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Methods for the phenotypic detection of HCV inhibitor resistant subpopulations

An inhibitor and resistance technology, applied in the field of phenotype detection of HCV inhibitor resistant subgroups, can solve the problem of reduced susceptibility

Inactive Publication Date: 2014-09-10
LAB OF AMERICA HLDG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This reduced susceptibility to a particular drug renders treatment with that drug ineffective for the infected individual

Method used

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  • Methods for the phenotypic detection of HCV inhibitor resistant subpopulations
  • Methods for the phenotypic detection of HCV inhibitor resistant subpopulations
  • Methods for the phenotypic detection of HCV inhibitor resistant subpopulations

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0152] [Example 1: Preparation of samples for phenotypic analysis]

[0153] 【Sample preparation and amplification】

[0154] Most samples were received as frozen plasma with information including HCV subtype (ie, 1a or 1b) and viral load. Thaw samples, store in frozen aliquots if necessary, and process 200 μL aliquots. Viral particles are disrupted by adding a lysis buffer containing a chaotropic agent. Genomic viral RNA (vRNA) was extracted from viral lysates using oligonucleotide-linked magnetic beads. Purified vRNA was used as a template for first-strand cDNA synthesis in a reverse transcriptase (RT) reaction. The resulting cDNA was used as template for the first round of nested polymerase chain reaction (PCR), resulting in amplification of the entire NS5B region. Due to the sequence variation between subtypes 1a and 1b, specific 1a and 1b RTs and round 1 and 2 PCR primers were used. If no isoform information is available, the two primer sets can be used sequentially or...

Embodiment 2

[0159] [Example 2: Phenotypic assay to determine susceptibility to HCV inhibitors]

[0160] RTV RNA was electroporated into the Huh7 cell line and electroporated cells were incubated in the absence and presence of serially diluted inhibitors. RNA input was monitored by measuring the amount of luciferase activity produced in electroporated cells 4 hours after electroporation. Luciferase activity is expressed as relative light units (RLU). Replication competence (RC) was determined by evaluating luciferase activity in the absence of inhibitor relative to RNA input and a control reference replicon RTV (Con1) 72-96 hours after electroporation. Assay background was determined using a replication defective Con1 replicon (Con1 polymerase deficient) (data not shown). Inhibitor susceptibility was determined by evaluating the ability of RTV to replicate in the absence and presence of inhibitors 72-96 hours after electroporation. The % inhibition at each serially diluted inhibitor con...

Embodiment 3

[0165] [Example 3: For IC 95 Measurement of FC improves sensitivity for detection of inhibitor susceptibility]

[0166] for evaluation Sensitivity of HCV NS5B assays to detect subpopulations of drug-resistant variants using data from reference viruses (wild-type, WT) containing Con1 or H77 and containing specific SDMs conferring reduced susceptibility to one or more NS5B inhibitors C o RNA of RTV from the NS5B region of n1 or H77 (mutant, MT). WT and MT RTV (100% WT or 100% MT) were evaluated independently or as defined MT:WT mixtures (20:80, 40:60, 60:40 and 80:20%). Samples were assessed for susceptibility to specific NS5B inhibitors, as well as INF as a control (SDM is not expected to affect INF susceptibility). Inhibitor susceptibility data were obtained for all samples tested. Evaluation on IC 50 -FC and IC 95 - Observed differences in FC values ​​to define the relationship between the percentage of each MT RTV in the mixture and the IC-FC susceptibility parameter...

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Abstract

Methods and compositions for the efficient and accurate determination of MIsceptibility of a hepatitis C virus (HCV) population to an HCV inhibitor are provided. In certain aspects, the methods involve introducing into a cell a patient derived segment, wherein the cell or the patient derived segment comprises an indicator nucleic acid that produces a detectable signal that is dependent on the HCV; measuring the expression of the indicator gene in the presence of varying concentn'ltions of the HCV inhibitor: determining a standard curve of susceptibility: comparing the IC95 fold change, slope, or maximum inhibition percentage of the HCV population to that of a control HCV population.

Description

[0001] This application claims priority to US Provisional Application No. 61 / 566,595, filed December 2, 2011. The entire content of this application is incorporated herein by reference. 【Technical field】 [0002] Embodiments of the invention relate to methods of determining the susceptibility of hepatitis C virus ("HCV") or a population of HCVs to HCV inhibitors. Also provided are methods of determining the replication capacity of an HCV or population of HCVs. 【Background technique】 [0003] HCV affects an estimated 170 million people worldwide, including 4 million Americans, or about 1% of the US population, making it the most common blood-borne disease. HCV infection becomes a chronic condition in approximately 55-85% of patients. Late complications of chronic HCV infection include cirrhosis, hepatocellular carcinoma, and death. There is no effective vaccine for the prevention of HCV infection. [0004] HCV is an enveloped virus containing a positive-sense linear singl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70
CPCG01N33/5023C12Q1/707G01N33/576C12Q2600/106C12Q2600/158G01N33/5767
Inventor J·D·李维斯C·J·派特罗普罗斯W·黄
Owner LAB OF AMERICA HLDG
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