METHOD OF INHIBITING INDUCTION OF LATENT OR CHRONIC VIRAL INFECTIONS

Abstract: Disclosed are methods of inhibiting or controlling the induction of a viral infection from a latent or chronic infection to an active replicating infection. Oligomers complementary to a viral enhancer site inhibit such induction.

Description: DESCRIPTION Method of Inhibiting Induction of Latent or Chronic Viral Infections Background of the Invention The present invention is directed to processes for the inhibition of induction of viruses in latently or chronically infected cells into the actively replicating form. Retroviruses were so named because in their life cycle the normal flow of genetic information (DNA to mRNA to protein) is reversed. In cells the genetic material is DNA; when genes are expressed, the DNA is first tran- scribed into messenger RNA (mRNA) which then serves as a template for the production of proteins. However, the genes of a retrovirus are encoded in RNA; the genes must be converted into DNA before they can be expressed. Only then are the viral genes transcribed into mRNA and trans- lated into proteins in the usual sequence. A retrovirus infects a cell by binding to the outside of the cell and injecting its core. The core contains the RNA encoding the viral genome as well as structural proteins (gag gene) , viral enzymes and regulatory proteins (tat, rev, nef) . One enzyme is responsible for converting the viral genetic information into DNA. This DNA polymer- ase (pol gene) first makes a single-stranded DNA copy of the viral RNA. An associated enzyme, ribonuclease, destroys the original RNA, and the polymerase makes a second DNA strand, using the first one as a template. (The polymerase and ribonuclease together are often called "reverse transcriptase") . Thus, the viral genome, now in the form of double-stranded DNA (the same form in which the cell carries its own genes) , migrates to the cell nucleus. A third viral enzyme, called an integrase, may then splice • the viral genome — its full complement of genetic information — into the host cell's DNA. Oncef so incorporated, the viral DNA (the "provirus") will be duplicated together with the cell's own genes every time the cell divides. Thus established, the infection is a permanent part of the cell. During this latent or chronic infection phase, new virus particles are not generally produced. The production of new virus particles (virions) begins when nucleotide sequences in the long terminal repeat (LTR) region of the viral DNA direct enzymes belonging to the host cell to copy the DNA of the inte¬ grated virus into RNA. Some of the RNA will provide the genetic information for a new generation of virus. Certain other RNA strands serve as mRNAs that guide cellular machinery in producing the structural proteins and enzymes of the new virus. The conversion of the virus from the latent proviral stage to the active replicating stage is called induction or rescue. Other viruses, besides retroviruses, such as Herpes viruses and the Hepatitis B virus have similar cycles with latent or chronic infection phases and active replicating phases. With many viruses, especially retroviruses, after initially infecting a cell the viral genome is integrated into the DNA in the cell's nucleus, in what is called the provirus form. The provirus may remain integrated in the cell's DNA during a long period of chronic or latent infection. The latent or chronic infection period ends when the provirus (virus) is induced and enters an active replicating phase. Mammalian viruses which exhibit such a life cycle often having long periods of latency (or chronic infection) as well as active replication phases include retroviruses such as the AIDS virus (human immuno¬ deficiency virus or HIV) , the various Herpes viruses and Hepatitis B. Since an individual (or animal) may be virtually asymptomatic during this chronic or latent infection phase and suffer few of the ravages of the active replicating phase, once an individual was infected. it would be advantageous to maintain the virus in provirus form and inhibit its conversion (induction and rescue) into the active replicating phase. The AIDS virus, HIV, is an example of a virus that would be advantageous to maintain in the provirus form. After initial infection with HIV, the virus often repli¬ cates abundantly, and free virus may appear in the brain and spinal cord and in the bloodstream. Fevers, rashes, flu-like symptoms and sometimes neurological complaints can accompany this first wave of HIV replication. Then, within a few weeks, the amount of virus in the circulation and the cerebrospinal fluid drops precipitously and the initial symptoms disappear. However, the virus is still present (primarily in the provirus form) . It can be found not only in the T4 lymphocytes, the subset of immune system cells originally thought to be its only target, but also in other classes of immune system cells, in cells of the nervous system and the intestine and probably in some bone marrow cells. From about two to ten years after the start of this asymptomatic period, replication of the virus flares again and the infection enters an active replicating phase, generally the final stage. Underlying this variable course of infection are complex interactions between HIV and its host cells. The virus behaves differently depending on the kind of host cell and the cell's own level of activity. In T cells, HIV can lie dormant indefinitely, inextricable from the cell but hidden from the victim's immune system. When the cells are stimulated (induced) , however, it can destroy them in a burst of replication. In other cells, such as the immune system cells called macrophages and their precursors, called monocytes, the virus may be latent or grow continuously, but slowly, sparing the cell but probably altering its function. In HIV, a regulatory gene known as tat, for trans- activator, is one gene responsible for the burst of viral replication seen, for example, in T4 cells that have been stimulated by an encounter with an antigen. ' The tat gene is unusual in both its structure and its effects. It is made up of two widely separated sequences of nucleotides; after it is transcribed into mRNA the intervening genetic material must be spliced out before the transcript can be made into protein. The tat gene has sites termed splice/- acceptor ("S/A") and splice/donor ("S/D") which allow the splicing out of non-transcribed material. The effect of the resulting small protein (from tat) is dramatic; it can increase the expression level of the viral genes to 1,000 times the baseline expression level. Nonionic oligonucleoside alkyl- and aryl-phosphonate analogs complementary to a selected foreign nucleic acid sequence can selectively inhibit the expression or func- tion or expression of that particular nucleic acid without disturbing the function or expression of other nucleic acids present in the cell, by binding to or interfering with that nucleic acid. (See, e.g. U.S. Patent No. 4,469,863 and 4,511,713). The use of complementary nuclease-resistant nonionic oligonucleoside methylphos- phonates which are taken up by mammalian cells to inhibit viral protein synthesis in certain contexts, including Herpes simplex virus-1 is disclosed in U.S. Patent No. 4,757,055. The use of anti-sense oligonucleotides or phosphoro- thioate analogs complementary to a part of viral mRNA to interrupt the transcription and translation of viral DNA into protein has been proposed. The anti-sense constructs can bind to viral RNA and were thought to obstruct the cells ribosomes from moving along the RNA and thereby halting the translation of mRNA into protein, a process called "translation arrest" or "ribosomal-hybridization arrest." (See, Yarchoan et al., "AIDS Therapies," Scientific American, pp. 110-119 (October 1988)). The inhibition of infection of cells by HTLV-III by administration of oligonucleotide complementary to highly conserved regions of the HTLV-III genome necessary for HTLV-III replication and/or expression is disclosed in U.S. Patent No. 4,806,463. The oligonucleotides were found to affect viral replication and/or gene expression as assayed by reverse transcriptase activity (replication) and production of viral proteins pl5 and p24 (gene expression) . The ability of some antisense oligodeoxynucleotides containing internucleoside methylphosphonate linkages to inhibit HIV-induced syncytium formation and expression has been studied (See Sarin, et al., PNAS 85:7448-7451 (1988) .) Summary of the Invention The present invention is directed to methods of inhibiting or controlling of the induction of a viral infection from a latent or chronic infection to an active replicating infection. The present invention is also directed to novel methylphosphonate nucleoside oligomers which are useful for inhibiting the induction of such viruses. Accordingly, the present invention is directed to a method of treating virally infected cells to inhibit induction of a latent or chronic viral infection into an active replicating infection which comprises treating said cells or their growth environment with an Oligomer which is complimentary to an Enhancer Site as defined herein- below. Suitable nucleoside oligomers include oligonucleo¬ tides, nonionic oligonucleoside alkyl- and aryl- phosphonate analogs, phosphorothioate analogs, neutral phosphate ester analogs of oligonucleotides, phosphoro- amidate analogs or other oligonucleotide analogs and modified oligonucleotides. The present invention is also directed to the treat¬ ment of isolated cells, or individuals or animals whose cells or fluids have such latent or chronic viral infec- tions or contain viruses capable of entering a latent or chronic infection state. In particular, the method of the present ' invention is especially suited to treating infections which typically have periods of latent or chronic infection followed by especially virulent active replicating phases. Such viruses include retroviruses such as HIV, HTLV-I, HTLV-II and the like; the Herpes viruses; Hepatitis B virus; and the like. Although Oligomers having various internucleo- side phosphorous linkages may be used according to the present invention, due to their increased resistance to enzymatic metabolism, it is particularly preferred to use oligonucleoside alkyl-and aryl-phosphonate analogs in to the methods of the present invention. The present invention also provides certain novel oligonucleoside methyl phosphonate analogs ("MP- Oligomers") which are particularly active in inhibiting induction of virus replication in latent and/or chronic virus infections. The present invention also provides Oligomers capable of hybridizing to an Enhancer Site of viral DNA of a virus which maintains or is capable of maintaining a chronic or latent infection or to a viral RNA sequence which corresponds to said viral DNA. The present invention also provides hybridization probes for a virus which maintains or is capable of main- taining a chronic or latent infection which comprises an Oligomer of at least 8 nucleosides wherein said Oligomer is substantially complementary to an Enhancer Site of said virus. Also provided herein are diagnostic methods; includ- ing methods for detecting the presence of in a test sample of a virus which maintains or is capable of maintaining a chronic or latent infection. Definitions \ As used herein, the following terms have the follow- ing meanings unless expressly stated to the contrary. The term "latent infection" refers to a " viral infec¬ tion wherein the provirus is integrated into the genetic nuclear structure (DNA) of the host cell and wherein there is no unintegrated viral DNA, no viral RNA and no viral proteins. The term "chronic infection" refers to a viral infec¬ tion having the provirus or virus material in the nucleus or cytoplasm of the host cell and which, until induced, has little or no detectable viral RNA or protein. The term "Enhancer Site" refers to all sequences of U3 of a retrovirus on the viral DNA or to the correspond¬ ing sequences of the viral RNA which affect viral replica¬ tion and to similar sites in Herpes or Hepatitis B viruses which have equivalent activity. The term "Oligomer" refers to oligonucleotides, non¬ ionic oligonucleoside alkyl- and aryl-phosphonate analogs, phosphorothionate analogs of oligonucleotides, phospho- amidate analogs of oligonucleotides, neutral phosphate ester oligonucleotide analogs, and other oligonucleotide analogs and modified oligonucleotides. The term "methylphosphonate Oligomer" (or "MP- oligomer") refers to nucleotide oligomers (or oligonucleo¬ tide analogs) having internucleoside phosphorus group linkages wherein at least one methylphosphonate inter- nucleoside linkage replaces a phosphodiester internucleo¬ side linkage. The term "nucleoside" includes a nucleosidyl unit and is used interchangeably therewith. In the various oligomer sequences listed herein "p" in, e.g., as in ApA represents a phosphodiester linkage, and "p_" in, e.g., as in CpG represents a methylphosphonate linkage. Brief Description of the Drawings FIG. 1 depicts a generalized genetic map of a retro- virus whose induction may be inhibited according to the present invention. FIG. 2 depicts a genetic map of HIV. FIG. 3 shows the position in the HIV genome of the sequences complimentary to some of the oligomers tested. Detailed Description of the Invention The present invention is directed to methods of inhibiting the induction of a viral infection using Oligomers which are complimentary to and which bind to an Enhancer Site on the viral DNA or a corresponding sequence of the viral RNA. Since these Oligomers are complimentary to the (+) or "sense" strand of the viral nucleic acid, they are called "anti-sense Oligomers". These Oligomers are constructed to be complimentary to a specific region of the viral nucleic acid. Accordingly, such anti-sense oligomers when complimentary to the first splice acceptor region (S/A-l) of the tat gene may be termed "anti-tat S/A-l" or anti- S/A-l, when complimentary to the secord splice acceptor region (S/A-2)of the tat gene may be termed anti-tat-s/A- 2 or anti-S/A-2, when complimentary to the target region of tat (TAR) may be termed "anti-TAR" or when compliment¬ ary to the initiator region of env may be termed "anti- env" . and so forth. Preferred are Oligomers having at least 8 nucleo- sides, which is usually a sufficient number to allow for specific binding to a desired nucleic acid sequence. More preferred are Oligomers having from about 8 to about 40 nucleotides; especially preferred are Oligomers having from about 10 to about 25 nucleosides. Due to a combina¬ tion of ease of synthesis, with specificity for a selected sequence, coupled with minimization of intra-Oligomer, internucleoside interactions such as folding and coiling, it is believed that Oligomers having from about 12 to about 15 nucleosides comprise a particularly preferred group. Preferred Oligomers These Oligomers may comprise either oligoribonucleo- sides or oligodeoxyribonucleosides; however, oligodeoxy- ribonucleosides are preferred. 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 analogs, phosphoroamidate analogs and neutral phosphate ester oligonucleotide analogs. However, especially pre¬ ferred 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 dis- closures of which are incorporated herein by reference. A particularly preferred class of those phosphonate analogs are methylphosphonate Oligomers. Preferred synthetic methods for methylphosphate Oligomers ("MP-Oligomers") are described in Lee, B.L. et al. Biochemistry 27:3197-3203 (1988) and Miller, P.S., et al. , Biochemistry 25:5092-5097 (1986), the disclosures of which are incorporated herein by reference. Preferred are oligonucleoside alkyl- and aryl- phosphonate analogs wherein at least one of the phospho- diester internucleoside linkages is replaced by a 3' - 5' linked internucleoside methylphosphonyl (MP) group (or "methylphosphonate"). The methylphosphonate linkage is isosteric with respect to the phosphate groups of oligo¬ nucleotides. . Thus, these methylphosphonate oligomers ("MP-oligomers") should present minimal steric restric¬ tions to interaction with complimentary polynucleotides or single-stranded regions of nucleic acid molecules. These MP-oligomers 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, chemi- luminescent groups, cross-linking agents, intercalating agents such as acridine, or groups capable of cleaving the targeted portion of the viral nucleic acid such as molecular scissors like o-phenanthroline-copper or EDTA- iron may be incorporated in the MP-Oligomers. Preferred are MP-oligomers having at least about 8 nucleosides which is usually sufficient to allow for specific binding to the desired nucleic acid sequence. More preferred are MP-oligomers having from about 8 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 of intra-Oligomer, internucleoside interactions such as folding and coiling, particularly preferred are MP-oligomers of from about 12 to 15 nucleosides. Especially preferred are MP-oligomers where the 5'- internucleoside linkage is a phosphodiester linkage and the remainder of the internucleoside linkages are methyl- phosphonyl linkages. Having a phosphodiester linkage on the 5' - end of the MP-oligomer permits kinase labelling and electrophoresis of the oligomer and also improves its solubility. Target regions of the viral nucleic acid were sequenced and MP-oligomers complimentary to the sense strands of those regions were prepared by the methods disclosed in the above noted patents. In particular, in retroviruses we have found that MP- oligomers complimentary to an Enhancer Site of U3 of the viral DNA or the corresponding sequence in viral RNA are especially preferred. One preferred group of MP-oligomers may include a sequence complimentary to all or a portion of a tract immediately 5' to U3 having the sequence: AAAAGAAAAGGGGGGACT I- U3 In one embodiment of the present invention, we have found that MP-oligomers which are complimentary to the portion of an Enhancer Site of U3 region which binds the NF- B protein in the viral DNA or the corresponding sequence in the viral RNA to be especially effective in inhibiting the induction of HIV in chronically infected cells, even when the cells are treated with a strong inducer such as the protein kinase C activator phorbol myristate acetate (PMA) . Such MP-oligomers were effective in inhibiting viral induction without significantly inhib- iting cell proliferation or other normal cellular func¬ tions. This is in contrast to MP-oligomers complimentary to some other regions of the HIV genome wherein activity in decreasing infection by HIV appeared to correspond to activity in inhibiting cell proliferation and other normal cell functions. Accordingly, in one embodiment, the present invention is directed to novel MP-oligomers which are complimentary to an Enhancer Site in HIV and which are capable of inhib¬ iting induction of chronically infected cells without sig- nificantly inhibiting cell proliferation. In particular, examples of such preferred MP-oligomers of the present invention include: dTpA.ApAβGE pCECECECEAEG wherein p = phosphodiester linkage p. = methylphosphonate linkage Inhibition of HIV In one embodiment, the present invention is directed to a method of inhibiting the induction of HIV virus in cells which have a chronic or latent HIV infection. People have been looking at different ways of modify¬ ing nucleotides complimentary to different regions of the AIDS virus HIV. Previously two strategies have been tried, either to make the nucleotide oligomers or modified oligomers complimentary to structural genes of the virus or compli¬ mentary to regulatory genes. Oligomers complimentary to viral regulatory genes were directed to two possible targets: (a) the initiation codon AUG of a particular gene and the sequence that followed, or (b) the sequences around the site known as the splice/acceptor site, abbre¬ viated S/A, which would give the added advantage of possibly being able to block expression of two different messages simultaneously. It has been shown that sequences complimentary to the particular sites can bind and inhibit expression of the virus. However, it has been found that oligomers which bind to those sites and inhibit the virus inhibit normal cellular functions, including cell divi¬ sion. For example, when a T-cell line or macrophage cell line that proliferates autonomously (i.e. without added growth factors) is used, if viral replication is blocked by 50%, it has been found that the growth of those cells is blocked by 50%. In normal T-cells or other normal blood components, these anti-S/A oligomers, even though they are supposed to be specific for viral nucleic acid sequences, seem to effect important cell functions. We believe that these oligomers, particularly ones compli¬ mentary to regulatory regions of the viral genome, also inhibit consensus S/A sequences in the normal human genome that are similar to viral S/A sequences. That effect would explain why such oligomers inhibit important house¬ keeping functions of the cell, while inhibiting viral replication. According to the method of the present invention, we are able to inhibit induction and, thus, replication of the virus using oligomers capable of binding both to the viral RNA and DNA. The genetic material of HIV and other retroviruses is RNA. Once the virus infects a cell, it directs synthesis of a double-stranded DNA version of its genome which is incorporated into the cell•s genome as the provirus. We decided to target the region of the viral genome known as the U3 region, which is the farthest down- stream part of the viral RNA, since oligomers compliment¬ ary to the DNA Enhancer Site represented once in RNA would directly bind to the viral message and block reverse transcription, and if the viral DNA synthesis had begun, the oligomers would bind to the DNA copy (when one of these viruses infects the cell, DNA synthesis commences within the first two to three hours after infection) . In virus-directed DNA synthesis in retroviruses, the U3 region is duplicated and copies form on either end of the viral gene sequence. The region including U3 is called the long terminal repeat or LTR, and includes an important enhancer site for the virus. Certain cellular or viral proteins bind to the enhancer sites in viral DNA, and cause the amount of viral replication to increase tremen¬ dously. Thus, Oligomers complimentary to these enhancer sites which can block binding of those proteins to the enhancer sites would inhibit the conversion of a chronic or latent virus infection into an active replicative state. Also by blocking this earlier step in the viral replication scheme, we would also block reverse transcrip- tion as well. Since the LTR region is long, up to about 400 bp of information, a DNase I protection reaction was used to determine what parts of the U3 region of the viral DNA were open and not protected by proteins (See, Wu, F., et al. J. Virol. .62.: 218-225 (1988)) and thus were avail- able to be bound by oligomers. By comparing those open regions with regions we believed were related to viral replication in other cells, we selected certain regions and made Oligomers complimentary to those regions. We chose regions including the enhancer site that had the capacity to bind Oligomers in the both DNA form and the RNA form. Previous work on oligomers complimentary to viral nucleic acid only targeted messenger RNA sequences in an attempt to block transcription. Normally, the viral DNA sequences are not available for binding by oligomers, since the DNA is unavailable due to supercoiling, being double stranded, or being bound by protein. However, we found a sequence that would be available for oligomer binding in the RNA form as many of the other sequences are, but would be available for binding in the DNA form as well. According to the method of present invention, these Oligomers are complimentary to and capable of binding to the binding region for enhancer proteins rather than just bind to the RNA to block transcription. Thus, these oligomers complimentary to the enhancing region are cap- able of inhibiting viral replication by two means. First they can block transcription by targeting the sequence that is represented in the RNA, and, second they can inhibit induction and, thus, replication by binding to the viral DNA in the enhancer site of U3 in order to block the binding of a regulatory protein to that region and thus inhibiting induction. We have found that when one of these Oligomers, in concentrations which are non-cytotoxic and which do not significantly depress cell replication, is put in a culture of cells with the addition of a viral activator, the Oligomer blocked the induction, of HIV- associated tat activity by greater than 90% and of viral replication by greater than 50% at a 50μm concentration (a number of different viral activators may be used but due to its strong and general effects, preferred is the protein kinase C-activator known as phorbol ester) . It was generally thought that the most important controlling region for HIV was the tat gene, and that by inhibiting expression of that gene, viral ' replication could be inhibited. However, as noted above, the anti-S/A oligomers that have been found to inhibit expression of that gene were also found to inhibit normal cellular pro- cesses. In an attempt to overcome the problem of inhibi¬ tion of cellular functions, we also attempted to inhibit the target for that gene ("TAR") rather than the gene itself using one oligomer complimentary to TAR. This oligomer complimentary to TAR did not inhibit viral replication. In an additional aspect, the present invention is directed to the use of these Oligomers in determining the presence or absence of a virus which maintains or is cap¬ able of maintaining a chronic or latent infection state or the presence of such a chronic or latent viral infec¬ tion in samples including isolated cells, tissue samples or bodily fluids. For example, these Oligomers may be used in determining the presence or absence of HIV or infection by HIV. (See, e.g. US Patent No. 4,806,463). Thus, in one aspect, the present invention is directed to hybridization assay probes comprising these Oligomers and to detection assays using these Oligomers. These probes may also be used in diagnostic kits. These Oligomers may be labelled by any of several well known methods. Useful labels include radioisotopes as well as non-radioactive reporting groups. Isotopic labels include 3 H, 35 S, 32 P, 125 I, Cobalt and U C. Most methods of isotopic labelling involve the use of enzymes and include the known methods of nick translation, end labelling, second strand synthesis, and reverse transcrip¬ tion. When using radio-labelled probes, hybridization can be detected by autoradiography, scintillation counting, or gamma counting. The detection method selected will depend upon the hybridization conditions and the particular radioisotope used for labelling. Non-isotopic materials can also be used for label¬ ling, and may be introduced by the incorporation of modi- fied nucleosides or nucleoside analogs through the use of enzymes or by chemical modification of the Oligomer, for example, by the use of non-nucleotide linker groups. Non- isotopic labels include fluorescent molecules, chemilumi- nescent molecules, enzymes, cofactors, enzyme substrates, haptens or other ligands. One preferred labelling method is acridinium esters. Such labelled Oligonucleotides are particularly suited as hybridization assay probes and for use in hybridization assays. To assist in understanding the present invention, the following examples are included which describe the results of a series of experiments. The following examples relat¬ ing to this invention should not, of course, be construed 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 the art are considered to fall within the scope of the present inven¬ tion as hereinafter claimed. Examples Example 1 Activity of Certain MP-01igomers in Inhibiting HIV Replication Oligonucleotide methylphosphonate analogs compli- mentary to the initiator codon regions of the gag-pol polypeptide and of tat were tested for their effect as specific inhibitors of HIV replication. An Oligomer specific to B-globulin mRNA and a d-AT polymer were used as negative controls. Varying concentrations of the Methylphosphonate ("MP") oligomers were incubated with phytohemagglutin ("PHA") stimulated human peripheral blood mononuclear cells (PBMC) for two hours of 37"C and then exposed to 100 ID-50 of Stock HIV from HTLV-IIIB-infected H9 cells. Eighteen hours later, fresh medium and addi- tional oligomer were added. Cells were harvested after seven days and evaluated for HIV-specific proteins in an indirect immunofluorescence assay employing IgG isolated from the serum of an HIV-infected individual. In parallel, reverse transcriptase (RT) activity was measured in the culture supernatants by standard techniques (See, Laurence, J., et al.. Science 235:1501-1504 (1987)). Results are reported in Table I. As may be seen from Table I, oligomers specific to the initiator codon region of gag-pol and tat were ineffective at inhibiting HIV replication, although they were non-cytotoxic to activated PBMC at concentrations up to 200 μM. Example 2 Effect of Anti-Tat MP-Oligomers on HIV-Associated Trans- Activation Methylphosphonate oligomers complimentary to the tat initiation and splice/acceptor regions were tested for their ability to inhibit HIV-associated transactivation as measured by the ability of tat to enhance expression of the chloramphenicol acetyl transfer gene (CAT) upon trans- fection of a human T4+ ϊymphoblastoid T cell line with either a CAT plasmid alone or together with a tat plasmid (See: Laurence, J., e£ al.. , J. Clin. Invest. 80:1631-1639 (December 1987)) . The CAT plasmid pC15CAT and the control tat plasmid pCV-1, which lacked the splice/acceptor sites, were provided by Dr. Wong-Staal of the NCI. The experi- mental tat plasmid containing the two tat-associated exons, pBR322/pIIIextat. jj was provided by Dr. Haseltine of Harvard Medical School. CAT activity was assessed by TLC analysis of acetylated forms of [ 14 C]-chloramphenical by standard techniques. (See, Laurence, J. , et al., J. Clin. Invest. 80:1631-1639 (1987)). Results are shown in Table II. The MP-oligomer directed against the tat initiation site had no effect, while the MP-oligomer directed against the splice/acceptor site showed inhibition of transactivation at 50 μM. Example 3 Effect of MP-Oligomers on HIV-Replication In Vitro Anti-tat MP-oligomers were tested for their ability to block HIV infection of target cells (in order to see if that activity paralleled activity against trans- activation) . Cells and oligomer were incubated in the presence of HIV as described in Example 1. Effects of oligomer on normal cell function, T- lymphocyte viability and PHA-mediated proliferation were measured (See, Laurence, et al. , J. Clin. Invest. 80:1631- 1639 (1987)). No adverse effect on viability, measured by the ability to exclude trypan blue was noted. However, three different anti-tat-splice/acceptor oligomers (S/A- 1, S/A-IA and S/A-2, see below) depressed PHA-mediated blastogenesis by about 40 to 50% at concentrations >25 μM in 3 separate experiments. Results of oligomers in inhibiting HIV infection are reported in Table III. Oligomer identification MP oligomer Sequence anti-tat S/A-l dCpApCp.Cp.CEAp.AETETp.Cp.TpG anti-tat S/A-IA dApA AEAETE G EGE A ETEAEAEA anti-tat S/A-2 dTpGEGEGE EGEGE τ where p = phosphodiester linkage where E β methylphosphonate linkage Example 4 Effect of MP-Oligomers on PMA-Induction of HIV MP-oligomers were assayed for their effect on phorbol-ester (13-phorbol-12-myristate acetate or "PMA") mediated induction of HIV. In certain cells (including T4+ T-cells) , NF-κB, a factor that regulates transcription and binds to the HIV enhancer, also mediates, phorbol-ester and itogen or antigen activation. MP-oligomers complimentary to the tat-1inked target element TAR and to the enhancing region in U3 which binds NF-zcB were tested for their ability to inhibit PMA- induction of HIV replication-related effects. A stock solution of lOOμg/ml PMA (Sigma) was prepared in absolute ethanol diluted in RPMI-1640 and used in final concentrations of 5 to 500 ng/ml. Ul.l cells were obtained from T.M. Folks of the NIH; they were subcloned from Ul, a clone of the human monocytic cell line U937 which had been infected with the lymphadenopathy- associated virus (LAV) strain of HIV. H9, a human CD3+, CD4+ lymphoblastoid cell line permissive for the replica¬ tion of HIV annd partially resistant to its cytotoxic effects was obtained from R.C. Gallo of the NIH. Stock Samples of these cells were cultured in RPMI-1640 (Flow Laboratories, McLean, VA) ; plus 10% fetal bovine serum (FBS) , at a concentration of 5 x 10 5 cells/ml. Varying concentrations of Oligomer (2 to 100 μM) were incubated with Ul.l cells (a chronically infected macro- phage cell line) at 37 ' C for one hour prior to exposure of the cells to PMA (5 ng/ml) . Cultures were evaluated for HIV activity after 48 hours. HIV antigens were quantitated in supernatants by an ELISA-based assay for viral p24 core protein. Human immunoglobulin directed against p24 epitopes (Abbott Labs, Chicago, IL) contained in polystryene beads was added to supernatants and maintained overnight at room temperature. Plates were washed with citrate phosphate buffer; and rabbit anti-HIV IgG, followed by addition of horseradish peroxidase-labeled goat anti-rabbit antibody (Abbott) . Color was developed with O-phenylenediamine as a sub- strate, followed by IN H 2 S0 4 to stop the reaction. Absorb- ance was read at 492 n ; data were expressed as pg/10 4 cells. The sensitivity of this assay was < 60 pg/ml. Results are tabulated in Table IV. As may be seen, an 11-mer MP-oligomer complimentary to the enhancing sequence in U3 (which binds NF-cB) was able to block induction of HIV by almost 60% at 50 μM. That concentra- tion inhibited replication of Ul.l cells as measured by 3 H- Thymidine incorporation by less than 20 percent. DNA synthetic response was assayed according to the following procedure. Cells were collected, washed three times with PBS, and viability assessed by trypan blue dye exclusion. 1 x 10 4 viable cells were resuspended in 0.2 ml of medicine in polystryene flat-bottom microwell plates. Selected cultures were treated with inducing agent. All groups were assayed in triplicate. Cells were incubated for 48 hours; eighteen hours before culture termination, they were pulsed with 0.1 Ci of [ 3 H- methyl]-thymidine (1.9 Ci/μM sp. act.. New England Nuclear). The contents of each well were harvested and incorporation of radio¬ activity was measued by liquid scintillation counting. The cells were also assayed for PMA-induced enhance¬ ment of HIV-LTR driven CAT activity. The ability of the tat transcription unit of HIV to enhance the expression of the chloramphenical acetyl transferase (CAT) gene when CAT is linked to the LTR of HIV was measured as described in Laurence, J. et al. , J. Clin. Invest. .80:1631-1639 (1987) with the following modifications. 2 X 10 6 Ul.l cells per condition were washed with serum-free RPMI-1640 and resus¬ pended in 1ml of 5mM Tris (pH7.3) containing 250μg/ml DEAE-dextran (Sigma) and 2 or 4 μg of total plasmid DNA. Two plasmids (See Arya, S.K., et al., Science 229:69 (1985) ; plasmids were obtained from the sources noted in Example 2) were used either singly (HIV-LTR-CAT alone) or together (co-transfeetion of CAT and tat containing vectors). The tat plasmid pCV-1 contains a 1.8 kb frag- ment of HIV-1 cDNA encompassing the tat gene. The CAT plasmid pC15CAT contains SV-40 regulatory sequences, and the LTR and a portion of nef (3'-orf) of HIV-1. After transfection, cells were washed with serum-free RPMI-1640 and incubated in 0.5 ml of culture medium at 37 β C. Cer- tain cultures also contained PMA (50 ng/ml) . Cells were harvested, washed with PBS, resuspended in 100 μl of 0.25 M Tris (pH 7.08), and cellular extracts were prepareed by three cycles of freezing (in ethanol and dry ice) and thawing at 37 β C. CAT activity was determined by incubat¬ ing 50 μl aliquots of cell extracts with [ 14 C]-chloramphen- icol (New England Nuclear) and 2.5M acetyl coenzyme A (P- L Biochemicals, Inc., Piscataway, NJ) at 37"C for 2 hours and extracting with ethyl acetate. The acetylated forms of chloramphenicol were separated from the unacetylated form by ascending thin layer chromatography using a chromatogram sheet (Eastman Kodak, Rochester, NY) in a chamber containing chloroform and methanol (19:1, v/v). The chromatogram was then autoradiographed. Areas of radioactivity were marked, cut from the sheet, and counted in scintillation fluid. Results are tabulated in Table V. Mp-oligomer Nucleotide sequence position Identification dCpAECECECEAEAETETECETEG- 5366-5355 tat splice- a c c e p t o r site=anti-tat S/A-l dApAEAEAETEGEGEAETEAEAEA 5356-5343 tat splice- a c c e p t o r site= anti-tat S/A-IA dTpAEA AEGET CE E E EAEG -83 to -94 enhancer (NF-/cB repeated element of U3 of HIV- 1) =anti-enhancer dGETECETEGEGE G E τ E E τ E A E A TAR = anti-TAR p=phosphodiester linkage E=methylphosphonate linkage Example 5 Effect of Oligomers on Spontaneous Cell Growth and DNA Synthetic Response Spontaneous growth and DNA synthetic response, as measured by ^-Thymidine incorporation, of an immortal T4+cell line (SK7) and normal, mitogen-exposed peripheral blood mononuclear cells (PBMC) in the presence of a MP- oligomer complimentary to the S/A-l region of HIV and a MP-oligomer complimentary to the initiator codon of env was measured, as described in Example 4. Results are tabulated in Tables VI-A (SK7) and VI-B (PMBC) . As may be seen, the anti-S/A-1 oligomer signifi¬ cantly inhibited cellular proliferation in both cell lines. The anti-env oligomer inhibited cell proliferation to a lesser effect, however, we have found that anti-env MP-oligomers have poor anti-viral activity. Identification MP-01igomer Sequence anti-S/A-1 dCpCECEAEAETETECETE G anti-env dCpAE G E G ECE A E A E G E A E A E τ E c Table I Effect of MP-Oligomers Complimentary to HIV-Specific Initiation Codons on Viral Infection A s s a y s for H I V Replication Percent Inhibition Oligomer Immuno- MP-Oligomer MP- Concen- fluores- RT seguence oligomer tration cence Activity d-CpAETET£CETE G ET B-globin 20μM 0 0 d-CpAEAETE ECETE G ET B-globin 200μM 0 0 d-Ap(TEAE)n none 200μM 0 0 d-TpTETECETETE G E ETEC tat 200μM ND 0 d-TpCETECETECETE- CECETETEC pol 200μM 22.7 Table II Effect of Anti-Tat MP-Oligomers on HIV-Associated Trans- Activation Percent Inhibition of CAT MP-Oligomer MP-oligomer Oligomer Control Exper. seguence specificity Cone. Plasmid Plasmid d-TpTE ECETfi TEGECETEC tat-initiation 50μM 0 0 d-CpCECEAE pT TECE G tat-S/A-1 50μM 0 >50 Table III Effect of Anti-Tat MP-Oligomers oni HIV-Associated Replication In Vitro HIV Reverse Transcriptase Activity MP-Oligomer MP-oligomer Oligomer Mean cpm %Inhi- sequence specificity Cone. + SD* bition d-TpTETfiCETETE tat-initiation 50μM 69,575 1.0 GECETEC ±11,595 d-CpCfiCEAEAETE tat-S/A-1 50μM 8,674 87.7 TECET£ G ± 2,245 None - 70,293 ±16,101 - * Mean of two experiments Table IV Effect of MP-Oligomers on Induction of HIV from Chronically Infected Cells* HIV p24 antigen/ 10 4 cells HIV inducer Concen¬ MP-Oligomer MP- ■oligomer Cone. PMA, tration % Inhi- sequence specificity (ul ) 5ng/ml (pq/ml) bition - - - - <30 - — + 1,915 ™ ±308 anti-tat-S/A-1 tat S/A 50 - 0 - anti-enhancer enhancer 50 - 0 - anti-tat-S/A-1 tat S/A 50 + 1,250 34.7 anti-enhancer enhancer 50 + 785 59.0 * 1 x 10 Ul.l cells were plated in flat-bottom micro- wells in 0.2ml RPMI 1640 + 10% FBS. Selected cultures were exposed to PMA (5ng/ml phorbol myristate acetate) for 30 minutes prior to addition of MP-oligomer. Cell-free supernatants were harvested at 48 hours for determination of p24 antigen determination (Abbott Lab Kit) . Table V Effect of MP-Oligomers on UP Regulation of HIV-Ltr CAT- Driven Activity bv Tat* Plasmid CAT PMA (10 Oligomer Oligomer transfected acti- Exp't. ng/ml) specificity cone. (uMi HIV-CAT/LTR tat vity - - - + - 1.9 - - - + + 9.8 - anti-tat-s/A-1 50 + + 10.3 - anti-tat-S/A-1 100 + + 4.7 - anti-TAR 100 + + 9.4 B + - - + + 14.5 + anti-TAR 100 + + 14.7 + anti-enhancer 100 + + 0.3 Table VI Effect Of Oligomers On Cellular Proliferation Measured By The Incorporation Of 3 h-Thymidine Table VI-A - Effect On Sk7 T-Cell Hvbridoma Line CAT/TAT Transection CAT Oligomer S/A-l S/A-l env Amount - lOμM 50μM 50μM CPM* 780 770 670 410 660 % Inhibition 14 50 16 TABLE VI-B - EFFECT ON PMBC HTLV-III - + + + Oligomer (50μM) - - S/A-l env CPM* 8600 9530 5240 8500 % Inhibition - - 45 11 * Average of duplicate samples. * Average of duplicate samples.

Claims: Claims: 1. A method of preventing induction of a latent or chronic virus infection into an actively replicating form in cells having a chronic or latent infection of said virus which comprises treating said cells or their growth environment with an Oligomer which is complimentary to and which can bind to or interact with an Enhancer Site of the viral DNA or a corresponding sequence in the viral RNA. 2. A method according to claim 1 wherein said virus is a retrovirus, a Herpes virus or a Hepatitis B virus. 3. A method according to claim 2 wherein said Oligomer comprises a nonionic oligonucleoside alkyl- or aryl-phosphonate analog, a phosphorothioate oligonu¬ cleotide analog, a phosphoramidate oligonucleotide analog, or a neutral phosphate ester oligonucleotide analog. 4. A method according to claim 3 wherein said virus comprises a retrovirus. 5. A method according to claim 4 wherein said Oligomer comprises an oligonucleoside alkyl- or aryl- phosphonate analog. 6. The method according to claim 5 wherein said Oligomer comprises a methylphosphonate Oligomer. 7. The method according to claim 6 wherein said virus is HIV. 8. The method according to claim 7 wherein said Oligomer comprises at least 10 nucleosides. 9. The method according to claim 8 wherein said Oligomer comprises from about 10 to about 25 nucleosides. 10. The method according to claim 9 ' wherein the Oligomer has a phosphodiester internucleoside linkage at its 5'-end and wherein the remainder of the inter¬ nucleoside linkages are methylphosphonate linkages. 11. The method according to claim 10 wherein said Oligomer is complementary to the Enhancer Site of U3 which binds to NF-/cB. 12. The method according to claim 11 wherein said Oligomer has from about 12 to about 15 nucleosides. 13. The method according to claim 12 wherein said Oligomer has the sequence: TpAEAEA£GE ECECE ECEAE G wherein p is a phosphodiester internucleoside linkage and E is a methylphosphonate linkage. 14. A methylphosphonate Oligomer which is complementary to an Enhancer Site of viral DNA of a virus which maintains or is capable of maintaining a chronic or latent infection or to a viral RNA sequence which corresponds to said viral DNA. 15. An Oligomer according to claim 14 wherein said virus comprises a retrovirus, or Herpes virus or a Hepatitis B virus. 16. An Oligomer according to claim 15 which comprises an oligodeoxyribonucleoside. 17. An Oligomer according to claim 16 which comprises at least about 8 nucleosides. 18. An Oligomer according to claim 17 which comprises from about 10 to about 25 nucleosides. 19. An Oligomer according to claim 18 wherein said virus is a retrovirus. 20. An Oligomer according to claim 19 wherein said virus is HIV. 21. An Oligomer according to claim 20 which is complementary to at least a portion of a tract immediately 5' to U3. 22. An Oligomer according to claim 21 wherein said tract has the sequence: AAAAGAAAAGGGGGGCT U3 23. An Oligomer according to claim 18 which comprises from about 12 to about 15 nucleosides. 24. An Oligomer according to claim 23 which has the following sequence: dTpAEAEAEGETECE c E c E c EAE G wherein p is a phosphodiester internucleoside linkage and E is a methylphosphonate internucleoside linkage. 25. A methylphosphonate Oligomer which is complimentary to the enhancer site which is capable of binding NF-κB in U3 of the LTR of a retrovirus. 26. An oligomer according to claim 25 wherein said virus is HIV. 27. An oligomer according to claim 26 having a 5'- phosphodiester internucleoside linkage at its 5'-end and wherein the remainder of internucleoside linkages are methylphosphonate linkages. 28. An oligomer according to claim 27 which comprises a deoxynucleoside oligomer. 29. An oligomer according to claim 28 having at least about 8 nucleosides. 30. A method of treating an organism or isolated cells thereof having a chronic or latent viral infection or a fluid from that organism or cells having a virus capable of entering a chronic or latent infection in order to maintain said infection in a chronic or latent phase and to inhibit its induction to a active replicating phase which comprises the administration to said organism of a therapeutically effective amount of an Oligomer which is complimentary to the base sequence of an Enhancer Site of said virus, effective to inhibit induction of said virus. 31. A method according to claim 30 where said virus comprises a virus which maintains a chronic or latent infection. 32. A method according to claim 31 wherein said virus comprises a retrovirus, a Herpes virus or a Hepatitis B virus. 33. A method according to claim 32 wherein said Oligomer comprises a nonionic oligonucleoside alkyl- or aryl-phosphonate analog, a phosphorothioate oligo¬ nucleotide analog a phosphoramidate oligonucleotide analog, or a neutral phosphate ester oligonucleotide analog. 34. A method according to claim 33 wherein said virus comprises a retrovirus. 35. A method according to claim 34 wherein said Oligomer comprises an oligonucleoside alkyl- or aryl- phosphonate analog. 36. The method according to claim 35 wherein said Oligomer comprises a methylphosphonate Oligomer. 37. The method according to claim 36 wherein said virus is HIV. 38. The method according to claim 37 wherein said Oligomer comprises at least 8 nucleosides. 39. The method according to claim 38 wherein said Oligomer comprises from about 10 to about 25 nucleosides. 40. The method according to claim 39 wherein the Oligomer has a phosphodiester internucleoside linkage at its 5'-end and wherein the remainder of the inter- nucleoside linkages are methylphosphonate linkages. 41. The method according to claim 40 wherein said Oligomer is complementary to the Enhancer Site of U3 which binds to NF-ΛB. 42. The method according to claim 41 wherein said Oligomer has from about 12 to about 15 nucleosides. 43. The method according to claim 42 wherein said Oligomer has the sequence: dTpAEAEAE G E τ E c ECECECEAE G wherein p is a phosphodiester internucleoside linkage and E is a methylphosphonate linkage. 44. An Oligomer capable of hybridizing to an Enhancer Site of viral DNA of a virus which maintains or is capable of maintaining a chronic or latent infection or to a viral RNA sequence which corresponds to said viral DNA. 45. An Oligomer according to claim 44 wherein said virus comprises a retrovirus, or Herpes virus or a Hepatitis B virus. 46. An Oligomer according to claim 45 which comprises an oligodeoxyribonucleoside. 47. An Oligomer according to claim 46 which comprises at least about 8 nucleosides. 48. An Oligomer according to claim 47 which comprises from about 10 to about 25 nucleosides. 49. An Oligomer according to claim 48 wherein said virus is a retrovirus. 50. An Oligomer according to claim 49 wherein said virus is HIV. 51. An Oligomer according to claim 50 which is complementary to at least a portion of a tract immediately 5' to U3. 52. An Oligomer according to claim 51 wherein said tract has the sequence: AAAAGAAAAGGGGGGCT U3 53. An Oligomer according to claim 48 which comprises from about 12 to about 15 nucleosides. 54. An Oligomer according to claim 53 which has the following sequence: dTAAAGTCCCCAG 55. An Oligomer according to claim 53 which is complementary to the Enhancer Site which is capable of binding NF-*cB in U3. 56. A hybridization probe for a virus which maintains or is capable of maintaining a chronic or latent infection which comprises an Oligomer of at least 8 nucleosides wherein said Oligomer is substantially complementary to an Enhancer Site of said virus. 57. A probe according to claim 56 wherein said virus comprises a retrovirus, or Herpes virus or a Hepatitis B virus. 58. A probe according to claim 57 which comprises an oligodeoxyribonucleoside. 59. A probe according to claim 58 which comprises at least about 8 nucleosides. 60. A probe according to claim 59 which comprises from about 10 to about 25 nucleosides. 61. A probe according to claim 60 wherein said virus is a retrovirus. 62. A probe according to claim 61 wherein said virus is HIV. 63. A probe according to claim 62 which is complementary to at least a portion of a tract immediately 5' to U3. 64. A probe according to claim 63 wherein said tract has the sequence: AAAAGAAAAGGGGGGCT U3 65. A probe according to claim 60 which comprises from about 12 to about 15 nucleosides. 66. A probe according to claim 65 which is complementary to the Enhancer Site which is capable of binding NF-κB. 67. A method for detecting the presence in a test sample of a virus which maintains or is capable of maintaining a chronic or latent infection which comprises: (a) bringing together test sample nucleic acid and an Oligomer sufficiently complementary to hybridize with an Enhancer Site of viral DNA or a viral RNA sequence which corresponds to said viral DNA; (b) incubating said Oligomer and test sample under specified hybridization conditions such that said Oligomer hybridizes only to viral nucleic acid and does not detectably hybridize with cell nucleic acid; and (c) assaying for hybridization of Oligomer to test sample. 68. A method according to claim 67 wherein said virus is a retrovirus, a Herpes virus or a Hepatitis B virus. 69. A method according to claim 68 wherein said virus comprises a retrovirus. 70. A method according to claim 69 wherein said Oligomer comprises an oligonucleoside alkyl- or aryl- phosphonate analog. 71. The method according to claim 72 ' wherein said Oligomer comprises a methylphosphonate Oligomer. 72. The method according to claim 71 wherein said virus is HIV. 73. The method according to claim 72 wherein said Oligomer comprises at least about 8 nucleosides. 74. The method according to claim 73 wherein said Oligomer comprises from about 10 to about 25 nucleosides. 75. The method according to claim 74 wherein the Oligomer has a phosphodiester internucleoside linkage at its 5'-end and wherein the remainder of the inter¬ nucleoside linkages are methylphosphonate linkages. 76. A detectably labelled Oligomer capable of hybridizing to an Enhancer Site of viral DNA of a virus which maintains or is capable of maintaining a chronic or latent infection or to a viral RNA sequence which corresponds to said viral DNA. 77. A detectably labelled methylphosphonate Oligomer which is complementary to an Enhancer Site of viral DNA of a virus which maintains or is capable of maintaining a chronic or latent infection or to a viral RNA sequence which corresponds to said viral DNA. 78. A diagnostic kit for detecting the presence or absence of a virus which maintains or is capable of maintaining a latent or chronic infection which comprises an Oligomer according to claim 14, 44, 76 or 77.

Inventors: LAURENCE JEFFREY, MILLER PAUL S, TS O PAUL O P, LAURENCE, JEFFREY, MILLER, PAUL, S., TS'O, PAUL, O., P.
Applicants: CORNELL RES FOUNDATION INC, UNIV JOHNS HOPKINS, CORNELL RESEARCH FOUNDATION, INC., THE JOHNS HOPKINS UNIVERSITY
Application year: 1990
Main classification: A61K31
IPC codes: A61K31, A61K48, A61P31, C07H21, C12N15, C12Q1