Intrahepatic hepatitis C virus RNA quantification in microdissected hepatocytes

Vona, Giovanna; Tuveri, Rosella; Delpuech, Oona; Vallet, A.; Canioni, Danielle; Ballardini, G.; Trabut, Jean Baptiste; Bail, Brigitte Le; Nalpas, Bertrand; Carnot, Françoise; Pol, Stanislas; Bréchot, Christian; Thiers, Valérie

doi:10.1016/j.jhep.2003.12.012

Cited by 18 publications

(18 citation statements)

References 42 publications

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“…We note that the average HCV copy number in the HCV subgenomic replicon is 5,000 to 10,000, whereas it has been estimated that the number of HCV genomes in an infected hepatocyte in vivo is Ͻ10 copies per hepatocyte (9,30,34,39). Thus, the molarity of HCV target sites in our experimental system is much higher than that which might be encountered in vivo.…”

mentioning

confidence: 68%

Selection, Optimization, and Pharmacokinetic Properties of a Novel, Potent Antiviral Locked Nucleic Acid-Based Antisense Oligomer Targeting Hepatitis C Virus Internal Ribosome Entry Site

Laxton

Brady

Moschos

et al. 2011

Antimicrob Agents Chemother

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We have screened 47 locked nucleic acid (LNA) antisense oligonucleotides (ASOs) targeting conserved (>95% homology) sequences in the hepatitis C virus (HCV) genome using the subgenomic HCV replicon assay and generated both antiviral (50% effective concentration [EC 50 ]) and cytotoxic (50% cytotoxic concentration [CC 50 ]) dose-response curves to allow measurement of the selectivity index (SI). This comprehensive approach has identified an LNA ASO with potent antiviral activity (EC 50 ‫؍‬ 4 nM) and low cytotoxicity (CC 50 >880 nM) targeting the 25-to 40-nucleotide region (nt) of the HCV internal ribosome entry site (IRES) containing the distal and proximal miR-122 binding sites. LNA ASOs targeting previously known accessible regions of the IRES, namely, loop III and the initiation codon in loop IV, had poor SI values. We optimized the LNA ASO sequence by performing a 1-nucleotide walk through the 25-to 40-nt region and show that the boundaries for antiviral efficacy are extremely precise. Furthermore, we have optimized the format for the LNA ASO using different gapmer and mixomer patterns and show that RNase H is required for antiviral activity. We demonstrate that RNase H-refractory ASOs targeting the 25-to 40-nt region have no antiviral effect, revealing important regulatory features of the 25-to 40-nt region and suggesting that RNase H-refractory LNA ASOs can act as potential surrogates for proviral functions of miR-122. We confirm the antisense mechanism of action using mismatched LNA ASOs. Finally, we have performed pharmacokinetic experiments to demonstrate that the LNA ASOs have a very long half-life (>5 days) and attain hepatic maximum concentrations >100 times the concentration required for in vitro antiviral activity.Hepatitis C virus (HCV) infection is a major cause of liver disease, with Ͼ170 million infected people worldwide being at risk from liver failure and hepatocarcinoma. Current standard of care (SOC) using pegylated alpha 2a interferon (IFN-␣2a)-ribavirin is failing 40 to 50% of treated HCV patients, so new therapies are urgently required. New directly acting antiviral therapies for hepatitis C are becoming available, but these will eventually fail many HCV patients due to emerging drugresistant mutations in HCV strains (6, 7). HCV genomic RNA is an attractive antiviral target because it holds genetic information for viral proteins and contains regions of highly conserved sequence required for HCV replication/translation. Many groups have identified antisense oligonucleotides (ASOs) capable of inhibiting HCV RNA replication and viral polyprotein synthesis in vitro. Clinical trials on chronically HCV-infected patients show that modified ASOs targeting HCV can result in Ͼ2-log-unit decreases in viral loads, although the mechanism driving clinical antiviral activity has yet to be fully validated as antisense (14,28,33). Moreover, locked nucleic acid (LNA) represents a new generation of ASOs with improved affinity of binding to RNA targets, increased sequence specificity, greater biostability ag...

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mentioning

confidence: 68%

Selection, Optimization, and Pharmacokinetic Properties of a Novel, Potent Antiviral Locked Nucleic Acid-Based Antisense Oligomer Targeting Hepatitis C Virus Internal Ribosome Entry Site

Laxton

Brady

Moschos

et al. 2011

Antimicrob Agents Chemother

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“…Nonetheless, highly sensitive detection methods are continuously being developed and should facilitate a more accurate assessment of the true extent of HCV infection/ replication in a variety of cell types and tissues. 37,38 To date, extrahepatic replication of HCV has been explored using a plethora of cell culture and molecular biological assays. The use of "strand-specific" reverse transcriptase polymerase chain reaction (RT-PCR) to detect negativestrand HCV RNA is often cited as evidence for viral replication.…”

Section: Detection Of Extrahepatic Replicationmentioning

confidence: 99%

“…37,65 The relevance of each of these strategies to extrahepatic replication of HCV is not currently known and can only be determined through extensive validation and cross-comparison amongst all available methodologies.…”

Section: Detection Of Extrahepatic Replicationmentioning

confidence: 99%

Extrahepatic replication of HCV: Insights into clinical manifestations and biological consequences

2006

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“…The liver is the primary site of HCV replication, and previous work revealed a direct correlation between liver and serum HCV RNA levels (16,(20)(21)(22). Here, we sought to investigate the applicability of the assay to quantitation of HCV Ag in paired serum and liver biopsy specimens from treatment-naïve, HCV-infected patients.…”

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confidence: 99%

Strong Correlation between Liver and Serum Levels of Hepatitis C Virus Core Antigen and RNA in Chronically Infected Patients

et al. 2012

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HCV core antigen (Ag) and HCV RNA levels were evaluated in matched liver biopsy samples and sera from 22 patients with hepatitis C infection by using the quantitative Architect HCV Ag immunoassay and a real-time RT-qPCR assay, respectively. The data showed a strong correlation between liver and serum compartments of HCV Ag levels (r ‫؍‬ 0.80) and HCV RNA levels (r ‫؍‬ 0.87). In summary, the serum HCV Ag and RNA levels reflect the intrahepatic values. Hepatitis C virus (HCV) is an important human pathogen; worldwide, over 170 million people are chronically infected. The standard virologic diagnosis of infection is based on the detection of specific anti-HCV antibodies and cannot distinguish between past and active infections. Therefore, assays of the HCV RNA load in serum or plasma have become essential for confirming active infections and guiding the initiation and continuation of treatment with pegylated interferon and ribavirin or other antivirals. At present, real-time PCR is the benchmark technique for detecting and quantitating HCV RNA in clinical practice, with limits of detection of 10 to 15 IU/ml (4, 7, 15). However, these assays require high levels of technical skill and are labor-intensive in routine use.Hepatitis C virus particles contain a core antigen (Ag) which encapsidates the viral RNA. The core Ag is present in the serum of infected individuals, probably in both complete virions and RNAfree core protein structures (18). Recently, a highly sensitive and quantitative immunoassay for HCV Ag was developed (the Architect HCV Ag test; Abbott Diagnostics, Rungis, France). It has a cutoff of 3 fmol/liter (0.06 pg/ml). Previous studies using this assay have indicated that (i) the time course of HCV Ag levels is similar to those of HCV RNA for all phases of infection (11,17) and (ii) the serum concentrations of HCV Ag and RNA are closely correlated (8-10, 12, 17).However, the HCV Ag assay has not previously been applied to the measurement of intrahepatic HCV Ag levels. The liver is the primary site of HCV replication, and previous work revealed a direct correlation between liver and serum HCV RNA levels (16,(20)(21)(22). Here, we sought to investigate the applicability of the assay to quantitation of HCV Ag in paired serum and liver biopsy specimens from treatment-naïve, HCV-infected patients. We also investigated the relationship between the intrahepatic viral load and the serum viral load, as determined by Ag and RNA assays.We retrospectively evaluated liver biopsy specimens and sera collected from 22 chronically HCV-infected patients and 3 HCVnegative patients. For each patient, a percutaneous liver biopsy specimen and a serum sample were simultaneously collected and stored at Ϫ80°C. Epidemiological, clinical, and biochemical data (age, gender, HCV genotype, fibrosis, and alanine aminotransferase levels [IU/liter]) were also recorded (Table 1).Each sample of frozen liver tissue was weighed and cut into two similar portions. One portion was used for total protein extraction and homogenized in passive ly...

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Intrahepatic hepatitis C virus RNA quantification in microdissected hepatocytes

Cited by 18 publications

References 42 publications

Selection, Optimization, and Pharmacokinetic Properties of a Novel, Potent Antiviral Locked Nucleic Acid-Based Antisense Oligomer Targeting Hepatitis C Virus Internal Ribosome Entry Site

Selection, Optimization, and Pharmacokinetic Properties of a Novel, Potent Antiviral Locked Nucleic Acid-Based Antisense Oligomer Targeting Hepatitis C Virus Internal Ribosome Entry Site

Extrahepatic replication of HCV: Insights into clinical manifestations and biological consequences

Strong Correlation between Liver and Serum Levels of Hepatitis C Virus Core Antigen and RNA in Chronically Infected Patients

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