NS5A Inhibitor Research Articles

Prevalence of NS5A resistance associated substitutions in patients with hepatitis C virus genotypes 1a and 3: Impact on current therapeutic strategies.

The presence of resistance-associated substitutions (RASs) at NS5A region might compromise the efficacy of Direct Acting Antiviral agents (DAAs). HCV resistance at NS5A region is mainly focused on patients with hepatitis C virus (HCV) genotypes 1a (G1a) and 3 (G3) with other factors of poor treatment response (ie cirrhosis, prior treatment-exposure, or HCV-RNA >800 000 IU/mL). Herein, we evaluated in a cohort of HCV G1a and G3 infected patients the prevalence of RASs at domain I NS5A using population-based sequencing and the impact of RASs on the optimization of current therapeutic strategies. The RASs considered as clinically relevant were: M28A/G/T, Q30D/E/H/G/K/L/R, L31M/V/F, H58D, and Y93C/H/N/S for G1a and Y93H for G3. A total of 232 patients naïve to NS5A inhibitors were included (166 G1a, 66 G3). The overall prevalence of NS5A RASs for G1a and G3 patients was low (5.5%) or null, respectively. A high proportion of patients harbored, at least, one factor of poor response (78.9% for G1a, and 75.8% for G3). Overall, the rates of patients harboring NS5A RASs in combination with any of the other factors were low and the vast majority of patients (G1a> 94% and G3 100%) could be treated with standard treatments of 12 weeks without ribavirin. In conclusion, testing NS5A RASs in specific HCV-infected populations (ie G1a & G3, cirrhosis, prior treatment experienced, HCV-RNA >800 000 IU/mL) might be useful to optimize current NS5A-based therapies avoiding ribavirin-related toxicities, and shortening treatment duration in the majority of patients.

Glecaprevir + pibrentasvir for treatment of hepatitis C

ABSTRACTIntroduction: Glecaprevir/pibrentasvir is a fixed-dose combination regimen of a new generation NS3/4A inhibitor and an NS5A inhibitor with potent antiviral activity against all hepatitis C virus (HCV) genotypes. This regimen offers a shorter course of therapy (8 weeks) for selected patients regardless of genotype and has demonstrated high virological efficacy for retreatment of individuals who previously failed an NS5A containing regimen. Glecaprevir and pibrentasvir are minimally excreted by the kidneys; thus this regimen can safely be used in individuals with severe chronic kidney disease (CKD), including those undergoing hemodialysis.Areas covered: This review covers the mechanism of action, pharmacokinetics, clinical applications, efficacy, and safety profile of glecaprevir/pibrentasvir. It also covers key phase 2 and 3 clinical trials that led to licensure of this regimen.Expert opinion: Glecaprevir/pibrentasvir is the latest antiviral regimen licensed in the United States for treatment of HCV infection. Although several other direct-acting antiviral agents (DAAs) are currently available, glecaprevir/pibrentasvir has some unique characteristics that expand treatment options for HCV infection, including patients with comorbidities such as advanced stage CKD or prior treatment failure to antiviral regimens containing other DAAs.

Efficacy and safety of ledipasvir/sofosbuvir for genotype 1b chronic hepatitis C patients with moderate renal impairment.

To evaluate the efficacy and safety of ledipasvir and sofosbuvir therapy for genotype 1b in chronic hepatitis C patients with chronic kidney disease (CKD) stage 3. In a multicenter collaborative retrospective study, 706 patients who have received ledipasvir which is NS5A inhibitor, and sofosbuvir 400mg which is NS5B nucleoside polymerase inhibitor daily for 12weeks between September 2015 and January 2017 were subjected to this analysis. Virologic response and adverse events in patients with CKD stage 3 were compared with those in patients with CKD stages 1 and 2. The rates of sustained virologic response (SVR) were 97.0% in patients with CKD stage 1, 97.1% in patients with CKD stage 2, and 94.7% in patients with CKD stage 3, respectively. There were no significant differences in the SVR rates between CKD stages 1 and 2, and CKD stage 1 and stage 3. The incidence of adverse events over than grade 2 was 0% in patients with CKD stage 1, 0.5% in patients with CKD stage 2, and 3.0% in patients with CKD stage 3, respectively. For treatment and follow-up period, eGFR levels in the patients with CKD stage 3 were not worsened compared to those at baseline. This study suggested that the virologic response of ledipasvir and sofosbuvir in patients with CKD stage 3 was not inferior to those with CKD stages 1 and 2. In addition, administration of ledipasvir and sofosbuvir did not affect eGFR levels in the patients with CKD stage 3.

Efficacy of ledipasvir/sofosbuvir plus ribavirin for 12 weeks in patients with chronic hepatitis C genotype 3 and compensated liver disease.

In the era of direct-acting antivirals, hepatitis C virus (HCV) genotype (GT) 3 remains as the most difficult-to-treat HCV-GT. Currently, data on the efficacy of ledipasvir/sofosbuvir plus ribavirin (SOF/LDV+RBV) in GT3-infected patients are limited. We investigated the efficacy of this regimen in a real-life cohort from Austria. A total of 55 patients with HCV-GT3 and compensated liver disease (20% treatment-experienced, 33% with cirrhosis, 7% with HIV coinfection) from four Austrian hepatitis centers received treatment with SOF/LDV+RBV for 12 weeks. The primary endpoint was sustained virological response 12 weeks after end of therapy (SVR12). In the modified intention-to-treat analysis - excluding patients lost to follow-up - the overall SVR12 rate was 94% (95% confidence interval: 84-99%). In treatment-naive and treatment-experienced patients, SVR12 rates were 95 and 89%, respectively. SVR12 rate was 91% in patients without cirrhosis and 100% in patients with cirrhosis. There were no serious adverse events. Viral sequencing did not show the presence of any resistance-associated substitutions in any of the three relapsed patients. Despite a very weak antiviral activity of ledipasvir against HCV-GT3 in vitro, a 12-week course of SOF/LDV+RBV was highly effective, with a 94% SVR12 rate in our cohort of compensated HCV-GT3-infected patients. Thus, if pangenotypic NS5A inhibitors are not available or not reimbursed by insurances, SOF/LDV+RBV seems to be an effective alternative in patients with HCV-GT3 infection.

HCV Genotype 6a Escape From and Resistance to Velpatasvir, Pibrentasvir, and Sofosbuvir in Robust Infectious Cell Culture Models

Chronic liver diseases caused by hepatitis C virus (HCV) genotype 6 are prevalent in Asia, and millions of people require treatment with direct-acting antiviral regimens, such as NS5A inhibitor velpatasvir combined with the NS5B polymerase inhibitor sofosbuvir. We developed infectious cell culture models of HCV genotype 6a infection to study the effects of these inhibitors and the development of resistance. The consensus sequences of strains HK2 (MG717925) and HK6a (MG717928), originating from serum of patients with chronic HCV infection, were determined by Sanger sequencing of genomes amplified by reverse-transcription polymerase chain reaction. Invitro noninfectious full-length clones of these 6a strains were subsequentlyadapted in Huh7.5 cells, primarily by using substitutions identified in JFH1-based Core-NS5A and Core-NS5B genotype 6a recombinants. We studied the efficacy of NS5A and NS5B inhibitors in concentration-response assays. Weexamined the effects of long-term culture of Huh7.5 cells incubated with velpatasvir and sofosbuvir singly or combined following infection with passaged full-length HK2 or HK6a recombinant viruses. Resistance-associated substitutions (RAS) were identified by Sanger and next-generation sequencing, and their effects on viral fitness and in drug susceptibility were determined in reverse-genetic experiments. Adapted full-length HCV genotype 6a recombinants HK2cc and HK6acc had fast propagation kinetics and high infectivity titers. Compared with an HCV genotype 1a recombinant, HCV genotype 6a recombinants of strains HK2 and HK6a were equallysensitive to daclatasvir, elbasvir, velpatasvir, pibrentasvir, and sofosbuvir, but less sensitive to ledipasvir, ombitasvir, and dasabuvir. Long-term exposure of HCV genotype 6a-infected Huh7.5 cells with a combination of velpatasvir and sofosbuvir resulted in clearance of the virus, but the virus escaped the effects of single inhibitors via emergence of the RAS L31V in NS5A (conferring resistance to velpatasvir) and S282T in NS5B (conferring resistance to sofosbuvir). Engineered recombinant genotype 6a viruses with single RAS mediated resistance to velpatasvir or sofosbuvir. HCV genotype 6a viruses with RAS NS5A-L31V or NS5B-S282T were however, able to propagate and escape in Huh7.5 cells exposed to the combination of velpatasvir and sofosbuvir. Further, HCV genotype 6a with NS5A-L31V was able to propagate and escape in the presence of pibrentasvir with emergence of NS5A-L28S, conferring a high level of resistance to this inhibitor. Strains of HCV genotype 6a isolated from patients can be adapted to propagate in cultured cells, permitting studies of the complete life cycle for this important genotype. The combination of velpatasvir and sofosbuvir is required to block propagation of original HCV genotype 6a, which quickly becomes resistant to single inhibitors via the rapid emergence and persistence of RAS.These features of HCV genotype 6a could compromise treatment.

Design, synthesis and identification of silicon-containing HCV NS5A inhibitors with pan-genotype activity

Modification of a HCV NS5A inhibitor, ombitasvir, led to the identification of 10d with improved pan-genotype NS5A inhibition and better pharmacokinetic properties. The key structural changes to ombitasvir include bioisosteric replacement of carbon with silicon atom. Compared with ombitasvir, the activity of anti-HCV genotypes (GT 1 to 6) of 10d is increased to some extent, especially the inhibitory activity against genotype 3a and 6a is increased by more than seven times, and the dog's in vivo pharmacokinetics properties were also superior to ombitasvir. Further drug evaluation showed that 10d was similar to ombitasvir on plasma protein binding and liver distribution profiles, with no cytotoxicity and no inhibitory effect on both CYP 450 and hERG ligand binding. However, permeability assay results indicated that 10d was not the substrate of P-gp or BCRP transporter, which is different from that of ombitasvir. The results of a 14-day repeat-dose toxicity study identified no toxicity with 10d. Our findings in preclinical tests suggest that the silicon-containing compound 10d could be worthy of continued study as a potential drug candidate.

Covering the Cover

Covering the Cover

Prevalence of pre-treatment hepatitis C virus NS5A resistance associated amino-acid substitutions in genotype 1A infected patients in Scotland

BackgroundHepatitis C (HCV) NS5A resistance associated amino-acid substitutions (RAS) can exist at baseline in treatment naïve individuals and have been shown to be associated with lower rates of sustained virological response (SVR) for patients infected with HCV genotype 1A (G1A) following treatment with NS5A inhibitors. ObjectivesThe aim of this study was to measure the prevalence of baseline NS5A resistance in Scotland. Study designThe study population consisted of 531 treatment naïve, G1A infected patients. The patient samples were collected between March and September 2017. The NS5A region was amplified and sequenced. ResultsBaseline NS5A resistance in Scotland is high (16.8%) and is comparable to rates reported by a number of previously published studies. The high rate of baseline RAS, together with the high cost of direct-acting antivirals (DAAs), supports resistance testing to guide current patient treatment. However, given the rate at which new DAAs are currently being licensed with ever broader genotype efficacy and higher SVR rates, baseline resistance testing may not be required in the near future. ConclusionsBaseline NS5A inhibitor resistance is high. The results of the present study support performing resistance testing at baseline for current regimens.

Hepatitis C virus re-treatment in the era of direct-acting antivirals: projections in the USA.

The introduction of oral direct-acting antivirals (DAAs) has dramatically changed the landscape of HCV treatment. However, a small percentage of patients fail to achieve sustained virologic response (SVR). Understanding the number of people who fail on DAAs and require re-treatment is important for budget impact and disease burden projections. To quantify the number of HCV patients who fail to achieve SVR on oral DAAs (NS5A vs. non-NS5A) and require re-treatment. We used a mathematical model to simulate clinical management of HCV in the USA, which included the implementation of HCV screening, treatment, and disease progression. We simulated different waves of DAA treatment and used real-world data to extract SVR rates and market shares of available therapies. Our model projected that the number of people living without viraemia (i.e. cured) would increase from 0.70 million in 2014 to 1.78 million by 2020. Between 2014 and 2020, 1.50 million people would receive treatment with DAAs, of whom 124 000 (8.3%) are projected to fail to achieve SVR. Among those treatment failures, 66 600 (53.7%) patients would fail treatment with NS5A inhibitors and 69 600 (56.1%) would have cirrhosis. During the same period, 34 200 people would progress to decompensated cirrhosis and 27 300 would develop hepatocellular carcinoma after failing to achieve SVR. Even in the era of highly effective DAAs, a significant number of patients will fail to achieve SVR and will require re-treatment options. Timely and effective re-treatment is essential to prevent the long-term sequelae of HCV.

Efficacy of direct-acting antiviral treatment for chronic hepatitis C: A single hospital experience.

AIMTo evaluate the efficacy of direct-acting antivirals (DAAs) in Kanto Rosai Hospital.METHODSAll patients with hepatitis C virus (HCV) who underwent DAA prescription were enrolled in this study. The present study was a single center retrospective analysis using patients infected with HCV genotype 1 or 2. Resistance analysis was performed by using direct sequencing and cycleave PCR in genotype 1 patients treated with interferon (IFN)-free DAA. The primary endpoint was sustained virologic response at 12 wk after therapy (SVR12).RESULTSA total of 117 patients participated in the study, including 135 with genotype 1 and 42 with genotype 2. Of the 135 patients with genotype 1, 16 received protease inhibitor + IFN + ribavirin and all achieved SVR. Of the 119 patients who received IFN-free DAA (in different combinations), 102 achieved SVR and 9 failed (7/9 were on daclatasvir/asunaprevir and 2/9 on ledipasvir/sofosbuvir). Efficacy analysis was done only for 43 patients who received daclatasvir/asunaprevir. From this analysis, Y93 resistance-associated substitutions were significantly correlated with SVR.CONCLUSIONThe SVR rate was 98% for genotype 1 and 100% for genotype 2. However, caution is needed for HCV NS5A resistance-associated substitutions that are selected by HCV NS5A inhibitors because cerebrovascular adverse events are induced by some DAA drugs.

Glecaprevir/pibrentasvir for the treatment of chronic hepatitis C virus infection in adults

The fixed-dose combination of glecaprevir (GLE), a nonstructural protein 3/4A (NS3/4A) protease inhibitor, and pibrentasvir (PIB), an NS5A inhibitor, was recently approved for the treatment of adult patients with chronic hepatitis C virus (HCV) genotypes 1-6 (GT-1-6) without cirrhosis or with compensated cirrhosis, and for the treatment of HCV GT-1 patients who have failed treatment with either NS5A inhibitors or NS3/4A protease inhibitors, but not both. This combination, administered over 8 or 12 weeks, has resulted in high cure rates in all six HCV genotypes, including patients with HIV coinfection. GLE/PIB was well tolerated, with the most common adverse events being headache and fatigue. GLE/PIB is recommended to be taken as three tablets (total daily dose: GLE 300 mg and PIB 120 mg) orally once daily with food. No dose adjustment is required in patients with any degree of renal impairment or in patients undergoing hemodialysis. Dose adjustment is also not required in patients with Child-Pugh A liver disease. However, the use of GLE/PIB is not recommended in patients with moderate (Child-Pugh B) or severe (Child-Pugh C) hepatic impairment.

Interferon-Free Regimen for Hepatitis C: Insight and Management.

The rapid development of direct-acting antiviral agents (DAA) for hepatitis C virus (HCV) therapy dramatically altered the treatment landscape of this disease. The DAA regimen is associated with various advantages including high sustained virological response (SVR) with minimum side effects and low pill load and specific inhibition of viral replication, which lowers dependence on the host cell. This regimen has substantially replaced conventional (interferon) therapy with high cure rates (> 90%) in most HCV populations. This review provides insight into clinical studies of NS3/4A protease inhibitors, NS5B viral polymerase inhibitor (nucleotide and non-nucleotide), and NS5A inhibitors, alone and in combination.

Meta-Analysis of Grazoprevir plus Elbasvir for Treatment of Hepatitis C Virus Genotype 1 Infection

Background and aim. Grazoprevir is an NS3/4A protease inhibitor (PI), while elbasvir is an NS5A inhibitor. We performed this meta-analysis to directly compare grazoprevir plus elbasvir and ribavirin regimen vs. grazoprevir and elbasvir without ribavirin in the treatment of hepatitis C virus genotype 1 infection and to precisely evaluate the efficacy of the latter regimen in cirrhotic, IL28 CC genotype patients and those coinfected with human immunodeficiency virus.Material and methods. A computer literature search of PubMed, Scopus, EBSCO, Embase, and Cochrane central was conducted. Studies were screened for eligibility. Sustained virologic response (SVR) rates were pooled using OpenMeta [Analyst] software for windows. A subgroup analysis was performed to stratify the treatment efficacy according to the different baseline characteristics of HCV patients.Results. Eight randomized controlled trials (n = 1,297 patients) were pooled in the final analysis. The overall SVR rate was 96.6% with 95% CI [95.5% to 98%]. For cirrhotic patients, the SVR rate was 95.7% with 95% CI [93.9% to 97.5%] and for non-cirrhotic patients, the SVR rate was 97% with 95% CI [95.9% to 98.4%]. Furthermore, the addition of ribavirin (RBV) to the treatment regimen did not significantly improve the SVR (RR 1.003, 95% CI [0.944 to 1.065]).The dual regimen was effective in patient populations with NS3 resistance-associated substitution (RAS). However, this regimen achieved lower SVR rates (< 90%) in patients with NS5A RAS.Conclusions. We conclude that the 12-week treatment regimen of the fixed dose combination of grazoprevir plus elbasvir achieved high SVR rates in patients with HCV genotype 1 infection. The addition of ribavirin to this regimen did not add a significant benefit.

Ravidasvir. Second-generation NS5A inhibitor, Treatment of hepatitis C virus infection

Ravidasvir. Second-generation NS5A inhibitor, Treatment of hepatitis C virus infection

Inhibitory Effects of Amentoflavone and Orobol on Daclatasvir-Induced Resistance-Associated Variants of Hepatitis C Virus.

Hepatitis C virus (HCV) is recognized as a major causative agent of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Despite rapid progress in the development of direct-acting antivirals (DAA) against HCV infection in recent years, cost-effective antiviral drugs with more affordable prices still need to be developed. In this study, we screened a library of natural compounds to identify natural HCV inhibitors. The library of the pure compounds extracted from Chinese herbs deposited in the chemical bank of National Research Institute of Chinese Medicine (NRICM), Taiwan was screened in the cell culture-derived HCV (HCVcc) system. We identified the flavone or flavan-based compounds amentoflavone, 7,4[Formula: see text]-dihydroxyflavanone, and orobol with the inhibition of viral entry, replication, and translation of the HCV life cycle. Amentoflavone and orobol also showed inhibitory effects on resistant-associated variants to the NS5A inhibitor daclatasvir. The results of this study have the potential to benefit patients who are intolerant to the adverse effect of pegylated interferon or who harbor resistant strains refractory to treatment by current direct-acting antiviral agents.

Safety, tolerability and pharmacokinetics of a novel oral hepatitis C virus NS5A inhibitor DAG181PA Capsules in Chinese healthy volunteers

Safety, tolerability and pharmacokinetics of a novel oral hepatitis C virus NS5A inhibitor DAG181PA Capsules in Chinese healthy volunteers

The global origins of resistance-associated variants in the non-structural proteins 5A and 5B of the hepatitis C virus.

New, costly, fast acting, therapies targeting the non-structural proteins 5A and 5B (NS5A and NS5B) regions of the hepatitis C virus (HCV) genome are curative in the majority of cases. Variants with certain mutations in the NS5A and NS5B regions of HCV have been shown to reduce susceptibility to direct-acting NS5A and NS5B therapy and are found in treatment naïve patients. Despite this, the ease with which these variants evolve is poorly known, as are their evolutionary and geographic origins. To address this crucial gap we inferred the evolutionary and geographic origins of resistance-associated variants (RAVs) in the HCV NS5A and NS5B regions of subtypes 1a, 1b, and 3a sequences available from global databases. We found that RAVs in the NS5A region of HCV, when prevalent, were widely dispersed throughout the phylogenetic tree of HCV with multiple independent origins and that these variants are globally distributed. In contrast, most of the NS5B C316N variants came from one of two clades in the phylogenetic tree of HCV subtype 1b. The presence of serine (S) at codon 218 of HCV NS5B appears to facilitate the evolution of the C316N RAV. Other NS5B RAVs did not arise very frequently in our data set, except for S556G in subtype 1b and with respect to geography NS5B RAVs were also globally distributed. The inferred distribution of RAVs in the NS5A region and frequency of their origin suggest a low fitness barrier without the need for co-evolution of compensatory mutations. A low fitness barrier may allow rapid selection of de novo resistance to NS5A inhibitors during therapy.

Successful retreatment with sofosbuvir plus ledipasvir for cirrhotic patients with hepatitis C virus genotype 1b, who discontinued the prior treatment with asunaprevir plus daclatasvir: A case series and review of the literature.

BackgroundInterferon-free treatment results in higher sustained virologic response (SVR) rates, with no serious adverse events in hepatitis C virus (HCV)-infected patients. However, in some patients with treatment-failure in HCV NS5A inhibitor-including interferon-free regimens, the treatment-emergent HCV NS5A resistance-associated variants (RAVs), which are resistant to interferon-free retreatment including HCV NS5A inhibitors, are observed. In HCV-infected Japanese patients with daclatasvir and asunaprevir treatment failure, retreatment with sofosbuvir and ledipasvir could lead to only ∼70% SVR rates.Case summaryThree HCV genotype (GT)-1b-infected cirrhotic patients who discontinued the combination of daclatasvir and asunaprevir due to adverse drug reactions within 4 weeks; retreatment with sofosbuvir and ledipasvir combination could result in SVR in these patients without RAVs. One HCV GT-1b-infected cirrhotic patient who discontinued the combination of daclatasvir and asunaprevir due to viral breakthrough at week 10; retreatment with sofosbuvir and ledipasvir combination for this patient with the treatment-emergent HCV NS5A RAV-Y93H resulted in viral relapse at week 4 after the end of the treatment.ConclusionRetreatment with sofosbuvir and ledipasvir is effective for HCV GT-1b patients who discontinue the combination of daclatasvir and asunaprevir within 4 weeks. The treatment response should be related to the existence of treatment-emergent HCV NS5A RAVs, but may not be related to the short duration of treatment.

Efficacy of NS5A Inhibitors Against Hepatitis C Virus Genotypes 1–7 and Escape Variants

Inhibitors of the hepatitis C virus (HCV) NS5A protein are a key component of effective treatment regimens, but the genetic heterogeneity of HCV has limited the efficacy of these agents and mutations lead to resistance. We directly compared the efficacy of all clinically relevant NS5A inhibitors against HCV genotype 1-7 prototype isolates and resistant escape variants, and investigated the effects of pre-existing resistance-associated substitutions (RAS) on HCV escape from treatment. We measured the efficacy of different concentrations of daclatasvir, ledipasvir, ombitasvir, elbasvir, ruzasvir, velpatasvir, and pibrentasvir in cultured cells infected with HCV recombinants expressing genotype 1-7 NS5A proteins with orwithout RAS. We engineered HCV variants that included RAS identified in escape experiments, using recombinants with or without T/Y93H and daclatasvir, or thatcontained RAS previously reported from patients. NS5A inhibitors had varying levels of efficacy against original andresistant viruses. Only velpatasvir and pibrentasvir had uniform high activity against all HCV genotypes tested. RAS hotspots in NS5A were found at amino acids 28, 30, 31, and 93. Engineered escape variants had high levels of fitness. Pibrentasvir had the highest level of efficacy against variants;viruses with RAS at amino acids 28, 30, or 31 hadnoapparent resistance to pibrentasvir, and HCV with RASat amino acid 93 had a low level of resistance to this drug.However, specific combinations of RAS and deletion of amino acid 32 led to significant resistance to pibrentasvir. Forthe remaining NS5A inhibitors tested, RAS atamino acids28 and 93 led to high levels of resistance. Among theseinhibitors, velpatasvir was more effective against variants with RAS at amino acid 30 and some variants withRAS at amino acid 31 than the other agents. Variants withthepre-existing RAS T/Y93H acquired additional NS5Achanges during escape experiments, resulting in HCV variantswith specific combinations of RAS, showing high fitness and high resistance. We performed a comprehensive comparison of the efficacy of the 7 clinically relevant inhibitors of HCV NS5A and identified variants associated with resistance to each agent. These findings could improve treatment of patients with HCV infection.

Resistance analysis in patients with genotype 1–6 HCV infection treated with sofosbuvir/velpatasvir in the phase III studies

The fixed-dose combination of sofosbuvir/velpatasvir was highly efficacious in patients infected with genotype (GT)1-6 hepatitis C virus (HCV) in the ASTRAL studies. This analysis evaluated the impact of baseline resistance-associated substitutions (RASs) on treatment outcome and emergence of RASs in patients infected with HCV GT1-6 who were treated with sofosbuvir/velpatasvir. Non-structural protein 5A and 5B (NS5A and NS5B) deep sequencing was performed at baseline and at the time of relapse for all patients treated with sofosbuvir/velpatasvir for 12 weeks (n = 1,778) in the ASTRAL-1-3, ASTRAL-5 and POLARIS-2-3 studies. Patients with 37 known and 19 novel HCV subtypes were included in these analyses. Overall, 28% (range 9% to 61% depending on genotype) had detectable NS5A class RASs at baseline, using a 15% sequencing assay cut-off. There was no significant effect of baseline NS5A class RASs on sustained virologic response at week 12 (SVR12) with sofosbuvir/velpatasvir; the SVR12 rate in the presence of NS5A class RASs was 100% and 97%, in patients with GT1a and GT1b infection, respectively, and 100% in patients with GT2 and GT4-6 infections. In GT3 infection, the SVR rate was 93% and 98% in patients with and without baseline NS5A class RASs, respectively. The overall virologic failure rate was low (20/1,778 = 1.1%) in patients treated with sofosbuvir/velpatasvir. Single NS5A class resistance was observed at virologic failure in 17 of the 20 patients. Sofosbuvir/velpatasvir taken for 12 weeks once daily resulted in high SVR rates in patients infected with GT1-6 HCV, irrespective of baseline NS5A RASs. NS5A inhibitor resistance, but not sofosbuvir resistance, was detected in the few patients with virologic failure. These data highlight the high barrier to resistance of this regimen for the treatment of chronic HCV across all genotypes in the vast majority of patients. Sofosbuvir/velpatasvir taken once daily for 12 weeks resulted in high sustained virologic response rates in patients infected with HCV, irrespective of the presence of NS5A resistance-associated variants prior to treatment. Single class NS5A inhibitor resistance, but not sofosbuvir resistance, was detected in the few patients with virologic failure. These data highlight the high barrier to resistance of this regimen for the treatment of chronic HCV across all genotypes in the vast majority of patients.