IE913321A1 - Compositions and methods for inhibiting growth or¹replication of microbes, viruses and self-replicating¹nucleic acids - Google Patents

Compositions and methods for inhibiting growth or¹replication of microbes, viruses and self-replicating¹nucleic acids

Info

Publication number
IE913321A1
IE913321A1 IE332191A IE332191A IE913321A1 IE 913321 A1 IE913321 A1 IE 913321A1 IE 332191 A IE332191 A IE 332191A IE 332191 A IE332191 A IE 332191A IE 913321 A1 IE913321 A1 IE 913321A1
Authority
IE
Ireland
Prior art keywords
oligonucleotides
oligonucleotide
hybridizable
alkyl
nucleoside
Prior art date
Application number
IE332191A
Original Assignee
Univ Maryland
Univ Johns Hopkins
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Univ Maryland, Univ Johns Hopkins filed Critical Univ Maryland
Publication of IE913321A1 publication Critical patent/IE913321A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • 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
    • 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
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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
    • C12N15/1133—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 against herpetoviridae, e.g. HSV
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/702—Specific hybridization probes for retroviruses
    • C12Q1/703—Viruses associated with AIDS
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/705—Specific hybridization probes for herpetoviridae, e.g. herpes simplex, varicella zoster
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/312—Phosphonates
    • C12N2310/3125—Methylphosphonates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification

Abstract

The invention relates to compositions and methods for inhibiting the growth or replication of microbes, viruses or self-replicating nucleic acids. Antisense oligonucleotides that bind to strategic sites in the microbe, virus of self-replicating nucleic acid genome find particular utility in preventing proliferation and pathogenesis; and in detecting microbe, virus or self-replicating nucleic acid. Therapeutic compositions comprising at least two oligonucleotides and methods using the compositions are effective in inhibiting the growth or replication of homologous and heterologous microbes, viruses or self-replicating nucleic acids.

Description

COMPOSITIONS AND METHODS FOR INHIBITING GROWTH OR REPLICATION OF MICROBES, VIRUSES AND SELF-REPLICATING NUCLEIC ACIDS The invention described and claimed herein was supported in part by grants from the Department of Health and Human Services, National Institutes of Health.
FIELD OF THE INVENTION The invention relates to oligonucleotides; medicaments and pharmaceutic compositions comprising the oligonucleotides; methods of detecting microbes, viruses and self-replicating nucleic acids using the oligonucleotides; methods of inhibiting growth or replication of microbes, viruses and self-replicating nucleic acids using the oligonucleotides; and therapeutic methods using the oligonucleotides to inhibit homologous and heterologous growth or replication of microbes, viruses or self-replicating nucleic acids.
BACKGROUND OF THE INVENTION Antisense RNA that can inhibit selectively gene expression at the level of translation or mRNA processing has been proposed as a possible genetic approach for the prevention and treatment of disease. Antisense RNA introduced directly into cells or expressed off transfected DNA has been shown to repress the expression of endogenous eukaryotic genes. However, the inherent properties of RNA and problems of working with RNA, such as the near ubiquitous presence of RNases in cells, reagents and supplies, has stimulated a search for alternative means of genetically repressing the expression of specific genes using antisense oligonucleotides.
One such development is non-ionic nucleic acid analogues that contain a 3'-5’ methylphosphonate group in place of the negatively charged phosphodiester group found normally in oligonucleotides. The analogues are resistant to nuclease hydrolysis and penetrate the plasma membrane of cells in culture. Other means of improvement include the use of terminal blocking groups, intercalating agents or phosphorothioates.
Antisense oligonucleotides have found limited success in inhibiting the growth of Rous sarcoma virus, vesicular stomatitis virus, simian virus 40, influenza virus and human immunodeficiency virus.
SUMMARY OF THE INVENTION Accordingly, one object of the instant invention is to provide oligonucleotides that inhibit herpesvirus growth or replication in a superior manner.
A second object of the invention is to provide novel compositions and methods for inhibiting growth or replication of microbes, viruses or selfreplicating nucleic acids.
Another object of the invention relates to the use of multiple oligonucleotides directed to nonoverlapping sites in a microbe, virus or selfreplicating nucleic acid genome to inhibit growth or replication thereof.
A fourth object of the invention is to provide novel compositions and methods for inhibiting growth or replication of a primary microbe, virus or self-replicating nucleic acid, such as a herpesvirus, and of a secondary microbe, virus or self-replicating nucleic acid, such as human immunodeficiency virus, using one or more oligonucleotides hybridizable with nucleic acids of said primary microbe, virus or self-replicating nucleic acid.
These and other objects have been attained by providing novel oligonucleotides that hybridize to specific target sites in the genome of a microbe, virus or self-replicating nucleic acid.
The invention provides a method of detecting herpesvirus comprising the steps of: (i) obtaining a biologic sample containing nucleic acids; (ii) treating said sample so the nucleic acids contained therein are made single stranded; (iii) exposing said treated sample to a labelled oligonucleotide wherein said oligonucleotide is hybridizable with nucleic acids of herpesvirus; and (iv) detecting hybridized sequences.
In one aspect, the invention provides a composition for inhibiting herpesvirus growth or replication comprising an oligonucleotide hybridizable with nucleic acids of said herpesvirus.
In another embodiment, the invention provides a method for inhibiting herpesvirus growth or replication comprising the step of contacting nucleic acids of herpesvirus with an oligonucleotide hybridizable with said nucleic acids of herpesvirus.
In yet another embodiment, the invention provides therapeutic compositions for inhibiting 9 21 22 - 4 herpesvirus growth or replication comprising an oligonucleotide, or pharmaceutically acceptable salts thereof, hybridizable with nucleic acids of herpesvirus and a pharmaceutically acceptable carrier.
In an even further embodiment, the invention provides a therapeutic method for inhibiting herpesvirus growth or replication in a host carrying herpesvirus comprising administering a pharmaceutically effective amount of a therapeutic composition comprising an oligonucleotide, or pharmaceutically acceptable salts thereof, hybridizable with nucleic acids of herpesvirus.
In another embodiment the invention provides a composition for inhibiting growth or replication of a microbe, virus or self-replicating nucleic acid comprising at least two oligonucleotides hybridizable with non-overlapping sites in nucleic acids of said microbe, virus or self-replicating nucleic acid.
In a further embodiment the invention provides a method for inhibiting growth or replication of a microbe, virus or self-replicating nucleic acid comprising the step of contacting nucleic acids of a microbe, virus or self-replicating nucleic acid with at least two oligonucleotides hybridizable with non-overlapping sites in nucleic acids of said microbe, virus or self-replicating nucleic acid.
In an even further embodiment, the invention provides therapeutic compositions for inhibiting microbe, virus or self-replicating nucleic acid growth or replication comprising at least two oligonucleotides, or pharmaceutically acceptable salts thereof, hybridizable with non-overlapping sites in nucleic acids of said microbe, virus or self-replicating nucleic acid and a pharmaceutically acceptable carrier.
In another embodiment, the invention provides a therapeutic method for inhibiting microbe, virus or self-replicating nucleic acid growth or replication in a host carrying a microbe, virus or self-replicating nucleic acid comprising administering pharmaceutically effective amounts of at least two oligonucleotides, or pharmaceutically acceptable salts thereof, hybridizable with nonoverlapping sites in nucleic acids of said microbe, virus or self-replicating nucleic acid.
In yet a further embodiment, the invention provides a composition for inhibiting human immunodeficiency virus growth or replication in a specimen containing human immunodeficiency virus and a second virus comprising at least one oligonucleotide hybridizable with nucleic acids of said second virus.
The invention further provides a method for inhibiting human immunodeficiency virus growth or replication in a specimen containing human immunodeficiency virus and a second virus comprising the step of contacting nucleic acids of said second virus with at least one oligonucleotide hybridizable with nucleic acids of said second virus.
Another embodiment of the invention is a therapeutic composition for inhibiting human immunodeficiency virus growth or replication in a host carrying human immunodeficiency virus and a second virus comprising at least one oligonucleotide, or pharmaceutically acceptable salts thereof, hybridizable with nucleic acids of said second virus and a pharmacuetically acceptable carrier.
Finally, the invention provides a therapeutic method for inhibiting human immunodeficiency virus growth or replication in a host carrying human immunodeficiency virus and a second virus comprising administering a pharmaceutically effective amount of at least one oligonucleotide, or pharmaceutically acceptable salts thereof, hybridizable with nucleic acids of said second virus.
BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a schematic representation of the herpes simplex virus (HSV) genome with several genes preferred as targets for antisense oligonucleotides highlighted. In the figure, UL denotes the long unique sequence segment flanked by the terminal, TRL, and internal, IRL, repeat sequences. Us denotes the short unique sequence segment flanked by the internal, IRj, and terminal, TRj, repeat sequences. Above the gene map is a relative scale wherein 1 represents the entire HSV genome.
Figure 2 is another schematic representation of the HSV genome showing other relevant genes. The long region is divided into functional domains as determined by restriction enzymes and denoted as C, F, E and D. ORF size is the predicted open reading frame size in kilobases (K) . In the relative scale above the gene map, the solid boxes represent the long segment flanking repeats and the hollow boxes represent the short segment flanking repeats. The genes are identified numerically, such as UL5, or according to function and include dbp which encodes the major DNA binding protein, which is involved in DNA unwinding, and pol which encodes DNA polymerase.
Figure 3 depicts dose response curves of HSV-1 growth in cells exposed to antisense oligonucleotides. Vero cells were infected with 10 pfu per cell of HSV-1 and treated with increasing concentrations of oligonucleotide at the time of infection. The cultures were assayed for infectious virus 24 hours later. Results are expressed as the percent inhibition of virus titers compared to untreated HSV-l-infected cells. In the figure, the solid circles represent the oligonucleotide TTCCTCCTGCGG; the X's represent the oligonucleotide TCCTCCTG; the hollow squares represent the oligonucleotide GCGGGAAGGCAC; the solid squares represent the oligonucleotide TCCTGCGGGAAG; the solid triangles represent the oligonucleotide TTCCTCCT; and the hollow triangles represent the oligonucleotide TCCTGCGG.
Figure 4 depicts activation of HSV by the IE110 gene product and inhibition of IEllO-induced activation by specific oligonucleotides. Vero cells were transfected with pICPIO-cat, a plasmid carrying an HSV promoter and the CAT structural gene, and activating IE110. In the graph, the open bar represents cells grown in the absence of an oligonucleotide; the hatched bars denoted as (a) represent cells grown in the presence of the oligonucleotide TTCCTCCTGCGC; the stippled bars denoted as (b) represent cells grown in the presence of the oligonucleotide GCGGGGCTCCAT; and the dotted bars denoted as (c) represent cells grown in the presence of the oligonucleotide AGTCTGCTGCAA.
Figure 5 depicts dose response and inhibition of HSV-1 growth in cells by antisense oligonucleotides, as in Figure 3 solid circles represent the rTCCTCCTGCGG; solid triangles oligonucleotide GCTTACCCGTGC; X's In the figure oligonucleotide represent the represent the oligonucleotide GCGGGGCTCCAT; and hollow circles represent the oligonucleotide AGTCTGCTGCAA.
Figure 6 shows the effect of virus m.o.i. (multiplicity of infection) on dose response inhibition of HSV-1 growth by an oligonucleotide complementary to the translation initiation site of HSV-1 IE mRNA 1 with the sequence GCGGGGCTCCAT, designated IE110. In the figure solid circles represent Vero cells infected with 0.1 pfu per cell of HSV-1 and hollow circles represent cells infected with 10 pfu per cell of HSV-1.
Figures 7A and 7B depict dose response inhibition of HSV-1 growth in Vero cells exposed to antisense oligonucleotides.
In Figure 7A, HSV-1 growth in Vero cells infected with 0.1 pfu per cell of HSV-1 was monitored. In the figure, X's represent the oligonucleotide GCGGGGCTCCAT (designated as IE110) ί circles represent the oligonucleotide TTCCTCCTGCGG (designated as IE4,5); and squares designate the use of both oligonucleotides simultaneously.
In Figure 7B, the synergistic effect of IE110 and IE4,5 oligomers on the inhibition of HSV-2 replication is depicted. The concentration of each oligonucleotide was covaried and inhibition of plaque formation was determined. The term FIC denotes the ratio of the concentration of IE4,5 required to inhibit plaque formation by 60% in the presence of a fixed concentration of IE110 to the concentration required in the absence of IE110. The units of the X axis are the ratio of the fixed concentration of IE110 to the concentration of IE110 that produced 60% inhibition of plaque formation in the absence of IE4,5. The diagonal line (the X’s) shows the theoretical plot for each oligonucleotide used alone. The solid circles represent the synergistic effect of the combination.
Figure 8 depicts dose response curves of HSV-1 growth in cells exposed to antisense oligonucleotides. In the figure, the small circles represent oligomer specific for the IE5 initiation site and designated IE12 with the sequence GGCCCACGACAT. IE 12 does not show significant inhibition. Triangles represent the oligonucleotide AATGTCGGCCAT (designated as IE68 and specific for the IE4 initiation site) . The large circles represent inhibition obtained by combining the two oligonucleotides. At all points, the combination of oligonucleotides achieves a higher level of inhibition at a lower concentration of oligonucleotides than when either is used alone. In the combination treatment, the points represent total concentration of both oligonucleotides.
Figure 9 depicts inhibition of HIV activation by a herpesvirus-specific oligonucleotide. Cells were cotransfected with a plasmid carrying the HIV promoter upstream of the CAT structural gene and a plasmid encoding the HSV-1 IE110 protein. The transformants were then exposed to varying concentrations of oligonucleotides. Oligomer to the initiation site of IE110 is GCGGGGCTCCAT, to the second splice acceptor site of IE110 is AGTCTGCTGCAA and to the splice acceptor site of IE4,5 is TTCCTCCTGCGG.
Figure 10 depicts inhibition of HIV activation by a herpesvirus-specific oligonucleotide, GCGGGGCTCCAT (designated as IE110 oligomer); and activation by the IE110 protein of HSV-1 and the homologous gene product from cytomegalovirus (designated as CMV). The IE110 protein and the oligonucleotide were combined in one treatment. The curve denoted PBR is a control using pBR322. IFU is infectious forming unit. - 10 DETAILED DESCRIPTION OF THE INVENTION 9 21 22 The invention relates to inhibiting growth or replication of microbes, viruses or selfreplicating nucleic acids, or the translation or expression of microbe, virus or self-replicating nucleic acid genes that are essential for growth and proliferation, by interfering with the normal means of expression of critical genes by using antisense oligonucleotides that are complementary to specific targeted sites in the microbe, virus or self-replicaing nucleic acid genome.
The compositions and methods of the instant invention find utility in the detection, diagnosis and manipulation of the herpesviruses and in the treatment of disease caused directly or indirectly by the herpesviruses.
Particular advantages are obtained with any microbe, virus or self-replicating nucleic acid when more than one oligonucleotide is used wherein the oligonucleotides are directed to nonoverlapping sites in the microbe, virus or selfreplicating nucleic acid genome, that is, to separate target sites.
The term oligonucleotide is used herein to include oligomers comprised of deoxyribose and ribose, the sugars found normally in nucleic acids, and modified analogs thereof, such as 2'-O-methyl ribose. The oligonucleotide may bind to singlestranded nucleic acids or to double-stranded nucleic acids.
The term self-replicating nucleic acids is used herein to include nucleic acids found in organelles, such as chloroplasts and mitochondria, and alone, such as plasmids, viroids and the like.
The term virus is used herein to include intracellular agents that are characterized by a 9 21 22 - 11 lack of independent metabolism and by the ability to replicate only within living host cells.
The term microbe is used herein to include prokaryotes, such as bacteria, actinomycetes, chlamydiae, mycoplasmae and the like? and eukaryotes, such as protozoa, fungi, parasites and the like.
For example, the herpesviruses, whose natural host is the human species, comprise seven species, herpes simplex type 1 (HSV-1) and type 2 (HSV-2), varicella zoster (VSV), Epstein-Barr (EBV), cytomegalovirus (HCMV), human herpesvirus 6 (HHV-6) and 7 (HHV-7). (As used herein, the term herpesvirus includes the seven species described above.) They are DNA viruses with large genomes. Common genetic features include repeat sequences, long and short unique sequences and immediate-early regulatory genes that control the transcription and expression of downstream genes.
The oligonucleotides can be produced in any of a variety of art-recognized methods, for example the commonly used solid phase triester or phosphoramidite chemistry. Nucleic acid synthesis in vitro has become highly automated using equipment that is readily available to the artisan. Furthermore, customized oligonucleotides can be obtained under contract from a variety of commercial sources. Alternatively, the antisense nucleotides can be synthesized in situ by transfecting cells with an appropriate cloned sequence.
The oligonucleotides must be capable of bypassing membranous barriers, of resisting the degradative means present in the milieu and hybridizing the nucleic acids of the microbe, virus or self-replicating nucleic acid. Thus, unmodified oligodeoxyribonucleotides and oligoribonucleotides, 21 22 - 12 and other oligonucleotides can be used if introduced directly into cells by methods including electroporation, microimmunization, liposome fusion, precipitation and microparticle bombardment.
The antisense oligonucleotides also can be produced endogenously in host cells following transfection by an appropriate recombinant. Sequences encoding the antisense molecule are cloned into an appropriate site in an expression cassette carrying the necessary host RNA polymerase binding sites of a vector. The recombinant is then introduced into cells using standard techniques, such as those described above.
Chemically modified oligonucleotides (or as used herein modified oligonucleotides or modified oligonucleosides or modified nucleosides) may be used, for example, ο1igonuc1eoside methylphosphonates as described in U.S. Patent No. 4,469,863 or 4,511,713; phosphorothioate analogs as described in Matsukura et al. (PNAS 84, 7706-7710, 1987); oligonucleotides carrying terminal groups and oligomers that are dervitized, such as with psoralen. Modified nucleotides offer the advantages of increased hydrophobicity or nuclease resistance or both. Thus, oligonucleotides carrying certain modified bases are capable of penetrating the mammalian cell plasma membrane. Also, the termini of the oligonucleotide can be modified to render the oligomer resistant to intracellular exonucleases. The use of chemically modified oligonucleotides is preferred in practicing the instant invention, and especially perferred is the use of oligonucleoside alkyl or aryl phosphonates.
The oligonucleotides can vary in size depending upon the specificity desired, expected 9 - 13 intracellular stability and technical considerations. Generally, the longer the oligonucleotide the greater the specificity, at least in the range of 8 to 20 nucleotides. However, if the targeted sequences are part of a small microbe, virus or self-replicating nucleic acid genome, then the effective concentration of the driver DNA, that is the microbe, virus or selfreplicating nucleic acid genome, in the hybridization reaction between genome target site and oligonucleotide is high thereby enabling one to achieve adequate specificity using shorter oligonucleotides. Oligonucleotides of 8 to 20 bases are preferred, and those of 12-20 bases are particularly preferred. When introduced by means of an antisense vector, the oligonucleotides can be as large as 50 to 60 bases, if not larger. It is possible, in order to span a larger region, to employ two or more oligomers that hybridize to adjacent sites, for example two 15mers to span a 30 base pair site in situ.
Any means of contacting oligonucleotides with nucleic acids of a microbe, virus or selfreplicating nucleic acid is suitable for practicing the instant invention. In a therapeutic setting, a preferred means is to use oligonucleotides that alone or in a composition are stable in the peripheral circulation and can penetrate the cell membrane. The means of introducing the oligonucleotides in a therapeutically effective concentration can be any of those used currently for delivering biologicals, such as immunoglobulin, peptides and enzymes, for example, intravenous administration, intramuscular immunization, implants, intranasal administration, contained in liposomes, attached to a cell-binding agent, such as an antibody, attached to ribozymes and other 9 21 22 - 14 parenteral routes. Furthermore, because many microbes, viruses or self-replicating nucleic acids produce epithelial lesions, the oligonucleotide may be delivered in a vehicle comprising a cream or an ointment.
Suitable concentrations of oligonucleotides needed to inhibit growth or replication of a microbe, virus or self-replicating nucleic acid can be determined readily by the skilled artisan. For example, the animal studies disclosed herein provide a basis for extrapolating dosage in other species based on weight comparisons between the mouse and other species. Or the amounts used herein can provide a reference point for use in other species. The concentration required will depend in part, for example, on the chemistry of the oligonucleotides; having to traverse physiologic barriers, such as the blood-brain or blood-testis barrier; toxicity; route of administration, such as oral, transdermal and nasal; the disease requiring treatment, such as a verruca, an outbreak of shingles, an intestinal ascaritic infestation and bacteremia; and half life in the peripheral circulation. Examples of suitable therapeutic doses in terms of internal doses (that is the dose at the required site in the body) are between 10 nM and 10 μΜ.
Depending on the intended mode of administration, the oligonucleotides can be delivered in a variety of pharmaceutically accepted ways. The compositions may be in the form of a solid or semisolid, but it is most likely that the oligomers will be admininistered in liquid form. The compositions will include the oligomers, or pharmaceutically acceptable salts thereof, conventional excipients, such as a sterile buffered saline solution, and other agents, carriers, 9 21 22 - 15 adjuvants, diluents etc. for increasing the stability, enhancing wettability and the like of the composition.
Actual methods and doses are known, or will be apparent to those skilled in the art, for example see Remington1s Pharmaceutical Sciences. Mack Publ. Co., Easton, PA.
The invention also contemplates the use of at least two oligonucleotides directed to nonoverlapping sites in the microbe, virus or selfreplicating nucleic acid genome. Because targeted sites are not overlapping and the three-dimensional folding of the microbe, virus or self-replicating nucleic acid genome in a host cell or in situ may not be predictable, each oligonucleotide of a plurality of oligonucleotides contains sequences that are complementary to a single site. An unexpected enhanced inhibition of microbe, virus or self-replicating nucleic acid growth or replication is obtained when more than one oligonucleotide is used, the oligonucleotides being hybridizable to different sites in a single gene or in separate genes.
According to the instant invention, there are certain regions of the microbe, virus or selfreplicating nucleic acid genome that are preferred as targets for antisense oligonucleotides.
Preferred targets are those that have direct or indirect control over basic mechanisms in microbe, virus or self-replicating nucleic acid growth. Thus, regulatory genes expressed early in development that trigger major developmental pathways are suitable (known as master regulatory genes) as are other regulatory genes that control the expression of a smaller number of genes, as in an operon. Other suitable targets are genes involved with microbe, virus or self-replicating nucleic acid replication, such as DNA polymerase 21 22 - 16 and reverse transcriptase. Another set of suitable targets are genes that are involved in pathogenesis se such as those that confer tissue or cell specificity, virulence and latency.
Important sites within the targets are transcription initiation sites (also known as promoter or polymerase binding site), translation initiation sites, splice donor and acceptor sites, messenger cap sites and transcription initiation sites. For example in HSV-1 an oligonucleotide that is hybridizable to the IE2 translation initiation site, such as 51-ATATCAATGTCAGTCGCCAT-3' (hereinafter all the sequences are in the 5' to 3' orientation), is suitable, as is an oligonucleotide hybridizable to the IE5 translation initiation site, such as GGCCCACGACAT; oligonucleotides hybridizable to the IE4 mRNA cap site, such as GCGGGCGTCGGT, GGCGCCGTCTGC and GTGGCCGGCGCC; oligonucleotides hybridizable to the IE5 mRNA cap site, such as GCGGGCGTCGGT, GGCGCCGTCTGC and GTGGCCGGCGCC; an oligonucleotide hybridizable to the IE4,5 splice acceptor site, such as TTCCTCCTGCGG; an oligonucleotide hybridizable to the IE4,5 splice donor site, such as GCTTACCCGTGC; an oligonucleotide hybridizable to the IE1 translation initiation site, such as GCGGGGCTCCAT; an oligonucleotide hybridizable to the IE3 translation initiation site, such as GTTCTCCGACGCCAT; an oligonucleotide hybridizable to the IE3 cap site, such as CGCTCCGTGTGG; and an oligonucleotide hybridizable to the IE4 translation initiation site, such as AATGTCGGCCAT.
As mentioned above and will be exemplified further in the Examples following, the size of the oligonucleotide may vary and the oligonucleotide may include bases complementary to the target genomic site that flank the sequences recited - 17 herein or lack bases of the sequences recited herein. Thus, the IE4 translation initiation site sequence AATGTCGGCCAT may be extended with complementary, and thus hybridizable bases in either the 5' or 3' direction to obtain an oligonucleotide of more than 12 bases; may be truncated by one or more bases to yield an effective oligonucleotide of fewer than 12 bases; may be extended in either the 51 or 31 direction with a concommitant deletion of one or more bases from the non-extended end to yield an oligonucleotide containing only part of the recited sequence etc., so long as the oligonucleotide is capable of interfering with the normal transcription or translation of IE4.
Ideal targeting about a splice site can be achieved with an oligonucleotide that, as equally as possible, spans the exon/intron junction. Ideal targeting about a translation initiation site can be achieved with an oligonucleotide that in the complementary 3' to 5' orientation, begins at the ATG initiation codon and extends into the coding region (thus the oligonucleotide in the 5' to 3' orientation will have as the last three bases CAT).
Genes that are essential for synthesizing nucleic acids of a microbe, virus or selfreplicating nucleic acid are preferred targets for antisense oligonucleotides (Figure 2) . Examples of such genes are those that encode DNA binding proteins, gyrases, enzymes that relate to the synthesis of nucleotide triphosphates, DNA nucleases required for DNA replication and polymerases. Also included are transcrition initiation sites or promoter sequences. In the exemplary organism, HSV-1, suitable genes include the DNA polymerase gene, thymidine kinase gene (TK) , ribonucleotide reductase gene (RRI locus, - 18 also known as 1CP6, and RR2 locus, also known as 38K) and genes encoding enzyme stabilizing proteins such as the 18OK for ribonucletide reductase. An oligonucleotide hybridizable to the polymerase gene is CCGCCGCCACCGGAAAAGAT; an oligonucleotide hybridizable to TK is TGGCAGGGGTACGAAGCCAT; and an oligonucleotide hybridizable to RRI is GCGGCTGGGCGGCTGGCCAT.
Also preferred as targets are sequences which are crucial to transcription, such as promoter sequences. For example, in HSV there is a consensus sequence common to the promoters of the IE genes with the sequence TAATGARAT, wherein R can be any base including A, T, G, C, U or functional equivalents thereof, wherein a functional equivalent is a nitrogenous base that can be substituted and retain complementarity. Examples include inosine and 5-methyl-cytosine.
Furthermore, regulatory genes, many of which are not translated, that govern the orderly expression of downstream genes are especially suitable as targets for antisense oligonucleotides. Many of these early expressed regulatory genes are termed master genes. In the HSV-1 genome, the VmW65 locus is one such master gene. An hybridizable to the VmW65 oligonucleotide initiation site would be suitable, such as TCGTCGACCAAGAGGTCCAT, CAAACAGCTCGT, CCATGTCGGCAA, GTCCGCGTCCAT, GCCGTCCGCGTC and TGGCGAAGCGCC; and an oligonucleotide hybridizable to the VmW65 mRNA cap site would suffice, such as ACAGCCCGTGGT, CCGAGGAATGAC and CCGTTCCCGAGG.
Another set of genes that are preferred for targeting are those that are essential for pathogenesis. In a virus, for example, such genes are those involved in latency, cell tropism, the choice of specific cell to infect or determining - 19 1 9 21 22 virulence. In HSV-1, one such gene is LAT (latency-associated transcript) and suitable oligonucleotides are those hybridizable to the initiation site of LAT ORF-1, such as CACTGGCATGGA, GCATCCTGCCAC, CCCCGAAAGCAT, GATCCCCGAAAG and CTGACCACCGAT; and those hybridizable to the initiation site of LAT ORF-2, such as GCAGGCTCTGGT, CCATGTTGGGCA, TGGGGGTGCCAT, GAGTGGGGGTGC and GGTGCGTGGGAG.
As mentioned previously, IE genes are not restricted to HSV-1 and are a feature common to the herpesviruses. The IE gene homologues in related herpesviruses are suitable targets for antisense oligonucleotides. Thus in HCMV, oligonucleotides hybridizable to the HCMV IE2 initiation site are suitable, such as CAAGGACGGTGA, CCATCGTGTCAA, AGAGGACTCCAT, GGCAGAGGACTC and CTTTCTCTTGGC; as are those hybridizable to the IE2 homologue mRNA cap site, such as GACGGTTCACTA, CAGGCGATCTGA and CGTCTCCAGGCG.
In VZV, the HSV-1 IE110 gene homologue is known as gene 61. Oligonucleotides which are suitable are those that are hybridizable to the 61 translation initiation site, such as CAACTGGCTGTA, CCATGGTAACAA, TATGGTATCCAT, TAATATGGTATC and ACCGCCCGCTAA. The VZV 62 gene is the homologue of the HSV-1 IE3 gene. Oligonucleotides which are suitable are those that are hybridizable to the 62 translation initiation site, such as TGGGGTGAATTT, CCATCGCACTGG, CGGCGTATCCAT, CGGCGGCGTATC and GCGCTGCATCGG.
Especially preferred is to target two genes that complement the expression and/or function of each other, for example two genes that encode proteins of a metabolic pathway, two genes of a developmental pathway or two master regulatory genes that each control the expression of different - 20 developmental programs, or combinations thereof. Knowing the strategy, the skilled artisan can determine readily suitable portions of the microbe, virus or self-replicating nucleic acid to target and hence suitable combinations of oligonucleotides.
Although in a preferred embodiment the invention contemplates the use of two or more oligonucleotides, there are instances where a single oligonucleotide is more than 90% effective. For example, the oligonucleotides TTCCTCCTGCGG, GCTTACCCGTGC, GCGGGGCTCCAT and AATGTCGGCCAT each are effective in inhibiting HSV-1 growth when used alone. However, the activity of the oligonucleotides is enhanced, that is additive or synergistic, when used in combination with another oligonucleotide. The phenomenon is manifest as higher level of inhibition or effectiveness at a lower dose or both.
The nature in which an oligonucleotide can influence normal gene expression includes binding to existing single-stranded regions in the nucleic acids of a microbe, virus or self-replicating nucleic acid, which includes naturally occurring single-stranded nucleic acids and regions of double-stranded nucleic acids that are melted partially, and binding to double-stranded nucleic acids to form a nucleic acid triplex structure, which includes binding to cloverleaf and stem-loop structures commonly found amongst single-stranded nucleic acids that self base pair or fold back upon themselves. For a review of the topic see Uhlmann & Peyman (Chem Rev 90, 544-579, 1990).
Of related interest is the observation that many people infected with HIV (human immunodeficiency virus) do not develop AIDS (acquired immune deficiency syndrome). It is believed that factors other than HIV are essential for activation of HIV thereby enhancing its replication to the levels required to cause AIDS. Among those factors are the viruses including JC, adenoviruses and the herpesviruses including HSV-1, HSV-2, HCMV, VZV, EBV, HHV6 and HHV7. Specific genes of those viruses activate the expression of HIV either directly at the level of virus replication or indirectly by activating expression of a reporter gene driven by the HIV promoter. Thus, inhibition of herpesvirus growth or replication using herpes-specific antisense oligonucleotides prevents HIV activation and may indirectly forestall the progression of events from HIV exposure to the clinical manifestation of AIDS. HSV-1 genes involved in HIV activation include IE110 (encoded by IE1).
The invention will now be described in further detail by way of the following non-limiting examples.
REFERENCE EXAMPLE The following materials and methods were employed in the experiments described in the Examples.
Vero (African green monkey kidney) cells were grown in Eagle's minimal essential medium (MEM) supplemented with 25 mM HEPES and 10% (v/v) fetal bovine serum. HEp-2 (human epidermoid carcinoma No. 2) cells were grown in medium 199 with 10% calf serum. U937 (human histiocytic lymphoma) cells were grown in RPMI 1640 with heat-inactivated fetal calf serum.
Oligo(nucleoside-methylphosphonate)s were synthesized, for example, as described in either of Miller et al. (Biochem 18, 5134-5143, 1979), Miller et al. (Nucl Acids Res 10, 979-991, 1982), Miller et al. (Biochem 25, 5092-5097, 1986), U.S. Patent No. 4,469,863 or U.S. Patent No. 4,511,713. Briefly, the process is a solid phase technique using, for example polystyrene as a support. The process consists of esterifying a 5'-O-protected nucleoside having a 3'-hydroxyl group with an alkyl or arylphosphonic acid in the presence of a condensing agent, such as mesitylenesulfonyl tetrazolide. The result is a 5'-O-protected nucleoside-3'-O-alkyl or arylphosphonate. That resulting compound is then esterified with a 3'-Oprotected nucleoside having a 5'-hydroxyl group in the presence of a condensing agent to form a fully protected dinucleoside alkyl or arylphosphonate. The protecting groups are removed from the dinucleoside compound to form the dinucleoside alkyl or arylphosphonate. For example, in the case of dA-containing oligomers bearing N-benzoyl protecting groups, the protected dinucleoside was treated with hydrazine. The 3'-0-acetyl and 5'-0dimethoxytrityl protecting groups were removed by sequential treatment with ammonium hydroxide and 80% acetic acid.
The process is repeated the requisite number of steps using the appropriate protected nucleosides to form an oligonucleotide of defined length and sequence.
Alternatively, the process can also be carried out by esterifying a 3'-O-protected nucleoside having a 5'-hydroxyl group with an alkyl or arylphosphonate in the presence of an activating agent to form a 3'-O-protected nucleoside-5'-Oalkyl or arylphosphonate. That compound is then esterified with a 5'-O-protected nucleoside having a 3'-hydroxyl group in the presence of an activating agent to form a fully protected dinucleoside alkyl or arylphosphonate. As described above, the protecting groups are removed to form the dinucleoside alkyl or arylphosphonate. Further details can be obtained by referring to one of the above-mentioned references.
HSV-1 was grown and titered on Vero or HEp-2 cells as described in Strnad & Aurelian (Virol 87, 401-415, 1978).
Cytoplasmic RNA was extracted from virusinfected cells. In some cases, infected cells were treated with cycloheximide (50 μg/ml) to enhance mRNA production. At six hours postinfection, cells were washed and harvested in 100 mM tris/HCl (pH 7.4) with 150 mM NaCl. The cells at a concentration of 2 χ 107 per ml were resuspended in lysis buffer comprising 10 mM tris/HCl (pH 7.5)/150 mM NaCl/1.5 mM MgCl2/0.5% Nonidet P-40/20 mM vanadyl ribonucleoside complex. The suspension was vortexed gently and placed on ice for 10 minutes. After lysis, the suspension was centrifuged at 400 x g for 5 minutes, the supernatant was removed and added to 3 volumes of buffer containing 10 mM tris/HCl (pH 7.5), 5 mM EDTA and 0.5% sodium dodecyl sulphate. The solution was extracted with phenol/chloroform followed by chloroform, made 0.3 M in sodium acetate (pH 5.0) and precipitated with 2.5 volumes of cold ethanol.
SI nuclease analysis was performed by coprecipitating a labelled viral genomic probe (Kulka et al. PNAS 86, 6868-6872, 1989) with 10 gg of cytoplasmic RNA obtained as described above. The virus-infected cells were either treated or not treated with one or more oligonucleotides. The DNA/RNA pellet was resuspended in 20 μΐ of 90% (v/v) formamide in 0.4 M NaCl/40 mM PIPES, pH 6.8/1 mM EDTA. The mixture was heated at 90*C for 3 minutes, incubated at 57.5°C for 16 hours and then rapidly chilled on ice. Nuclease SI digestion was performed at 28’C for 1 hour in 300 μΐ of 0.25 M NaCl/30 mM sodium acetate, pH 4.5/2 mM ZnSO4 with 60 units of SI nuclease. The Sl-digested hybrids were extracted with phenol/chloroform and precipitated with ethanol. The products were analyzed by electrophoresis (room temperature, 16 hours, 50V) on 1.5% agarose gels under nondenaturing (90 mM tris/90 mM boric acid, pH 8.3/1 mM EDTA) or alkaline (30 mM NaOH/2 mM EDTA) conditions. Gels were dried on DE-18 paper and radioactivity visualized by autoradiography (at room temperature) using Kodak X-Omat-S film. Relative proportions of spliced and unspliced mRNA were determined by densitometric scanning on a UV/visible spectrophotometer.
Cell proteins were metabolically labelled with [35S] methionine (150 MCi/per ml) from 6 to 7 hours post-infection. The cells were harvested, washed in cold PBS (phosphate-buffered saline) and resuspended in lysis buffer (10 mM tris, pH 8.0/150 mM NaCl/1% Nonidet P-40/1% deoxycholate/0.1% sodium dodecyl sulphate/1 mM phenylmethylsulfonyl fluoride) at a concentration of 1 χ 105 cells per 25 μΐ. Suspensions were placed on ice for 15 minutes and then subjected to 5 rounds of freeze-thaw. The suspensions were cleared of cell debris by centrifugation at 5000 x g for 10 minutes. Electrophoresis was carried out under reducing conditions in 8.5% polyacrylamide gels.
For in vivo assays the antiviral activity of an oligonucleotide was examined in the mouse. Mice were anesthetized using, for example, ether or intraperitoneal injection of sodium pentobarbital. Animals were infected with about 2 χ 101 to 1 x 10 pfu of HSV-1. Animals were ear injected with a volume of approximately 10 μΐ or in the foot pad - 25 with about the same volume. Mice in experimental groups were treated dermally with oligomer in a PEG base at the site of injection on day 0 or on days 0-5 after infection or injected with oligomer at infection. Tissues were examined for virus titer on day 6 after infection. Virus titers were determined on Vero cells.
For chloramphenicol acetyl transferase (CAT) assay, constructs were prepared using the plasmid pCATB' and method described in Wymer et al. (J. Virol 63, 2773-2784, 1989). The plasmid contains the CAT structural gene without eukaryotic promoter sequences. The construct pICPIO-cat contains a 649 base pair fragment of HSV-2 DNA, the ICP10 promoter, inserted 5' to the CAT structural gene. A plasmid carrying the HIV promoter (HIV-LTR) driving the CAT gene was also used. With the appropriate transcription signals, the CAT mRNA is produced and translation of the bacterial CAT occurs.
CAT activity was monitored using an art. 14 recognized assay. Briefly, 0.2 μα of [ C] chloramphenicol substrate is mixed with the test solution and incubated for 1 hour. Radioactivity was monitored in a liquid scintillation counter. Generally, Vero cells were cotransfected with the target plasmid pICPIO-cat and a transactivating IE110 containing plasmid (pIGA-15) as described in Wymer et al., supra. The transfection mixtures contain 1 mg of target plasmid and 0.1 mg of transactivator DNA. Parallel cotransfections employ pBR322 as non-specific DNA to equalize concentration effects. The recombinant plasmid pSV2CAT was used as a positive control and the plasmid pCATB' was used as a negative control. Transfected cells were incubated in the presence or absence of increasing concentrations (0-250 μΜ) of oligomer added immediately after transfection (0 hours) and harvested 40-44 hours later.
HIV titers were determined by a syncytium formation assay. Briefly AA2 cells (2 χ 104) were grown in 96-well flat bottom microliter plates in RPMI 1640 with 10% heat-inactivated fetal calf serum, non-essential amino acids, 1 mM pyruvate and 1.5 Mg/ml polybrene (Sigma). HTLV-IIIB was obtained by clarifying the supernatant of an infected MOLT-3 cell line grown in RPMI 1640 with 10% heatinactivated fetal calf serum. Two-fold dilutions of MOLT-3 supernatant in 100 μΐ volumes were added to the AA2 cultures and incubated for 5-8 days. Titers, expressed as infectious units, represent the reciprocal of the highest dilution that gives rise to syncytium formation.
EXAMPLE 1 Vero cells were exposed to the dodecamer, TTCCTCCTGCGG (0-100 μΜ) at the time of infection with HSV-1 (10 pfu per cell) and virus titers were determined 24 hours later. Other oligomers were used in similar fashion. The results of those experiments are shown in Figure 3 and summarized in Table 1 on the following page.
Cultures exposed to the octomer TCCTCCTG and dodecamer showed a dose dependent decrease in HSV-1 titer. Significant reduction in virus growth (9098%) was seen in cultures exposed to the dodecamer in a concentration of 100 μΜ.
IE mRNA's 4 and 5 are col inear on the HSV-1 and HSV-2 genomes with 65% base sequence homology at the splice acceptor type. As shown in Table 1, some of the oligonucleotides were effective in both strains whereas other oligonucleotides were specific for HSV-1. 1 TABLE 1 2 Effect of the oligo(deoxynucleoside 3 4 methylphosphonate sequence) on HSV growth 5 6 Oligomer % inhibition 7 8 Exon Intron HSV-1 HSV-2 9 (3' AAGGAG GACGCC 5' ) 10 TCCTC CTG 85 40 11 TTCCTC CT 14 ND 12 GCGTTCCTC CTG 30 0 13 GTTCCTC CTGCG 26 0 14 TTCCTC CTGCGG 98 0 15 TC CTGCGG 0 0 16 TC CTGCGGGAAG 9 12 17 GCGGGAAGGCAC 0 0 18 TCCCT CTG 0 0 19 20 HSV-1- or HSV-2-infected Vero cells were 21 treated with the respective : oligomers (100 μΜ) at 22 0 hour postinfection. Infectious virus was 23 determined by plaque assay at 24 hour 24 postinfection. Results are the average of three to 25 six experiments for each oligomer. They are expressed as the percent inhibition of virus titers compared to the appropriate control (no oligomer).
HSV-1 IE mRNA 4 acceptor splice site is shown in parentheses. The HSV-2 IE mRNA 4 acceptor splice site is GGGCCG GACGCT. _ EXAMPLE 2 Cytoplasmic RNA (10 ug) extracted from HSV-linfected HEp-2 cells untreated or treated with 100 μΜ of the dodecamer TTCCTCCTGCGG at the time of infection was hybridized with a labelled HSV probe and then digested with SI nuclease.
On neutral gels the band pattern of nucleaseresistant products from untreated cells consisted of two major bands which were interpreted to represent the 5' leader to the Hindlll site (1200 base pairs, bp) and the splice junction to the Hindlll site (930 bp).
Nuclease-resistant products from the oligomertreated cells consisted of three bands. They included 93 0 bp and 12 00 bp products, equivalent to those identified in untreated cells, and an additional band that consisted of unspliced mRNA and the 145 bp intron joined to the 1200 bp leader.
Analysis of SI nuclease-resistant products on alkaline gels confirmed the presence of unspliced IE mRNA 4 in oligomer-treated cells. Thus, one band was observed in untreated HSV-l-infected cells representing a 3' terminal transcript extending from the splice junction to the Hindlll site (about 900 bp).
The nuclease-resistant products from oligomertreated cells revealed two bands including the 900 bp band and an additional 13 00 bp band that probably corresponded to an RNA transcript extending from the 5' leader through the intron to the 3' Hindlll site.
Approximately 16-20% of the IE mRNA 4 from oligomer-treated cells was present as unspliced message, as determined by densitometric scanning. The result was about a 20% decrease in the levels of spliced mRNA. - 29 EXAMPLE 3 9 HEp-2 cells were infected with HSV-1 (25 pfu per cell) or mock infected with PBS in the presence of 2.8 mM L-canavanine. (L-canavanine restricts gene expression to IE (a) and a subclass of Ε(β) proteins.) The cells were then treated with 100 mM of the dodecamer, TTCCTCCTGCGG. The cells were labelled with [35S] methionine (150 MCi/ml) at 6-7 hours postinfection. Cell extracts were adjusted to the same protein concentration and analyzed on sodium dodecyl sulphate polyacrylamide gels. The gel analysis revealed that the synthesis of viral but not cellular proteins was reduced significantly by oligomer treatment.
EXAMPLE 4 21 22 In two independent experiments for each oligomer, treatment of HSV-l-infected cells with the dodecamer GCGGGGCTCCAT, which is complementary to the translation initiation site of IE110, and the dodecamer AGTCTGCTGCAA, which is complementary to the IE110 second splice acceptor site, resulted in a dose dependent inhibition of IEllO-mediated transactivation in a CAT assay. In these experiments Vero cells were co-transfected with 1 mg of pICPIO-cat DNA and 0.1 mg activating IE110 DNA. The cells were grown for 40-44 hours in the absence or presence of increasing concentrations of oligomers. The cells were harvested and were assayed for CAT activity.
Maximal inhibition of 100% was observed in cells exposed to 250 mM of GCGGGGCTCCAT. The other dodecamer was somewhat less effective. Inhibition was specific, the dodecamer TTCCTCCTGCGG that is complementary to the IE mRNA 4 splice acceptor - 30 site did not inhibit IEllO-mediated transactivation (Figure 4).
EXAMPLE 5 The effect of an oligomer to the donor splice site of IE mRNA4,5 on HSV-1 growth was determined in comparison to the effect of the oligomer to the acceptor splice site shown in Example 1.
Vero cells were infected with 10 pfu per cell of HSV-1 and treated with four different dodecamers at a concentration of 0-200 μΜ. The dodecamers included the two of Example 4, the dodecamer of Example 1, and the dodecamer complementary to the IE4,5 mRNA splice donor site, GCTTACCCGTGC. Virus titers were determined 24 hours later.
As depicted in Figure 5, both of the dodecamers complementary to the IE mRNA4,5 acceptor and donor splice sites resulted in 85-98% inhibition of virus growth in the concentration range of 100-200 mM. However at the m.o.i. of 10, the dodecamer complementary to the IE mRNAl translation initiation site resulted in a maximum of about 15-20% inhibition of virus growth and the dodecamer complementary to the IE mRNA 1 second splice acceptor site resulted in no inhibition.
EXAMPLE 6 Although the dodecamers of Example 4 showed a dose dependent inhibition of IEllO-mediated transactivation of CAT activity, the oligomers had minimal or no affect on HSV-1 growth at a m.o.i. of .
The role of virus concentration and ability of the IE110 oligomers to inhibit HSV-1 growth were examined. Vero cells infected with either 10 pfu - 31 1 or 0.1 pfu per cell of HSV-1 were exposed to the oligomer GCGGGGCTCCAT in a concentration range of 0-200 mM at the time of infection. Virus titers were determined 24 hours later.
The results are summarized in Figure 6 for two independent experiments. The dodecamer resulted in significant inhibition of virus growth in Vero cells infected at a low m.o.i.
EXAMPLE 7 21 22 The effectiveness of using a plurality of oligonucleotides for the inhibition of virus growth in Vero cells was studied. Vero cells were exposed to the dodecamer GCGGGGCTCCAT and TTCCTCCTGCGG at a concentration of 0-200 mM at the time of infection with 0.1 pfu per cell of HSV-1. Virus titers were determined 24 hours later.
The results are summarized in Figures 7A and 7B. Either of the two oligonucleotides, denoted in the figure as IE110 and IE4,5, caused inhibition of virus growth in a dose dependent fashion similar to that found in Examples 1-6 above, with a maximal inhibition of 90-98% at 100-200 mM. When infected cells, however, were treated with both oligomers simultaneously, the effective concentration range for virus inhibition is greater (25-200 mM) than for treatment with either oligonucleotide alone. Additionally, treatment with a plurality of nucleotides resulted in significant inhibition (SO99. 8%) at 25-75 mM and a 50% decrease in virus titers at 10 mM.
In Figure 7B, the results of the data were calculated and presented according to formulae that verify synergistic interactions beteen the two oligomers. The data presented in this fashion reveal in a rather dramatic way that a combination of the two oligonucleotides resulted in a synergistic inhibition of viral replication.
EXAMPLE 8 In a similar study, oligonucleotide complementary to the initiation site of IE4 (designated in Figure 8 as IE68) and oligonucleotide complementary to the IE5 initiation site (designated in Figure 8 as IE12) were tested as described above.
The data summarized in Figure 8 reveal that the IE12 oligonucleotide was ineffective in inhibiting virus growth whereas the other nucleotides resulted in a 85-95% inhibition of viral growth over a range of 25-200 mM of the oligonucleotides. Mixing the two oligonucleotides did not diminish the binding and inhibitory activity of the IE5 initiation site oligonucleotide.
EXAMPLE 9 Mice were infected with HSV-1 (2000 pfu) , treated with oligomer on day 0 or on days 0-5 postinfection and the tissues were examined for virus titers on day 6 postinfection. While single treatment with oligomer (500 μΜ) had little effect on virus growth (27% inhibition), daily application resulted in 82% inhibition of virus titers. With a higher concentration of virus per dose, 2 χ 106 pfu, animals treated twice daily (receiving a total amount of 1000 μΜ per day) with TTCCTCCTGCGG did not develop lesions (0/5 as compared to 5/5 in untreated animals).
Consistent with these studies, animals which were inoculated in the foot pad with HSV-1 (1 χ 106 pfu) and then treated twice (days 1 and 2 postimmunization) with 50 μΜ of dodecamer, showed about an 86% reduction in virus titer.
EXAMPLE 10 Although it is unclear why not all people infected with HIV develop AIDS, it is becoming evident that many factors other than HIV are essential for activation of the virus. Notable among the factors that may enhance HIV activation are viruses including JC, adenoviruses and the herpesviruses including HSV-1, HSV-2, HCMV, VZV, EBV, HHV-6 and HHV-7. Specific genes of the herpesviruses activate the expression of HIV either directly at the level of viral replication or by activating expression of a reporter gene driven by the HIV promoter.
U937 cells were cotransfected with a construct carrying the HIV-LTR promoter upstream to the CAT structural gene and a construct carrying sequences encoding HSV-1 IE110. Those same transfected cells were then treated with oligomers complementary to the initiation or splice sites of the IE110 gene. After 40 hours of culture, the presence or absence of CAT was determined.
Results of a representative experiment are presented in Figure 9. In the experiment, three different oligomers were tested, two are complementary to IE110 sequences and the oligomer denoted as IE4,5 is complementary to an unrelated gene. The oligomers were used at two different concentrations. The two oligomers complementary to IE110 sequences resulted in a dose-related inhibition of the HIV promoter with the oligomer complementary to the initiation site showing a higher level of activity than the oligomer complementary to the second splice site. The effect is specific as the IE4,5 oligomer does not inhibit HIV activation to a significant degree.
In another experiment that monitored HIV activation by assaying virus growth (Figure 10), it was found that growth of HIV was enhanced 2500-fold by the addition of HSV IE110 and the activation was reduced significantly by the addition of the IE110 oligomer complementary to the initiation site.
In the figure, it will be appreciated that untreated cells and cells that were treated with the oligomer showed no HIV activation after 8 days whereas cells treated with the HSV gene product showed remarkable proliferation.
When the cells were treated with a combination of IE110 and the oligomer at a concentration of 150 mM, a moderate degree of HIV growth was noted. The level of virus growth did not differ however from the degree of HIV activation obtained when the cells were exposed to the control plasmid pBR322.
As testimony to the relatedness of the herpesviruses, U937 cells were exposed also to the homologue IE gene product of cytomegalovirus. Although the degree of activation was not as great as that found with the HSV gene product, there was a significant enhancement of HIV growth by the CMV gene product.
EXAMPLE 11 Additional animal studies revealed that dermal application of oligonucleotides was effective in inhibiting virus replication.
Mice were infected with HSV-1 and injected interdermally with the oligonucleotide TTCCTCCTGCGG at the time of infection. Then once daily thereafter the oligonucleotide suspended in a PEG 21 22 - 35 (polyethylene glycol) cream was applied to the site of infection. The concentration of oligonucleotide in the cream was 500 μΜ which is 5-fold higher than used for parenteral administration.
The results are summarized in Table 2.
TABLE 2 Effect of ΙΕ4,Λ5 oligomer on virus clearance Group (n = 10) Inhibition Av. virus titer/ear % (day 3) At re in do Al ar It sk of in on de ot sp in co no Virus dose (l x io6 PFU) / Vehicle (no oligomer) 2 x 10 Oligomer (500 μΜ) 5.0 X 10 Virus dose (1 χ 104 PFU) Vehicle (no oligomer) 8 x io2 Oligomer (500 μΜ) 6.8 X 10 At either of the two doses, virus titers were reduced significantly, however, somewhat better inhibition was obtained at an initial low virus dose of 1 x 10 pfu.
All publications cited in the specification are herein incorporated by reference.
It will be appreciated by one of ordinary skill in the art that the compositions and methods of the instant invention are capable of being incorporated in the form of variety of embodiments, only a few of which have been illustrated and described above. The invention may be embodied in other specific forms without departing from the spirit or the central characteristics of said invention. The described embodiments are to be considered in all respects only as illustrative and not restrictive. All changes that come within the - 36 1 meaning and range of equivalency of the claims are to be embraced within the scope of the claims in view of the teachings in the specification.

Claims (164)

1. IN THE CLAIMS:
1. An oligonucleotide selected from group consisting of: the (a) T T C C T C C T G C G G; (b) G c T T A C c c G T G C; 5 (c) G c G G G G C T C C A T; (d) A A T G T C G G C C A T; (e) A T A T c A A T G T CAG T c G c c A T; (f) G G C c c A C G A c A T; 10 (g) G C G G G C G T C G G T; (h) G G C G C C G T C T G C; (i) G T G G c C G G C G C C; (j) G C G G G C G T C G G T; (k) G G C G C C G T C T G C; 15 (1) G T G G C C G G C G C C; (m) G T T C T C C G A C G C C A T; (n) C G C T c C G T G T G G; (o) C C G c c G C C A C CGG A A A A c A T; 20 (P) T G G c A G G G G T ACG A A G C C A T; (q) G C G G C T G G G C GGC T G G C C A T; (r) T C G T C G A C C A AGA 25 G G T C c A T; (s) C A A A c A G C T C G T; (t) C C A T G T C G G C A A; (u) G T C C G C G T C C A T; (v) G c C G T C C G C G T C; 30 (w) T G G C G A A G C G C C; (x) A C A G c C C G T G G T? (y) C C G A G G A A T G A C; (Z) C C G T T C C C G A G G; (aa) C A C T G G C A T G G A; 35 (bb) G C A T C C T G C C A C? (cc) C C C C G A A A G C A T; - 38 10 (dd) GATCCCCGAAAG; (ee) CTGACCACCGAT; (ff) GCAGGCTCTGGT; (gg) CCATGTTGGGCA; (hh) TGGGGGTGCCAT; (ii) GAGTGGGGGTGC; (jj) GGTGCGTGGGAG; (kk) CAAGGACGGTGA; (11) CCATCGTGTCAA; (mm) AGAGGACTCCAT; (nn) GGCAGAGGACTC; (oo) CTTTCTCTTGGC; (pp) GACGGTTCACTA; (qq) CAGGCGATCTGA; (rr) CGTCTCCAGGCG; (SS) CAACTGGCTGTA; (tt) CCATGGTAACAA; (uu) TATGGTATCCAT; (vv) TAATATGGTATC; (w) ACCGCCCGCTAA; (XX) TGGGGTGAATTT; (yy) CCATCGCACTGG; (ZZ) CGGCGTATCCAT; (a·) CGGCGGCGTATC; (b* ) GCGCTGCATCGG; (o') AGTCTGCTGCAA; and (d·) T A A T G A R A T; or ribose containing oligonucleotides corresponding thereto, wherein T is replaced by U; and where A, T, G, U and C are adenine, thymidine, guanine, uracil and cytosine, and R is A, T, G, C, U or functional equivalents thereof.
2. The oligonucleotide or ribose containing oligonucleotide of claim 1 selected from the group consisting of: (a) TTCCTCCTGCGG; (b) G C T T A C C C G T G C; (c) (d) (e) GCGGGGCTCCAT AATGTCGGCCAT A T T c A T C C AAT G T C A G C A T; 5 (f) G G C C C A C G A C A T (g) G C G G G C G T C G G T (h) G G C G C C G T C T G c (i) G T G G C C G G C G C c (j) G C G G G C G T C G G T 10 (k) G G C G C C G T C T G c (1) G T G G C C G G C G C c (m) G T T C T C C G A C G c (n) C G C T C C G T G T G G (o) C C G c C G C C A C C G 15 A A A A C A T; (p) T G G C A G G G G T A C A A G C C A T; (q) G C G G C T G G G C G G T G G C C A T; 20 (r) T C G T C G A C C A A G G G T C C A T; (s) C A A A C A G C T C G T (t) C C A T G T C G G C A A (u) G T C c G C G T C C A T 25 (v) G c C G T C C G C G T C (w) T G G C G A A G C G C C (x) A C A G C C C G T G G T (y) C C G A G G A A T G A c (Z) C C G T T C C C G A G G 30 (aa) C A C T G G C A T G G A (bb) G C A T C C T G C C A C (cc) C C C C G A A A G C A T (dd) G A T c C C C G A A A G (ee) C T G A C C A C C G A T 35 (ff) G C A G G C T C T G G T (gg) C C A T G T T G G G C A (hh) T G G G G G T G C C A T - 40 (ii) GAGTGGGGGTGC; (jj) GGTGCGTGGGAG? (kk) AGTCTGCTGCAA; and (11) TAATGARAT.
3. The oligonucleotide or ribose containing oligonucleotide of claim 2 selected from the group consisting of: (a) TTCCTCCTGCGG; (b) GCTTACCCGTGC; (c) GCGGGGCTCCAT; and (d) AATGTCGGCCAT.
4. The oligonucleotide or ribose containing oligonucleotide of claim 1 selected from the group consisting of: (a) CAAGGACGGTGA; (b) CCATCGTGTCAA; (c) AGAGGACTCCAT? (d) GGCAGAGGACTC; (e) CTTTCTCTTGGC; (f) GACGGTTCACTA; (g) C A G G C G A T C T G A; and (h) CGTCTCCAGGCG.
5. The oligonucleotide or ribose containing oligonucleotide of claim 1 selected from the group consisting of: (a) CAACTGGCTGTA; (b) CCATGGTAACAA; (C) TATGGTATCCAT; (d) TAATATGGTATC; (e) ACCGCCCGCTAA? (f) TGGGGTGAATTT; (g) CCATCGCACTGG? (h) CGGCGTATCCAT? (i) CGGCGGCGTATC; and (j) GCGCTGCATCGG. - 41
6. The oligonucleotide or ribose containing oligonucletide of claims l, 2, 3, 4, or 5 which comprises one or more modified nucleosides.
7. The oligonucleotide of claim 6 wherein said modified nucleoside is an aryl or alkyl phosphonate nucleoside.
8. The oligonucleotide of claim 7 wherein said aryl or alkyl phosphonate nucleoside is methylphosphonate nucleoside.
9. A composition for inhibiting herpesvirus growth or replication comprising an oligonucleotide hybridizable with nucleic acids of said herpesvirus.
10. The composition of claim 9 wherein said oligonucleotide hybridizes to a cap site, a transcription initiation site, a translation initiation site or a splice site.
11. The composition of claim 10 wherein said oligonucleotides hybridizable to a splice site contain exon and intron sequences and the difference in the number of bases containing exon 5 sequences and the number of bases containing intron sequences is 0 or 1.
12. The composition of claim 10 wherein said oligonucleotides hybridizable to a translation initiation site are complementary to the ATG initiation codon and adjacent 3' bases in the 5 coding region, so that the last three bases of said oligonucleotide in the 5' to 3’ orientation are C, A and T, in that order.
13. The composition of claims 9, 10, 11 or 12 wherein said oligonucleotide hybridizes to a regulatory gene, a gene required for nucleic acid replication or a gene involved in pathogenesis.
14. The composition of claim 13 wherein said regulatory gene is an immediate-early (IE) gene. - 42
15. The composition of claim 13 wherein said regulatory gene is Vmw65.
16. The composition of claim 13 wherein said oligonucleotide is selected from the group consisting of: (a) T T c CTCCTGCGG; 5 (b) G C T TACCCGTGC; (c) G c G GGGCTCCAT; and A A T GTCGGCCAT; or ribose containing oligonucleotides corresponding thereto, wherein T is replaced by U; and where A, 10 T, G, U and C are adenine, thymidine, guanine, uracil and cytosine.
17. The composition of claim 9 wherein said oligonucleotide comprises one or more alkyl or arylphosphonate nucleosides.
18. The composition of claim 17 wherein said alkyl or arylphosphonate nucleoside is methylphosphonate nucleoside.
19. The composition of claim 16 wherein said oligonucleotide or ribose containing oligonucleotide comprises one or more alkyl or arylphosphonate nucleosides.
20. The composition of claim 19 wherein said alkyl or arylphosphonate nucleoside is methylphosphonate nucleoside.
21. An in vitro method of inhibiting herpesvirus growth or replication comprising the step of contacting nucleic acids of said herpesvirus with an oligonucleotide hybridizable with said nucleic 5 acids of herpesvirus.
22. The method of claim 21 wherein said oligonucleotide hybridizes to a cap site, a transcription initiation site, a translation initiation site or a splice site.
23. The method of claim 22 wherein said oligonucleotides hybridizable to a splice site - 43 contain exon and intron sequences and the difference in the number of bases containing exon sequences and the number of bases containing intron sequences is 0 or 1.
24. The method of claim 22 wherein said oligonucleotides hybridizable to a translation initiation site are complementary to the ATG initiation codon and adjacent 3' bases in the coding region, so that the last three bases of said oligonucleotide in the 5' to 3 1 A and T, in that order. orientation are c,
25. The method of claims 21, 22, 23 or 24 wherein said oligonucleotide hybridizes to a regulatory gene, a gene required for nucleic acid replication or a gene involved in pathogenesis.
26. The method of claim 25 wherein said regulatory gene is an immediate-early (IE) gene.
27. The method of claim 25 wherein said regulatory gene is Vmw65. 28. The method of claim 25 wherein said oligonucleotide is selected from the group consisting of: (a) TTCCTCCTG C G G; (b) GCTTACCCGTGC;
(C) GCGGGGCTCCAT; and (d) A A T G T C G G C C A T; or ribose containing oligonucleotides corresponding thereto, wherein T is replaced by U; and where A, T, G, U and C are adenine, thymidine, guanine, uracil and cytosine.
29. The method of claim 21 wherein said oligonucleotide comprises one or more alkyl or arylphosphonate nucleosides.
30. The method of claim 29 wherein said alkyl or arylphosphonate nucleoside is methylphosphonate nucleoside. - 44
31. The method of claim 28 wherein said oligonucleotide or ribose containing oligonucleotide comprises one or more alkyl or arylphosphonate nucleosides.
32. The method of claim 31 wherein said alkyl or arylphosphonate nucleoside is methylphosphonate nucleoside.
33. A therapeutic composition for inhibiting herpesvirus growth or replication comprising an oligonucleotide, or pharmaceutically acceptable salts thereof, hybridizable with nucleic acids of 5 said herpesvirus and a pharmaceutically acceptable carrier.
34. The therapeutic composition of claim 33 wherein said oligonucleotide hybridizes to a cap site, a transcription initiation site, a translation initiation site or a splice site.
35. The therapeutic composition of claim 34 wherein said oligonucleotides hybridizable to a splice site contain exon and intron sequences and the difference in the number of bases containing 5 exon sequences and the number of bases containing intron sequences is 0 or 1.
36. The therapeutic composition of claim 34 wherein said oligonucleotides hybridizable to a translation initiation site are complementary to the ATG initiation codon and adjacent 3' bases in 5 the coding region, so that the last three bases of said oligonucleotide in the 5’ to 3' orientation are C, A and T, in that order.
37. The therapeutic composition of claims 33, 34, 35 or 36 wherein said oligonucleotide hybridizes to a regulatory gene, a gene required for nucleic acid replication or a gene involved in 5 pathogenesis. - 45 10
38. The therapeutic composition of claim 37 wherein said regulatory gene is an immediate-early (IE) gene.
39. The therapeutic composition of claim 37 wherein said regulatory gene is Vmw65.
40. The therapeutic composition of claim 37 wherein said oligonucleotide is selected from the group consisting of: (a) TTCCTCCTGCGG; (b) GCTTACCCGTGC; (c) GCGGGGCTCCAT; and (d) A A T G T C G G C C A T; or ribose containing oligonucleotides corresponding thereto, wherein T is replaced by U; and where A, T, G, U and C are adenine, thymidine, guanine, uracil and cytosine.
41. The therapeutic composition of claim 33 wherein said oligonucleotide comprises one or more alkyl or arylphosphonate nucleosides.
42. The therapeutic composition of claim 41 wherein said alkyl or arylphosphonate nucleoside is methylphosphonate nucleoside.
43. The therapeutic composition of claim 40 wherein said oligonucleotide or ribose containing oligonucleotide comprises one or more alkyl or arylphosphonate nucleosides.
44. The therapeutic composition of claim 43 wherein said alkyl or arylphosphonate nucleoside is methylphosphonate nucleoside.
45. Use of a composition comprising an oligonucleotide, or pharmaceutically acceptable salt thereof, for inhibiting herpesvirus growth or replication and which is hybridizable with nucleic acids of said herpesvirus. - 46
46. use according to claim 45 wherein said oligonucleotide hybridizes to a cap site, a transcription initiation site, a translation initiation site or a splice site.
47. Use according to claim 46 wherein said oligonucleotides hybridizable to a splice site contain exon and intron sequences and the difference in the number of bases containing 5 exon sequences and the number of bases containing intron sequences is 0 or 1.
48. Use according to claim 46 wherein said oligonucleotides hybridizable to a translation initiation site are complementary to the ATG initiation codon and adjacent 3' bases irr 5 the coding region, so that the last three bases of said oligonucleotide in the 5' to 3 1 orientation are C, A and T, in that order.
49. Use according to claims 45, 46, 47 or 48 wherein said oligonucleotide hybridizes to a regulatory gene, a gene required for nucleic acid replication or a gene involved in pathogenesis.
50. Use according to claim 49 wherein said regulatory gene is an immediate-early (IE) gene.
51. Use according to claim 49 wherein said regulatory gene is Vmw65.
52. Use according to claim 49 wherein said oligonucleotide is selected from the group consisting of: (a) T T C C T c c T G C G G; (b) G C Τ T A C C C G T G C; (c) G C G G G G C T c CAT; and (d) A A T G T C G G C CAT; or ribose containing oligonucleotides corresponding thereto, wherein T is replaced by U; and where A, 10 T, G, U and C are adenine, thymidine, guanine, uracil and cytosine. - 47
53. Use according to claim 45 wherein said oligonucleotide comprises one or more alkyl or arylphosphonate nucleosides.
54. Use according to claim 53 wherein said alkyl or arylphosphonate nucleoside is methylphosphonate nucleoside.
55. Use according to claim 52 wherein said oligonucleotide or ribose containing oligonucleotide comprises one or more alkyl or arylphosphonate nucleosides.
56. Use according to claim 55 wherein said alkyl or arylphosphonate nucleoside is methylphosphonate nucleoside.
57. A method of detecting herpesvirus comprising the steps of: (i) obtaining a biologic sample containing nucleic acids; (ii) treating said sample so the nucleic acids contained therein are made single stranded; (iii) exposing said treated sample to a labelled oligonucleotide wherein said oligonucleotide is selected from the group consisting of: (a) TTCCTCCTGCGG; (b) GCTTACCCGTGC; (C) GCGGGGCTCCAT; (d) AATGTCGGCCAT; (e) ATATCAAT GTCAGTCG C C A T; (f) GGCCCACGACAT; (g) GCGGGCGTCGGT; (h) G G C G C C G T C T G C; (i) (j) (k) (l) 5 (m) (n) (o) 10 (p) (q) (r) (s) 20 (t) (u) (V) (w) (x) 25. (y) (z) (aa) (bb) (cc) 26. 30 (dd) (ee) (ff) (gg) (hh) 27. 35 (ii) (jj) (kk) - 48 GTGGCCGGCGCC; GCGGGCGTCGGT; GGCGCCGTCTGC; GTGGCCGGCGCC; GTTCTCCGACGCCAT; CGCTCCGTGTGG; CCGCCGCC ACCGGAAA A G A T; TGGCAGGG GTACGAAG C C A T; GCGGCTGG GCGGCTGG C C A T; TCGTCGAC CAAGAGGT C C A T; CAAACAGCTCGT? CCATGTCGGCAA? GTCCGCGTCCAT; GCCGTCCGCGTC; TGGCGAAGCGCC; ACAGCCCGTGGT; CCGAGGAATGAC; CCGTTCCCGAGG; CACTGGCATGGA; GCATCCTGCCAC; CCCCGAAAGCAT; GATCCCCGAAAG; CTGACCACCGAT? GCAGGCTCTGGT; CCATGTTGGGCA; TGGGGGTGCCAT; GAGTGGGGGTGC; GGTGCGTGGGAG; CAAGGACGGTGA? - 49 (11) CCATCGTGTCAA; (mm) AGAGGACTCCAT; (nn) GGCAGAGGACTC; (OO) CTTTCTCTTGGC; (pp) GACGGTTCACTA; (qq) CAGGCGATCTGA; (rr) CGTCTCCAGGCG; (SS) CAACTGGCTGTA; (tt) CCATGGTAACAA; (uu) TATGGTATCCAT; (vv) TAATATGGTATC; (ww) ACCGCCCGCTAA; (xx) TGGGGTGAATTT; (yy) CCATCGCACTGG; (zz) CGGCGTATCCAT; (a') CGGCGGCGTATC; (b·) GCGCTGCATCGG; (o') AGTCTGCTGCAA; and (d * ) TAATGARAT; or ribose containing oligonucleotides corresponding thereto, wherein T is replaced by U; and where A, T, G, U and C are adenine, thymidine, guanine, uracil and cytosine, and R is A, T, G, C, U or functional equivalents thereof; and (iv) detecting hybridized sequences.
58. The method of claim 57 wherein said oligonucleotide or ribose containing oligonucletide comprises one or more modified nucleosides.
59. The method of claim 58 wherein said modified nucleoside is an aryl or alkyl phosphonate nucleoside.
60. The method of claim 59 wherein said aryl or alkyl phosphonate is methylphosphonate nucleoside.
61. A composition for inhibiting microbe, virus or self-replicating nucleic acid growth or - 50 replication comprising at least two oligonucleotides hybridizable to non-overlapping 5 sites in the nucleic acids of said microbe, virus or self-replicating nucleic acid.
62. The composition of claim 61 wherein at least one of said oligonucleotides is hybridizable to a cap site, a transcription initiation site, a translation initiation site or a splice site.
63. The composition of claim 62 wherein said oligonucleotides hybridizable to a splice site contain exon and intron sequences and the difference in the number of bases containing exon 5 sequences and the number of bases containing intron sequences is 0 or 1.
64. The composition of claim 62 wherein said oligonucleotides hybridizable to a translation initiation site are complementary to the ATG initiation codon and adjacent 3' bases in the 5 coding region, so that the last three bases of said oligonucleotide in the 5' to 3’ orientation are C, A and T, in that order.
65. The composition of claims 61, 62, 63 or 64 wherein at least one of said oligonucleotides is hybridizable to a regulatory gene, a gene required for nucleic acid replication or a gene involved in 5 pathogenesis.
66. The composition of claim 65 wherein one of said oligonucleotides is hybridizable to a first portion of nucleic acid of said microbe, virus or self-replicating nucleic acid in which the 5 expression or function of said first portion is required for expression or function of a second portion of nucleic acid of said microbe, virus or self-replicating nucleic acid and another of said oligonucleotides is hybridizable to said second 10 portion. - 51
67. The composition of claim 66 wherein said virus is a herpesvirus.
68. The composition of claim 67 wherein said regulatory gene is an immediate-early (IE) gene.
69. The composition of claim 67 wherein said regulatory gene is Vmw65.
70. The composition of claim 67 wherein at least one of said oligonucleotides is selected from the group consisting of: (a) TTCCTCCTGCGG; (b) GCTTACCCGTGC; (C) G C G G G G C T C C A T; and (d) A A T G T C G G C C A T; or ribose containing oligonucleotides corresponding thereto, wherein T is replaced by U; and where A, T, G, U and C are adenine, thymidine, guanine, uracil and cytosine.
71. The composition of claim 61 wherein at least two of said oligonucleotides comprise one or more alkyl or arylphosphonate nucleosides.
72. The composition of claim 71 wherein said alkyl or arylphosphonate nucleoside is methylphosphonate nucleoside.
73. The composition of claim 70 wherein said oligonucleotide or ribose containing oligonucleotide comprises one or more alkyl or arylphosphonate nucleosides.
74. The composition of claim 73 wherein said alkyl or arylphosphonate nucleoside is methylphosphonate nucleoside.
7 5. An in vitro method for inhibiting microbe, virus or self-replicating nucleic acid growth or replication comprising the step of contacting nucleic acids of said virus or microbe with at least two oligonucleotides that are hybridizable with nonoverlapping sites in said nucleic acids of said microbe, virus or self-replicating nucleic acid. - 52
76. The method of claim 75 wherein at least one of said oligonucleotides is hybridizable to a cap site, a transcription initiation site, a translation initiation site or a splice site.
77. The method of claim 76 wherein said oligonucleotides hybridizable to a splice site contain exon and intron sequences and the difference in the number of bases containing exon 5 sequences and the number of bases containing intron sequences is 0 or 1.
78. The method of claim 76 wherein said oligonucleotide hybridizable to a translation initiation site is complementary to the ATG initiation codon and adjacent 3’ bases in the 5 coding region, so that the last three bases of said oligonucleotide in the 5' to 3' orientation are C, A and T, in that order.
79. The method of claims 75, 76, 77 or 78 wherein at least one of said oligonucleotides is hybridizable to a regulatory gene, a gene required for nucleic acid replication or a gene involved in 5 pathogenesis.
80. The method of claim 79 wherein one of said oligonucleotides is hybridizable to a first portion of nucleic acids of said microbe, virus or self-replicating nucleic acid in which the 5 expression or function of said first portion is required for expression or function of a second portion of nucleic acids of said microbe, virus or self-replicating nucleic acid and another of said oligonucleotides is hybridizable to said second 10 portion.
81. The method of claim 80 wherein said virus is a herpesvirus.
82. The method of claim 81 wherein said regulatory gene is an immediate-early (IE) gene. - 53
83. The method of claim 81 wherein said regulatory gene is Vmw65.
84. The method of claim 81 wherein at least one of said oligonucleotides is selected from the group consisting of: (a) TTCCTCCTGCGG; (b) GCTTACCCGTGC; (c) GCGGGGCTCCAT; and (d) A A T G T C G G C C A T; or ribose containing oligonucleotides corresponding thereto, wherein T is replaced by U; and where A, T, G, U and C are adenine, thymidine, guanine, uracil and cytosine.
85. The method of claim 75 wherein at least two of said oligonucleotides comprise one or more alkyl or arylphosphonate nucleosides.
86. The method of claim 85 wherein said alkyl or arylphosphonate nucleoside is methylphosphonate nucleoside.
87. The method of claim 84 wherein said oligonucleotide or ribose containing oligonucleotide comprises one or more alkyl or arylphosphonate nucleosides.
88. The method of claim 87 wherein said alkyl or arylphosphonate nucleoside is methylphosphonate nucleoside.
89. A therapeutic composition for inhibiting microbe, virus or self-replicating nucleic acid growth or replication comprising at least two oligonucleotides, or pharmaceutically acceptable salts thereof, that are hybridizable with nonoverlapping sites in nucleic acids of said microbe, virus or self-replicating nucleic acid and a pharmaceutically acceptable carrier.
90. The therapeutic composition of claim 89 wherein at least one of said oligonucleotides is hybridizable to a cap site, a transcription - 54 initiation site, a translation initiation site or 5 a splice site.
91. The therapeutic composition of claim 90 wherein said oligonucleotides hybridizable to a splice site contain exon and intron sequences and the difference in the number of bases containing 5 exon sequences and the number of bases containing intron sequences is 0 or 1.
92. The therapeutic composition of claim 90 wherein said oligonucleotides hybridizable to a translation initiation site are complementary to the ATG initiation codon and adjacent 3’ bases in 5 the coding region, so that the last three bases of said oligonucleotide in the 5' to 3' orientation are C, A and T, in that order.
93. The therapeutic composition of claims 89, 90, 91 or 92 wherein at least one of said oligonucleotides is hybridizable to a regulatory gene, a gene required for nucleic acid replication 5 or a gene involved in pathogenesis.
94. The therapeutic composition of claim 93 wherein one of said oligonucleotides is hybridizable to a first portion of nucleic acids of said microbe, virus or self-replicating nucleic 5 acid in which the expression or function of said first portion is required for expression or function of a second portion of nucleic acids of said microbe, virus or self-replicating nucleic acid and another of said oligonucleotides is 10 hybridazable to said second portion.
95. The therapeutic composition of claim 94 wherein said virus is a herpesvirus.
96. The therapeutic composition of claim 95 wherein said regulatory gene is an immediate-early (IE) gene.
97. The therapeutic composition of claim 95 wherein said regulatory gene is Vmw65. - 55 10
98. The therapeutic composition of claim 95 wherein at least one of said oligonucleotides is selected from the group consisting of: (a) TTCCTCCTGCGG; (b) GCTTACCCGTGC; (c) GCGGGGCTCCAT; and (d) AATGTCGGCCAT; or ribose containing oligonucleotides corresponding thereto, wherein T is replaced by U; and where A, T, G, U and C are adenine, thymidine, guanine, uracil and cytosine.
99. The therapeutic composition of claim 89 wherein at least two of said oligonucleotides comprise one or more alkyl or arylphosphonate nucleosides.
100. The method of claim 99 wherein said alkyl or arylphosphonate nucleoside is methylphosphonate nucleoside.
101. The method of claim 98 wherein said oligonucleotide or ribose containing oligonucleotide comprises one or more alkyl or arylphosphonate nucleosides.
102. The method of claim 101 wherein said alkyl or arylphosphonate nucleoside is methylphosphonate nucleos ide.
103. Use of a composition comprising at least two oligonucleotides, or pharmaceutically acceptable salts thereof, for inhibiting microbe, virus or self replicating nucleic acid growth or replication and which are hybridizable with non-overlapping sites in nucleic acids of said microbe, virus or selfreplicating nucleic acid.
104. Use according to claim 103 wherein at least one of said oligonucleotides is hybridizable to a cap site, a transcription - 56 initiation site, a translation initiation site or 5 a splice site.
105. Use according to claim 104 wherein said oligonucleotides hybridizable to a splice site contain exon and intron sequences and the difference in the number of bases containing 5 exon sequences and the number of bases containing intron sequences is 0 or 1.
106. Use according to claim 104 wherein said oligonucleotides hybridizable to a translation initiation site are complementary to the ATG initiation codon and adjacent 3’ bases in 5 the coding region, so that the last three bases of said oligonucleotide in the 5' to 3' orientation are C, A and T, in that order.
107. Use according to claims 103, 104, 105 or 106 wherein at least one of said oligonucleotides is hybridizable to a regulatory gene, a gene required for nucleic acid replication 5 or a gene involved in pathogenesis.
108. Use according to claim 107 wherein one of said oligonucleotides is hybridizable to a first portion of nucleic acids of said microbe, virus or self-replicating nucleic 5 acids in which the expression or function of said first portion is required for expression or function of a second portion of said nucleic acids of said microbe, virus or self-replicating nucleic acid and another of said oligonucleotides is 10 hybridizable to said second portion.
109. Use according to claim 108 wherein said virus is a herpesvirus.
110. Use according to claim 109 wherein said regulatory gene is an immediate-early (IE) gene.
111. Use according to claim 109 wherein said regulatory gene is Vmw65. - 57
112. Use according to claim 109 wherein at least one of said oligonucleotides is selected from the group consisting of: (a) T T C CTCCTGCGG; 5 (b) G C T TACCCGTGC; (c) G C G GGGCTCCAT; and (d) A A T GTCGGCCAT; or ribose containing oligonucleotides corresponding thereto, wherein T is replaced by U; and where A, 10 T, G, U and C are adenine, thymidine, guanine, uracil and cytosine.
113. Use according to claim 103 wherein at least two of said oligonucleotides comprise one or more alkyl or arylphosphonate nucleosides.
114. Use according to claim 113 wherein said alkyl or arylphosphonate nucleoside is methylphosphonate nucleoside.
115. Use according to claim 112 wherein said oligonucleotide or ribose containing oligonucleotide comprises one or more alkyl or arylphosphonate nucleosides.
116. Use according to claim 115 wherein said alkyl or arylphosphonate nucleoside is methylphosphonate nucleoside.
117. A method for inhibiting human immunodeficiency virus growth or replication in a specimen containing human immunodeficiency virus and a second virus comprising the step of 5 contacting nucleic acids of said specimen with at least one oligonucleotide that is hybridizable with nucleic acids of said second virus.
118. The method of claim 117 wherein at least one of said oligonucleotides is hybridizable to a cap site, a transcription initiation site, a translation initiation site or a splice site. - 58
119. The method of claim 118 wherein said oligonucleotides hybridizable to a splice site contain exon and intron sequences and the difference in the number of bases containing exon 5 sequences and the number of bases containing intron sequences is 0 or 1.
120. The method of claim 118 wherein said oligonucleotides hybridizable to a translation initiation site are complementary to the ATG initiation codon and adjacent 3' bases in the 5 coding region, so that the last three bases of said oligonucleotide in the 5' to 3' orientation are C, A and T, in that order.
121. The method of claims 117, 118, 119 or 120 wherein at least one of said oligonucleotides is hybridizable to a regulatory gene, a gene required for nucleic acid replication or a gene involved in 5 pathogenesis.
122. The method of claim 121 wherein said second virus is herpesvirus.
123. The method of claim 122 wherein said regulatory gene is an immediate-early (IE) gene.
124. The method of claim 122 wherein said regulatory gene is Vmw65.
125. The method of claim 122 wherein at least one of said oligonucleotides is selected from the group consisting of: (a) T T C C T C C T G C G G; (b) G C T T A C C c G T G C; (c) G c G G G G C T C c A T; and (d) A A T G T C G G C c A T; or ribose containing oligonucleotides corresponding thereto, wherein T is replaced by U; and where A, 10 T, G, U and C are adenine, thymidine, guanine, uracil and cytosine. - 59
126. The method of claim 117 wherein at least one of said oligonucleotides comprise one or more alkyl or arylphosphonate nucleosides.
127. The method of claim 126 wherein said alkyl or arylphosphonate nucleoside is methylphosphonate nucleoside.
128. The method of claim 125 wherein at least one of said oligonucleotides or ribose containing oligonucleotides comprise one or more alkyl or arylphosphonate nucleosides.
129. The method of claim 128 wherein said alkyl or arylphosphonate nucleoside is methylphosphonate nucleoside.
130. The method of claim 117 wherein said specimen is contacted with at least two oligonucleotides.
131. A therapeutic composition for inhibiting human immunodeficiency virus growth or replication in a host carrying human immunodeficiency virus and a second virus comprising at least one 5 oligonucleotide, or pharmaceutically acceptable salts thereof, that is hybridizable with nucleic acids of said second virus and a pharmaceutically acceptable carrier.
132. The therapeutic composition of claim 131 wherein at least one of said oligonucleotides is hybridizable to a cap site, a transcription initiation site, a translation initiation site or 5 a splice site.
133. The therapeutic composition of claim 132 wherein said oligonucleotides hybridizable to a splice site contain exon and intron seguences and the difference in the number of bases containing 5 exon seguences and the number of bases containing intron seguences is 0 or 1.
134. The therapeutic composition of claim 132 wherein said oligonucleotides hybridizable to a - 60 translation initiation site are complementary to the ATG initiation codon and adjacent 3’ bases in 5 the coding region, so that the last three bases of said oligonucleotide in the 5' to 3' orientation are C, A and T, in that order.
135. The therapeutic composition of claims 131, 132, 133 or 134 wherein at least one of said oligonucleotides is hybridizable to a regulatory gene, a gene required for nucleic acid replication 5 or a gene involved in pathogenesis.
136. The therapeutic composition of claim 135 wherein said second virus is herpesvirus.
137. The therapeutic composition of claim 136 wherein said regulatory gene is an immediate-early (IE) gene.
138. The therapeutic composition of claim 136 wherein said regulatory gene is Vmw65.
139. The therapeutic composition of claim 136 wherein said oligonucleotide is selected from the group consisting of: (a) T T CCT C C T G C G G; (b) G C TTA C C C G T G C; (c) G C GGG G C T C C A T; and (d) A A TGT C G G C C A T; or ribose containing oligonucleotides corresponding thereto, wherein T is replaced by U; and where A, 10 T, G, U and C are adenine, thymidine, guanine, uracil and cytosine.
140. The therapeutic composition of claim 131 wherein at least one of said oligonucleotides comprise one or more alkyl or arylphosphonate nucleosides.
141. The therapeutic composition of claim 140 wherein said alkyl or arylphosphonate nucleoside is methylphosphonate nucleoside.
142. The therapeutic composition of claim 139 wherein at least one of said oligonucleotides or - 61 ribose containing oligonucleotides comprise one or more alkyl or arylphosphonate nucleosides.
143. The therapeutic composition of claim 142 wherein said alkyl or arylphosphonate nucleoside is methylphosphonate nucleoside.
144. The therapeutic composition of claim 131 that comprises at least two oligonucleotides.
145. Use of a composition comprising at least one oligonucleotide, or pharmaceutically acceptable salt thereof, for inhibiting human immunodeficiency virus growth or replication in a host carrying human immunodeficiency virus and a second virus and which is hybridizable with nucleic acids of said second virus.
146. Use according to claim 145 wherein at least one of said oligonucleotides is hybridizable to a cap site, a transcription initiation site, a translation initiation site or 5 a splice site.
147. Use according to claim 146 wherein said oligonucleotides hybridizable to a splice site contain exon and intron sequences and the difference in the number of bases containing 5 exon sequences and the number of bases containing intron sequences is 0 or 1.
148. Use according to claim 146 wherein said oligonucleotide hybridizable to a translation initiation site are complementary to the ATG initiation codon and adjacent 3' bases in 5 the coding region, so that the last three bases of said oligonucleotide in the 5’ to 3' orientation are C, A and T, in that order.
149. USe according to claims 145, 146, 147 or 148 wherein at least one of said oligonucleotides is hybridizable to a regulatory - 62 gene, a gene required for nucleic acid replication or a gene involved in pathogenesis.
150. Use according to claim 149 wherein said second virus is herpesvirus.
151. Use according to claim 150 wherein said regulatory gene is an immediate-early (IE) gene.
152. Use according to claim 150 wherein said regulatory gene is Vmw65.
153. Use according to claim 150 wherein said oligonucleotide is selected from the group consisting of: (a) TTCCTCCTGCGG; (b) GCTTACCCGTGC; (c) GCGGGGCTCCAT; and (d) A A T G T C G G C C A T; or ribose containing oligonucleotides corresponding thereto, wherein T is replaced by U; and where A, T, G, U and C are adenine, thymidine, guanine, uracil and cytosine.
154. Use according to claim 153 wherein at least one of said oligonucleotides comprise one or more alkyl or arylphosphonate nucleosides.
155. Use according to claim 154 wherein said alkyl or arylphosphonate nucleoside is methylphosphonate nucleoside.
156. Use according to claim 145 wherein at least one of said oligonucleotides or ribose containing oligonucleotides comprise one or more alkyl or arylphosphonate nucleosides.
157. Use according to claim 156 wherein said alkyl or arylphosphonate nucleoside is methylphosphonate nucleoside.
158. Use according to claim 145 wherein said cell is contacted with at least two oligonucleotides. - 63
159. An oligonucleotide according to claim 1, substantially as hereinbefore described and exemplified.
160. A composition according to claim 9 or 33, substantially as hereinbefore described.
161. A method according to claim 57 of detecting herpesvirus, substantially as hereinbefore described.
162. A composition according to claim 61 or 89, substantially as hereinbefore described.
163. A method according to claim 117 for inhibiting human immunodeficiency virus growth or replication in a specimen, substantially as hereinbefore described.
164. A composition according to claim 131, substantially as hereinbefore described.
IE332191A 1990-09-21 1991-09-20 Compositions and methods for inhibiting growth or¹replication of microbes, viruses and self-replicating¹nucleic acids IE913321A1 (en)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
US58618590A 1990-09-21 1990-09-21

Publications (1)

Publication Number Publication Date
IE913321A1 true IE913321A1 (en) 1992-02-25

Family

ID=24344663

Family Applications (1)

Application Number Title Priority Date Filing Date
IE332191A IE913321A1 (en) 1990-09-21 1991-09-20 Compositions and methods for inhibiting growth or¹replication of microbes, viruses and self-replicating¹nucleic acids

Country Status (8)

Country Link
EP (1) EP0656942A1 (en)
JP (1) JPH06501162A (en)
AU (2) AU8756691A (en)
CA (1) CA2092711A1 (en)
IE (1) IE913321A1 (en)
IL (1) IL99543A0 (en)
NZ (1) NZ239883A (en)
WO (1) WO1992005284A1 (en)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU8617691A (en) * 1990-08-15 1992-03-17 Genta Incorporated Inhibition of herpesviridae infection by antisense oligonucleotides
CA2139339A1 (en) * 1992-07-02 1994-01-20 Julianna Lisziewicz Method of inhibiting viral replication
CA2105595A1 (en) * 1992-09-23 1994-03-24 Ramaswamy Narayanan Antisense polynucleotides
FR2708270B1 (en) * 1993-07-28 1995-10-20 Genset Sa Antisense oligonucleotides directed against Herpes Simplex virus types 1 & 2.
DE4331670A1 (en) * 1993-09-17 1995-03-23 Hoechst Ag Antisense oligonucleotides against HSV-1 and the preparation thereof
EP0763050B1 (en) * 1994-06-01 2000-01-05 Hybridon, Inc. Branched oligonucleotide as pathogen-inhibitory agents
US7176303B2 (en) 2003-11-06 2007-02-13 Isis Pharmaceuticals, Inc. Modulation of STAT5 expression

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NZ209840A (en) * 1983-10-17 1988-11-29 Kaji Akira A method of inhibiting viral propagation by hybridising dna with the viral rna thus blocking its action
US4806463A (en) * 1986-05-23 1989-02-21 Worcester Foundation For Experimental Biology Inhibition of HTLV-III by exogenous oligonucleotides
WO1989008146A1 (en) * 1988-02-26 1989-09-08 Worcester Foundation For Experimental Biology Inhibition of htlv-iii by exogenous oligonucleotides
US5004810A (en) * 1988-09-30 1991-04-02 Schering Corporation Antiviral oligomers

Also Published As

Publication number Publication date
NZ239883A (en) 1993-10-26
WO1992005284A1 (en) 1992-04-02
JPH06501162A (en) 1994-02-10
AU8756691A (en) 1992-04-15
CA2092711A1 (en) 1992-03-22
EP0656942A1 (en) 1995-06-14
AU2491095A (en) 1995-11-02
EP0656942A4 (en) 1998-04-15
IL99543A0 (en) 1992-08-18

Similar Documents

Publication Publication Date Title
US6265167B1 (en) Targeted RNA degradation using nuclear ribozyme RNA
US5567604A (en) Anti-viral guanosine-rich oligonucleotides
Boldogh et al. Transcriptional activation of cellular oncogenes fos, jun, and myc by human cytomegalovirus
US6015886A (en) Oligonucleotide phosphate esters
Zabolotny et al. The herpes simplex virus type 1 2.0-kilobase latency-associated transcript is a stable intron which branches at a guanosine
Kulka et al. Site specificity of the inhibitory effects of oligo (nucleoside methylphosphonate) s complementary to the acceptor splice junction of herpes simplex virus type 1 immediate early mRNA 4.
US6369038B1 (en) Closed antisense and sense oligonucleotides and their applications
US6028188A (en) Synthetic oligomers having chirally pure phosphonate internucleosidyl linkages mixed with non-phosphonate internucleosidyl linkages
US9902953B2 (en) Oligonucleotides comprising alternating segments and uses thereof
AU678628B2 (en) Oligonucleotide modulation of multidrug resistance-associated protein
Miller et al. Oligonucleotide inhibitors of gene expression in living cells: New opportunities in drug design
AU2003202376A1 (en) Oligonucleotides comprising alternating segments and uses thereof
US5849900A (en) Inhibition of viruses by antisense oligomers capable of binding to polypurine rich tract of single-stranded RNA or RNA-DNA hybrids
EP0832211B1 (en) Oligonucleotides specific for hepatitis b virus
JPH02276577A (en) Hiv transactivator target arrangement (tar) from i and antisense oligonucleotides for inhibiting synthesis of transactivator protein (tat) and use thereof
WO1994007367A9 (en) Anti-viral oligomers that bind polypurine tracts of single-stranded rna or rna-dna hybrids
EP1656453A2 (en) Eukaryotic expression systems for expression of inhibitory rna in multiple intracellular compartments
IE913321A1 (en) Compositions and methods for inhibiting growth or¹replication of microbes, viruses and self-replicating¹nucleic acids
US5801235A (en) Oligonucleotides with anti-cytomegalovirus activity
US6403302B1 (en) Methods and compositions for triple helix formation
US5550047A (en) Oligonucleotides with anti-Epstein-Barr virus activity
Adami et al. The length but not the sequence of the polyoma virus late leader exon is important for both late RNA splicing and stability
US5807838A (en) Oligonucleotide modulation of multidrug resistance-associated protein
WO2004060323A2 (en) Novel inhibitors of poxvirus replication
JPH0977670A (en) Antiviral agent

Legal Events

Date Code Title Description
FC9A Application refused sect. 31(1)