Antitumor effects of baculovirus-infected dendritic cells against human pancreatic carcinoma.

Recently, we showed that baculovirus (BV)-infected dendritic cells (DCs) (BV-DCs) induced antitumor immunity against established tumors in mice. These antitumor effects were CD8(+) T-cell and natural killer (NK) cell dependent but CD4(+) T-cell independent. In the current study, we examined the antitumor effect of BV-DCs on human pancreatic cancer cells (AsPC-1). After treatment with BV-infected bone marrow-derived dendritic cells (BMDCs), human pancreatic tumors caused by AsPC-1 cells in a nude mouse model were significantly reduced in size, and the survival of the mice was improved compared with that of non-immature BMDC (iDC)- and BV-DC-immunized mice. We also found that wild-type BV could activate human DCs (HDCs) and that NK cells were activated by BV-infected HDCs (BHDCs). Our findings show that BV-DCs can induce antitumor immunity, which paves the way for the use of this technique as an effective tool for DC immunotherapy against malignancies.

Baculovirus-mediated interferon alleviates dimethylnitrosamine-induced liver cirrhosis symptoms in a murine model.

The wild-type baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) infects a range of mammalian cell types in vitro but does not replicate in these cells. The current study investigated the in vivo effect of AcMNPV in the mouse model of liver cirrhosis induced by the mutagen dimethylnitrosamine. Intraperitoneal injection of AcMNPV induced an immune response. The baculovirus was taken up by the liver and spleen where it suppressed liver injury and fibrosis through the induction of interferons. This study presents the first evidence of the feasibility of using baculovirus to treat liver cirrhosis.

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.

Baculovirus directly activates murine NK cells via TLR9.

The importance of natural killer (NK) cells in innate immune responses against tumors or viral infections enhances the appeal of NK cell-based immunotherapeutic approaches. We have recently reported that baculovirus (BV)-infected dendritic cells (DCs; BV-DCs) induce antitumor immunity against established tumors in mice. These antitumor effects were CD8+ T-cell and NK cell dependent; however, they were found to be CD4+ T-cell independent. In this study, we investigated the involvement of Toll-like receptor 9 (TLR9) in the process of BV recognition by NK cells. We found that BV directly stimulated NK cells, induced the expression of the activation marker CD69 and promoted interferon-gamma (IFN-γ) production and cytotoxicity. Moreover, TLR9 knockout in mice (tlr9-/- NK cells) inhibited NK cell responses to BV, indicating that TLR9 may have a relevant role in the BV-induced upregulation of NK cell functions. Our data demonstrated for the first time that NK cells directly recognize BV via TLR9, which provides opportunities for the use of this technique as an effective tool for BV-based immunotherapies against malignancies.

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.

Potent growth inhibition of human tumor cells in culture by arginine deiminase purified from a culture medium of a Mycoplasma-infected cell line.

Two kinds of growth-inhibitory substances were found in culture of a Rous sarcoma virus-transformed rat liver cell line, RSV-BRL. The two substances were purified from the serum-free culture medium and identified as transforming growth factor beta 1 and Mycoplasma-derived arginine deiminase (EC 3.5.3.6), respectively. The arginine deiminase was an acid-labile but dithiothreitol-resistant protein with a molecular weight of 45,000 and pI 4.7. Its Km value for L-arginine was 0.3 mM, which is about 30 times lower than that of bovine liver arginase. It was stable and active under culture conditions. When added into cultures, the arginine deiminase inhibited the growth of various human cancer cell lines at a dose of 5 ng/ml or higher by depleting L-arginine in the culture media. This effective dose was about 1000 times lower than that of bovine liver arginase. These results suggested the possibility of chemotherapeutic use of arginine deiminase for human cancers.

Characterization of the secondary structure of an oligonucleotide corresponding to the autocleavage site of a precursor RNA from bacteriophage T4.

We studied the secondary structure of an RNA fragment (GUUUCGUACAAAC) (R1) having the sequence corresponding to the self-cleavage domain in a precursor RNA molecule from bacteriophage T4 infected Escherichia coli cells (p2Sp1 RNA). We synthesized an oligoribonucleotide (CAAACGUACAAAC) (R3) which contained the sequence (CGUACA) proposed for the p2Sp1 RNA self-cleavage site but did not form the hairpin loop structure. The self-cleavage ability of the single stranded RNA (R3) is significantly lower than that of R1. We have also designed a modified RNA fragment (R2), which contained a noncleavable RNA with 2'-O-methylcytidine or 2-O-methyluridine. R3 did not exhibit cleavage. To further investigate the structural requirements in the cleavage reaction, we synthesized mutant RNAs which contained different bases within consensus sequences and the cleavage sites were tested for self-cleavage. Guanosine and adenosine 3'-phosphates seemed not to be susceptible to transesterification at the cleavage site. The data from native gel electrophoresis, the CD spectra and the Tm suggested that the hairpin structure is necessary for the cleavage.

Properties of base-pairing in the stem region of hairpin antisense oligonucleotides containing 2'-methoxynucleosides.

We have designed a new type of antisense oligodeoxyribonucleotide. These oligonucleotides are able to form hairpin loop structures at the 3'-ends. The stability to nuclease degradation was observed by incubation of these hairpin oligonucleotides with snake venom phosphodiesterase, DNA polymerase, and fetal bovine serum. Of particular interest is the hairpin antisense oligonucleotide containing 2'-methoxynucleosides with base-pairing in the stem region at the 3'-end, which has increased nuclease resistance.

The stem hairpin loop structure of p2Sp1 RNA is required for RNA-cleaving activity.

We studied the hairpin-loop structure of an RNA fragment (GUUUCGUACAAAC) (R13) with the sequence corresponding to the self-cleavage domain in the precursor of an RNA molecule from bacteriophage T4-infected Escherichia coli cells (p2Sp1 RNA). In order to determine the influence of the hairpin-loop structure on these sequence-specific cleavage reactions, we have synthesized oligoribonucleotides containing hairpin-loop, double-helical stem-loop, and single-stranded RNA structures. The cleavage was affected by the hairpin-loop structure. Furthermore, the helix-stem, which retains the thermodynamically extrastable stem hairpin-loop structures, is also important for the cleavage activity. However, the thermodynamically extrastable helix-stem structure reduced the cleavage activity of the adjacent UA and CA sequences at the helix-stem site. For the cleavage reactions of the RNA cleavage products, the R6 (ACAAAC), R7 (GUUUCGU), and R9 (GUUUCGUAC) mers from the parent RNA, R13 (GUUUCGUACAAAC), a very slight amount of cleavage product (2%) from the RNA 9 was observed, but no reaction occurred for the R6 and R7. We also describe the influences of the sequences (UA and CA) on the cleavage activity.

Cleavage effect of oligoribonucleotides substituted at the cleavage sites with modified pyrimidine- and purine-nucleosides.

The precursor of an RNA molecule from T4-infected E. coli cells (p2Spl RNA) has the capacity to cleave itself at specific positions [UpA (139-140) and CpA (170-171)], within a putative loop and stem structure. This sequence-specific cleavage requires at least a monovalent cation and non-ionic detergents. In order to determine the influence of the pyrimidine and purine bases on these sequence-specific cleavage reactions, we studied the cleavage reactions of hairpin loop RNAs substituted at the cleavage sites with modified pyrimidine- and purine-nucleosides. The cleavage was affected by the 2'-hydroxyl groups and the bases of the pyrimidines, and the 6-amino group of the purine.

Site-specific cleavage of tRNA by imidazole and/or primary amine groups bound at the 5'-end of oligodeoxyribonucleotides.

Sequence specific RNA cleaving molecules were synthesized by attaching novel polyamine derivatives bearing imidazole and/or primary amine groups to the 5'-end of DNA oligonucleotides as the sequence-recognizing moieties. The actions of the molecules on a half-tRNA(Asp) were investigated. The oligonucleotides directed the nuclease activity (the imidazole and the primary amine are the catalytic groups) of the enzyme to the nucleotides directly adjacent to the complementary target sequence on the substrate RNA. The cleavage reaction shows a bell-shaped pH dependence with a maximum at pH 7.0, indicating the participation of protonated and non-protonated imidazoles residues in the process. The specificity of these hybrid enzymes can be easily altered, and they should prove to be useful tools for probing RNA structures in solution and as potential reactive groups in antisense oligonucleotide derivatives. We also describe the site-specific cleavage of tRNA(Asp) by the cleaving reagents bearing imidazole and/or primary amine groups at the 5'-end of oligodeoxyribonucleotides.

Characterization and biological activity of 8-substituted analogues of 2',5'-oligoadenylates.

Analogues of the 2',5'-linked adenylate trimer 5'-monophosphates (p5'A2'p5'A2'p5'A) containing 8-hydroxyadenosine and 8-mercaptoadenosine in the first, second, and third nucleotide positions were tested for their ability to bind to and activate RNase L of mouse L cells. The ability of p5'ASH2'p5'ASH2'p5'ASH (pASH3) (1c) to bind 2-5A dependent endonuclease was markedly decreased. On the other hand, an analogue of the 2',5'-linked adenylate trimer monophosphate substituted by 8-hydroxyadenosine in the first, second, and third nucleotide positions bound almost as well as parent 2-5A [pppA(2'p5'A)2] (p3A3) (1d) to RNase L. The 8-substituted analogues of 2-5A were more resistant to the degradation by the (2',5') phosphodiesterase. Of particular interest is monophosphate, pASH3 (1c) which possessed higher anti-HIV activity than pA3 (1a) or pAOH3 (1b).

Knocking out a specific tRNA species within unfractionated Escherichia coli tRNA by using antisense (complementary) oligodeoxyribonucleotides.

Methods for the preparation of an Escherichia coli tRNA mixture lacking one or a few specific tRNA species can be the basis for future applications of cell-free protein synthesis. We demonstrate here that virtually a single tRNA species in a crude E. coli tRNA mixture can be knocked out by an antisense (complementary) oligodeoxyribonucleotide. One out of five oligomers complementary to tRNA(Asp) blocked the aspartylation almost completely, while minimally affecting the aminoacylation with other 13 amino acids tested. This 'knockout' tRNA behaved similarly to the untreated tRNA in a cell-free translation of an mRNA lacking Asp codons.

Specific inhibition of influenza virus RNA polymerase and nucleoprotein gene expression by circular dumbbell RNA/DNA chimeric oligonucleotides containing antisense phosphodiester oligonucleotides.

We have designed a new class of oligonucleotides, 'dumbbell RNA/DNA chimeric oligonucleotides', consisting of a sense RNA sequence and its complementary antisense DNA sequence, with two hairpin loop structures. The reaction of the nicked (NDRDON) and circular (CDRDON) dumbbell RNA/ DNA chimeric oligonucleotides with RNase H gave the corresponding antisense phosphodiester oligodeoxynucleotide together with the sense RNA cleavage products. The liberated antisense phosphodiester oligodeoxynucleotide was bound to the target RNA, which gave RNA cleavage products by treatment with RNase H. The circular dumbbell RNA/DNA chimeric oligonucleotide showed more nuclease resistance than the linear antisense phosphodiester oligonucleotide (anti-ODN) and the nicked dumbbell RNA/DNA chimeric oligonucleotide. The CDRDON with four target sites (influenza virus A RNA polymerases (PB1, PB2, PA) and nucleoprotein (NP)) was synthesized and tested for inhibitory effects by a CAT-ELISA assay using the clone 76 cell line. The circular dumbbell DNA/ RNA chimeric oligonucleotide (CDRDON-PB2-as) containing an AUG initiation codon sequence as the target of PB2 showed highly inhibitory effects.

Cleavage reaction of a synthetic oligoribonucleotide corresponding to the autocleavage site of a precursor RNA from bacteriophage T4.

A fragment (GUUUCGUACAAAC) having a consensus sequence for the self-cleavage domain in a precursor of an RNA molecule from T4-infected Escherichia coli cells (p2Sp1; precursor of species 1) was chemically synthesized and found to be cleaved either between CA (139-140) or between UA (137-138) in the presence of monovalent cations and a non-ionic detergent. The cleaved products had 5'-hydroxyl and 3'-phosphate groups, of which some were in the form 2',3'-cyclic phosphates.

The influence of oligodeoxyribonucleotide phosphorothioate pyrimidine strands on triplex formation.

Analogues of the homopyrimidine oligonucleotide dT15 that contained phosphorothioate bonds of a mixture of diastereoisomers or one of the two stereoisomers (either Rp or Sp) were synthesized. The analogues were mixed under conditions conducive to the formation of triple-stranded assemblies. The mixtures were characterized by their thermal stabilities (Tm values), CD spectra, and gel electrophoresis pattern. The 34-mer duplexes containing 15 central purines on one strand and 15 complementary pyrimidines on the other strand gave no detectable triple helix upon combination with dT15S14. On the other hand, 34-bp duplexes with dT15S1, having Rp or Sp, formed triple helixes. This suggests that a steric factor plays an important role in triple helix formation.

pH-independent inhibition of restriction endonuclease cleavage via triple helix formation by oligonucleotides containing 8-oxo-2'-deoxyadenosine.

The ability of homopyrimidine oligonucleotides containing 8-oxo-2'-deoxyadenosine to form stable, triple helical structures with the sequence containing the recognition site for the class II-S restriction enzyme, Ksp632-I, was studied as a function of pH. The 8-oxo-2'-deoxyadenosine-substituted oligomers were shown to inhibit enzymatic cleavage and to bind within the physiological pH range in a pH-independent fashion without compromising specificity.

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.

A single uridine modification at the wobble position of an artificial tRNA enhances wobbling in an Escherichia coli cell-free translation system.

5-Methoxyuridine was introduced into the first position of the anticodon of the unmodified form of tRNA(1Ser) from Escherichia coli. The codon reading efficiencies of this tRNA (tRNA(5-methoxyuridine UGA)) relative to those of the unmodified counterpart (tRNA(UGA)) were measured in a cell-free translation system. tRNA(5-methoxyuridine UGA) was more efficient than tRNA(UGA) in the reading of the UCU and UCG codons and was less efficient in the reading of the UCA codon. Thus, the single modification of U to 5-methoxyuridine can enhance the wobble readings.