The effectiveness of gene therapy is essentially governed by the ability of the vector to reach the relevant tissue, and once there, to facilitate the expression of appropriate quantities of the gene product. Over the course of evolution, viruses have developed highly specialized and successful methods to achieve those goals. For this reason, advances in gene therapy have mainly focused on the development of viral systems such as adenovirus (Ad), retrovirus, adeno associated virus, herpes virus, and others. This approach has been successful, as demonstrated by the recent proliferation of publications and human gene therapy trials involving various virus-based vectors. In particular, first generation replication-defective adenoviral vectors based on human serotypes from subgroup C (such as types 2 and 5) have been and continue to be very popular as vehicles for delivery and expression of foreign genes. However, each virus has evolved to optimize its own life cycle and consequently endows vectors based on it with properties and limitations that affect their suitability as choices for various gene therapy applications. These properties need to be understood and taken into account in order to design vectors with improved characteristics, such as Ad vectors with improved regulation of transgene transcription.