Hepatitis C virus (HCV) genotype 4 (HCV-4) infection is considered to be difficult to treat and has become increasingly prevalent in European countriesHepatitis C virus (HCV) affects an estimated 170 million people worldwide. HCV infection persists in 50 to 85% of those infected and can, over decades, lead to cirrhosis and hepatocellular carcinoma (11). The HCV genome displays considerable sequence divergence, and HCV variants have been classified into seven major genotypes. Genotypes 1, 2, 3, 4, and 6 are further subdivided into numerous subtypes (subtypes a, b, c, etc.) (27). In the absence of complete genome sequences, the designation of a subtype is based mainly on consensus regions in the core/E1 and NS5B regions of the HCV genome (27). The HCV genotype distribution depends on the geographical region and the mode of transmission. As the distribution of HCV genotypes can change over time, genotyping provides a powerful tool that may be used to investigate the spread of HCV within a community (18).In Europe, North America, and Australia, most HCV-infected patients (Ͼ80%) are infected with genotype 1, 2, or 3 (10). HCV genotype 4 (HCV-4) is the most common genotype in the Middle East and in northern and central Africa, accounting for more than 20% of all chronic HCV infections worldwide (28). In Egypt, the country with the highest prevalence of HCV in the world, more than 90% of patients are infected with . HCV-4 is considered difficult to treat and has a sustained virological response rate of approximately 60% (28), where the rates are 40 to 50% for genotype 1 and 80 to 90% for genotypes 2 and 3 (17).Recent studies emphasize that the prevalence of HCV-4 in Europe has increased in the past few decades due to the immigration of HCV carriers and the subsequent spread of HCV-4 in European populations at risk for HCV infection (2,5,16,24,25,30). In southern Europe, HCV-4 is responsible for 10 to 24% of chronic HCV infections. In The Netherlands, HCV-4 accounts for an estimated 10% of chronic HCV infections (6, 32). Currently, the development of new genotypespecific antiviral agents is focused mainly on HCV genotype 1. The emergence of HCV-4 may require agents specific for HCV-4 to improve the response rates and decrease the future burden of HCV-4 disease. The population of the region around Amsterdam, The Netherlands, comprises many ethnicities and diverse groups at risk for HCV infection, providing

g Evolutionary analysis of hepatitis C virus (HCV) genome sequences has provided insights into the epidemic history and transmission of this widespread human pathogen. Here we report an exceptionally diverse set of 178 HCV genotype 2 (HCV-2) isolates from 189 patients in Amsterdam, comprising 8 distinct HCV subtypes and 10 previously not recognized, unclassified lineages. By combining study subjects' demographic information with phylogeographic and molecular clock analyses, we demonstrate for the first time that the trans-Atlantic slave trade and colonial history were the driving forces behind the global dissemination of HCV-2. We detect multiple HCV-2 movements from present-day Ghana/Benin to the Caribbean during the peak years of the slave trade (1700 to 1850) and extensive transfer of HCV-2 among the Netherlands and its former colonies Indonesia and Surinam over the last 150 years. The latter coincides with the bidirectional migration of Javanese workers between Indonesia and Surinam and subsequent immigration to the Netherlands. In addition, our study sheds light on contemporary trends in HCV transmission within the Netherlands. We observe multiple lineages of the epidemic subtypes 2a, 2b, and 2c (together 67% of HCV-2 infections in Amsterdam), which cluster according to their suspected routes of transmission, specifically, injecting drug use (IDU) and contaminated blood/blood products. Understanding the epidemiological processes that generated the global pattern of HCV diversity seen today is critical for exposing associations between populations, risk factors, and specific HCV subtypes and might help HCV screening and prevention campaigns to minimize the future burden of HCV-related liver disease.

Adeno-associated viruses (AAVs) are among the most efficient vectors for liver gene therapy. Results obtained in the first hemophilia clinical trials demonstrated the long-term efficacy of this approach in humans, showing efficient targeting of hepatocytes with both self-complementary (sc) and single-stranded (ss) AAV vectors. However, to support clinical development of AAV-based gene therapies, efficient and scalable production processes are needed. In an effort to translate to the clinic an approach of AAV-mediated liver gene transfer to treat Crigler-Najjar (CN) syndrome, we developed an (ss)AAV8 vector carrying the human UDP-glucuronosyltransferase family 1-member A1 (hUGT1A1) transgene under the control of a liver-specific promoter. We compared our construct with similar (sc)AAV8 vectors expressing hUGT1A1, showing comparable potency in vitro and in vivo. Conversely, (ss)AAV8-hUGT1A1 vectors showed superior yields and product homogeneity compared with their (sc) counterpart. We then focused our efforts in the scale-up of a manufacturing process of the clinical product (ss)AAV8-hUGT1A1 based on the triple transfection of HEK293 cells grown in suspension. Large-scale production of this vector had characteristics identical to those of small-scale vectors produced in adherent cells. Preclinical studies in animal models of the disease and a good laboratory practice (GLP) toxicology-biodistribution study were also conducted using large-scale preparations of vectors. These studies demonstrated long-term safety and efficacy of gene transfer with (ss)AAV8-hUGT1A1 in relevant animal models of the disease, thus supporting the clinical translation of this gene therapy approach for the treatment of CN syndrome.