Next generation quantitative HCV drug resistance assay

下一代定量 HCV 耐药性测定

• 批准号: 10324964
• 负责人: Richard Wagner
• 金额: $ 83.45万
• 依托单位: MEDOSOME BIOTEC, LLC
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-03-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10324964
• 关键词: Accreditation; Aftercare; American; Americas; Antiviral Agents; Antiviral Therapy; Biological Assay; CLIA certified; Chronic Hepatitis C; Clinical; Clinical Data; Clinical Trials; Communicable Diseases; Data; Development; Drug resistance; Evaluation; Florida; Genes; Genotype; Hepatitis C virus; In Vitro; Individual; Laboratories; Link; Liver diseases; Measures; Medical Genetics; Methods; Minor; Minority; Molecular Cloning; Morphologic artifacts; Patients; Pharmaceutical Preparations; Phase; Play; Population; Protease Inhibitor; Regimen; Replicon; Resistance; Retreatment; Role; Sampling; Societies; Specimen; Speed; Techniques; Technology; Testing; Therapeutic; Treatment Failure; Treatment Protocols; Treatment outcome; Universities; Validation; Variant; Viral; Virus; Work; assay development; commercialization; cost; deep sequencing; drug resistant virus; genetic testing; improved; inhibitor/antagonist; next generation; novel strategies; patient population; phase 1 study; resistance mutation; response; success; therapy resistant; treatment response; treatment strategy; viral resistance; virology

PROJECT SUMMARY / ABSTRACT The objective of this Phase II application is to complete the development of a next generation quantitative drug resistance linkage assay that can accurately determine the abundance and linkage of HCV variants resistant to direct-acting antiviral agents (DAAs). Drug resistance to HCV is a major threat to achieving sustained virologic response (SVR) in HCV-infected individuals. Resistance-associated substitutions (RASs) pre-exist in patients naïve to DAA therapy, and resistant variants are selected after treatment failures. Presence of HCV RASs is known to impact the efficacy of DAAs. The FDA has recommended baseline resistance testing prior to initiation of certain DAAs regimens. In addition, the American Association for the Study of Liver Diseases (AASLD) and the Infectious Diseases Society of America (IDSA) have recommended resistance testing in patients failing initial DAAs to guide the selection of re-treatment regimens. Emerging data from clinical trials suggest that the identity and the abundance of RASs may impact treatment outcome. Furthermore, our preliminary data suggest that linkage of RASs may also compromise clinical response to DAAs. However, commercially available HCV resistance assays cannot accurately determine the abundance or linkage of RASs. In Phase I studies, we have developed a quantitative Single Variant Sequencing (SVS) assay, which takes advantage of the speed and accuracy of the high-throughput MiSeq technology, and a random sequencing tags strategy that removes biases and technical artifacts known to obscure true representations of sequence variants. We successfully optimized the primers, amplification and sequencing conditions for the SVS method and conducted initial testing of the optimized SVS methods using in vitro and clinical samples. Building on our success, this Phase II application will complete the development of the assay by expanding coverage to HCV genotypes 2-6, and then experimentally validate and commercialize the SVS assay via four Specific Aims: 1) Complete development of an optimized SVS assay for linkage quantification of HCV GT 1-6 in NS3, NS5A and NS5B genes, 2) Experimentally validate the SVS assay using standard virus genotype and drug resistance virus panels, 3) Conduct pre-market field evaluation using real-world clinical samples, and 4) Validate the SVS assay in Medosome's CLIA certified and CAP accredited Florida licensed clinical genetic testing laboratory. An accurate and low cost SVS assay will have tremendous commercialization potential, given the global burden of HCV with millions of HCV-infected individuals who will require resistance testing to maximize treatment success during DAA therapy.

项目摘要/摘要 此二期申请的目标是完成下一代定量药物的开发 一种能准确确定丙型肝炎病毒耐药变异株丰度和连锁的抗药性连锁分析方法 直接作用抗病毒药物(DAA)。对丙型肝炎病毒的耐药性是实现持续病毒学的主要威胁 丙型肝炎病毒感染者的应答(SVR)。耐药相关替换(RAS)在患者中预先存在 天真的DAA疗法，在治疗失败后选择耐药变种。存在丙型肝炎病毒RASS是 已知会影响DAA的效力。FDA建议在启动前进行基线抗药性测试 某些DAA疗法。此外，美国肝病研究协会(AASLD)和 美国传染病学会(IDSA)建议对初次失败的患者进行耐药性测试 DaaS指导复治方案的选择。来自临床试验的新数据表明，这种身份 RAS的丰度可能会影响治疗结果。此外，我们的初步数据表明， RAS的连锁也可能影响对DAA的临床反应。然而，商业上可用的丙型肝炎病毒 抗性检测不能准确地确定RAS的丰度或连锁。在第一阶段的研究中，我们有 建立了一种定量的单变量测序(SVS)分析方法，该方法利用了快速和 高通量MiSeq技术的准确性，以及消除偏差的随机测序标签策略 以及已知的模糊序列变体真实表示的技术人工制品。我们成功地优化了 对SVS方法的引物、扩增和测序条件进行了初步检测 利用体外样品和临床样品优化SVS方法。在我们成功的基础上，这一第二阶段的应用 将通过将覆盖范围扩大到丙型肝炎病毒2-6型来完成检测的开发，然后 通过四个具体目标通过实验验证SVS分析并将其商业化：1)完成 优化的SVS方法用于丙型肝炎病毒NS3、NS5A和NS5B基因GT1-6的连锁定量 使用标准病毒基因型和耐药病毒面板对SVS检测进行实验验证，3) 使用真实的临床样本进行上市前现场评估，以及4)在 Medosome的CLIA认证和CAP认证的佛罗里达州注册的临床基因测试实验室。一个准确的 考虑到丙型肝炎病毒在全球的负担，低成本的SVS检测将具有巨大的商业潜力 数以百万计的丙型肝炎病毒感染者将需要进行耐药性测试，以最大限度地提高治疗成功率 DAA疗法。