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Type of Document Dissertation Author Pruitt, Amy Forister, Author's Email Address amy_pruitt@ncsu.edu URN etd-03282008-095037 Title Heat Induced Reporter and Therapeutic Gene Expression in Cancer Gene Therapy Degree PhD Graduate Program Comparative Biomedical Sciences Advisory Committee
Advisor Name Title Marlene Hauck Committee Chair Jorge Piedrahita Committee Co-Chair Jill Barnes Committee Member Mark Dewhirst Committee Member Keywords
- Heat Induced Reporter Gene Expression
Date of Defense 2007-12-14 Availability unrestricted Abstract Gene therapy for cancer is an important research focus, with most approved gene therapy clinicaltrials involving cancer patients. This reflects, in part, the inadequacies of conventional treatment for advanced, refractory solid tumors. However, significant problems remain to be solved before gene therapy will become a first line modality in the clinical setting. Two challenges to overcome are the need for targeted transgene expression and noninvasive evaluation of transgene
expression. In this study, we hypothesized that the use of the heat inducible heat shock protein
70 promoter (HSP) in combination with the human somatostatin receptor type 2 (hSSTr2)
reporter gene will allow for the in vivo, non-invasive monitoring and quantification of targeted
therapeutic gene (murine Interleukin 12) expression. Replication-incompetent adenoviral vectors containing either the therapeutic gene (AdHSPmIL-12) or the reporter gene (AdHSPhsstr2)
under control of the HSP were constructed and their function first assessed in vitro. Cells were
infected with both vectors and then heated at temperatures that ranged from 38-42o C.
Uninfected cells and infected cells at 37o C served as controls. Twenty-four hours after heating,radiolabeled somatostatin analogue was added to the wells, and radioligand receptor binding was measured with a gamma counter. A murine interleukin-12 (mIL-12) ELISA kit was used to measure mIL-12 levels in the supernatants. The adenoviral vectors function was also assessed in
vivo. Balb/c and C57BL/6 mice with subcutaneous mammary adenocarcinoma and murine melanoma tumors respectively received intratumoral co-injection of 1 X 108 pfu each of AdHSPhsstr2 and AdHSPmIL-12 followed by either no hyperthermia, hyperthermia for 40 minutes at 41.5°C or hyperthermia for 40 minutes at 42.5°C. Additional animals not receiving intratumoral vector injection served as controls. Radioligand was injected via the tail vein 48 h after heating, and animals were sacrificed one hour after tail vein injection. Tissues were collected and counted in a gamma counter. Tumors were also disaggregated in lysis buffer using a Mini-Beadbeater, and murine IL-12 concentrations in tumor extracts were determined using a commercially available murine interleukin-12 (mIL-12) ELISA kit. In the in vitro experiments,
a statistically significant heat-dose response of radioligand binding for the reporter gene and
mIL-12 production for the therapeutic gene was detected, with minimal expression observed in
the controls. Results demonstrated a strong correlation between radioligand uptake and mIL-12
production in response to heat treatment with r2 = 0.9886. A statistically significant heat-dose
response of tumor radioligand uptake for the reporter gene and tumor mIL-12 production for the
therapeutic gene was also detected in vivo, with minimal expression observed in the controls.
Normal tissue biodistribution of radioligand was similar between all treatment groups with no
significant difference between the infected groups and the non-infected control. Results
demonstrated a strong correlation between tumor radioligand uptake and tumor mIL-12
production in response to heat treatment with r2 = 0.9937. The results of this study indicate that
gene expression from both vectors could be induced in vitro and in vivo by hyperthermia in a
temperature dependent manner and that induced expression from the reporter and therapeutic
gene vectors were highly related. Normal tissue biodistribution of radioligand was similar
between all treatment groups with no significant difference between the infected groups and the
non-infected controls demonstrating the ability of the HSP promoter to limit transgene
expression to the tumor. Results have the potential to pave the way for evaluation of heat
induced gene therapy with hSSTr2 reporter monitoring in animals and humans.
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