Construction and characterization of helper-dependent adenoviral vectors for sustained in vivo gene therapy

A helper-dependent adenoviral (HDAd) vector is the most recently developed adenoviral vector. It does not contain any viral coding sequences except the inverted terminal repeat for replication origin and the packaging signal. Its safety profile and duration of transgene expression in vivo have improved substantially compared to early generation adenoviruses. Despite its usefulness for experimental gene therapy, technical difficulties in producing the HDAd vector have hampered its wide application. This chapter illustrates important considerations in vector design, unique features of this system, and an overview of vector production, which is followed by a step-by-step protocol for vector production. Vector characterization and troubleshooting are also provided at appropriate steps.