A new class of anti-HIV-1 oligonucleotide targeted to the polypurine tract of viral RNA.

The PPT is highly conserved among the known HIV-1 strains, and is a possible target for triplex formation. We show triple-helix formation by a two-strand-system (FTFOs, DsDGloopT5-37) targeted to the polypurine tract (PPT) of HIV-1. In HIV-1 infected MOLT-4 cells, the FTFOs containing phosphorothioate groups at the antisense strand and guanosine rich parts within the third Hoogsteen base pairing sequence inhibit the replication of HIV-1 more effectively than the antisense phos-phorothioate oligonucleotides indicating sequence-specific inhibition of HIV-1 replication for 62 days. However, AZT, treated cells expressed high levels of p 24 products after 46 days.

Anti-HIV-1 activity of an antisense phosphorothioate oligonucleotide bearing imidazole and primary amine groups.

We have previously shown that RNA cleaving reagents with imidazole and primary amine groups on the 5'-end of antisense oligodeoxyribonucleotides could site-specifically cleave CpA as the target sequence of the substrate tRNA in vitro. In this study, a RNA cleaving reagent, composed of imidazole and primary amine groups on an antisense phosphorothioate oligonucleotide (Im-anti-s-ODN), was synthesized and evaluated for anti-HIV-1 activity in MT-4 cells. The sequence of the Im-anti-s-ODN was designed to be complementary to the HIV-1 gag-mRNA and to bind adjacent to the CpA cleavage site position. Im-anti-s-ODN encapsulated with the transfection reagent, DMRIE-C, had higher anti-HIV-1 activity than the unmodified antisense phosphorothioate oligonucleotide (anti-s-ODN) at a 2 microM concentration. Furthermore, the Im-anti-ODN encapsulated with DMRIE-C conferred sequence-specific inhibition.

Antisense therapy of influenza.

The liposomally encapsulated and the free antisense phosphorothioate oligonucleotides (S-ODNs) with four target sites (PB1, PB2, PA, and NP) were tested for their abilities to inhibit virus-induced cytopathogenic effects by a MTT assay using MDCK cells. The liposomally encapsulated S-ODN complementary to the sites of the PB2-AUG initiation codon showed highly inhibitory effects. On the other hand, the inhibitory effect of the liposomally encapsulated S-ODN targeted to PB1 was considerably decreased in comparison with those directed to the PB2 target sites. The liposomally encapsulated antisense phosphorothioate oligonucleotides exhibited higher inhibitory activities than the free oligonucleotides, and showed sequence-specific inhibition, whereas the free antisense phosphorothioate oligonucleotides were observed to inhibit viral absorption to MDCK cells. Therefore, the antiviral effects of S-ODN-PB2-AUG and PA-AUG were examined in a mouse model of influenza virus A infection. Balb/c mice exposed to the influenza virus A (A/PR/8/34) strain at dose of 100 LD(50)s were treated i.v. with various doses (5-40 mg/kg) of liposomally (Tfx-10) encapsulated PB2-AUG or PA-AUG before virus infection and 1 and 3 days postinfection. PB2-AUG oligomer treated i.v. significantly prolonged the mean survival time in days (MDS) and increased the survival rates with a dose-dependent manner. We demonstrate the first successful in vivo antiviral activity of antisense administered i.v. in experimental respiratory tract infections induced with influenza virus A.

Inhibition of human telomerase activity by antisense phosphorothioate oligonucleotides encapsulated with the transfection reagent, FuGENE6, in HeLa cells.

Telomerase, a ribonucleoprotein, synthesizes telomeric repeats (TTAGGG) onto the ends of chromosomes to maintain the constant length of the telomere DNA, and its activity is detectable in approximately 85%-90% of primary human cancers. Thus, it is postulated that human telomerase might be associated with malignant tumor development and could be a highly selective target for antitumor drug design. Antisense phosphorothioate oligonucleotides (S-ODN) were investigated for their abilities to inhibit telomerase activity in the HeLa cell line. The S-ODN were designed to be complementary to nucleotides within the RNA active site of telomerase. As a transfection reagent, FuGENE6 (Boehringer Mannheim, Mannheim, Germany) was used to enhance the cellular uptake of the oligonucleotides in cell cultures. The S-ODN encapsulated with FuGENE6 clearly inhibited telomerase activity in HeLa cells and showed sequence-specific inhibition. The encapsulated S-ODN-3 with a 19-nucleotide, (nt) chain length had inhibitory effects similar to those of the 21-mer and 23-mer S-ODN sequences (S-ODN-4 and 5), but the 15-mer and 17-mer S-ODN sequences (S-ODN-1 and 2) failed to satisfactorily prevent telomerase activity. However, apoptotic HeLa cell death was not associated with telomerase inhibition. Furthermore, the encapsulated S-ODN did not appear to be cytotoxic in terms of the cell growth rate. The oligonucleotides encapsulated with the transfection reagent had enhanced cellular uptake, and cytoplasmic and nuclear localizations were observed. However, weak fluorescent signals were observed within the cytoplasms of HeLa cells treated with the free S-ODN-3. Thus, the activities of the S-ODN were effectively enhanced by using the transfection reagent. The transfection reagent, FuGENE6, may thus be a potentially useful delivery vehicle for oligonucleotide-based therapeutics and transgenes and is appropriate for use in vitro and in vivo.

Anti-human immunodeficiency virus type 1 of a two-strand-system targeted to the polypurine tract.

The polypurine tract (PPT) is highly conserved among the known human immunodeficiency virus (HIV)-1 strains, and is a possible target for triplex formation. We show the effects of triple-helix formation by assays of primer extension inhibition in vitro, using a two-strand-system (FTFOs) targeted to the PPT of HIV-1. The two-stranded composition of a triple-helix is thermodynamically and kinetically superior to the three-strand-system. The FTFOs inhibited the RT activity in a sequence-specific manner, i.e., the triplex actually formed at the PPT and blocked the RT. The FTFOs containing the phosphorothioate groups at the antisense sequences showed greater 3'-exonuclease resistance. In the observation of the FITC-DsDGloopT5-37 with MOLT-4 cells by a confocal laser scanning microscope, diffuse fluorescence was apparently observed in the cytoplasm and nucleus. However, weak fluorescence was observed within the cytoplasm and nucleus of MOLT-4 cells treated with the antisense phosphorothioate oligonucleotides (S-ODN-gag-AUG). In HIV-1 infected MOLT-4 cells, the FTFOs containing the phosphorothioate groups at the antisense sequence sites and guanosine rich parts within the third Hoogsteen base pairing sequence inhibit the replication of HIV-1 more effectively than the antisense oligonucleotides, indicating sequence-specific inhibition of HIV-1 replication.

Anti-HIV-1 activity by a triple-helix forming oligonucleotides targeted to polypurine tract on viral RNA.

Reverse transcription of HIV-1 into double-stranded DNA involves initiation of plus-strand DNA synthesis at the polypurine tract, PPT, by reverse transcriptase (RT). The PPT is a possible target for triple-helix formation. We show the effects of triple-helix formation by assays of RNase H cleavage inhibition in vitro using two systems (two-strand-system (FTFOs) or three-strand-system (TFOs)) targeted to the polypurine tract (PPT) of HIV-1. The two-stranded composition of a triple-helix is thermodynamically and kinetically superior to the three-strand-system. The FTFOs inhibited the RNase H activity in a sequence-specific manner, i.e., the triplex actually formed at the PPT and blocked the RNase H. The FTFOs containing the phosphorothioate groups at the antisense strand showed greater 3'-exonuclease resistance. In HIV-1 infected MT-4 cells, the FTFOs containing the phosphorothioate groups at the antisense strand and guanosine rich parts within the third Hoogsteen base pairing sequence inhibit the replication of HIV-1 more effectively than the antisense phosphorothioate oligonucleotides, indicating sequence-specific inhibition of HIV-1 replication.

Inhibition of HIV-1 replication by a two-strand system (FTFOs) targeted to the polypurine tract.

Reverse transcription of HIV-1 vRNA into the double-stranded DNA provirus involves initiation of plus-strand DNA synthesis at the polypurine tract (PPT) by reverse transcriptase (RT). The PPT is highly conserved among the known human immunodeficiency virus (HIV-1) strains and is a possible target for triplex formation. We show the effects of triple-helix formation by assays of primer extension inhibition in vitro, using a two-strand system (foldback triplex-forming oligonucleotides (FTFOs)) targeted to the PPT of HIV-1. The two-stranded composition of a triple-helix is thermodynamically and kinetically superior to the three-strand system. The FTFOs inhibited the RT activity in a sequence-specific manner, i.e. the triplex actually formed at the PPT and blocked the RT. The FTFOs containing the phosphorothioate groups at the antisense sequences showed greater 3'-exonuclease resistance. In HIV-1-infected MOLT-4 cells, the FTFOs containing the phosphorothioate groups at the antisense sequence sites and guanosine rich parts within the third Hoogsteen base-pairing sequence inhibit the replication of HIV-1 more effectively than the antisense oligonucleotides, indicating sequence-specific inhibition of HIV-1 replication.

Specific inhibition of human telomerase activity by transfection reagent, FuGENE6-antisense phosphorothioate oligonucleotide complex in HeLa cells.

Human telomerase might be associated with malignant tumor development and could be a highly selective target for antitumor drug design. Antisense phosphodiester (ODNs) and phosphorothioate (S-ODNs) oligonucleotides were investigated for their abilities to inhibit telomerase activity in the HeLa cell line. The ODNs and S-ODNs were designed to be complementary to nucleotides within the RNA active site of telomerase. As a transfection reagent, FuGENE6 was used to enhance the cellular uptake of oligonucleotides in cell cultures. The results showed that S-ODN-3 (19-mer) encapsulated with FuGENE6 clearly inhibited the telomerase activity in HeLa cells, and the inhibitory efficiency increased with an increase in the S-ODN-3. However, free S-ODN-3 showed no inhibitory activity. On the other hand, ODN-3 encapsulated with FuGENE6 had no detectable inhibitory activity. The encapsulated S-ODNs exhibited higher inhibitory activities than the free S-ODNs, and showed sequence specific inhibition. Thus, the activities of the S-ODNs were effectively enhanced by using the transfection reagent. The transfection reagent, FuGENE6, may thus be a potentially useful delivery vehicle for oligonucleotide-based therapeutics and transgenes, and is appropriate for use in vitro and in vivo.

[Drug delivery system (DDS) for the use of antisense oligodeoxynucleotide phosphorothioate in controlling gene expression].

Synthetic antisense oligodeoxynucleotide phosphorothioates (ODNs) are widely used as therapeutic tools in various in vitro and in vivo systems. Here, we applied ODNs to inhibit viral gene expression. Human T-cell leukemia virus type I (HTLV-I) is a retrovirus, and is closely linked to adult T-cell leukemia (ATL), HTLV-I-associated myelopathy/tropical spastic paraparesis(HAM/TSP), and other HTLV-I-associated diseases. With an attempt to control viral replication in vitro, ODNs to HTLV-I tax gene were synthesized and applied. In addition, 1,2-dioleoyloxy-3-(trimethylammonio) propane, DOTAP as a drug delivery system, was exploited to increase the cellular uptake of ODNs. Combination of ODNs and DOTAP was more effective to suppress viral antigen expression than ODNs only. Therefore this combination method may be useful in clinical trials for HTLV-I-associated diseases.

Inhibition of human T-cell leukemia virus type 1 replication by antisense env oligodeoxynucleotide.

Human T-cell leukemia virus type 1 (HTLV-1) infection is associated with adult T-cell leukemia and HTLV-associated myelopathy/tropical spastic paraparesis. Inhibition of HTLV-1 transmission is important to prevent the above HTLV-1-associated diseases. We used the antisense oligodeoxynucleotides (oligos) complementary to the first splice junction, rex responsive site, gag, env, tax, rex, and p21 and evaluated the effects on the syncytium formation between HTLV-1 producing human T-cell line, C9/PL cells, and HTLV-1-uninfected human glioma cell line, U251-MG cells. The syncytium formation was significantly inhibited the virion production assayed by antisense oligos to env, tax, gag, p21, and rex, with antisense oligo to env being the most inhibitory. Antisense oligos to env and tax also inhibited reverse transcriptase activity. Antisense oligo to env may have a potential as a preventive measure of HTLV-1 replication and transmission in vivo.

Long-term, high dose interferon-alpha treatment in HTLV-I-associated myelopathy/tropical spastic paraparesis: a combined clinical, virological and immunological study.

The efficacy of long-term, high dose interferon-alpha (IFN-alpha) therapy was studied in seven patients with HTLV-I-associated myelopathy (HAM)/tropical spastic paraparesis (TSP). IFN-alpha was administered at a dose of 6 x 10(6) international units daily for the initial 2 weeks and thereafter 3 times a week for the following 22 weeks. Five patients showed a sustained improvement in motor performance during and up to 6 months after the completion of IFN-alpha. The other patient who responded to IFN-alpha initially dropped out at 3 months because of depression, while another patient first deteriorated and thereafter dropped out. In the six responders, the absolute number of peripheral blood lymphocytes (PBL) harboring the HTLV-I genome as evaluated by the quantitative polymerase chain reaction method decreased significantly during the therapy period (28.6 +/- 16.6% reduction, P = 0.0083), whereas the one deteriorated patient showed a 2.5-fold increase in HTLV-I-infected cells. The autoproliferation of CD4+ T clone cells from a single cell culture was markedly depressed even after the cessation of IFN-alpha in the responders who completed long-term IFN-alpha therapy. In addition, the CD8+DR+ T cells in the peripheral blood and soluble IL-2 receptor levels in the sera increased significantly during the therapy in all patients (P = 0.0431 and P = 0.0041, respectively). Therefore, the results of our study suggested that both the reduction of HTLV-I proviral DNA load and immunomodulation by long-term IFN-alpha therapy contributed to its sustained clinical benefits.

Inhibition of syncytium formation by antisense oligonucleotide phosphorothioates complementary to tax mRNA of human T-cell leukemia virus type 1 (HTLV-1).

HTLV-1 infection is known as the factor to cause adult T-cell leukemia (ATL). Antisense oligonucleotide phosphorothioates against tax gene and control oligonucleotide phosphorothioates were synthesized. Antisense oligonucleotide was complementary to the region of initiation codon of tax gene. Two control oligonucleotides were tax sense and random. HTLV-1-positive human T-cell line, C91/PL and HTLV-1 non-infected human glioma cell line, U251-MG were co-cultured in the presence of antisense or control oligonucleotides for 24 hours. Oligonucleotides used in this study were not toxic at 10 microM concentration. Antisense oligonucleotide against tax gene inhibited 59% the syncytium formation assay at 10 microM concentration.

Inhibition of HTLV-I induction and virus-induced syncytia formation by oligodeoxynucleotides.

HTLV-I is an exogenous human retrovirus that is a causative agent of adult T cell leukemia (ATL). In addition to the structural genes (gag, pol and env), a gene termed pX is postulated to be associated with leukemogenesis in ATL. Since no effective chemotherapy is currently available, it is important to find suitable therapeutic means against ATL. Here, we tested the inhibitory effect of antisense oligodeoxynucleotides (ODNs) on HTLV-I infection in different systems. ODNs were synthesized with the phosphorothioate backbone targeted to either structural genes or transactivator genes. The phosphorothioate ODNs were found to have two distinct target sites to exert their effect on HTLV-I infection: 1) Several ODNs, including sense ODNs and random oligomers, blocked syncytium formation induced by HTLV-I at a concentration of 0.1 microM. Their inhibitory effect on syncytium formation seemed to be exerted in a nonantisense manner, most probably due to their interaction with the cell membrane. 2) Efficient suppression by ODNs of gag gene expression after chemical induction was observed in HTLV-I-transformed T cells in an antisense manner. In this suppression, tax-antisense ODN showed virtually complete inhibition of gag protein expression, but not RNA expression, at the concentration of 0.1 microM, whereas tax-sense ODN displayed a weak inhibitory effect. Our results suggest that the influence of the phosphorothioate compound should be considered from the aspect of two separated mechanisms of antiviral activity, the effects on early (viral adsorption) and late (translation) phase infection.

Induction of antibody responses that neutralize human T-cell leukemia virus type I infection in vitro and in vivo by peptide immunization.

In order to define neutralization regions on the envelope antigen of human T-cell leukemia virus type I (HTLV-I), we have generated a number of new anti-envelope gp46 monoclonal antibodies from rats and mice. Epitopes recognized by new monoclonal antibodies which could neutralize HTLV-I in syncytium and transformation inhibition assays were localized to sequences in gp46 from amino acids 186 to 193, 190 to 195, 191 to 195, 191 to 196, and 194 to 199. Ovalbumin-conjugated synthetic gp46 peptides containing these neutralization epitopes, pep190-199 (a synthetic gp46 peptide containing amino acids 190 to 199) and pep180-204, but not pep185-194 or pep194-203, could give rise to HTLV-I-neutralizing antibody responses in rabbits. These immune or nonimmune rabbits were then challenged with HTLV-I by intravenous inoculation with 5 x 10(7) live HTLV-I-producing ILT-8M2 cells. By a PCR assay, it was revealed that HTLV-I provirus was detected in peripheral blood lymphocytes from nonimmune and pep288-312-immunized rabbits, whereas the provirus was not detected in peripheral blood lymphocytes from pep190-199- and pep180-204-immunized rabbits over an extended period. These results suggest that the induction of anti-gp46 neutralizing antibody responses by immunization with synthetic peptides has the potential to protect animals against HTLV-I infection in vivo.