Next generation quantitative HCV drug resistance assay

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.