Date of Award

2015

Degree Name

Biomedical Sciences

College

Joan C. Edwards School of Medicine

Type of Degree

Ph.D.

Document Type

Dissertation

First Advisor

Pier Paolo Claudio

Second Advisor

Travis Salisbury

Third Advisor

W. Elaine Hardman

Fourth Advisor

Jagan Valluri

Fifth Advisor

Piyali Dasgupta

Abstract

A major challenge for effective gene therapy is systemic delivery of viruses carrying therapeutic genes into affected tissue. The immunogenic nature of human adenoviruses (Ads) limits their use for intratumoral (IT) injection in gene therapy. Ads transfection is further hampered by the fluctuating presence of Coxsackie and Adenovirus Receptor (CAR) and integrins on the cells’ surface. To circumvent these limitations we developed a novel approach wherein Ads are encapsulated inside the shell of lyophilized, lipid-encapsulated, perfluorocarbon microbubbles (MBs)/ultrasound (US) contrast agents, which act as delivery vehicles for a sitespecific gene transfer system.

We performed infection studies with Ad.GFP (Green Fluorescent Protein), Ad.mda-7 (melanoma differentiation associated gene 7) and CTV.mda-7 on human DU145 and mouse prostate cancer cells as well as observed enhanced GFP expression when Ad.GFP was delivered by MBs and US. Our results show that US breaks open the MB/Ads complexes by undergoing cavitation at the sonoporated site, which allows Ads to transfer their transgene only to the sonoporated region. Cavitation collapse of the MBs creates small shockwaves that increase cell permeability by forming temporary micropores on the cell surface bypassing the receptormediated dependence of Ads for transfection. Fetal bovine serum (FBS) containing complement did not allow the unprotected Ads to infect the cells; however, MBs complexed with Ad.GFP did infect DU145 and TRAMP-C2 cells in a FBS rich media.

We studied MB assisted gene delivery of reporter (GFP) and therapeutic genes (p53, Rb, Rb2 (p130) and Mda-7/IL-24) into prostate cancer (PC) xenografted in immune-compromised athymic mice. The results demonstrated that MBs protect the host from unspecific viral immune response thus protecting the viral payload and allowing for intravenous (IV) injection rather than IT injection. Additionally, Ad gene transfer was enhanced at the targeted/sonoporated mice tumor xenografts. This research demonstrated mda-7’s efficacy in reducing primary (treated) and untreated tumors that simulated the presence of metastasis in athymic mice xenograft models bearing human PC cells. Bystander anti-tumor activity of mda-7, a secreted cytokine was noted for non-targeted tumors.

Earlier in vitro studies on the combination of radiation and gene therapy (Ad.p53, Ad.Rb, and Ad.p130) demonstrated an increase in the percentage of cell death for DU145 cells. We also studied UTMD (ultrasound targeted microbubble destruction) gene therapy in combination with external beam radiation for radiation resistant PC. The results demonstrated an enhanced therapeutic benefit of tumor suppressor genes in radiation resistant PC. We also demonstrated an increase in the expression of tumor suppressor genes at the tumor site due to MBs and US.

These findings highlight the potential therapeutic benefit of this novel image guided gene transfer technology alone or in combination with external beam radiation for prostate cancer patients with therapy resistant disease.

Subject(s)

Prostate -- Cancer -- Research.

Gene therapy -- Research.

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