of RSV (3' to 5') , wherein gene boundaries are indicated by vertical lines and gene lengths of intergenic, gene overlap, and proposed leader and trailer regions are indicated in number of bases beneath the map; FIG. 2 is a schematic representation of the partial nucleotide sequences of the RSV genes NSl, NS2, N, P, M, SH, G, F, 22K, and L; FIG. 3 is a graphic representation of the effect of various oligonucleotides on RSV replication in vitro : -D-, unrelated oligonucleotide, -0-, vRNA 1-20 (SEQ ID N0:1) ; -o-, vRNA 590-609 (SEQ ID N0:4) ; -Δ-, vRNA 41-60 (SEQ ID NO:3) ; FIG. 4 is a graphic representation of the effect of ribavirin (-D-) and various oligonucleotides: -0-, NS2 1-20 (SEQ ID NO:6) ; -o-, NS2 aug (SEQ ID NO:5); -Δ-, vRNA 31-50 (SEQ ID NO:2) on RSV replication in vitro ; FIG. 5 is a graphic representation of the effect of ribavirin (-□-) and various oligonucleotides: -0-, vRNA 590-609 (SEQ ID NO:4) ; -o-, vRNA 41-60 (SEQ ID NO:3) ; -Δ-, vRNA 31-50 (SEQ ID NO:2) ; and (-D-) , unrelated oligonucleotide on RSV replication in vitro,- FIG. 6 is a graphic representation of the percent difference between the expected inhibition of RSV replication and the measured inhibition of RSV replication obtained when two different oligonucleotides of the invention are used in combination; and FIG. 7 is a graphic representation of the percent difference between the expected inhibition of RSV replication and the measured inhibition of RSV replication obtained when one oligonucleotide of the invention and ribavirin are used in combination. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. The issued U.S. patent and allowed applications cited herein are hereby incorporated by reference. The present invention relates to oligonucleotides which are sufficiently complementary to regions of the RSV genome such that, under normal physiological conditions existing in the cell, they hybridize to those regions, rendering the genome unavailable to serve as a template for production of another genome strand or mRNA. As a result, RSV replication is inhibited. The oligonucleotides may be targeted to any region of the vRNA, such as those depicted in the simplified genomic map of FIG. 1, and schematically in FIG. 2. Examples of such oligonucleotides include those complementary to a portion of the leader region including nucleotides 1-20 (from the 3' end of the vRNA) , regions of the NSl gene, including nucleotides 31-50, and 41-60, and regions of the NS2/P genes, including nucleotides 590-609. These oligonucleotides are shown in TABLE 1 below and have the nucleotides sequences set forth herein as SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively. Oligonucleotides of the invention are composed of deoxyribonucleotides, ribonucleotides, or a combination of both, with the 5' end of one nucleotide and the.3' end of another nucleotide being covalently linked. . These oligonucleotides are at least 6 nucleotides in length, but are preferably 12 to 60 nucleotides long, with 15 to 25mers being the most common. They can be prepared by art-recognized methods such as phosphoramidate or H-phosphonate chemistry which can be carried out manually or by an automated synthesizer as described in Uhlmann et al . { Chem. Rev. (1990) 90:534-583) . The oligonucleotides of the invention may also be modified in a number of ways without compromising their ability to hybridize to RSV vRNA. For example, the oligonucleotides may contain other than phosphodiester internucleotide linkages between the 5' end of one nucleotide and the 3' end of another nucleotide in which the 5' nucleotide phosphate has been replaced with any number of chemical groups. Examples of such chemical groups include alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters. Oligonucleotides with these linkages can be prepared according to known methods (see, e.g., Uhlmann et al . (1990) Chem. Rev. 90:543-583) . Other modifications include those which are internal or at the end(s) of the oligonucleotide molecule and include additions to the molecule of the internucleoside phosphate linkages, such as cholesteryl or diamine compounds with varying numbers of carbon residues between the amino groups and terminal ribose, deoxyribose and phosphate modifications which cleave, or crosslink to the opposite chains or to associated enzymes or other proteins which bind to the viral genome. Examples of such modified oligonucleotides include oligonucleotides with a modified base and/or sugar such as arabinose instead of ribose, or a 3' , 5'- substituted oligonucleotide having a sugar which, at both its 3' and 5' positions is attached to a chemical group other than a hydroxyl group (at its 3' position) and other than a phosphate group (at its 5' position) . Other modified oligonucleotides are capped with a nuclease resistance-conferring bulky substituent at their 3' and/or 5' end(s) , or have a substitution in one nonbridging oxygen per nucleotide. Modifications of the oligonucleotides can be at some or all of the internucleoside linkages, as well as at either or both ends of the oligonucleotide and/or in the interior of the molecule. The preparation of these modified and unmodified oligonucleotides is well known in the art (reviewed in Agrawal et al . (1992) Trends Biotechnol. 10:152-158; Agrawal in Protocols for Oligonucleotides and Analogs, Synthesis and Properties (Agrawal, ed.) , Humana Press, Totowa, New Jersey (1993) , Chapter 20) . For example, nucleotides can be covalently linked using art- recognized techniques such as phosphoramidate, H- phosphonate chemistry, or methylphosphoramidate chemistry ( see, e.g., Uhlmann et al . (1990) Chem. Rev. 90:543-584; Agrawal et al . (1987) Tetrahedron. Lett. 28: (31) :3539-3542) ; Caruthers et al . (1987) Meth. Enzymol. 154:287-313; U.S. Patent 5,149,798) . Oligomeric phosphorothioate analogs can be prepared using methods well known in the field such as methoxyphosphoramidite {see , e.g. , Agrawal et al. (1988) Proc. Natl. Acad. Sci. (USA) 85:7079- 7083) or H-phosphonate (see, e.g. , Froehler (1986) Tetrahedron Lett. 27:5575-5578) chemistry. The synthetic methods described in Bergot et al. {J. Chromatog. (1992) 559:35-42) can also be used. The present invention further provides therapeutic compositions having antiviral activity against RSV, and including the oligonucleotides of the present invention, along with a physiologically acceptable carrier. As used herein, a "physiologically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions . This therapeutic formulation may be used to inhibit, reduce or prevent the replication of RSV in cells through the activity of the oligonucleotides in the composition. RSV-infected cell are then treated with the therapeutic formulation in an amount sufficient to enable the binding of the oligonucleotide to the vRNA in the infected cells. In this way, the binding of the oligonucleotide to the RSV RNA inhibits the expression and replication of the virus. Thus the oligonucleotides according to the invention can be used to control RSV infection in infected cell cultures, thus allowing cultures to be used for other purposes. The oligonucleotides of the invention may also be used to treat RSV infection in mammals. In this method, the composition is administered once in a therapeutically effective amount or repeatedly in less than therapeutic amounts. Administration may be by intravenous or intraperitoneal injection, or by intranasal, oral, transdermal, or subcutaneous administration. Effective dosages of the oligonucleotide and modes of its administration in the treatment of RSV can be determined by routine experimentation. The pharmaceutical forms suitable for injectable or other use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile. It must be stable under the conditions of manufacture and storage and may be preserved against the contaminating action of microorganisms, such as bacterial and fungi. The carrier can be a solvent or dispersion medium. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents. Prolonged absorption of the injectable therapeutic agents can be brought about by the use of the compositions of agents delaying absorption. A demonstration of the antiviral effect of individual oligonucleotides of the invention is described below in the examples and is depicted in FIGS. 3 - 5. Briefly, the phosphorothioate oligonucleotides targeted to mRNA and vRNA as set forth in TABLE 1 below were synthesized and administered at several different concentrations to human epidermal carcinoma cells which had been preinfected with RSV. Virus was removed and infected cells were incubated for 3 days with either an oligonucleotide, ribavirin, or no additional treatment. The effect of the treatment was determined by plaque assay, ELISA, or virus yield assay. The ELISA results are shown in TABLE 1, where "EC S0 " is the ribavirin or oligonucleotide concentration which inhibits virus replication by 50%. The oligonucleotides referred to as "NSl aug," "NS2 (1-15) ," and "NS2 (1-20)" are complementary to mRNA transcribed from the NSl and NS2 genes. The vRNA oligonucleotides are complementary to genomic RNA nucleotides 1-20 of the leader sequences and those regions encoding portions of the NSl, NS2, and P genes. TABLE 1 SEQ ID EG so¬ Name Sequence NO. Expt. # → 1 ribavarin N/A 10.0 il.0 7.0 vRNAl-20 ACGCGAAAAAATGCGTACAA 1 16 5 3.2 VRNA31-50 TAAACCAAAAAAATGGGGCA 2 1.1 1.1 0.7 VRNA41-60 AAATGGGGCAAATA....HATT 3 1.1 1.1 1 VRNA590-609 AAAAATGGGGCAAATAAATC 4 1.2 1.1 1 I H NSl aug CTCAATGAATTGCTGCCCAT 5 >30 >30 NS2 (1-15) TGATTTATTTGCCCC 25 30 NS2 (1-20) TGAATTGATTTATTTGCCCC >30 30 As shown in the table above, EC S0 values measured -for mRNA-targeted oligonucleotides were greater than or equal to 10 μM. Antisense oligonucleotides targeted to vRNA were more potent inhibitors (EC 50 values were about 1 μM) . Under the same experimental conditions, ribavirin inhibited RSV replication with an EC 50 of about 10 μM. These results indicate that the oligonucleotides of the invention targeted to RSV vRNA can effectively inhibit RSV replication. Under experimental conditions where the multiplicity of infection (MOI) was 10 fold lower, the antiviral activity of the oligonucleotides increase (see FIG. 5) . In addition, two different oligonucleotides or one oligonucleotide and ribavirin were tested in combination. Data was analyzed according to the dissimilar site inhibitor model of Prichard et al. (Antiviral Res. (1990) 14:181-206) . Three dimensional graphs of percent inhibition above expected for all drug combinations were generated. The results are shown in FIGS. 6 and 7, which represent the average data from two individual experiments. These figures show that oligonucleotides of the invention can interact synergistically with ribavirin (FIG. 7) or another oligonucleotide (FIG. 6) . At low concentrations, two oligonucleotides together produce as much as 50% more inhibition than expected by adding the inhibition of the individual oligonucleotides (FIG. 6) . At higher concentrations, the interactions are somewhat antagonistic. Similarly, when ribavirin was combined with an oligonucleotide, 20-30% more inhibition than expected was detected (FIG. 7) . At higher concentrations, the interactions were additive. These results suggest that combinations of oligonucleotides or one oligonucleotide plus ribavirin have a therapeutic advantage over the individual drugs alone. The following examples illustrate the preferred modes of making and practicing the present invention, but are not meant to limit the scope of the invention since alternative methods may be utilized to obtain similar results. EXAMPLES 1. Synthesis and Purification of Oligonucleotides Phosphodiester-linked oligonucleotides and oligonucleotide phosphorothioates were synthesized on an automated synthesizer (Gene Assembler, Pharmacia Biotech, Inc., Piscataway, NJ) . They were assembled by phosphoramidate chemistry. The synthesis and purification of were carried out by the method of Agrawal et al. (Proc. Natl. Acad. Sci. (USA) (1989) 86:7790-7794) . Oligonucleotide concentrations were determined by absorbance at 260 nm, taking into account the molar extinction coefficient of the nucleotides present in each sequence (Ausubel et al . , eds. (1987) Current t l Otocols in Molecular Biology (Wiley, New York)) . 2 . Cells HEp-2 cells, obtained from the American Type Culture Collection (Rockville, MD, ATCC No. CLL23) were cultured in Minimum Essential Medium with Earl's balanced salts (EMEM) (JRH Biosciences, Lenexa, KA) supplemented with 10% (volume: olume) fetal bovine serum (FBS) AT 37°C in 5% C0 2 . 3. Virus Respiratory syncytial virus (RSV) strain A2 obtained from American Type Culture Collection, (Rockville, MD, ATCC No. VR-1302) was grown in HEp-2 cells. Virus is harvested from supernatant 4 to 6 days after infection of the cells . The supernatant was stored at -80°C and used as stock in all experiments. 4. Ribavirin The compound was obtained from Pharmacia, Inc. (Alachua, FL) . 5. Inhibition Studies with Single Drug HEp-2 cells (20,000 cell/well of a microtiter plate) were incubated with RSV at a multiplicity of infection (moi) of 0.01 for 60 to 90 minutes at 37°C. Virus was removed and oligonucleotides (diluted in culture medium) or ribavirin are/is added to the infected cells. Oligonucleotides were tested at 4 to 6 concentrations from 30 to 0.01 μM. Infected cells were incubated for 3 days at 37°C. 6. Inhibition Studies with Drug Combinations Two oligonucleotides or one oligonucleotide and ribavirin (individually or collectively called "drugs") were evaluated in combination according to the methods of Prichard et al . (Antiviral Res. (1990) 14:181-206) . HEp-2 cells were infected with RSV as described above. Drugs were added to the RSV-infected HEp-2 cells in a 96 well plate. The same concentration of one drug was added to 10 wells in the top row of the plate. Serial dilutions of the drug were made into the other rows of the plate. Serial dilutions of the second drug were prepared in the columns of another 96 well plate, then transferred to the plate containing the RSV-infected cells. This established a checkerboard matrix of concentrations on the plate, containing 9 concentrations of one drug in the presence of 7 concentrations of the second drug (a total of 63 different drug combinations) . In addition, the drugs were titrated alone to determine their individual effects on virus replication. Data from drug combinations were analyzed according to the following equation for dissimilar site inhibitors as described by Prichard et al . (ibid. ) Z=X + Y(l-X) This equation states that the total inhibition (Z) produced by a combination of drugs is equal to the sum of the inhibition produced by one drug (X) plus the inhibition by a second drug (Y) on the populations unaffected by the first drug (1-X) . Using this equation, the inhibition data for the individual drugs at each concentration was used to calculate expected levels of inhibition for each combination. The difference between measured inhibition and expected inhibition was determined. The percent difference between measured and expected was plotted as a function of both drugs in a 3- dimensional graph. The results are shown in FIGS. 5 and 6. Values greater than 0 suggest synergistic interaction between drugs. Values less than 0 suggest that two drugs are antagonistic. Values equal to 0 suggest that the interactions are additive. Plaque Assay Plate HEp-2 cells were seeded in 24-well plate (s) at 250,000 cells/well and incubated at 37°C overnight. Cells were washed in serum-free media. Virus was diluted in media with 2% FBS at half-log increments. Each dilution of virus (50 μl) was added to replicate wells and incubated 1-2 hours at 37°C, 5% C0 2 . Each well was overlayed with 1 ml of 0;8% methylcellulose in media with 2% FBS and incubated at 37°C, 5% C0 2 . When plaques developed (3-4 days) , overlay was discarded and cells were stained with 5% glutaraldehyde/0.1% crystal violet (0.5 ml/well) . Cells were incubated at room temperature for 45 minutes. Stain was discarded and plaques were counted under a dissecting microscope. 8. ELISA The enzyme immunoassay procedure is based on the method of Kang and Pai (A.J.C.P. (March, 1989) 323-326) which is a modification of the methods of Berkowitz et al. (Antimicrob. Agents Chemother. (1985) 28:207-210)) and Rabalais et al . (Antimicrob. Agents Chemother. (1987) 31:946-948) , and used monoclonal antibodies specific for RSV F glycoprotein (provided by the FDA) . These RSV antibodies were diluted 1:10,000 in 1% bovine serum albumin (BSA) in phosphate buffered saline (PBS) and reacted for 1 hour at 37°C. Secondary antibody (horse radish peroxidase labelled goat anti-mouse IgG) was also diluted 1:10,000. Color was developed with 3, 3', 5, 5' -tetramethyl-benzidine (TMB) substrate, stopped with 0.1 N H 2 S0 4 , and the color read at 450 nm with a 620 nm reference filter. Virus replication as determined by this ELISA in "drug"-treated cultures was compared to untreated, infected cultures to obtain the per cent inhibition. The EC S0 , or the drug concentration which inhibits viral replication by 50%, was determined graphically. 9. Virus Yield Assay Supernatant from RSV-infected HEp-2 cells was titrated onto uninfected HEp-2 cells. The cells were then cultured for 3 to 7 days (or until syncytia were present in the culture) , and the virus measured by the immunoassay described in Example 7 above. Virus titer of oligonucleotide or ribavirin-treated cells was compared to that of untreated cells. EQUIVALENTS Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention, and are covered by the following claims. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Kilkuskie, Robert E. Brown-Vargas, Patrick E. (ii) TITLE OF INVENTION: Oligonucleotides Having Anti- Respiratory Syncytial Virus Activity (iii) NUMBER OF SEQUENCES: 7 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Lappin & Kusmer (B) STREET: 200 State Street (C) CITY: Boston (D) STATE: Massachusetts (E) COUNTRY: USA (F) ZIP: 02109 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE : Patentin Release #1 .0, Version #1 .25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Kerner, Ann-Louise (B) REGISTRATION NUMBER: 33,523 (C) REFERENCE/DOCKET NUMBER: HYZ-016PCT (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 617-330-1300 (B) TELEFAX: 617-330-1311 (2) INFORMATION FOR SEQ ID NO: 1 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 : ACGCGAAAAA ATGCGTACAA 20 (2) INFORMATION FOR SEQ ID NO:2 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2 : TAAACCAAAA AAATGGGGCA 20 (2) INFORMATION FOR SEQ ID NO:3 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3 : AAATGGGGCA AATAAGAATT 20 (2) INFORMATION FOR SEQ ID NO:4 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4 : AAAAATGGGG CAAATAAATC 20 (2) INFORMATION FOR SEQ ID NO:5 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5 : CTCAATGAAT TGCTGCCCAT 20 (2) INFORMATION FOR SEQ ID NO: 6 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6 : TGATTTATTT GCCCC 15 (2) INFORMATION FOR SEQ ID NO: 7 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7 : TGAATTGATT TATTTGCCCC 20

Claims: What is claimed is: 1. A synthetic oligonucleotide which is complementary to and hybridizes with a portion of respiratory syncytial virus genomic RNA under physiological conditions. 2. The oligonucleotide of claim 1 which includes at least 12 nucleotides. 3. The oligonucleotide of claim 2 which includes from 12 to 60 nucleotides. 4. The oligonucleotide of claim 1, which is modified. 5. The oligonucleotide of claim 2, which is modified. 6. The oligonucleotide of claim 3, which is modified. 7. The oligonucleotide of claim 4 comprising at least one internucleotide linkage selected from the group consisting of alkylphosphonate, phosphorothioate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, phosphate ester, carbamate, carbonate, phosphate triester, acetamidate, carboxymethyl ester, and combinations thereof. 8. The oligonucleotide of claim 5 comprising at least one internucleotide linkage selected from the group consisting of alkylphosphonate, phosphorothioate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, phosphate ester, carbamate, carbonate, phosphate triester, acetamidate, carboxymethyl ester, and combinations thereof. 9. The oligonucleotide of claim 6 comprising at least one internucleotide linkage selected from the group consisting of alkylphosphonate, phosphorothioate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, phosphate ester, carbamate, carbonate, phosphate triester, acetamidate, carboxymethyl ester, and combinations thereof. 10. The oligonucleotide of claim 7 comprising at least one phosphorothioate internucleotide linkage. 11. The oligonucleotide of claim 8 comprising at least one phosphorothioate internucleotide linkage. 12. The oligonucleotide of claim 9 comprising at least one phosphorothioate internucleotide linkage. 13. The oligonucleotide of claim 1 which comprises at least one deoxyribonucleotide 14. The oligonucleotide of claim 2 which comprises at least .one deoxyribonucleotide 15. The oligonucleotide of claim 3 which comprises at least one deoxyribonucleotide 16. The oligonucleotide of claim 1 which comprises at least one ribonucleotide. 17. The oligonucleotide of claim 2 which comprises at least one ribonucleotide. 18. The oligonucleotide of claim 3 which comprises at least one ribonucleotide. 19. The oligonucleotide of claim 1 having a nucleotide sequence selected from the group consisting of SEQ ID N0:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. 20. The oligonucleotide of claim 2 having a nucleotide sequence selected from the group consisting of SEQ ID N0:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. 21. The oligonucleotide of claim 3 having a nucleotide sequence selected from the group consisting of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. 22. A synthetic oligonucleotide which has antiviral activity against respiratory syncytial virus effected by hybridization with a portion of the genomic RNA of the virus. 23. A pharmaceutical composition comprising the oligonucleotide of claim 1 and a pharmaceutically acceptable carrier. 24. A pharmaceutical composition comprising the oligonucleotide of claim 2 and a pharmaceutically acceptable carrier. 25. A pharmaceutical composition comprising the oligonucleotide of claim 3 and a pharmaceutically acceptable carrier. 26. A pharmaceutical composition comprising the oligonucleotide of claim 4 and a pharmaceutically acceptable carrier. 27. A pharmaceutical composition comprising the oligonucleotide of claim 5 and a pharmaceutically acceptable carrier. 28. A pharmaceutical composition comprising the oligonucleotide of claim 6 and a pharmaceutically acceptable carrier. 29. The pharmaceutical composition of claim 23 comprising at least one oligonucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. 30. The pharmaceutical composition of claim 24 comprising at least one oligonucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3 , and SEQ ID NO:4. 31. The pharmaceutical composition of claim 25 comprising at least one oligonucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. 32. A pharmaceutical composition comprising at least two different oligonucleotides of claim 4 and a pharmaceutically acceptable carrier. 33. A pharmaceutical composition comprising at least two different oligonucleotides of claim 5 and a pharmaceutically acceptable carrier. 34. A pharmaceutical composition comprising at least two different oligonucleotides of claim 6 and a pharmaceutically acceptable carrier. 35. A pharmaceutical composition comprising at least two different oligonucleotides of claim 19 and a pharmaceutically acceptable carrier. 36. A pharmaceutical composition comprising at least two different oligonucleotides of claim 20 and a pharmaceutically acceptable carrier. 37. A pharmaceutical composition comprising at least two different oligonucleotides of claim 21 and a pharmaceutically acceptable carrier. 38. The pharmaceutical composition of claim 23 further comprising ribavirin. 39. The pharmaceutical composition of claim 24 further comprising ribavirin. 40. The pharmaceutical composition of claim 25 further comprising ribavirin. 41. The pharmaceutical composition of claim 26 further comprising ribavirin. 42. The pharmaceutical composition of claim 27 further comprising ribavirin. 43. The pharmaceutical composition of claim 28 further comprising ribavirin. 44. A method of inhibiting RSV replication in a cell comprising the step of administering to the cell a therapeutic amount of the pharmaceutical composition of claim 23. 45. A method of inhibiting RSV replication in a cell comprising the step of administering to the cell a therapeutic amount of the pharmaceutical composition of claim 24. 46. A method of inhibiting RSV replication in a cell comprising the step of administering to the cell a therapeutic amount of the pharmaceutical composition of claim 25. 47. A method of inhibiting RSV replication in a cell comprising the step of administering to the cell a therapeutic amount of the pharmaceutical composition of claim 32. 48. A method of inhibiting RSV replication in a cell comprising the step of administering to the cell a therapeutic amount of the pharmaceutical composition of claim 33. 49. A method of inhibiting RSV replication in a cell comprising the step of administering to the cell a therapeutic amount of the pharmaceutical composition of claim 34. 50. A method of inhibiting RSV replication in a cell comprising the step of administering to the cell a therapeutic amount of the pharmaceutical composition of claim 38. 51. A method of inhibiting RSV replication in a cell comprising the step of administering to the cell a therapeutic amount of the pharmaceutical composition of claim 39. 52. A method of inhibiting RSV replication in a cell comprising the step of administering to the cell a therapeutic amount of the pharmaceutical composition of claim 40. 53. A method of treating respiratory syncytial virus infection comprising the step of administering to an infected mammal or cell the pharmaceutical composition of claim 23. 54. A method of treating respiratory syncytial virus infection comprising the step of administering to an infected mammal or cell the pharmaceutical composition of claim 24. 55. A method of treating respiratory syncytial virus infection comprising the step of administering to an infected mammal or cell the pharmaceutical composition of claim 25. 56. A method of treating respiratory syncytial virus infection comprising the step of administering to an infected mammal or cell the pharmaceutical composition of claim 32. 57. A method of treating respiratory syncytial virus infection comprising the step of administering to an infected mammal or cell the pharmaceutical composition of claim 33. 58. A method of treating respiratory syncytial virus infection comprising the step of administering to an infected mammal or cell the pharmaceutical composition of claim 34. 59. A method of treating respiratory syncytial virus infection comprising the step of administering to an infected mammal or cell the pharmaceutical composition of claim 38. 60. A method of treating respiratory syncytial virus infection comprising the step of administering to an infected mammal or cell the pharmaceutical composition of claim 39. 61. A method of treating respiratory syncytial virus infection comprising the step of administering to an infected mammal or cell the pharmaceutical composition of claim 40.

Inventors: BROWN-VARGAS PATRICK E, KILKUSKIE ROBERT E, BROWN-VARGAS, PATRICK, E., KILKUSKIE, ROBERT, E.
Applicants: HYBRIDON INC, HYBRIDON, INC.
Application year: 1995
Main classification: C07H21
IPC codes: A61K31, A61P31, C07H21, C12N15