Graduation Year

2010

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Molecular Medicine

Major Professor

Richard Heller, Ph.D.

Co-Major Professor

Kenneth Ugen, Ph.D.

Committee Member

Sophie Dessureault, M.D., Ph.D.

Committee Member

Thomas Klein, Ph.D.

Committee Member

Kenneth Wright, Ph.D.

Keywords

HARV Bioreactor, Gene Delivery, B16.F10, HaCaT, 3D Spheroid

Abstract

Melanoma is an aggressive disease with few effective treatment options. Non-toxic, anti-tumor therapies and prophylactic approaches are currently being investigated to identify treatment options that will control and remove late-stage melanoma.

The overall goal of this project was to establish an effective delivery method for a plasmid encoding human interleukin (phIL-15) into mouse melanoma cells (B16.F10) using the gene transfer technique electroporation (EP)1. The EP delivery phIL-15 was optimized using an in vitro 3D tumor model. The purpose was to translate these IL-15 delivery conditions into an in vivo mouse melanoma model to study IL-15 signal transduction and stimulate immune cells to destroy tumor antigens as well as promote an anti-tumor immune memory response.

The in vitro 3D tumor model and the mouse model demonstrated similar expression patterns when delivering phIL-15 with different EP conditions. Intra-tumoral delivery using 500V/cm 20ms enhanced gene delivery and increased IL-15 protein expression compared to 1300V/cm 100μs. There was also a visible increase in transfection efficacy between tumor cells compared to skin cells when delivering pmIL-12 and phIL-15 plasmid constructs in vivo. The plasmid+EP groups 1300V/cm and 500V/cm stimulated increased expression of cytokines IL-1β, IL-6, INFγ, MIP-1β and TNFα. These EP groups also promoted tumor regression by up-regulating CD8+ T cells and CD4+ T cells which targeted melanoma. Regression and survival studies demonstrated that 73.3% of mice cleared B16.F10 cells when treated with phIL-xi15+1300V/cm and pVax+500V/cm. In addition, 53% of the mice responded to the phIL-15+500V/cm treatment group. Furthermore, 75% of the mice from group phIL-15+500V/cm survived secondary inoculation and tumor challenge. In conclusion, plasmid with encoding gene insert phIL-15 delivered by EP has the potential to act as an anti-tumor therapy because it promotes melanoma regression and enhances mouse survival through innate and adaptive cell-mediated immune responses.

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