Biomedical subjects

Naoko Miyano-Kurosaki

Publications and source records attributed to Naoko Miyano-Kurosaki.

At least 19 recordsLinked to original sources

PubMed2006

Inhibition of HIV-1 replication by vesicular stomatitis virus envelope glycoprotein pseudotyped baculovirus vector-transduced ribozyme in mammalian cells.

The baculovirus has recently emerged as a promising vector for in vivo gene therapy. To investigate its potential as a delivery vector for an anti-virus ribozyme targeting HIV-1, we constructed recombinant baculovirus vectors bearing a ribozyme-synthesizing cassette driven by the tRNA(i)(Met) promoter with enhanced transduction efficiency by displaying vesicular stomatitis virus glycoprotein (VSV-G) on the viral envelope. Transduction of HeLa CD4(+) cells with a recombinant baculovirus delivering the HIV-1 U5 gene-specific ribozyme dramatically suppressed HIV-1 expression in this cell line. The VSV-G pseudotyped baculovirus vector-transduced ribozyme potently inhibited HIV-1 replication compared to a recombinant baculovirus vector-transduced ribozyme lacking VSV-G. The use of a baculovirus vector might be beneficial for application in gene therapy.

Baculoviridae↗

PubMed2006

Inhibition of hepatitis C virus RNA replication by short hairpin RNA synthesized by T7 RNA polymerase in hepatitis C virus subgenomic replicons.

RNA interference (RNAi) is a cellular process that induces gene silencing by which small duplexes of RNA specifically target a homologous sequence for cleavage by cellular ribonucleases. Here, to test the RNAi method for blocking hepatitis C virus (HCV) RNA replication, we created four short hairpin RNAs (shRNAs) targeting the HCV internal ribosome entry site/Core gene transcript using T7 RNA polymerase. shRNA suppressed the replication of HCV RNA in the HCV replicon. On the other hand, short interfering RNAs synthesized using the T7 RNA polymerase system trigger a potent induction of interferon-alpha and -beta in a variety of cells. We examined whether the shRNAs synthesized using the T7 RNA polymerase system activated double-stranded RNA-dependent protein kinase, 2'-5' oligoadenylate synthetase, or interferon-regulatory factor-3. Our results demonstrated that the T7-transcribed shRNA did not activate these proteins in Huh-7 cells and the HCV replicon. These shRNAs are a promising new strategy for anti-HCV gene therapeutics.

Base Sequence↗

PubMed2006

Characterization of baculovirus Autographa californica multiple nuclear polyhedrosis virus infection in mammalian cells.

The baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) is used as a vector in many gene therapy studies. Wild-type AcMNPV infects many mammalian cell types in vitro, but does not replicate. We investigated the dynamics of AcMNPV genomic DNA in infected mammalian cells and used flow cytometric analysis to demonstrate that recombinant baculovirus containing a cytomegalovirus immediate early promoter/enhancer with green fluorescent protein (GFP) expressed high levels of GFP in Huh-7 cells, but not B16, Raw264.7, or YAC-1 cells. The addition of butyrate, a deacetylase inhibitor, markedly enhanced the percentage of GFP-expressing Huh-7 and B16 cells, but not Raw264.7 and YAC-1 cells. The addition of 5-aza-2'-deoxycytidine, a DNA methylation inhibitor, had no enhancing effect. Polymerase chain reaction analysis using AcMNPV-gp64-specific primers indicated that AcMNPV infected not only Huh-7 and B16 cells, but also Raw264.7 and YAC-1 cells in vitro. The genomic DNA was detected in Huh-7 and B16 cells 96 h after infection. Genomic AcMNPV DNA in YAC-1 cells was not transported to the nucleus. Luciferase assay indicated that AcMNPV p35 gene mRNA and p35 promoter activity were clearly expressed only in Huh-7 and B16 cells. These results suggest that viral genomic DNA expression is restricted by different host cell factors, such as degradation, deacetylation, and inhibition of nuclear transport, depending on the mammalian cell type.

Animals↗

PubMed2006

Suppression of HIV-1 replication by a combination of endonucleolytic ribozymes (RNase P and tRNnase ZL).

We examined the combinatorial action of RNase P and tRNase ZL-mediated specific inhibition of HIV-1 in cultured cells. We designed two short extra guide sequences (sEGS) that specifically recognize the tat and vifregions of HIV-1 mRNA and mediate the subsequent cleavage of hybridized mRNA by the RNase P and tRNase ZL components. We constructed an RNase P and tRNase ZL-associated vif and tat sEGS expression vector; which used the RNA-polymerase III dependent U6 promoter, as an expression cassette for EGS. Together, the RNase P and tRNase ZL-associated sEGS molecules allow more efficient suppression of HIV-1 mRNA production when separately applied. The possibilities offered by the vector to encode sEGS will provide a powerful tool for gene therapy.

Animals↗

PubMed2006

Silencing of HIV-1 gene expression by siRNAs in transduced cells.

The RNA interference (RNAi) phenomenon is a recently observed process in which the introduction of a double-stranded RNA (dsRNA) into cells causes the specific degradation of an mRNA containing the same sequence. To study dsRNA-mediated gene interference targeted to the env gene (NL4-3: 7490-7508) in HIV-1 infected cells, we constructed tandem-type and hairpin-type siRNA expression vectors, which were under the control of two U6 promoters. We also constructed lentiviral-based siRNA expression vectors for further assessment of their antiviral activity in transduced cells. At both the transient plasmid and lentiviral-mediated RNA expression levels, the siRNA encoding the env fragment exhibited sequence-specific suppression of target gene expression and strongly inhibited (> or = 90%) HIV-1 infection in the cells, as compared to the antisense RNA expression vector. Targeting the HIV-1 env gene with siRNAs encoding the env gene fragment (7490-7508) might be an effective strategy for gene therapy applications in HIV-1/AIDS treatment and management.

Animals↗

PubMed2006

Inhibition of hcv replication in HCV replicon by shRNAs.

We show that the vector-derived long dsRNA specifically inhibits the replication of HCV RNA in HCV replicon. We designed a long dsRNA targeted to the full-length HCV IRES/core elements (1-to 377-nt). Our results revealed that the replication of HCV RNA was reduced to near background levels in a sequence-specific manner by the long dsRNAs in the HCV replicon. We also designed four shRNAs against several regions (120- to 139-nt, 260- to 279-nt, 330- to 349-nt, and 340- to 359-nt) of the HCV IRES/Core elements. The two HCV IRES/core-specific shRNAs, 330- to 349-nt and 340- to 359-nt, containing the AUG initiation codon sequence showed stronger HCV inhibitory effects than the other two shRNAs, 120- to 139-nt and 260- to 279-nt.

Hepacivirus↗

PubMed2006

HIV gene therapy using RNA virus systems.

We designed a vector to produce single-stranded DNA (ssDNA). We used HIV-1 reverse transcription for the purpose of constructing a DNAzyme expression vector against the HIV-1 env V3 loop. Initiation of HIV-1 reverse transcription requires the formation of a complex containing the viral RNA, tRNALys and reverse transcriptase. The expression vector contains the HIV-1 primer binding site (PBS) and tRNALys at the 3' end of its RNA transcript, thus enabling to an ssDNA would be synthesized by HIV-1 reverse transcriptase. We have demonstrated that the DNAzyme expressed by the lentiviral vectors suppressed HIV-1 replication in SupT1 cells.

Cell Line↗

PubMed2006

Silencing of HIV-1 gene expression by two types of siRNA expression systems.

The RNA interference (RNAi) phenomenon is a recently discovered process in which the introduction of a double-stranded RNA (dsRNA) into cells causes the specific degradation of mRNA containing the same sequence. We designed mammalian expression vectors that direct the synthesis of small interfering RNA (siRNA)-like transcripts and examined them for their siRNA-mediated gene interference targeting the env gene (NL4-3:7490-7508, E7490). We constructed siRNA expression vectors for two different strands (sense and antisense; tandem promoter) and for siRNA expressed from the short hairpin RNA (shRNA). The inhibition efficacy on HIV-1 replication differed between these two vectors. Notably, the shRNA vector pU6-env-shRNA inhibited p24 production more effectively than the tandem promoter expression vector pU6-env-siRNA. Furthermore, we examined the ability of lentiviral vectors expressing shRNA to suppress HIV-1 expression in HIV-1-infected SupT1 cells. The env-shRNA (E 7490) almost completely suppressed HIV-1 expression in infected cells for up to 15 days.

Animals↗

PubMed2006

2-aminophenoxazine-3-one suppresses the growth of mouse malignant melanoma B16 cells transplanted into C57BL/6Cr Slc mice.

Since phenoxazine is an essential structure of actinomycin D, which exerts a strong anticancer effect, we examined the anticancer effect of 2-aminophenoxazine-3-one (Phx-3) on mouse malignant melanoma B16 cells in vitro and in vivo. Phx-3 inhibited proliferation of the B16 cells in a dose-dependent manner in vitro. We furthermore studied the in vivo effects of Phx-3 on mouse malignant melanoma B16 cells transplanted in female C57BL/6Cr Slc mice. Treatment with Phx-3 (0.5 mg/kg) completely suppressed the growth of mouse malignant melanoma B16 cells transplanted in mice as compared with the control group. Phx-3 was found to exert few adverse effects, in terms of bodyweight loss, changes in serum levels of blood biochemical parameters such as aspartate transaminase (AST), alanine transaminase (ALT), blood urea nitrogen (BUN) and creatinine, dysfunction of the liver and the kidney examined by pathological methods, piloerection and wasting, when mice were treated with a dose of 0.5 mg/kg. These results suggest that Phx-3 may be used to treat patients affected by malignant melanoma in future.

Alanine Transaminase↗

PubMed2005

Resistance to influenza A virus infection by antigen-conjugated CpG oligonucleotides, a novel antigen-specific immunomodulator.

Oligodeoxynucleotides (ODN) containing CpG motifs (CpG) act as modulators that bias the immune response towards a Th1-dominant phenotype. To investigate this effect further, we examined the protective effects of a covalently linked conjugate between CpG-ODN and HA-2kd antigen in mice infected with influenza A virus. The conjugated form of CpG-ODN and HA-2kd was more efficient in regulating influenza A virus than the unconjugated mixture of CpG-ODN and HA-2kd. The antigen-conjugated CpG-ODN induced an immune response with a Th1-dominant cytokine pattern characterized by the secretion of high levels of HA-2kd-specific interferon-gamma and IgG2a (Th1), which were only slightly induced by HA-2kd alone. These findings support the use of CpG-ODN-Ag conjugates as novel Ag-specific immunomodulators and suggest that CpG-ODN-HA-2kd might be a promising immune therapy for patients with influenza virus.

Animals↗

PubMed2005

Inhibition of HIV-1 gene expression by retroviral vector-mediated small-guide RNAs that direct specific RNA cleavage by tRNase ZL.

The tRNA 3'-processing endoribonuclease (tRNase Z or 3' tRNase; EC 3.1.26.11) is an essential enzyme that removes the 3' trailer from pre-tRNA. The long form (tRNase ZL) can cleave a target RNA in vitro at the site directed by an appropriate small-guide RNA (sgRNA). Here, we investigated whether this sgRNA/tRNase ZL strategy could be applied to gene therapy for AIDS. We tested the ability of four sgRNA-expression plasmids to inhibit HIV-1 gene expression in COS cells, using a transient-expression assay. The three sgRNAs guide inhibition of HIV-1 gene expression in cultured COS cells. Analysis of the HIV-1 mRNA levels suggested that sgRNA directed the tRNase ZL to mediate the degradation of target RNA. The observation that sgRNA was localized primarily in nuclei suggests that tRNase ZL cleaves the HIV-1 mRNA when complexed with sgRNA in this location. We also examined the ability of two retroviral vectors expressing sgRNA to suppress HIV-1 expression in HIV-1-infected Jurkat T cells. sgRNA-SL4 suppressed HIV-1 expression almost completely in infected cells for up to 18 days. These results suggest that the sgRNA/tRNase ZL approach is effective in downregulating HIV-1 gene expression.

Animals↗

PubMed2005

The middle to 3' end of the HIV-1 vif gene sequence is important for vif biological activity and could be used for antisense oligonucleotide targets.

The human immunodeficiency virus type-1 (HIV-1)-encoded vif protein is essential for viral replication, virion production, and pathogenicity. HIV-1 Vif interacts with the endogenous human APOBEC3G protein (an mRNA editor) in target cells to prevent its encapsidation into virions. Some studies have established targets within the HIV-1 vif gene that are important for its biologic function; however, it is important to determine effective therapeutic targets in vif because of its critical role in HIV-1 infectivity and pathogenicity. The present study demonstrates that virions generated in transfected HeLa-CD4+ cells, especially from HIV-1 vif frame-shift mutant (3' delta vif; 5561-5849), were affected in splicing and had low infectivity in MT-4 cells. In addition, HIV-1 vif antisense RNA fragments constructed within the same region, notably the region spanning nucleic acid positions 5561-5705 (M-3'-AS), which corresponds to amino acid residues 96-144, significantly inhibited HIV-1 replication in MT-4 and reduced the HIV-1 vif mRNA transcripts and reporter gene (EGFP) expression. The generated virions showed low secondary infection in H9 cells. These data therefore suggest that the middle to the 3' end of vif is important for its biological activity in the target cells.

3' Flanking Region↗

PubMed2005

Suppression of human immunodeficiency virus type 1 (HIV-1) replication by an HIV-1-dependent double locked vector with the Cre/loxP system.

We previously demonstrated the function of an HIV-1-dependent ribozyme expression vector, with which the site-specific excision of loxP sequences can be achieved by using the Cre-loxP system (ON/OFF) as a molecular switch in an acute HIV-1 infection. However, this expression system also revealed the lower, non-specific expression of the anti-H1V-1 ribozyme in the absence of tat. To circumvent this problem, we used the more efficient HIV-1-dependent Cre recombinase gene expression vector, encoding the LTR-gag-p17 (extending from the 5'-LTR to the middle of the gag gene (pLTR-gag-p17-Cre)). Comparatively, the pLTR-gag-p17-Cre induces a higher Cre-protein expression level in an HIV-1 infection-dependent manner than the minimal pLTR-Cre. Furthermore, we constructed the ploxP-Rz-U5 and pLTR-gag-p17-Cre plasmids and also combined them into a single vector, pLTR-gag-p17-Cre/loxP-Rz-U5, for a comparison of their anti-HIV-1 activities. The resultant simultaneous expression of the Cre protein and the homologous recombination of the two loxP sequences induced a high level of HIV-1 replication inhibition (95%). Significantly, a high steady-state of ribozyme expression was observed in the RT-PCR analysis. These data imply that targeting the HIV-1 genes with the pLTR-gag-p17-Cre/loxP-Rz-U5 vector, which mediates HIV-1-dependent ribozyme expression, would be a useful tool for HIV-1 gene therapy applications.

Gene Expression↗

PubMed2005

Lentiviral-mediated delivery of combined HIV-1 decoy TAR and Vif siRNA as a single RNA molecule that cleaves to inhibit HIV-1 in transduced cells.

RNA interference (RNAi) silences gene expression via short interfering 21-23 mer double-stranded RNA (siRNA) segments that guide cognate mRNA degradation in a sequence-specific manner. On the other hand, HIV-1 decoy TAR RNA are known to competitively interact with the HIV-1 Tat protein, to downregulate the enhanced gene expression from the long terminal repeat (LTR) promoters. Here we report that a novel expression construct, encoding both HIV-1 decoy TAR and Vif siRNA, as a single RNA substrate, was expressed under the control of the human U6 promoter, and later the TAR and siRNA were cleaved into their respective separate RNA by the endogenous RNase III-like enzyme. Each of the cleaved HIV-1 anti-genes then synergistically contributed toward enhancing the inhibition efficacy (>80%) of HIV-1 replication in transduced Jurkat cells. These results suggest that targeting HIV-1 mRNA with simultaneously expressed intracellular decoy TAR and Vif-siRNA could lead to an effective gene therapy strategy for the control and management of HIV-AIDS.

Acquired Immunodeficiency Syndrome↗

PubMed2005

Anti-tumor effect of intravenous TNFalpha gene delivery naked plasmid DNA using a hydrodynamics-based procedure.

High levels of foreign gene expression in mouse hepatocytes can be achieved by the rapid injection of a large volume of naked plasmid (pDNA) into animals via the tail vein, the so-called hydrodynamics-based procedure. In this study, we evaluated the efficacy of hydrodynamics-based tumor necrosis factor alpha (TNFalpha) transfer for tumor treatment, in which the naked pDNA encoding TNFalpha was administered into the tail vein following an intravenous injection of B16 melanoma cells. The mice treated with TNFalpha-expressing pDNA displayed a profound reduction in lung metastasis. These results suggest that the hydrodynamics-based transfer of naked pDNA is a convenient and efficient method of TNFalpha gene therapy against metastatic tumors.

Animals↗

PubMed2005

Suppression of HCV RNA replication by baculovirus-mediated shRNA expression.

We describe a HCV core-short-hairpin RNA (shRNA) expressed from a human U6 Pol II promoter, which synergistically enhanced the inhibition of HCV replication in HCV replicon. We also describe the construction of recombinant baculovirus expressing shRNAs of the HCV core gene under the control of the U6 promoter. The anti-HCV shRNAs may provide a practical basis for applying shRNA-based gene therapy to the treatment of HCV.

Baculoviridae↗

PubMed2004

Effective inhibition of HIV-1 replication in cultured cells by external guide sequences and ribonuclease P.

We examined the suppressive effect of HIV-1 RNA gene cleavage on HIV-1 expression, using the catalytic RNA subunit RNase P and the 3'-half tRNA(Try) [external guide sequence (EGS)] in cultured cells. HIV-1 expression was inhibited by the tRNA(met)-EGS-U5 and U6-EGS-U5 from the tRNA(met) and U6 promoters, respectively. There was no difference in the inhibitory effects on HIV-1 expression between the tRNA(met) and U6 promoters.

Anti-HIV Agents↗

PubMed2004

Intracellular expression of antisense RNA transcripts complementary to the human immunodeficiency virus type-1 vif gene inhibits viral replication in infected T-lymphoblastoid cells.

The human immunodeficiency virus type-1 (HIV-1)-encoded vif protein is essential for viral replication, virion production, and pathogenicity. HIV-1 vif interacts with the endogenous human APOBEC3G protein (an mRNA editor) in target cells to prevent its virions from encapsidation. Although some studies have established targets within the HIV-1 vif gene that are important for its biologic function, it is however important to further screen for effective therapeutic targets in the vif gene that could interfere with the HIV-1 vif-dependent infectivity and pathogenicity. This report demonstrates that HIV-1 vif antisense RNA fragments constructed within mid-3' region, notably the region spanning nucleic acid positions 5561-5705 (M-3'-AS), significantly inhibited HIV-1 replication in MT-4 and H9-infected cells and reduced the HIV-1 vif mRNA transcripts. These data clearly suggest that the above vif fragment, which corresponds to amino acid residues 96-144, could be an effective novel therapeutic target site for gene therapy applications, for the control and management of HIV-1 infection, due to its strong inhibition of HIV-1 replication in cells.

Base Sequence↗