Title page for ETD etd-04062006-102829


Type of Document Master's Thesis
Author Craig, Ryan
URN etd-04062006-102829
Title Genetically Modified IFN-Alpha for Gene Therapy Treatment of Squamous Cell Carcinoma
Degree Master of Science (M.S.)
Department Comparative Biomedical Sciences (Veterinary Medical Sciences)
Advisory Committee
Advisor Name Title
Shulin Li Committee Chair
Shisheng Li Committee Member
Steven Barker Committee Member
Keywords
  • tumor-targeted peptide
  • squamous cell carcinoma
  • ifn-alpha
  • cdgrc
Date of Defense 2006-03-21
Availability unrestricted
Abstract
In the last twenty years, interferon-α (IFN-α) has gained success as an immunotherapy for treating such cancers as hairy cell leukemia, malignant melanoma, and renal cell cancer. Our goal was to improve the effectiveness of IFN-α therapy by genetically modified the IFN-α gene to encode a tumor-targeting peptide fused to a functional IFN-α protein. To ensure the targeting peptide worked, a genetically modified reporter gene encoding a secreted alkaline phosphatase (SEAP) gene and different mini-peptides were used to determine distribution and targeting ability. The DNA fragment encoding the most effective peptide was selected to modify the IFN-α gene construct for therapeutic trials. This fusion gene encoded the peptide with the amino acid sequence of C-D-G-R-C, and demonstrated a higher localization of the genetically modified gene product in the tumor local area. Tumor volume and animal survival was measured over several weeks to compare the anti-tumor effects of the IFN-α to CDGRC-IFN-α treatments. Results indicate an increase in therapeutic efficacy due to treatment with the CDGRC-IFN-α gene over the wild-type IFN-α gene. Flow cytometry was performed and it was determined that both of the tumor targeted gene products, CDGRC-SEAP and CNGRC-SEAP share a high affinity for the receptor, Aminopeptidase N (CD13). In order to determine the mechanism responsible for the enhanced anti-tumor effect by CDGRC-IFN-α gene therapy, the T cell infiltration, subsequent CTL activity, and tumor vessel density were confirmed through immunostaining. An increase in number of CD8+ T cells was seen, as well as an increase in activity of cytotoxic T cells. Decreased vessel density in CDGRC-IFN-α treated animals suggest that this therapy enhanced anti-angiogenisis. A high level of non-specific activity was detected in the CTL assay, suggesting involvement of other immune cells, such as NK cells. Overall, this study describes the first example of using a genetically modified immunostimulatory gene encoding tumor-targeted IFN-α for treating tumors. This novel concept may have the potential for increasing therapeutic efficacy of several current cancer treatments.
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