765 Validation of Encephalapp_stroop for Covert Hepatic Encephalopathy Screening: A Multi-Center US Study

761

71% IL28B non-CC. All patients completed 12 weeks of therapy. HCV RNA was undetectable in 21/21 (100%) patients at the end of treatment. SVR at posttreatment week 12 (SVR12) was achieved by 10/11 patients in the ‘325 75mg group (mITT analysis; 1 missing patient achieved SVR24) and 9/10 patients in the ‘325 150mg group (1 missing patient remains in follow-up). There were no on-treatment virologic failures or posttreatment relapses. There were no serious adverse events (AEs), grade 3/4 AEs, or grade 3/4 liver-related lab abnormalities. The most frequent AEs were headache (29%), insomnia (19%), nausea (14%), and pain (14%). Conclusions: Twelve weeks of all-oral treatment with DCV+ASV+BMS-791325 achieved SVR12 in all patients with observed posttreatment data, with no virologic failures. These results extend the potent antiviral activity of this interferon- and ribavirin-free regimen to patients with HCV GT4 infection, while maintaining the positive tolerability and safety profile documented previously in GT1 patients.

AASLD Abstracts

Zebrafish Studies Show That Add3, Not Xpnpep1, Is a Biliary Atresia Risk Gene Vivian Tang, Shuang Cui, Randolph P. Matthews Biliary atresia (BA) is a progressive fibro-inflammatory cholangiopathy affecting the intraand extrahepatic bile ducts of neonates. Although BA is the most common identifiable cause of obstructive jaundice in infants, and is the leading indication for pediatric liver transplantation, the etiology remains elusive. We investigated the importance of genes identified in genome-wide association studies (GWAS) of BA patients using zebrafish. The Matthews's lab has demonstrated the utility of the zebrafish system to test the functionality of genes identified in GWAS of BA by demonstrating that knockdown of gpc1 leads to biliary defects. A prior GWAS examined single-nucleotide polymorphisms in 200 Han Chinese BA patients and 481 ethnically matched controls. The strongest association was found for a region located between the XPNPEP1 and ADD3 genes on 10q24.2. Our genetic analysis confirmed the importance of this region in a separate cohort of patients. To determine whether loss of xpnpep1 and/or add3a leads to biliary defects we performed knockdown studies of the respective genes using morpholino antisense oligonucleotides (MO). We then examined their biliary function using the lipid reporter PED6, biliary development using cytokeratin immunostaining, and gene expression using quantitative PCR. We confirmed that xpnpep1 and add3a are expressed in the developing zebrafish liver. Knockdown of add3a led to decreased biliary function by PED6 screening, while xpnpep1 knockdown had only a mild effect. There were developmental biliary defects in the add3a morphants, as demonstrated by cytokeratin immunostaining. The transcription factor vhnf1, implicated in biliary development, was significantly downregulated in add3a morphants but not xpnpep1 morphants. add3a morphants also demonstrated increased expression of gli2a, a Hedgehog target, which is consistent with prior studies of BA patients and of zebrafish models of BA such as gpc1 knockdown. Interestingly, knockdown of both add3a and gpc1 resulted in a synergistic disruptive effect on biliary development. While GWAS identified ADD3 and XPNPEP1 as potential BA susceptibility genes, our results suggest that ADD3 is likely the more important gene in this regard. Like gpc1, add3a acts via Hedgehog signaling, supporting a role for this important pathway in BA pathogenesis.

764 Sofosbuvir/Ledipasvir With and Without Ribavirin for 8 Weeks Compared to Sofosbuvir/Ledipasvir for 12 Weeks in Treatment-Naive Non-Cirrhotic Genotype-1 HCV-Infected Patients: The Phase 3 Ion-3 Study Kris V. Kowdley, Stuart C. Gordon, K. Rajender Reddy, Lorenzo Rossaro, David Bernstein, Di An, Evguenia Svarovskaia, Rob H. Hyland, Phil Pang, William T. Symonds, John G. McHutchison, Andrew J. Muir, Paul J. Pockros, David Pound, Michael W. Fried Background and Aims: ION-3 is an open-label, Phase 3 study designed to evaluate the efficacy and safety of a fixed-dose combination of sofosbuvir (SOF) 400 mg / ledipasvir (LDV) 90 mg with or without ribavirin (RBV) in treatment-naïve non-cirrhotic patients with genotype 1 HCV-infection. Given preliminary data, the aim of the study was to determine if a shorter, 8 week treatment duration was sufficient for GT1 infected patients without cirrhosis. Methods: Treatment-naïve, non-cirrhotic patients infected with HCV genotype 1 were randomized 1:1:1 to receive SOF/LDV±RBV for 8 weeks or SOF/LDV for 12 weeks. Randomization was stratified by HCV subtype. The primary endpoint was SVR12. Results: 647 patients were randomized and treated. 58% were male, 78% were white, 75% carried the non-CC IL28B allele, and 80% had genotype 1a infection. Treatment was well tolerated: 3 patients discontinued due to adverse events. 10 patients had SAEs: none were related to treatment. Common adverse events are tabulated. All patients achieved HCV RNA 1% of total subjects included hemoglobin < 9 g/dL (6%; SOF/LDV+RBV group), asymptomatic lipase elevations (2% SOF/LDV 12 week group), hyperglycemia (1% and 2% in the SOF/LDV 8 and 12 week groups respectively), and hyperbilirubinemia (1% SOF/LDV+RBV group only). Conclusions: All three sofosbuvir/ ledipasvir regimens were safe and well tolerated. Consistent with the safety profile of ribavirin, adverse events and ≥ Grade 3 abnormal laboratory findings were more common in the SOF/ LDV+RBV group. SVR12 data for all patients will be presented.

762 Control of Microenvironmental Cues Enables the Differentiation of Pluripotent Stem Cell Derived Hepatocyte-Like Cells to an Adult Phenotype Robert E. Schwartz, Jing Shan, Stephen A. Duncan, Wolfram Goessling, Sangeeta Bhatia Liver disease is an important clinical problem, impacting over 30 million Americans and over 600 million people worldwide and is the 12th leading cause of death in the United States and 15th worldwide. Several thousand Americans die yearly while waiting for liver transplantation due to liver organ paucity. Pluripotent stem cell derived (human embryonic stem (hES) cells and induced pluripotent stem cells (iPS)) derived hepatocyte-like cells would enable the study of the mechanisms of human disease and human development as well as provide a platform for pharmacology and toxicology drug screening in addition to cell based therapeutics. iPS cells were generated and then differentiated in a step-wise fashion with high efficiency and reproducibility into hepatocyte-like cells not only with the morphologic and phenotypic characteristics of hepatocytes but only with the functional characteristics as well. iPS derived hepatocyte-cell cells secrete urea, alpha-fetoprotein (AFP), alpha-1-antitrypsin, and albumin (~4 μg/million cells/day). However, molecular and functional analysis reveals that iPS derived hepatocyte-like cells exhibit an immature hepatic phenotype more closely resembling fetal hepatocytes rather than adult hepatocytes. iPS derived hepatocyte-like cells express fetal markers such as AFP and CYP3A7 and lack key mature hepatocyte functions, as reflected by drastically reduced activity (0.1%) of many detoxification enzymes (i.e. CYP2A6, CYP3A4). These key differences between iPS derived hepatocyte-like cells and adult hepatocytes have limited the use of stem cells as a renewable source of functional adult human hepatocytes for in vitro and in vivo applications. We hypothesized that pluripotent stem cell derived hepatocyte-like cells maturation was limited due to missing microenvironmental cues. To identify these missing cues, different approaches were undertaken including nonparenchymal cell coculture, cellular micropatterning, and a small molecule screening platform. Through the manipulation of the microenvironment we were able to differentiate pluripotent stem cell derived hepatocyte-like cells into adult hepatocyte-like cells as evidenced by improved albumin secretion, loss of AFP secretion (a fetal marker) and acquisition of CYP2A6 and CYP3A4 enzyme activity (adult cytochrome P450 enzyme).Our results demonstrate that microenvironmental cues are able to mature iPS derived hepatocyte-like cells into adult hepatocyte-like cells enabling new in vitro and in vivo applications including cell based therapeutics, ADME testing, and mechanistic studies of human development and disease.

765 Validation of Encephalapp_stroop for Covert Hepatic Encephalopathy Screening: A Multi-Center US Study Sanath Allampati, Andres Duarte-Rojo, Melanie B. White, Nicole Noble, Robert O'Shea, Christopher Flud, Jagpal S. Klair, Jasmohan S. Bajaj The EncephalApp_stroop App can screen for covert HE (CHE) but needs multicenter validation. EncephalApp has two parts; easier "Off" and a difficult "On" state. Time required to complete both parts, OffTime+OnTime, determines impairment. Aim: to define normative values for EncephalApp and perform sensitivity analyses of the MHE diagnosis in a multicenter US study. Methods: Age-matched healthy controls from three sites (Virginia, Arkansas and Ohio) and outpatient cirrhotics (Virginia and Ohio) were administered the gold standard (PHES,psychometric hepatic encephalopathy score: 6 tests) and EncephalApp. Results impaired>-4SD than controls was deemed CHE on PHES as recommended while 2SD beyond controls on OffTime+OnTime was considered impaired on EncephalApp. Controls were free of chronic diseases and cirrhotics did not have any neuropsychiatric diagnoses. Concordance between sites regarding EncephalApp cut-offs and PHES diagnosis of MHE was performed using Kappa. Sensitivity/specificity of EncephalApp cut-offs compared to PHES was calculated as a whole and in each center using ROC. Results: 134 controls (56 Virginia,50 Ohio,28 Arkansas, age 55±7yrs,education 14±2.5yrs) and 176 cirrhotics (137 Virginia,39 Ohio,57±4yrs,education 13±2yrs) were included. Based on control+2SD OffTime+OnTimes ,EncephalApp cut-offs were 206 seconds for all controls, 202 seconds using Virginia controls only, 211 seconds for Arkansas controls and 209 seconds for Ohio controls only. There was a high concordance of abnormal EncephalApp between these 3 sites (Kappa range 0.800.90 Table). The specificity/sensitivity were 81% using PHES as the gold standard at 183 seconds cut-off with AUC of 0.90. Conclusion: EncephalApp OffTime+OnTime is a valid method to screen for and exclude covert HE in this multi-center US study. Multi-Center Study of EncephalApp for Covert HE

763 All-Oral Therapy With Daclatasvir in Combination With Asunaprevir and Bms-791325 for Treatment-Naive Patients With Chronic HCV Genotype 4 Infection Tarek Hassanein, Gregory T. Everson, Karen Sims, Michael T. Bennett, Norman Gitlin, Eric Lawitz, Tuan T. Nguyen, Lynn R. Webster, Zobair M. Younossi, Howard I. Schwartz, Paul J. Thuluvath, Helen Zhou, Bhaskar Rege, Fiona McPhee, Megan Wind-Rotolo, Ellen Chung, Amber M. Griffies, Dennis M. Grasela, David F. Gardiner Introduction: The all-oral, ribavirin-free combination of daclatasvir (DCV; NS5A inhibitor), asunaprevir (ASV; NS3 inhibitor), and BMS-791325 (‘325; non-nucleoside NS5B inhibitor) achieved sustained virologic response (SVR12) in 92% of patients with chronic HCV genotype (GT)1 infection. This regimen has now been evaluated in patients with GT4 infection in a study expansion (AI443-014). Methods: Treatment-naïve, HCV GT4-infected patients were randomly assigned (1:1) to receive a twice-daily regimen of DCV 30mg, ASV 200mg, and ‘325 75mg (n=11) or 150mg (n=10) for 12 weeks. Results: Baseline characteristics were comparable across groups; overall, patients were 62% male, 91% white, non-cirrhotic, and

AASLD Abstracts

S-926