Each platform employs different sequencing chemistries that contribute to differences in assay performance, including read lengths achieved and base calling accuracy. Multiple NGS platforms are currently available (e.g., 454, Illumina, Ion Torrent, and PacBio), and these technologies continue to evolve even as newer technologies emerge. In contrast to Sanger population sequencing, NGS is an emerging technology that presents many potential data analysis and data integrity issues that must be considered when conducting a regulatory review. (GSI), utilized next-generation sequencing (NGS) for genotypic resistance analyses and included raw NGS data as requested in the SOF NDA, representing the first time NGS was used for genotypic resistance analyses of pivotal trials to support an antiviral drug NDA. The sponsor of the SOF NDA, Gilead Sciences, Inc. Historically, genotypic resistance data from pivotal trials of antiviral drugs have been generated using Sanger population sequencing for which data analysis is relatively standardized and straightforward. The FDA Division of Antiviral Products (DAVP) conducts independent analyses of resistance data in review of new drug applications (NDAs) for antiviral drugs. In addition, resistance data were assessed from a phase IIb liver pretransplant study, P7977-2025, which evaluated the efficacy of SOF/RBV in a subgroup of subjects with hepatocellular carcinoma meeting the Milan criteria before receiving a liver transplant (LT) to prevent HCV recurrence. The approval of SOF was based on data obtained in four pivotal phase III clinical trials P7977-1231 (FISSION), GS-US-334-0107 (POSITRON), GS-US-334-0108 (FUSION), and GS-US-334-0110 (NEUTRINO) for which drug resistance analyses were performed. When used in combination with ribavirin (RBV) with or without pegylated interferon-alpha (Peg-IFN-α), SOF treatment resulted in high sustained virological response (SVR) rates 12 weeks after end of treatment (SVR12) in subjects with chronic HCV genotype 1, 2, 3, or 4 infection. Food and Drug Administration (FDA)-approved nucleotide analog prodrug that targets the hepatitis C virus (HCV) nonstructural protein 5B (NS5B) polymerase and inhibits HCV replication. sustained virological response at 12 weeks after end of treatment.probabilistic variant detection reduced from a default value of 90 to 75 to increase the number of variant calls.Conclusion: SOF has a high barrier to resistance however, low-frequency NS5B substitutions associated with treatment failure were identified that may contribute to resistance of this important drug for chronic HCV infection. Analyses of these variants modeled in NS5B crystal structures indicated that all four substitutions could feasibly affect SOF anti-HCV activity. Moreover, baseline polymorphisms at position 316 were potentially associated with reduced response rates in HCV genotype 1b subjects. The NS5B substitutions, L159F (sometimes in combination with 元20F or C316N) and V321A, emerged in 2.2%-4.4% of subjects who failed SOF treatment across clinical trials. Low-frequency, treatment-emergent substitutions occurring at conserved NS5B amino acid positions in subjects who experienced virological failure were also noted and further evaluated. Using protocols we developed, independent analyses of the NGS data reproduced results that were comparable to those reported by Gilead Sciences, Inc. Additionally, structural bioinformatics approaches were used to characterize potential resistance-associated substitutions. The FDA Division of Antiviral Products developed an NGS analysis pipeline and performed independent analyses of NGS data from five SOF clinical trials. Food and Drug Administration (FDA)-approved antiviral drug for which genotypic resistance analyses were based almost entirely on next-generation sequencing (NGS), an emerging technology that lacks a standard data analysis pipeline. Potential mechanisms of HCV resistance to SOF and other nucleos(t)ide analog NS5B polymerase inhibitors are not well understood. High sustained virological response rates are achieved when SOF is used in combination with ribavirin with or without pegylated interferon in subjects with chronic HCV infection. Sofosbuvir (Sovaldi, SOF) is a nucleotide analog prodrug that targets the hepatitis C virus (HCV) nonstructural protein 5B (NS5B) polymerase and inhibits viral replication.
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