Improved system for helper-dependent adenoviral vector production.

Helper-dependent adenoviral vectors (HDAds) are devoid of all viral coding sequences and have demonstrated tremendous potential for gene therapy by providing increased cloning capacity (up to 37 kb) and long-term, high-level transgene expression in vivo with negligible toxicity. Currently, the most widely used method of producing HDAds is the Cre/loxP system developed by Graham and co-workers. However, two major obstacles currently hinder progress of this promising technology: (1) the difficulty of large-scale vector production and (2) helper virus (HV) contamination. We have developed an improved producer cell line, HV, and protocols that have successfully addressed these problems. With this system, >1 x 10(13) viral particles (vp) can be easily produced from 3 liters of cells within 2 weeks of vector rescue, with specific yields of >10,000 vp/cell and with exceedingly low HV contamination of 0.4-0.1% without relying on density-based vector purification and 0.02-0.01% following CsCl purification. This new system represents a major improvement over the original method in terms of simplicity, speed, vector yield, and purity, and it will significantly improve our ability to assess this promising gene therapy technology, especially in large animal models and, ultimately, for clinical applications.