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Biomedical subjects

Catherine H Wu

Publications and source records attributed to Catherine H Wu.

14 recordsLinked to original sources
PubMed2006

siRNA-resistance in treated HCV replicon cells is correlated with the development of specific HCV mutations.

RNA interference (RNAi) has been extremely effective against hepatitis C viral (HCV) gene expression in short-term cell culture. Our aim was to determine whether long-term RNAi might result in HCV-resistant mutants. Huh7 HCV subgenomic replicon cells were transfected with short interfering RNAs (siRNAs). HCV-RNA was quantified by real-time RT-PCR, and HCV NS5A levels were assayed by Western blots using specific antibody. Treatment with HCV-siRNA resulted in a 50% inhibition of HCV-RNA levels compared with pretreatment levels after 4 weeks (P < 0.05). HCV-RNA returned to 85% of pretreatment levels after cessation of HCV-siRNA treatment. Sequencing of the HCV-siRNA target and upstream region was performed on 10 colonies from subcloning using PCR products, each before, during and after siRNA treatment. All colonies except one from HCV-siRNA-treated cells during and after treatment had mutations. There were no mutations in the HCV-siRNA target region following control HBV-siRNA treatment. Subcloned replicon cells containing the point mutations in the target region were found to be resistant to HCV-siRNA inhibitory effects. In conclusion, even after 4 weeks of treatment of replicon cells with HCV-siRNA, HCV-RNA and HCV-NS5A protein expression could not be completely eliminated. HCV replicons isolated during or after treatment were associated with mutations in the siRNA target region, while controls were not.

Base Sequence↗
PubMed2006

Potential applications of siRNA in hepatitis C virus therapy.

Small interfering RNAs (siRNAs) are short RNA duplexes approximately 21 nucleotides long. When introduced into mammalian cells, siRNA can silence specific gene expression. Hepatitis C virus (HCV) replicates in the cytoplasm of liver cells without integration into the host genome. Because the HCV genome is a single-stranded RNA that functions both as a messenger RNA and as a viral replication template, destruction of HCV RNA could eliminate not only virally directed protein synthesis, but also viral replication. It has been demonstrated that siRNAs interfere with HCV gene expression and replication, and this review will describe the use of RNAi as a tool to inhibit HCV gene expression.

Animals↗
PubMed2005

A novel immunocompetent rat model of HCV infection and hepatitis.

BACKGROUND & AIMS: Hepatitis C virus (HCV) infects millions of people worldwide. Therapy is limited, and treatment does not produce a sustained response in the majority of patients. Development of new agents has been hampered by the lack of a convenient animal model. The aim of this study was to determine whether an immunocompetent rat, tolerized and transplanted with a human hepatoma cell line (Huh 7 cells), could be used to sustain an HCV infection. METHODS: Fetal rats were tolerized in utero with 10(5) Huh 7 cells. One day after birth, rats were transplanted with 5 x 10(6) Huh 7 cells and, a week later, inoculated with HCV, genotype 1. RESULTS: In tolerized, transplanted, and HCV-infected rats, Huh 7 cells were found in the liver, and HCV viral replication was detected by the presence of negative strand HCV RNA. HCV levels in serum were measured at 11,000 copies/mL at week 4, peaked at 22,500 copies/mL by week 12. In tolerized, transplanted, inoculated rats, but not controls, serum alanine aminotransferase (ALT) values increased to 60 IU/L by week 4 and reached a peak of approximately 120 IU/L by week 13. Histology showed foci of mononuclear infiltrates in portal and central regions. CONCLUSIONS: HCV-inoculated immunocompetent rats tolerized and transplanted with Huh 7 cells support HCV gene expression, viral replication, and develop biochemical and histologic evidence of hepatitis.

Animals↗
PubMed2005

Novel delivery methods for treatment of viral hepatitis: an update.

Viral hepatitis represents the most common cause of chronic liver disease worldwide. Currently approved therapies for chronic hepatitis B include IFN, an immune modulator, and nucleoside analogues lamivudine and adefovir. For chronic hepatitis C, a combination of pegylated IFN-alpha and ribavirin represents the standard treatment. However, currently available treatments for both these viruses are effective only in a limited number of patients, are costly, prolonged, associated with significant side effects and require a substantial commitment from the patients and healthcare providers. A number of novel antiviral treatments, together with strategies to enhance the response to current therapies, are being explored at present. For all new therapies, as well as for improving existing treatments, selective delivery of medications into liver cells would be desirable to enhance antiviral activity and avoid systemic side effects. New achievements in the field of drug and gene delivery against chronic hepatitis to the liver are reviewed here.

Antiviral Agents↗
PubMed2003

Inhibition of HBV replication by siRNA in a stable HBV-producing cell line.

Potent inhibition of endogenous gene expression by RNA interference has been achieved by using sequence-specific posttranscriptional gene silencing through the action of small interfering RNA molecules (siRNA). In these reports, the natural function of genes could be deduced through the ensuing loss of function. Based on the extraordinary effectiveness in silencing endogenous genes, we wondered whether siRNA could be applied against viral replication in a hepatitis B virus (HBV) model using HBV-specific siRNA. To test this idea, HepG2 2.2.15, a human hepatoblastoma cell line that constitutively produces infectious HBV particles, was transfected with HBV-specific siRNAs and controls. HBV surface antigen (HBsAg) secretion into culture media was inhibited by 78%, 67%, and 42% with siRNA against the polyadenylation (PA), precore (PreC), and surface (S) regions, respectively, compared with controls as detected by enzyme-linked immunosorbent assay. After exposure to HBVPA siRNA, Northern blot analysis showed that HBV pregenomic RNA levels were decreased by 72%, and levels of HBV RNA containing the polyadenylation signal sequence were suppressed by 86%, as detected by RNase protection assay. Levels of HBV core-associated DNA, a replication intermediate, also decreased by 71%. Immunocytochemistry revealed that 30% to 40% of the cells transfected with HBVPA siRNA were completely negative for detectable HBsAg levels. Controls consisting of treatment with HBV-specific siRNA alone, lipofection reagent alone, or random double-stranded RNA (dsRNA) lipofection complex failed to decrease HBV surface antigen, HBV messenger RNA (mRNA), or core-associated HBV-DNA levels. In conclusion, siRNA inhibits hepatitis B viral replication in a cell culture system. Future studies are needed to explore the specific delivery of siRNA to liver cells in vivo and the applicability of this approach.

Cell Line↗
PubMed2003

Hepatitis B virus infection of transplanted human hepatocytes causes a biochemical and histological hepatitis in immunocopetentent rats.

AIM: To characterize the host response to hepatitis B virus (HBV) infection in human hepatocytes transplanted into immunocompetent rodent rats tolerized by, and transplanted with primary human hepatocytes. METHODS: One week after the transplantation, rats were inoculated with HBV, and viral gene expression, replication, and host response was monitored. RESULTS: HBV DNA was detectable in serum for at least 60 days. HBsAg levels rose steadily for 3 weeks post-inoculation and then plateaued at a level of about 0.6 pg/ml. HBV RNA was also found in liver at levels that remained constant through the time course. Immunofluorescence revealed clusters of hepatocytes that stained positive for HBcAg. The presence of HBV covalently closed circular DNA (cccDNA) in liver was demonstrated using nuclease digestion of single-stranded DNA followed by PCR. Serum ALT levels rose and reached a peak level of 180 IU/L on day 18, but remained elevated for 60 days. Histology revealed a progressive predominantly mononuclear lobular hepatitis. CONCLUSION: These data indicate that human hepatocytes transplanted into rats rendered tolerant to these cells, when infected by HBV, results in biochemical as well as histological evidence of hepatitis that accompanies viral gene expression, and DNA replication.

Animals↗
PubMed2002

Human RhoGAP domain-containing proteins: structure, function and evolutionary relationships.

Proteins containing a RhoGAP (Rho GTPase activating protein) domain usually function to catalyze the hydrolysis of GTP that is bound to Rho, Rac and/or Cdc42, inactivating these regulators of the actin cytoskeleton. Using database searches, at least 53 distinct RhoGAP domain-containing proteins are likely to be encoded in human DNA. Phylogenetic analysis of only the RhoGAP domains divides these proteins into distinct families that appear to be functionally related. We also review the current understanding of the structure and likely functions of these human proteins. The presence of RhoGAP domains in a number of different human proteins suggests that cytoskeletal changes, regulated by Rho GTPase, may be integrated with many different signaling pathways.

Evolution, Molecular↗
PubMed2002

Evaluation of multicomponent non-viral vectors for liver directed gene delivery.

Multicomponent, non-viral gene delivery vehicles are designed to have as a minimum, a DNA binding component, and a cell recognition component for specific delivery to target cells. The DNA binding component cannot only bind, but also protect DNA from serum degradation, and tends to condense DNA to sizes that can be taken up by receptor-mediated processes of target cells. Generally, cationic peptides, single chained, e.g. poly-L-lysine or branched polymers or synthetic peptides with DNA binding properties are used for DNA binding components. Ligands for binding to receptors on cell surfaces can be covalently linked to the DNA binding component. Multicomponent, non-viral vectors have been successfully used to deliver genes into cells in vitro and in vivo. Improvements have been made to the non-viral carriers resulting in increased solubility of DNA/carrier complexes and longer survival in serum. Improvements have also been made by incorporating fusogenic/lysosomolytic components that enable DNA/carrier complexes to escape intracellular degradation and enhance the levels and duration of expression of genes in vitro and in vivo.

Animals↗
PubMed2002

Secondary structure and hybridization accessibility of hepatitis C virus 3'-terminal sequences.

The 3'-terminal sequences of hepatitis C virus (HCV) positive- and negative-strand RNAs contribute cis-acting functions essential for viral replication. The secondary structure and protein-binding properties of these highly conserved regions are of interest not only for the further elucidation of HCV molecular biology, but also for the design of antisense therapeutic constructs. The RNA structure of the positive-strand 3' untranslated region has been shown previously to influence binding by various host and viral proteins and is thus thought to promote HCV RNA synthesis and genome stability. Recent studies have attributed analogous functions to the negative-strand 3' terminus. We evaluated the HCV negative-strand secondary structure by enzymatic probing with single-strand-specific RNases and thermodynamic modeling of RNA folding. The accessibility of both 3'-terminal sequences to hybridization by antisense constructs was evaluated by RNase H cleavage mapping in the presence of combinatorial oligodeoxynucleotide libraries. The mapping results facilitated identification of antisense oligodeoxynucleotides and a 10-23 deoxyribozyme active against the positive-strand 3'-X region RNA in vitro.

5' Untranslated Regions↗
PubMed1999

A DNA delivery system containing listeriolysin O results in enhanced hepatocyte-directed gene expression.

AIM:To determine whether incorporation of the pH dependent bacterial toxin listeriolysin O (LLO) into the DNA carrier system could increase the endosomal escape of internalized DNA and result gene expression.METHODS:A multi component delivery system was prepared consisting of asialoglycoprotein (ASG), poly L-lysine(PL), and LLO.Two marker genes, luciferase and beta galactosidase in plasmids were complexed and administered in vitro to Huh7(ASG receptor (+) and SK Hep1(ASG receptor (-) cells. Purity, hemolytic activity, gene expression, specificity, and toxicity were evaluated.RESULTS:An LLO containing conjugate retained cell target-ing specificity and membranolytic activity. In ASG receptor (+) cells,luciferase gene expression was enhanced by more than 7 fold over that of conjugates without the incorporation of listeriolysin O. No significant expression occurred in ASG receptor (-) cells. Enhancement of betagalactosidase gene expression was less, but still significantly increased over controls. There was no detectable toxicity at concentrations shown to be effective in transfection studies.CONCLUSIONS:ASOR-PL can be coupled to LLO using disulfide bonds, and successfully target and increase the gene expression of foreign DNA.

Journal Article↗
PubMedDate not supplied

Conjugation of an antisense oligodeoxynucleotide to ribonuclease h results in sequence-specific cleavage and intracellular inhibition of HCV gene expression.

A recombinant E. coli ribonuclease H (RNase H) was chemically coupled to an antisense oligodeoxynucleotide (ODN) against the 5'-noncoding region (5'-NCR) of the hepatitis C virus. Purity of the conjugates was confirmed by sodium deodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as a band corresponding to approximately 23 kDa. Conjugate function was tested by the cleavage of a HCV RNA transcript including the 5'-NCR and core region and showed HCV sequence-specific cleavage by the appearance of an expected approximately 1000 nt fragment of RNA. Cleavage was not seen by RNase H alone, or ODN alone. Delivery studies using (32)P- and (125)I-labeling showed that while RNAse H failed to enter cells, the conjugate was efficiently taken into the cells. To assess intracellular effects, a cell line, Huh-7/CMV-NCRCDeltaluc, which expresses HCV mRNA (nt 1-585) fused to a marker gene, was transfected with the conjugate. Reporter gene expression was suppressed by 51.2% with the conjugate compared to only 39.7% by ODN alone, 35.8% by a mixture of RNase H plus ODN, and not at all by RNase H alone. In conclusion, the RNase H-ODN conjugate effectively cleaved an HCV transcript in vitro and inhibited expression of an HCV-marker fusion construct in a liver-derived cell line.

Cell Line↗