A variety of options, including surgery, radiation, chemotherapy, and biopharmaceuticals, or a combination of these therapies, have been developed and are commonly used for the treatment of cancer. However, these treatments often result in undesirable side effects for the patient and may not always be efficacious (1-3). As a result, the use of gene therapy as an alternative therapy for cancer has been developed and attempted by several investigators (4, 5). The adenovirus (Ad) is the most common vehicle used for the gene therapy of cancer, both as a vector and as an oncolytic agent (4, 5). The Ad is a non-enveloped DNA virus that binds to the Coxsackie-Ad receptor (CAR) on cells and has been studied for more than half a century. After binding to CAR, the virus interacts with the host cell integrins (which serve as secondary receptors) and is internalized by receptor-mediated endocytosis (6). A detailed description of Ad structure, gene composition, and biology is available elsewhere (7). Ad is one of the best-characterized viruses and, depending on the infecting serotype, is known to cause several different illnesses such as conjunctivitis, gastroenteritis, the common cold, and other respiratory ailments in humans (8, 9). There are ~50 serotypes of Ad, and among these Ad2 and Ad5 are the serotypes most commonly used for gene therapy. This is because Ad can be easily engineered to target a specific ailment such as cancer, can be made replication-deficient, does not integrate into the mammalian genome, can accommodate large transgenes, and can be modified to have a reduced immunological responses in mammals (10).

To make recombinant Ad suitable for use as a gene therapy vector, several strategies are used by investigators in the field (11). A common approach to generate an Ad vector is by substituting a viral gene(s) with the gene(s) of interest (11). For selective infection of cancerous tumors, conditionally replicating Ads (CRAds) have been generated that replicate only in the neoplastic cells and locally amplify the virus, which ultimately results in lysis of the host cell (thus the virus is oncolytic) (12). The lack of replication in normal tissue restricts the infection of CRAds to the cancerous cells, and the CRAds would replicate only until the cancerous cells are depleted. Other advantages of using CRAds for cancer therapy are discussed in detail by Kanerva and Hemminki (12). Although CRAds are being used in several clinical trials in the United States, still little is known regarding their replication, spreading ability, persistence, and interaction with the host system (13). According to Ono et al., this is probably because a non-invasive monitoring system was not available to investigate these CRAd parameters (13). Ono et al. envisioned that CRAds could probably be studied in an in vivo setting if the vector could be monitored with a fluorescent reporter gene (13).

This chapter details the development and in vitro and in vivo evaluation of a CRAd vector bearing the enhanced green fluorescent protein (EGFP) as the reporter gene (13).