Next generation quantitative HCV drug resistance assay

项目总结/摘要 第二阶段申请的目的是完成下一代定量药物的开发 耐药连锁分析，可以准确地确定抗HCV变异体的丰度和连锁， 直接作用的抗病毒剂（DAA）。HCV耐药性是实现持续病毒学治疗的主要威胁。 HCV感染者的SVR。患者中预先存在耐药相关置换（RAS） 对DAA治疗不敏感，并且在治疗失败后选择耐药变体。HCV RAS的存在是 已知会影响DAA的疗效。FDA建议在启动前进行基线耐药性测试 某些DAA疗法的副作用此外，美国肝病研究协会（AASLD）和 美国传染病协会（IDSA）建议对初次失败的患者进行耐药性测试， DAA指导再治疗方案的选择。来自临床试验的新数据表明， 并且RAS的丰度可能影响治疗结果。此外，我们的初步数据表明， RAS的连锁也可能损害对DAA的临床应答。然而，商业上可获得的HCV 抗性测定不能准确地确定RAS的丰度或连锁。在I期研究中，我们有 开发了一种定量单变异体测序（SVS）测定法，该测定法利用了速度和 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 以及已知模糊序列变体的真实表示的技术人工制品。我们成功地优化了 SVS方法的引物、扩增和测序条件，并对 使用体外和临床样品优化SVS方法。在我们成功的基础上， 将通过将覆盖范围扩大到HCV基因型2-6来完成检测试剂盒的开发，然后 通过以下四个具体目标实验验证和商业化SVS测定：1）完成 用于NS 3、NS 5A和NS 5 B基因中HCV GT 1-6的连锁定量的优化SVS测定，2） 使用标准病毒基因型和耐药病毒样本组对SVS检测进行实验验证，3） 使用真实世界临床样本进行上市前现场评价，以及4）在 Medosome的CLIA认证和CAP认证的佛罗里达许可的临床基因检测实验室。准确 低成本的SVS检测将具有巨大的商业化潜力， 数百万HCV感染者需要进行耐药性检测，以最大限度地提高治疗成功率， DAA疗法。

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.