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Enhancing transduction of breast and ovarian cancer using EGF and herceptin complexed adenoviral vectors
Adams, Kristen E.
Barry, Michael A.
Doctor of Philosophy
Successful gene therapy for breast and ovarian cancer will likely require that anti-cancer genes be delivered specifically to primary and metastatic tumor sites while avoiding normal tissues. Adenoviral vectors are attractive for cancer gene therapy, since they can deliver transgenes to many different tumors. While adenovirus is quite potent at gene delivery, it is also non-specific and delivers genes into tumor and non-tumor cells in vivo. For effective gene therapy, the natural tropism of adenovirus must be removed and the virus re-targeted to tumor cells using cancer-specific ligands. To identify new cell binding ligand, peptide presenting phage libraries were selected against human breast cancer cell lines. Displayed on phage, these peptides bound specifically to their selection target, cross-reacted to varying degrees on other breast cancer cell lines, and did not bind to normal breast epithelial cells. The binding properties of these peptides were compared with those of commercially available antibodies such as Herceptin and binding proteins such as EGF to determine viable candidates for vector targeting. Viral targeting methods developed in our laboratory show promise in both ablating the natural tropism of adenovirus and retargeting the virus. The targeting ligands were complexed to biotinylated adenovirus through avidin bridges and chemically cross-linked to adenovirus using bifunctional PEG molecules. The viral complexes were tested in vitro before delivery was evaluated in the in vivo xenograft tumor models. Fluorescent and luminescent reporter genes were used to determine the location of vector delivery through gene expression in vivo. Targeted adenovirus had reduced background transduction and somewhat increased breast and ovarian cancer transduction. Finally to better evaluate ligand performance, real time, dynamic imaging was used to track ligand distribution and kinetics in vivo in tumor models. Fluorescent conditions were first evaluated in mouse models, demonstrating that imaging in the near infrared had superior signal to noise profiles over fluorescence in the visual range. Therefore ligands were labeled with the near infrared dye IR800 and their distribution was tracked in real time. To evaluate the feasibility of tracking virus (not transgene products), adenovirus was labeled with IR800, given to mice and virion trafficking was successfully imaged.