To date, there is no a good way to measure the distribution of the recombinant virus on a living system or determine the success of such efforts with high spatial resolution. The basic understanding of the fate of recombinant adenovirus (Adv) (or any virus for that matter) would have a significant impact on our understanding and the design and use of recombinant viruses. In this proposal we will develop a contrast agent to be labeled to the Adv system, so that the in vivo distribution of the virus can be monitored with magnetic resonance imaging (MRI) over time. In the synthesis of the virus-Gd complex, we will use starburst dendrimer as the carrier of Gd ions. It has also been shown that dendrimer can be conjugated to antibody IgG. Then we will chemically attach the dendrimer-Gd-DTPA to a functional virus via a non-neutralizing monoclonal antibody. Furthermore, we hypothesize that the use of an antibody (Ab) (LM609) that has a high affinity to the alpha-v-beta-3 integrin can enhance the localization of the virus in the neovasculature of cancer. To test the hypothesis, we will synthesize bispecific antibodies composed of an antibody to attach to virus and another mAb, LM609 to target the alpha-v-beta-3 integrin expressed in the angiogenic hot spot of the tumor. We will compare the localization and retention of the two Gd-virus systems synthesized with bispecific antidodies (with LM609) and that synthesized without LM609. The delivery of a sufficient amount of the virus-Gd complex into the tumor not the normal tissues is a major concern. Therefore, in the next step we will investigate the bio-distribution of Gd- virus after administration via three routes: intravenous, intra- arterial, and intra-tumoral, and determine which route of injection can achieve the best localization of viruses in the tumor. To study the relationship of the gene expression with respect to the bio-distribution, we will quantitatively measure the expression of beta-gal by immunohistochemical staining with image analysis methods, and correlate the results with the amount of virus distribution measured by MRI. Upon successful completion, we should be able to encode anti-angiogenic genes into the recombinant Ad virus to serve as therapeutic agents. With the labeled imaging agent, we can determine the distribution of the viruses in the host and the amount localized in the tumor with MRI. Furthermore, MRI can be used to assess the treatment outcome for investigation of the relationships between the delivery of the gene therapeutics and the therapy efficacy. Due to the limited scope of the R21 award, we do not propose to proceed to the therapy studies in this proposal. However, the proposed studies are the important foundations for the future work. The success of the current proposed study will provide a better understanding of the Ad systems and have a great impact on the design of the anti-angiogenic gene therapy systems for cancer treatment.
