Antisense oligonucleotides directed against the viral RNA polymerase gene enhance survival of mice infected with influenza A.

We have investigated the ability of antisense phosphorothioate oligonucleotides to enhance the survival of mice infected with influenza A virus. The oligonucleotides were complementary to sequences surrounding the translation initiation codons of the viral PB2 or PA genes (PB2-as or PA-as, respectively) of the influenza A virus RNA polymerases. Intravenous administration of PB2-as in a complex with a cationic liposome, Tfx-10, significantly prolonged the mean survival time in days and increased overall survival rates of mice infected with the influenza A virus. Liposomally encapsulated PB2-as inhibited viral growth in lung tissues and reduced pulmonary consolidations. Liposomally encapsulated PB2-as could be an effective therapeutic agent against influenza A virus.

Gene silencing of virus replication by RNA interference.

Small interfering RNAs (siRNAs) are as effective as long double-stranded RNAs (dsRNAs) at targeting and silencing genes by RNA interference (RNAi). siRNAs are widely used for assessing gene function in cultured mammalian cells or early developing vertebrate embryos. They are also promising reagents for developing gene-specific therapeutics. The specific inhibition of viral replication is particularly well suited to RNAi, as several stages of the viral life cycle and many viral and cellular genes can be targeted. The future success of this approach will depend on the recent advances in siRNA-based clinical trials.

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.

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.

Inhibition of the human chemokine receptor CXCR4 by antisense phosphorothioate oligodeoxyribonucleotides.

The CXC chemokine receptor CXCR4/fusion, a major coreceptor for the T-cell line T-tropic (X4) HIV-1 virus, plays a critical role in T-tropic virus fusion and entry into permissive cells. In the present study, we describe the effects of an antisense phosphorothioate oligodeoxyribonucleotide (anti-S-ODN) on the inhibition of CXCR4 gene expression in X4 HIV-1 infected HeLa-CD4 cells, to find more efficacious therapeutic possibilities for human immunodeficiency virus type 1 (HIV-1) infection. The naked antisense phosphorothioate oligodeoxyribonucleotide (anti-S-ODN-1), containing the AUG initiation codon at the center of the oligodeoxyribonucleotide, showed a slightly higher inhibitory effect on HIV-1 gag p24 production among all sequences tested. We also examined the concomitant use of a basic peptide transfection reagent, nucleosomal histone proteins (RNP), for the delivery of the anti-S-ODN-1. The anti-S-ODN-1 encapsulated with RNP had higher inhibitory effects on p24 products than the naked anti-S-ODN-1. When the anti-S-ODN-1 encapsulated with RNP was incubated with HeLa-CD4 cells, the surface levels of this chemokine receptor showed high suppression, indicating sequence-specific inhibition. The activities of unmodified oligodeoxyribonucleotide are effectively enhanced by using a basic peptide, RNP.

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.

Sequence-specific inhibition of a transcription factor by circular dumbbell DNA oligonucleotides.

The inhibition of specific transcription regulatory proteins is a new approach to control gene expression. The transcriptional activities of DNA-binding proteins can be inhibited by the use of double-stranded oligonucleotides that compete for the binding to their specific target sequences in promoters and enhancers. We used nicked (NDODN-kappaB) and circular (CDODN-kappaB) dumbbell DNA oligonucleotides containing a NF-kappaB binding site to analyze the inhibition of the NF-kappaB-dependent activation of the human immunodeficiency virus type-1 (HIV-1) enhancer. The dumbbell DNA oligonucleotides are stable, short segments of double-stranded DNA with closed nucleotide loops on each end, which confer resistance to exonucleases. The dumbbell and other oligonucleotides (decoys) with the NF-kappaB sequence were found to compete with the native strand for NF-kappaB binding. The circular dumbbell and double-stranded phosphorothioate oligonucleotides competed with the native strand for binding to the NF-kappaB binding proteins, while the nicked NF-kappaB dumbbell was a less effective competitor. In Jurkat T-cells, the dumbbell and other oligonucleotides were tested for their ability to block the activation of the plasmid HIV-NL4-3 Luc. The CDODN-kappaB strongly inhibits the specific transcriptional regulatory proteins, as compared with the NDODN-kappaB and the double stranded phosphodiester oligonucleotides. On the other hand, the double stranded phosphorothioate oligonucleotides could also block this activation, but the effect was non-specific. The circular (CDODN) dumbbell oligonucleotides may efficiently compete for the binding of specific transcription factors within cells, thus providing anti-HIV-1 or other therapeutic effects.

Acquisition of HIV type 1 resistance by beta-chemokine-producing CD4+ T cells.

The CD4+ T cell is a major target cell type for human immunodeficiency virus type 1 (HIV-1) infection. In this study, we provide evidence that the susceptibility to HIV-1 infection is variable in individual CD4+ T cells. Five CD4+ T cell clones were isolated from an HIV-1-seronegative donor and were investigated for their susceptibility to HIV-1 infection. Four CD4+ T cell clones were resistant to infection by a macrophage-tropic (R5) HIV-1 isolate whereas one clone was fully permissive. The level of susceptibility to HIV-1 correlated inversely with beta-chemokine production, including RANTES (regulated on activation, normally T cell expressed and secreted), macrophage inflammatory protein 1alpha (MIP-1alpha), and MIP-1beta. Resistance to HIV-1 infection was abrogated by the combined use of neutralizing antibodies against these three beta-chemokines. Interestingly, a complete inhibition of HIV-1 infection was observed in peripheral blood mononuclear cells on infection induced by adding the culture supernatant or a small number of HIV-1-resistant cell clones. Our results suggest the presence of a clonal self-defense mechanism within the CD4+ T cell population in vivo that involves the secretion of beta-chemokines.

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.

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.

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.

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.

Inhibition of HIV-1 replication by the Cre-loxP hammerhead ribozyme.

Antiviral strategies to suppress productive human immunodeficiency virus type 1 (HIV-1) replication have included the generation of gene products that provide intracellular inhibition of an essential viral protein or RNA. The potential of such a molecular genetic intervention was examined by using the Cre/loxP recombination system. In this study, we constructed a loxP-casstte vector (LTR-ribozyme) and a Cre recombinase expression vector (LTR-Cre). The transcription of the ribozyme and Cre genes was designed to be driven from the LTR promoter. These vectors were transiently transfected into COS cells along with the viral expression vector, and inhibited the expression of viral protein in COS cells. These data further support the potential of this system as a therapeutic agent for HIV-1.

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.

Antisense phosphorothioate oligonucleotides targeted to the human chemokine receptor CXCR4.

The CXC chemokine receptor CXCR4 is used as a major co-receptor for fusion and entry by syncytia-inducing T-tropic (X4) isolates of HIV-1. In the present study, we report the effects of an antisense oligodeoxyribonucleotide on the inhibition of CXCR4 gene expression in X4 HIV-1 infected HeLa-CD4 cells, to find more efficacious therapeutic possibilities for Human Immunodeficiency Virus type 1 (HIV-1) infection. Antisense phosphorothioate oligodeoxyribonucleotides (anti-S-ODNs) corresponding to the sequence of bases 69 to 88 of the human CXCR4 mRNA gene were synthesized. When the naked anti-S-ODN was incubated with HeLa-CD4 cells, the surface levels of this chemokine receptor were reduced up to 50%, indicating sequence-specific inhibition. We also examined the concomitant use of a basic peptide transfection reagent, nucleosomal histone proteins (RNP), for delivery of anti-S-ODNs. The anti-S-ODN encapsulated with RNP had higher inhibitory effects on p24 products than the naked anti-S-ODN.

Effective suppression of HIV-1 gene expression by a mammalian tRNA 3' processing endoribonuclease and external guide sequence oligozymes.

We examined the suppression of virus expression by cleaveage of the HIV-1 RNA gene using a mammalian tRNA 3' processing endoribonuclease and an External Guide Sequence Oligozyme (EGS) in vivo. We constructed an EGS expression vector that used the tRNA(met) promoter as an expression cassette for EGS. The EGS expression vector was targeted to the upstream region of gag, region. The EGS expression vector was co-transfected into COS cells with the HIV-1 gene plasmid vector. As compared with the EGS non-expressing cells and the EGS expressing cells, the EGS expressing cells with the targeted gag start codon had a clearly decreased amount of the HIV-1 gag p24 protein. The EGS expressing cells with the targeted gag start codon showed effective suppression of HIV-1 gene expression. Thus, these studies describe novel gene targeting agents for the inhibition of gene expression and antiviral activity.

[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 HIV-1 replication by a new type of circular dumbbell RNA/DNA chimeric oligonucleotides.

We have designed a new class of oligonucleotides, "dumbbell RNA/DNA chimeric phosphodiesters," containing two alkyl loop structures with RNA/DNA base pairs [sense (RNA) and antisense (DNA)] in the double helical stem. The reaction of nicked (NDRDON-gag-AUG) and circular (CDRDON-gag-AUG) dumbbell RNA/DNA chimeric oligonucleotides with RNaseH gave the corresponding antisense phosphodiester oligonucleotide together with the sense RNA cleavage products. The liberated antisense phosphodiester oligodeoxynucleotide was bound to the target RNA. The circular dumbbell RNA/DNA chimeric oligoncleotide showed more nuclease resistance and cellular uptake than the linear antisense phosphorothioate oligodeoxynucleotide (S-ODN-gag-AUG) and nicked dumbbell RNA/DNA chimeric oligonucleotide. The CDRDON-gag-AUG with an AUG initiation codon sequence, as the target of the HIV-1 gag-gene (779-801), was synthesized and tested for inhibitory effects using peripheral blood mononuclear cells. The circular dumbbell RNA/DNA chimeric oligonucleotide (CDRDON-gag-AUG) showed highly inhibitory effects compared to the antisense phosphorothioate oligonucleotide (S-ODN-gag-AUG), indicating sequence-specific inhibition of HIV-1 replication without the inhibition of reverse transcriptase and/or the viral entry process such as antisense phosphorothioate oligonucleotides.

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.

Properties of circular dumbbell RNA/DNA chimeric oligonucleotides containing antisense phosphodiester oligonucleotides.

We have designed a new class of oligonucleotides, "dumbbell RNA/DNA chimeric phosphodiesters", containing two alkyl loop structures with RNA/DNA base pairs (sense (RNA) and antisense (DNA) in the double helical stem. The reaction of nicked (NDRDON) and circular (CDRDON) dumbbell RNA/DNA chimeric oligonucleotides with RNaseH gave the corresponding antisense phosphodiester oligonucleotide together with the sense RNA cleavage products. The liberated antisense phosphodiester oligodeoxynucleotide was bound to the target 35mer RNA, which gave 35mer RNA cleavage products by treatment with RNaseH. The circular dumbbell RNA/DNA chimeric oligonucleotide showed more nuclease resistance than the linear antisense phosphodiester oligodeoxynucleotide(anti-ODN) and the nicked dumbbell RNA/DNA chimeric oligonucleotide.