CA2089476A1 - Inhibition of herpesviridae infection by antisense oligonucleotides - Google Patents

Abstract

Antisense oligomers which are complementary to vital regions of a viral genome or mRNA transcripts thereof which exhibit antiviral activity are provided. Methylphosphonate oligomers complementary to particular sequences of herpes simplex virus type 1("HSV-1") messenger RNA have demonstrated antiviral activity. Methods of inhibiting viral replication using these oligomers are provided.

Description

W 0 ~2/03051 PCT/US91/057 DESCRIPTION ~S ~ il 1 b INHIBITION OF HERPESVIRIDAE INFECTION BY ANTISENSE
OLIGONUCLEOTIDES.

Backaround Of The Invention The present invention is directed to antisense oligomers which are complementary to a vital region of a viral genome which are active as antiviral agents.
The present invention is also directed to methods of interfering with replication of a virus after infection of host cells by the virus and to antisense oligomers which are useful in interfering with viral replication.
In viral replication, viral genes are typically activated and expressed in phases. In the replication of a virus such as a virus o~ the Herpes family, genes are expressed in three pha~es. Phase I involves the expression of about five genes. 'rhese genes are mostly regulatory in nature and are term~d "alpha genes." The lS second set of genes expressed are called "beta genes;"
~heir function is to make protelns that a~act nucleic acid metaboli~m and synthesis or replication of viral DNA.
The third set o~ genes, the gamma genes, comprise about 30 to 40 genes that control the synthesis sf structural proteins of the virus. The gamma yenes are induced after the onset of viral DNA synthesis. In the usual course of viral infection and replication, the infected cell is "killed", it may be killed outright, but alternatively may be constructively "killed" by being unable to divide or exprass its own genes. In order to prevent production of viral progeny and/or minimize the number of viral progeny produced, the viral replication process should be interrupted at a s~age prior to where viral progeny are produced. At a time soon after infe~tion, viral components reprogram the infected cell's metabolism for the production of viral progeny, and at this time, the cell is slated for eventual death.

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W0~2/03051 PCT/US91/OS7~6 The use of antisense oligonucleotides that are complementary to and bind to specific t-~rget nucleic sequences, particularly specific messenger RNA's, has been suggest~d as a means to deactivate specific genes. (See Weintraub, "Antisense RNA and DNA, Scientific Americ~n, pages 40 to 46 (January 1990)).
The use of antisense oligomers which are complementary to certain splice junction mRNA's of herpes simplex virus type 1 ("HSV-1") to specifically inhibit virus replication has been reported (See Kulka et al., Proc. Nat. Acad. Sci. (USA) 86:6868-6872 (September, 1989))-Summary Of The Invention Accorcling to the present invention, antisense oligomers are provided that are complementary to a vitalregion of a viral genome which act as antiviral agents.
Such vital regions comprise nucleic acid sequences necessary for viral replicati~n and are included in one or more essential genes. Thus, in one aspect, the present invention is directQd to an oligo~ler complementary to such a vital region or mRNA transcri.pt thereo~, which when hybridized to said target sequence!, inhibits or interferes with viral DNA synthesis or replication. In one preferred aspect the target se~uence comprises a portion of a mRNA
transcript of a gene essential for viral DNA synthesis or replication. Suitable target sequences include sequences at or proximate to a 5'-terminal translational s~tart or a 3'-terminal polyadenylation signal of the gene.
The present invention is also directed to methods of interfering with replication of a virus after it has infected host cells. The present invention is also directed to oligomers which are useful in interfering with and/or inhibiting such viral replication. According to methods of t~e present invention, said virus or viral DNA
3S or their environment is contacted with an oligomer which is complementary to a target seguence which comprises a SUBSTi ~` UT ~ r .
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vital region of the viral genome or mRNA transcript thereof. Optionally, two or more oligomers may be used wherein each oligomer is complementary to a different target sequence. Target sequences may be portions of the same gene or of different genes.
In one aspect, the present invention is directed to a method of interfering with replication of a virus after infection of host cells by the virus wherein the cells or their growth environment i5 contacted with an amount of an oligomar which is complementary to and which hybridizes with a messenger RNA sPquence for a gene essential for viral DNA synthesis and/or replication, that is effective to interfere with expression or function of said gene.
In one preferred aspect of the present invention, the method~ of the present invention are especially useful in interferin~ with viral replicatior1 in infections resulting from viruses of the family HerF\esviridae, particularly human herpes viruses, especially Herpes Simplex viruses.
Thus, the present invention is als~o directed to methods of in~ibiting or interfering with replication of a human herpes virus, especially a Herpes Simplex vlrus, by contacting the virus viral DNA or cells infected therewith with an oligomer complemantary to a nucleic acid target seguence essential for viral DNA synthesis or replication and wherein the oligomer can selectively hybridize with said target sequenced. For Herpes Simplex viruses, preferably the target sequence comprises a mRNA transcript of an essential ~-gene. Suitable ~ genes include UL5, UL8, UL9, ULl5, UL29, UL30, UL42 and UL52. Suitable regions of these genes for selection of a target sequence include a sequence at or proximate to a 5'-translational start or a 3'-polyadenyl~tion signal.
Among other factors, in one preferred aspect, the present invention is based on our finding tha~ oligomers complementary to mRNA transcripts o~ genes that code for the ~ group of polypeptides that are essential for viral 5~1T~TE SHEE~T

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WO~2/03~)5l PCT/US91/057~6 replication are especially effective in decreasing and/or inhibiting viral replication in Herpes Simplex viruses.
According to an additional aspect of the present Invention, methods of treating an organism infec~ed with a Herpes-viradae virus are provided using these antisense oligomers and methods.

Definitions As used herein, the following terms have the following meanings, unless expressly stated to the contrary:
The term "nuclecside" includes a nucleosidyl unit and is used interchangeably therewith.
The term "nucleotide" refers to a subunit of a nucleic acid consisting of a phosphate group, a S carbon sugar and a nitrogen containing base. In RNA the 5 carbon sugar is ribose. In DNA, it is a 2-deoxyribose. The term also includes analogs of such subunits.
The term "nucleotide multimer" refers to a chain of nucleotides linked by phosphodiester bonds, or analogs therQof.
An "oligonucleotide" is a nucleotide multimer generally about 3 to about lO0 nucleotides in length, but which may be greater than lOO nucleotides in length. They ara usually considered to be synthesized from nucleotide monomers.
A "deoxyribooligonucleotide" is an oligonuclaotide consisting of deoxyribonucleotide monomers.
A "polynucleotide~' refers to a nucleotide multimer generally about lOO nucleotides or more in length. These are usually of biological origin or ara obtained by enzymatic means.
A "nucleotide multimer probe" is a nucleotide multimer having a nucleotide sequence complementary with a target nucleotide sequence contained within a second nucleotide multimer, usually a polynucleotide. Usually the probe is selected to be perfectly complementary to the T`~ ~ ~

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A "non-nucleotide monomeric unit" refers to a monomeric unit which does not signi~icantly participate in hybridization of an oligomer. Such monomeric units must not, for example, participate in any significant hydrogen bonding with a nucleotide, and optionally include groupings capable of interacting after hybridization o~
oligomer to the target sequence, such as crosslinking alkylation, intercalating and chelating agents.
A nucleotide/non-nucleotide polymer" refers to a polymer comprised of nucleotide and non-nucleotide monomeric uni_s.
An "oligonucleotide/non-nucllsotide multimer" is a multimer generally of synthetic ori~in having less than 100 nucleotides, but which may contain in excess of 200 nucleotides and which contains one or more non-nucleotide monomeric units.
A "monomeric unit" ro~ers to a unit of either a nuclQotid~ r~agent or a non-nucleotide reagent o~ the present invention, which the realgent contributes to a polymer.
A "hybrid" is the complex formed ~etween two nucleotide multimers by Watson-Crick base pairing s between the complementary bases.
The term "oligomer" refers to oligonuc7eotides, nonionic oligonucleoside alkyl- and aryl-phosphonate analogs, phosphorothioate analo~s of oligonucleotides, phosphoamidate analogs of oligonucleotides, neutral phosphate ester oligonucleotide analogs, such as phosphotriesters and other oligonucleotide analogs and modified oligonucleotides, and also includes nucleotide/non-nucleotide poly~ers. The term also includes nucleotide/non-nucleotide polymers wherein one or more of the phosphorous group linkages between monomeric ., -..

WC~2/~)3051 PCT/US91/05756 7~ 6 units has been replaced by a non-phosphorous linXage such as a formacetal linkage or a carbamate linkage.
The term "alkyl- or aryl-phosphonate oligomer~' refers to nucleotide oligomers (or nucleotide/non-nucleotide polvmers) having internucleoside (or intermonomPr) phosphorus group linkages wherein at least one alkyl- or aryl- phosphonate linkage replaces a phosphodiester linkage.
The term "methylphosphonate oligomer~ (or "MP-oligomer") refers to nucleotide oligomers (ornucleotide/non-nucleotide polymer) having internucleoside (or intermonomer) phosphorus group linkages wherein at least one methylphosphonate internucleoside linkage replaces a phosphodiester i~ternucleoside linkage.
In some of the various oligomer sequences listed herein "p" in, e.g., as in ApA represents a phosphate diester linkage, and "~" in, e.g." as in C~G represents a methylphosphonate linkage. Certain other sequences are depicted without the use of p or ~ to indicate the type of 20 phosphorus diester linkage. In such occurrences, A as in r ATC indicates a phosphate diester linkage between the 3'-carbon of A and the 5' carbon of T, whereas ~, as in ATC
or ATC indicates a methylphosphonate linkage between the 3'-carbon of A and the 5'-carbon of T or T.
The term "antisense oligomer~ refers to an oligomer which is complementary ~o the "sense" strand of a DNA
duplex and to the mRNA transcript synthesized ~rom that sequence. A DNA duplex is comprised of two co~plementary DNA strands, one termed the "sense" strand and one termed the "antisense" strand. Messenger RNA transcripts ar~
synthesized using the antisense DNA strand as a template and hence are homologous ~with the replacement of T with A) to the sense strand.
ThQ term "vital region" of a viral geno~e or viral DNA refers to a nucleic acid sequence which is necessary for viral replication such that if the sequence is deleted Sl '~3STITU ~ F SHE~T

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or rendered nonfunctional, the virus is incapable of replication.
The term "deblocking conditions" describes the conditions used to remove the blocking (or protectin~) S group from the 5'-OH group on ~ ribose or deoxyribose group.
The term "deprotecting conditions" describes the conditions used to remove the protecting groups from the nucleoside bases.
The term "tandem oligonucleotide~' or "tandem oligomer" refers to an oligonucleotide or oligomer which is complemlentary to a sequence 5' or 3' to a target nucleic acid sequence and which is co hybridized with the oligomer complementary to the target sequence. Tandem lS oligamers may improve hybridization of these oligomers to the target by helping to make the target sequence more accessible to such oligomers, such as by decreasing the secondary structure of the target nucleic acid sequence.
The melting temperature or "Tm" of a duplex ~such as a dou~le strand~d nucleic acid DNA:DNA or RNA:DNA) is de~ined a the temperature at which half ~he helical structure is lost.

Detailed De,scri~tion Of The Invention Th~ present invention is directed to antisense oligomers useful as antiviral agents and to methods of interfering with viral replication in a host cell after its infe~tion using such antisense oligomers, wherein said oligomers are complementary to (and which hybridi~e with) a target nucleic acid sequence of a gene essential for viral replication or a viral messenger RNA transcript of said gene.

~re~errad ~arget Sequences In general, pre~erred are target nucleic acid seguences which comprise a ~ital region of the viral genome. Thesa target sequences may comprise portions of r~T~ 3~ET

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transcript thereof which are "available", i.e. are in a state where the complementary oligomer is able to hybridize with the target sequence. Thus, these target sequences are preferably single stranded and relatively free of secondary structure and bound protein.
Preferred target sequences include mRNA transcripts o~ genes which are "essential" for DNA replication.
Moreover, mRNA transcripts which are present in low numbers comprise particularly advantageous target sequences for this antisense therapy. With fewer mRNA
transcripts, a lower con~entration of oligomer can hybridize with and interfere with the function of a larger percentage of the mRNA from a particular gene.
Certain g~nes which code for ~RNAIs which are present in large amounts comprise less pre~erred target sequences, since if the function of these ~NA's is only partially blocked, the unhybridized mRNA's may proceed with the normal replicative cycle of th~! virus. Moreover, a proportionally larger amount of oligomer would ~e required ~o block an equivalent frac~lon oi8 the mRNA.
Preferred are essential genes which are expressed during the earlier stages of DNA replication, but after the cell is "committed" to death due to infection by the 2S virus. By blocking DNA replication at such an earlier stage, few if any functional virus particles are made.
Since the cell has already been "committed" to death it will die whether or not functional virus particles are made and after death, will be dealt with by the host organism's immune system. These cells include cells which, due to the viral infection, are unable to divide and/or express their own genes. If viral DNA replication is blocked before the cell has been committed to death, the viral DNA in the cell will not be destroyed and viral DNA replication may recommence later on.

SUBSTITUTE SHEET

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Suitable target sequences include sequences which are at or proximate to a 5~-terminal translational start or a 3'-terminal polyadenylation signal.
Preferred target sequencas include those which have a relatively high loc'al G-C base content. Sequences having a relatively high local G-C content are preferred in part because they tend to hybridize more tightly to the complementary oligomer and exhibit a correspondingly higher Tm. Especially preferred target sequences hav~ a high G-C base content on both ends of the target sequence that is complementary to and hybridizes with the complementary oligomer, which enables the ends of the oligomer to hybridize more tightly to the target sequence.

Pr~ferred Oliqome~s These oligomers may comprise either ribonucleoside or deoxyribonucleoside monomeric: units; however, deoxyribonucleoside monomeric unil:s are preferred.
Preferred are oliqomers which comprise from about 6 to about 40 nucleotides, more prei.erably ~rom about 12 to about 20 nucleo~ides. Although oligomers which comprise more than a~out 20 nucleotidec3 may be used, where complementarity to a longer sequence is desired, it may be advantageous to employ shorter tandem oligomers to maximize solubility and transpor~ across cell membranes while competing for the development of a secondary structure of the target nucleic acid, such as a mRNA.
Alternatively, it may be advantageous to use moro than one oligomer, each oligomer comple~entary to a distinct target sequence which may be part of the same gene or a different gen~.
Although nucleotide oligomers (i.e., having the phosphodiester internucleoside linkages present in natural nucleotide oligomers, as well as other oligonucleotide analogs) may be used according to the present invention, preferred oligomers comprise oligonucleoside alkyl and aryl~phosphonate analogs, phosphorothioate oligonucleoside SUeSTi T UTE SHEET

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analogs, phosphoro-amidate analogs and phosphotriester oligonucleotide analogs. However, especially preferred are oligonucleoside alkyl- and aryl-analogs which contain phosphonate linkages replacing the phosphodiester linkages which connect two nucleosides. The preparation of such oligonucleoside alkyl and aryl-phosphonate analogs and their use to inhibit expression of preselected nucleic acid sequences is disclosed in U.S. Patent Nos. 4,469,863;
4,511,713; 4,757,055; 4,507,433; and 4,591,614, the disclosures of which are incorporated herein by reference.
A particularly preferred class of those phosphonate analogs are methylphosphonate oligomers.
Such alkyl- and aryl-phosphonate oligomers advantageously have a nonionic phosphorus backbone -~hich l!j allows better uptake of oligomers by cells. Also, the alkyl- and aryl-phosphonate int~srmonomQric linkages of such alkyl- and aryl-phosphonate oligomers are advantageously resistant to nuclQases.
WherQ the oligomers comprise alkyl- or aryl-phosphona~c oligomers, it may~ be advanta~eous toincorporate nuclQosidQ monomeric units having modi~ied ribosyl moieties. The use of nucleotide units having 2'-O-alkyl- and in particular 2'-O-methyl-, ribosyl moieties, in these alkyl or aryl phosphonate oligomers may advantageously improve hybridization of the oligomer to its complementary target nucleic acid sequence.
Synthetic methods for preparing methylphosphate oligomers ("MP-oligomers") are described in Lee, BL. et al., ~iochemistxy 27:3197-3203 (1988) and Miller, P.W., et al., Biochemistry 25:5092-5097 (1986), the disclosure of which are incorporated herein by reference.
Preferred are oligonucl~oside alkyl- and aryl-phosphonate analogs wherein at least one of the phosphodiester internucleoside linkages is replaced by a 3' - 5' linked internucleoside methylphosphonyl (MP) group (or "methyl-phosphonate"). The methylphosphonate linkage is isosteric with respect to the phosphate groups of $~B~r~

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oligonucleotides. Thus, these methylphosphonate oligomers ("MP-oligomers") should present minimal steric restrictions to interaction with complementary polynucleotides or single-stranded regions of nucleic acid molecules. These MP-oliyomers should be more resistant to hydrolysis by various nuclease and esterase activities, since the methylphosphonyl group is not found in naturally occurring nucleic acid molecules. It has been found that certain MP-oligomers are more resistant to nuclease hydrolysis, are taken up in intact form by mammalian cells in culture and can exert specific inhibitory effects on cellular DNA and protein synthesis (See, e.g., U.S. Patent No. 4,469,863).
If desired, labeling groups such as psoralen, chemiluminescent groups, cross-linking agents, intercala~ing agents such as acricline, alkylating agents or groups capable of cleaving the targeted portio~ of the viral nucleic acid such as molecular scis90rs like o-phenanthroline-copper or EDTA-iron may be incorporated in the MP-oligomers.
Preferred are MP-oligomers h~lving at least about 6 nucleosid~s which i~ usually~ su~e~icient to allow for specific binding to the desired nucleic acid sequence.
More preferred are MP-oligomers having from about 6 to about 40 nucleosides, especially preferred are those having from about 10 to about 25 nucleosides. Due to a combination of ease of preparation, with specificity for a selected sequence and minimization o~ intra-oligomer-internucleoside interactions such as folding and coiling, particularly preferred are MP;oligomers of from about 12 to 20 nucleosides.
One group of preferred MP-oligomars includes MP-oligomers where the 5'-internucleoside linkage is a phosphodiester linkage and the remainder of the internucleoside linkages are methylphosphonyl (or methylphosphonate~ linkages. Having a phosphodiester ~E~IT~TE 5~EET

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W092/030sl PCT/~S91/OS7~6 linkage on the 5'-end of the MP-oligomer pe~mits kinase labelling and electrophoresis of the oligomer.

Preferred Embodiment of the Invention .
Infections due to viruses of the family Herpesvirdae comprise particularly suitable targets for ~herapy using the antisense oligo~ers and methods of the present invention. Herpes viruses vary greatly in their biological properties. Some have a wide host cell range, multiply efficiently and rapidly destroy the cells which they infect (HSV-l, HSV-2). Others have a narrow host cell range. A ubiquitous property of these herpes viruses ~ ~ is their capacity to remain latent in the host in which they multiply. The mechanism by which the virus perpetuates itself appears to re~lect a function of dedicated viral genes as well as association with appropriate cells. In general, infections caused by herpes viruses have been found to be persistant. Herpes viruses for which therapy using these anti~ense oligomers appears promising include human herpes viruses 1 to 7 which include Herpes Simplex Virus Type 1, Herpes Simplex Virus Type 2, Varicella-Zoster virus, Epstein-Barr virus, Cytomegalovirus and human herpes viruses 6 and 7.
In one preferred embodiment, ~le present invention is directed to antisense oligomers which are useful as antiviral agents against herpes simplex virus ("HSV"), particularly type 1 ~"HSV-l"), and to methods of controlling HSV-l infections by inhibiting~ and/or interfering with replication of HSV-l.
According to the present invention, antisense oligomers are provided that are complementary to "essential" genes.
In Herpes Yiruses, as ~uch as about one half of viral genes are non essential; ~hat is, they may be deleted or at least reduced in expres~ion or treated with antisense oligomers and not effect viral replication.

SUBSTITUTE SHEET

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Preferred target sequences for these antisense oligomers comprise essential genes, that is genes which when deleted or their function is compromised, significantly affect viral replication, particularly the synthesis and/or replication of DNA. Also, as noted, preferred target sequences inclu~e mR~A transcripts of such essential genes, wherein copies are present only in low n~mbers. For this reason, we have found that essential ~ genes of HSV-l to comprise particularly suitable target sequences for these antisense oligomers.
HSV-l has about 15 ~ genes, of which at least about 8 have ~een reported to be essential. These essential genes include the genes termed UL5, UL8, UL9, UL15, UL29, UL30, UL42 and UL52. These genes have been reported to l'; code ~or protelns which are necessary for viral DNA
synthesis and/or replication. Seven of these genes have been reported to be required for viral-origin-dependent DNA synthesis and to map in the I, component of the viral DNA. These seven genes have been reported to speci~y the ~ollowing: a DNA polymera~e ~UL30) with an apparent molecular wQi~h~ of 140,000; a singl~-strand speci~ic-DNA-binding protein designated ~s ICP8 (UL29) with an apparent molecular weight of 124,000; a protein binding to the origin of viral DNA synthesis (uLs) with a translated molecular weight of 94,000; a protein that binds to double-stranded DNA (UL42) with a molecular weight of 62,000; and three additional proteins (UL5, predicted molecular weight of 99,000; UL8, predicted ~olecular weight of 80,000; and UL52, predicted molecular weight of 114,000). ~hese three proteins form a complex in which each protein is present in equimolar ratios and which functions as a primase and helicase. The protein specified by UL5 has independently been shown to act as a DNA dependent ATPase.
The above noted seven proteins app~ar to be all that is necessary for ori5-dependent amplification of DNA
transfected into cells. For this reason, oligomers which S"~ST.TUTE S~

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W0')2/0305l PCT/US91/057~6 are complementary to the mRNA of one of these seven genes are particularly preferred and comprise especially suitable antiviral agents against HSV-1.
Of the above noted seven essential genes, preferred are the genes denoted UL5, UL8 and UL52. It is believed that the mRNA transcripts of these genes comprise target se~lences which are particularly susceptible to inhibition using these antisense oligomers.
Portions of these essential genes which ~ay be relatively more available to these antisense oligomers comprise esp~ecially suitable target sequences. It is believed that sequences that are proximate to the 5'-terminal translational start of these mRNA transcripts or to the 3'-terminal polyadenylation signal comprise especially suitable target sequences in view of their demonstrated susceptibility to inhibition of viral function due to hybridization of an antisense oligomer.
Preferred target sequences include ~portions of these mRNA
transcripts in which it appears that secondary structure of the mRNA does not inter~ere with its ability to hybridize to a complementary oligomer.
Antisense oligomers complement2lry to selected xegions of mRNA transcripts of these seven genes have been assayed for antiviral activity using a Virus Titer Reduction Assay (see Example A) and a Direct Plaque Assay (Example B) and have been found to demonstrate antiviral activity (see Tables II, III and IV).
To assist in understanding tha present invention, the following examples are included which described the results of a series of experiments. The following examples relating to this invention should not, of course, be co~strued in specifically limiting the invention and such variations of the invention, now known or later developed, which would be within the purview of one skilled in ~he art are considered to fall within the scope of the present invention as hereinafter claimed.

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WO(~2/03~SI PCT/US91tO575 ExamPle 1 PreParation Of Phos~hate Diester Oli~omers Phosphate diester oligomers are prepared using a Biosearch model 8750 DNA synthesizer using standard phosphoro-amidite chemistry (M.H. Caruthers, et al., Methods of Enzymol. 154:287-313 (1985)) according to the manufacturers recommendations. The 5'-dimethoxytrityl protecting group is left on at the end of the synthesis to permit purification on a Sep-Pak~ C18 cartridge (Millipore/Waters, Bedford, MA) as described by X.M. Lo et al. (Proc. Natl. Acad. Sci. (USA) 81:2~85-22~9 (1984)).
During this procedure, the dimethoxytrityl protecting group was removed.

Exam~le 2 PreParation Of Meth~lphos~honate Oli omers Methylphosphonate oligomers are synthesized using me~hylphosph~namidite monomers~ according to the chemical m~thods described by P.S. ~iller et al. (Nucleic Acids Re~ 6225-6~242 (1983)), A. Jager and J. Engels (Tetrahedron Letters 25:1437-1440 tl984)) and M.A. Dorman et al. (Tetrahedron Letters 40:95-102 ~1984)). Solid phase synthesis is per~ormed on a Biosearch Model 8750 DNA
synthesizer according to the manufacturer's recommenda-tions with the following modifications: 'IG'' and 'IC'' monomers are dissolved in 1:1 acetonitrile/dichloromethane at a concentration of 100 mM. "A" and "T" monomers are dissolved in acetonitrile at a concentration of 100 mM.
DEBLOCK reagent = 2~5% dichloroacetic acid in dichloromethane. OXIDIZER reagent = 25 g/L iodine in 2~5%
water, 25% 2,6-lutidine, 72.5% tetrohydrofuran. CAP A=
10% acetic anhydride in acetonitrile. ~AP B = 0.625% N,N-dimethylaminopyridine in pyridine. The 5'-dimethoxytrityl protecting group is left on at the end of the synthesis to facilitate purification of the oligomers, as described below.

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WO92/03051 PCTtUS91/0~756 ~ 16 The crude, protected methylphosphonate oligomers are removed from the solid support by mixing with concentrated ammonium hydroxide for two hours at room temperature. The solution is drained from the support using an Econo-ColumnT~ (Bio-Rad, Richmond, CA) and the support is washed five times with 1:1 acetonitxile/water. The eluted oligomer is evaporated to dryness under vacuum at room temperature. Next, the protecting groups are removed from the bases with a solution of ethylenediamine/ethanol/
acetonitrile/water (50:23.5:23.5:2.5) for 6 hours at room temperature. The resulting solutions are then evaporated to dryness under a vacuum.
The 5'-dimethoxytrityl ("trityl") containing oligomers are purified from non-tritylated failure sequences using a Sep-PakT~ C18 cartridge (Millipore/Waters Bed~ord, MA) as follows: The cartridge is washed with acetonitrile, 50% acetonitrile in 100 mM, triethylammonium bicarbonate (TEAB, pH 7.5) and 25 mM TEAB. Next, the crude methylphosphonate oligomer is dissolved in a small volume of 1:1 acetonitrile/water and then diluted with 25 mM TEAB to a final concentration of 5% acetonitrile. This solution is then passed through th~ cartridgs. N~xt, the cartridge is washed with 15-20% acetonitrile in 25 mM TEA~3 to elute failure sequences from the cartridge. The trityl-on oligomer remaining bound to the cartridge is then detritylatPd by washing with 25 mM TEAB, 2 trifluoroacetic acid, and 2S mM TEAB, in that order.
Finally, the trityl-selected oligomer is eluted~from the cartridge with 50~ acetonitrile/water and evaporated to dryness under vacuum a~ room temperature.
The methylphosphonate oligomers are further purified by reverse-phase HPLC chromatography as follows: A
Beckman System Gold HPLC is used with a Hamilton PRP-l column (Reno, NV, 10 ~, 7 mm i.d. x 30S mm long). Buffer A = 50 mM triethylammonium acetate (pH 7); Buffer B - 50%
acetonitrile in 50 mM triethylammonium acetate (pH 7).
The sample, dissolved in a small volume of 10-50 .~

:~ ' W092tO3~1 PCT/US9t/~ 6 acetonitrile/water, is loaded onto the column while flowing at 2.5-3 ml/minute with 100% Buffer A. Next, a linear gradient of 0-70% Buffer B is run over about 30-50 minutes at a flow rate of about 2.5-30 ml/minute.
Fractions containing ~ull length methylphosphonate oligomer are collected, evaporated under vacuum and resuspended in 50~ acetonitrile/water.

Example 3 Inhibition Of Herpes Sim~lex Virus-l Replication Oligomers complementary to designated sequences o~
c~rtain~ genes of herpes simplex virus-l ("HSV-l") were prepared. Sequences are reported in Tables I and IV.
These oligomers were assayed for their ability to inhibit HSV-l replication according to the Virus Titer Rf~duction Assay (Example A) and/or ~hel Direct Plaque Assay (Example B). Results are reported in Tables II, III, IV
and V.

Ex~mple ~
Vixus Titer Reduction ~0 The Virus Titer Reduction assay measures the ability of an oligomer to inhibit the total yield of virus produced by a group of cells. This test consists of two parts. First, a plate of cells is infected with virus;
second, oligomer in media or media alone is added; and third, the cells are incubated for one to several replications of virus. The cells are then harvested ~rom the well and various dilutions of this harvest are used to infect frèsh monolayers of cells. The amount of virus ~ound, measured by plaque foxmation, is the total amount of virus produced by the initial cells. A comparison of the total virus produced by treated and untreated cells is a measure of the i~hibition of virus by the test drug.
P~esult~ are reported in Tables II, III, IV (under "VTR"), and V.
SIJ~STITUTE SHEET

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oliyomers complementary to designated sequence~ of certain genes of herpes simplex virus-1 ("HSV-1") were prepared.
Sequences are reported in Tables I and IV.
These oligomer~ werc assayed ~or their ability to inhibit HSv-l replication according to the Virus Titar Reduction A say (Example A) and/or ~he Direct Plaque A~ay (Example B).
Results ara reported in Table~ II, III, IV and V.

VIRUS TITER REDUCTION
The Virus Titer ~eduction as~ay mQaaure~ tho ability of an oligomer to inhlblt the total yield o~ virus produced by a group of cQlls. This te3t con~ists of two parts. Firqt, a plate 0~ cnllg i9 in~ected w$th vi~uss secon~, ollgomer in m~dia or medla alono is addeds and thlrd, the cl~lls are incubated ~or one to s~veral replications o~ virus. Th~ colls aro thon harvested ~rom th~ woll and various dilutions o~ thls h~rv~t ar~ used to infect freqh monolayer~ Or cells. The acount of virus ~ound, mea3urad by plaqus rOrmation, is the total ~ount of virus produced by the initial cell~. A co~parlson o~ thQ total viru~
produced ~y tr~ated and untreatad c~lls i9 a m~a~uro of th~
inhibltion o~ viru~ by th~ te~t drug. Results ar~ report~d in Ta~le~ II, III, rv (undar "VT~, and V.

~ h~ Dlrec~t Plaqu~ ~s~ay maa ur~- tha ability of an oli~o~er to inhiblt in~ection and virus r~plic~tlon. C~lls are in~ected with enough virus to in~ect only a f~actlon o~ the cells. Th~n an ov~rlay with or wl~hout ollgom~, i3 add~d. This SUEISTITUTE SHEET
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WO g2/030~1 PCT/US91~05756 ' ~ ~ Y ~

overlay limits all spread of virus from cell to cell ~hrough the extracellular fluid, but not by direct contact or ~xtension. The resultant plaques can th~n be counted and obsarvQd ~or ~iZB- A
co~parison of plaqu~ number and 8iZQ in tr~at~d ~nd untrQated cells provides tha infor~ation deslrad. R~ults ara reported in Table IV under "PLAQUE."

SUE3STiTUTE SHEET

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W() '~2/03(151 PCI/US~l/OS7~6 ) U ~
TA~LE I

A. COM~LEMENTARY TO AREA AROUND TR~NSL~LoNAL S~ART
OLIG0~ER_~O~ LOCATION SE0UENCE
0015 UL5 -15 +3 5'-CAT-~C-C~C-~Ç_- CG-CTC-3' 0013 UL8-5 ~ +13 5l-CTa-ÇQQ-~9~-ÇC~-TC~-ÇAC-3' 0021 UL8~1 - +18 5'-GA~-~5_-$QQ-9~-GTC-ÇAT-3' 0028 UL8ll9 - +35 s'-C~Ç-~Ç- ÇC-~ÇQ-C~C-AC-3~
0046 UL8 5~-cc~-~5~-~ç~-aça-GC~ 3' 0047 --~ UL8 5'-GGC-ççC-~a-9~ ça-cGa-3' 0007 UL9_5 - +13 5'-C~-C~A-~a~-Ç~-~ga-Ç-3~
0056 UL15-6 - +12 5'-C -ACC~ a~-CCC-Q~-3' 0054 UL29-5 ~ +13 sl-Gç~-~3Q-~Q~-Qça-~9~--ç~-3 0039 UL30_3 _ +15 sl-GÇQ-~ÇQ-Cga-aaA-Ç~-Ç9--3' 0016 UL42-7 - +11 5'-G~ ÇQ-aTC-a3Q-ÇQ~-Ag~-3' 0052 ULS2-21 - -4 5~-GIQ-ÇgQ-9ÇQ-ÇCÇ-~aQ-aQC-3' 0019 ULS2-3 - +lS 5l-G~ç~5~Q-ç$Q-~QQ-sa~-~sç-3 0053 UL52+16 - +33 5l-c~-Q3~-~cç-~ca-s~ ç~-3 A, C, G or T ~ Pho~phate diester linkage ~ S~ ~ or ~ ~ Methylphosphon~te linkage TITUTE SH~ET

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WO ~2/~3051 PCl`~US9t/0~756 2û ~ u ~

8. CoMPTFMENT~RY TO PO~Y ~ SIGNAL
OLIGOMER N0. ~IÇ5~UQ~ ~S~IEY~
0005 UL5-3 - +15 5'-T~-g~-a~C-5~-Aa~-GGA-3' 0006 UL5+5 - +22 5~-C~-9~-95~-~~-TGT-C~-3' 0020 UL5+12 - +29 5'-Aa~-~5Q-GCT-GG~-TG~-~$~-3' 0008 UL5+23 - +40 5'-Aa~aQ-~~ a-a3~-~Q9-3' 0023 UL5+41 - +58 5-_GçQ_~g~_5~ _~a_AT~_3~
0059 UL5+59 - +76 5'-AC~-Ç~ -GCC-ÇGT-CGC-3' 0051 UL8-15 - +3 5'-A~ -aa~-G~C-~3~-5~-3' 0024 UL8+4 - +21 5'-A$~-GG5-_~a-~ a_-GCA-3' 0014 UL8+22 - +39 5'-TÇ~-C~G-G~C-Aa~-95~-r3~-3' 0022 UL8+40 - +57 5'-C~Q-9Qg-q9~-~ÇQ-CTC~ 3' OOS7 UL15-13 - +5 5'-T~A-55g-ggQ-9Q~-CAC-9~Q-3' 0055 U~29+4 - +21 5'-A5Q-5CQ-5aQ-~ a-55~-3' 0040 UL30+3 - ~20 s'-G~-9Q~-Ç9Q-~A-Ç~ 3~-3' 0025 UL42-6 - ~12 5'-AÇ~-~aQ-33~-25~-TAC-~5~-3' 0017 UL42+4 - +21 S'-T~Q-9g~-A~ S~-~a9-~-3' 0018 UL42+22 ~ +39 5~-GS9-~a-CGC-gQg-~a-g~Q-3l 0026 ULA2+40 - +58 5'-Ca~ a-9~-9~a-CCA-C~C-3' 0151 UL42~59 - +76 5l-GA9-gg~-~G~cqc-Gcc-A~o-3l 0027 UL52+3 - +20 5'-G~-CG~ C-AT~-~a~ -3' 0038 UL52~21 - ~38 5'~~ ~ag~AQa~Ça~$a~~3' 0035 U~52+39 - ~56 s~-c~-çc~-gç~ Q-Aça-~aa-3 A, C, G or T ~ Pho~p~ate diester linXage ~, Ç, Q os ~ ~ Methylp~osp~onata linkaga glJ~3$TlTUTE St;EET

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W O ~2/030~1 PCTtUS91/0S756 TAB~E II
ANTI-HERPES SI~PLEX VIRUS TYPE 1 ACTIVITY
(TRANSLATIONAL START) OLIGoME~ NO ! GE~ OCATION % IN~I~ITIoN
(Gene Product) IATG STA~ +1) 1~8I~9~1 +l 0015 DNA DNP. ATP~e -15--~ 3 63%
u~a ~ELICASE~PRIMASE
0021 +1-------~18 _.45%
002a . ~ +19---------~35 62 ~2 0007 -5--------+1:1 49 SPLICE JUNCTION

U~2 0054 -S--------~l'l 68 ~Q
DNA PO~YMERA5E
0039 -3---------~15 50%
U~42 DNA SYXTHE525 pRorEIN
00~6 -7------~ 11 26 U~2 HELICASE/PRIMASE

0019 -3--~ --+15 79~
0053 ~16--------+33 74%

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WO !)2t03051 PCI`/US91/05756 ~L~
ANTI-HERPES SIMPT~X VIRUS TYPE 1 ACTIVITY
(POLY A SIGNAL) OLIGOMER ~0.G~E LOC~TION S INHIBITION
(Gene Pr~duct) +l ~$AxI~uM) A(A)TAAA~
~L~
DNA CNP. ATPa~e 0005 -3-------+lS
0006 +5-~ +22 69%
0020 +12-------+29 18S
0008 +23-------+40 17~
0023 +41-------+58 96%
0059 +53-------+~6 18% ._ _ HEL:tCASE/PRIMASE
0051 -15-~ +3 0024 +4------+21 7816 0014 ~22--------+39 10~
00;!2 . +40-------~57 58S
~ .
SPLICE JUNCTION
0057 -13------+5 32%
S~2 DNA BINDING PRoTEIN
003Y +4-~ 21 10%
~3Q ` ' DNA PO~YMERASE
0040 . +3-------+20 55%
3ZL~L2 DNA SYNTHESIS PROTEIN
0025 -6-------+12 1~
001~ +4-------+21 3~t 0018 +22--------+39 0026 ~40-------~58 93 01Sl +59--------+76 HELIC~5EJP~I~ASE
002~ +3-------+20 8~%
0038 +21--------+38 - 98~
0035 +39-------+S6 66%

SUBSTITUTE SHEET

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WO ~2/03051 PCT/US91/057~6 TA~L~ IV

~ REDUCTION
OLIGOMER OF HSV-l NO. GENESEQUEN~ CONC. VTR PLAQUE
0002 UL85'-C~-Ç9Q-~9~-ÇÇ~-TC~-CAC-3' 100~M 35%
0002 UL85'-ç~-çg~-~9~-çça-59~-ç~_-3' 200~M 63 0002 UL85l-cTa-~99-~g~--ça-~çQ-ça~-3l 100uM 49 0002 UL85l-cTa-cGa~9~-ccA~ -cAc-3l 200~M 76~
0002 UL85'-3Q-Ç~ -CC~-~CG-CAC-3' 200~M 29%
0002 UL8- 5'-C~3-CG~-TGT-ÇÇa-~Ç~-C~C-3~ 200~M 23%
0004 UL85'-9a~ Q5-GaT-GTC-Ça~-3' 200~M 32%

A, C, G or T ~ Pho~phat~ dlest~r linkage ~, ~, 9 or ~ ~ Mothylpho~phonate linkago ~A~E~ ' ' A. ~ aEY~
01i~o~or ~O. 99n~ U Qn~ ~Q~h~
~1) 0021 U~3 ~32t) 82%
0028 U~8 (62%) ~2) 0013 UL8 0047 U~8 B. ~
QUL99~ 9~ Q~n~ Toaoth~r 0013 U~8 67%

a'`JQ~'T~ T

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Claims (50)

Claims

1. A method of interfering with replication of a virus after infection of host cells by said virus which comprises contacting said cells or their growth environment with an amount of an oligomer which is complementary to and which hybridizes with a messenger RNA
sequence for a gene essential for viral DNA replication, effective to interfere with expression or function of said gene.

2. A method according to claim 1 wherein said virus is a Herpesviridae virus.

3. A method according to claim 2 wherein said virus is selected from Herpes Simplex Virus Type 1, Herpes Simplex Virus Type 2, Varicella-Zoster virus, Epstein-Barr Virus, Cytomegalovirus, human herpes virus 6 and human herpes virus 7.

4. A method according to claim 3 wherein said virus comprises a Herpes Simplex Virus.

5. A method according to claim 4 wherein said gene comprises an essential beta gene.

6. A method according to claim 5 wherein said gene is selected from UL5, UL8, UL15, UL9, UL29, UL30, UL42 and UL52.

7. A method according to claim 6 wherein said oligomer is complementary to a sequence at or proximate to the 5'-terminal translational start or the 3'-terminal polyadenylation signal of said gene.

8. A method according to claim 6 wherein said gene is selected from UL5, UL8 or UL52.

9. A method according to claim 8 wherein said oligomer is complementary to a sequence at or promixate to the 5'-terminal translational start or the 3'-terminal polyadenylation signal of said gene.

10. An oligomer which is complementary to a target sequence which comprises a vital region of a viral genome or a mRNA transcript thereof which when hybridized to said target sequence inhibits or interferes with viral DNA
synthesis or replication.

11. An oligomer according to claim 10 which comprises an alkyl- or aryl-phosphonate oligomer.

12. An oligomer according to claim 11 which comprises a methylphosphonate oligomer.

13. An oligomer according to claim 12 wherein said target sequence comprises a portion of a mRNA transcript of a gene essential for viral DNA synthesis or replication.

14. An oligomer according to claim 13 wherein said target sequence is at or proximate to a 5'-terminal translational start or a 3'-terminal polyadenylation signal of said gene.

15. An oligomer which is complementary to a target sequence of a mRNA transcript of an essential HSV-1 .beta.
gene.

16. An oligomer according to claim 15 wherein said gene is selected from UL5, UL8, UL9, UL15, UL29, UL30, UL42 and UL52.

17. An oligomer according to claim 16 which comprises an alkyl- or aryl-phosphonate oligomer.

18. An oligomer according to claim 16 wherein said target sequence is at or proximate to a 5'-terminal translational start or a 3'-terminal polyadenylation signal of said gene.

19. An oligomer according to claim 16 wherein said oligomer comprises a methylphosphonate oligomer.

20. An oligomer according to claim 19 wherein said oligomer comprises a methylphosphonate oligomer.

21. An oligomer according to claim 19 wherein said target sequence is at or proximate to a 5'-terminal translational start or a 3'-terminal polyadenylation signal of said gene.

22. A method of inhibiting or interfering with DNA
synthesis or replication of a virus which comprises contacting said virus or viral DNA or their environment with an oligomer which is complementary to a target sequence which comprises a vital region of the viral genome or a mRNA transcript thereof.

23. A method according to claim 22 wherein said vital region comprises a gene essential for viral DNA
synthesis or replication.

24. A method according to claim 23 wherein said virus is a Herpesviridae virus.

25. A method of inhibiting or interfering with replication of a human herpes virus which comprises contacting said virus, viral DNA or cells infected therewith with an oligomer complementary to a nucleic acid target sequence essential for viral DNA synthesis or replication wherein said oligomer can selectively hybridize with said target sequence.

26. A method according to claim 25 wherein said human herpes virus comprises a Herpes Simplex Virus and said target sequence comprises a mRNA transcript of an essential .beta. gene.

27. A method according to claim 26 wherein said gene is selected from UL5, UL8, UL9, UL15, UL29, UL30, UL42 and UL52.

28. A method according to claim 27 wherein said oligomer comprises a methylphosphonate oligomer.

29. A method according to claim 27 wherein said target sequence is proximate to a 5'-terminal translational start or a 3'-terminal polyadenylation signal.

30. A method according to claim 27 wherein said gene is selected from UL5, UL8 and UL52.

31. A method according to claim 30 wherein said oligomer comprises a methylphosphonate oligomer.

32. A method according to claim 30 wherein said target sequence is proximate to a 5'-terminal translational start or a 3'-terminal polyadenylation signal.

33. A method according to claim 32 wherein said oligomer comprises a methylphosphonate oligomer.

34. A method of treating an organism infected with a Herpesviradae virus which comprises contacting said organism or cells thereof with a therapeutically effective amount of an oligomer which is complementary to a target sequence which comprises an essential gene for DNA
synthesis or replication or a mRNA transcript thereof.

35. A method according to claim 34 wherein said virus is selected from a Herpes Simplex Virus, type 1 or 2, Epstein-Barr virus, Cytomegalovirus, Varicella-Zoster virus, humn herpes virus 6 and human herpes virus 7.

36. A method of treating an organism or cells thereof infected with Herpes Simplex Type 1 Virus which comprises the administration to said organism or cells of a therapeutically effective amount of an oligomer which is sufficiently complementary to selectively hybridize to a target sequence which comprises a gene essential for viral DNA replication or synthesis or mRNA transcript thereof.

37. A method according to claim 36 wherein said gene is selected from UL5, UL8, UL9, UL15, UL29, UL30, UL42 and UL52.

38. A method according to claim 37 wherein said oligomer comprises only methylphosphonate internucleoside linkages.

39. A method according to claim 38 wherein said oligomer comprises from about 6 to about 30 nucleosides.

40. A method of inhibiting or interfering with DNA
synthesis or replication of a virus which comprises contacting said virus or a viral DNA with two or more oligomers wherein each oligomer is complementary to a different specific target sequence and wherein each target sequence comprises a vital region of the viral genome or a mRNA transcript thereof.

41. A method according to claim 40 wherein each target sequence comprises a portion of a mRNA transcript complementary to an essential gene.

42. A method according to claim 41 wherein each target sequence comprises a portion of the same essential gene.

43. A method according to claim 42 comprising three or more oligomers.

44. A method according to claim 42 wherein each target sequence comprises a portion of a different essential gene.

45. A method according to claim 44 comprising three or more oligomers.

46. A method of treating an organism infected with a Herpesviradae virus which comprises contacting said organism or cells thereof with a therapeutically effective amount of two or more oligomers, wherein each oligomer is complementary to a different target sequence which comprises a portion of an essential gene for DNA synthesis or a mRNA transcript thereof.

47. A method accoridng to claim 46 wherein each target sequence comprises a portion of the same essential gene.

48. A method according to claim 47 comprising three or more oligomers.

49. A method according to claim 46 wherein each target seuqence comprises a portion of a different essential gene.

50. A method according to claim 49 comprising three or more oligomers.