Hurdles and Improvements in Therapeutic Gene Transfer for Cancer

Over the past decades, gene therapy has emerged as representing a potential treatment modality for cancer. One promising cancer gene therapy approach is based on introducing a gene that encodes for an enzyme called a suicide protein into tumor. This enzyme converts a normally harmless prodrug into a toxic form that induces tumor cell death. Cell killing is observed also in the surrounding, non-transduced cells, which is a benefit since all tumor cells do not need contain the therapeutic gene. This phenomenon is called the bystander effect. Nevertheless, true success in clinical trials has not been achieved mainly due to insufficient gene delivery rate of the current vectors and inadequate bystander effect in many tumors. In the present study we evaluated various methods to overcome these problems; first by characterizing factors that may influence efficient therapeutic gene transfer and further by modifying viral vectors and the therapeutic gene with the aid of cell penetrating peptides. A number of factors, including host cell immune responses, can influence the gene transfer efficiency of viral and non-viral vectors. For that reason, we studied the contribution of the type I interferon response, an arm of innate immune system, to the therapeutic gene transfer. The commonly used viral vectors, with the exception of Semliki Forest virus, succeeded in avoiding the induction of the type I IFN response. However, the delivery of plasmid DNA and particularly most forms of RNA triggered the response in a variety of studied cell lines. In order to improve delivery of therapeutic gene into the tumor cells, we evaluated the feasibility of using cell penetrating peptides derived from Drosophila Antennapedia homeodomain and HIV-1 transactivator protein (TAT). These cationic peptides enhanced transduction efficiency of adenoand lentiviral vectors significantly in most of the tested human tumor cells. However, the property of a commonly used commercial transduction enhancer was found to be even better at boosting efficacy than the cell penetrating peptides. In another study included in this thesis, the cell penetrating peptide TAT was linked to suicide-marker fusion gene (TAT-TK-GFP and TK-GFP) to extend the cytotoxic impact of suicide gene therapy to adjacent cells and thus to compensate for the poor gene delivery rate. Against our original hypothesis, we found that the TAT containing fusion proteins were not trafficking between the cells. Despite the lack of intercellular movement, TAT-mediated increased cell killing was observed in some of the tested human tumor cell lines. However, in many cell lines the killing efficiencies of TAT-TK-GFP and TK-GFP were similar and in some cell lines the efficiency of TK-GFP was even better. In conclusion, these results indicate that the gene delivery can induce undesired immune responses in target cells and thus may represent a barrier against efficient therapeutic gene transfer. Although cell penetrating peptides improved the viral transduction rate, the utility of these peptides as general enhancers will most likely be limited by their high manufacturing costs compared to commercially available and clinically approved compound. Even though TAT -containing suicide fusion protein showed some enhancement of cell killing in certain tumor cell lines, no overall difference in efficacy between TAT-TK-GFP and TK-GFP was seen. Therefore, this concept needs to be further refined if it is to be considered as a potential supplement for cancer suicide gene therapy. National Library of Medicine Classification: QZ 52, QZ 266, QU 470, QU 475, QU 68 Medical Subject Headings: Neoplasms/therapy; Gene Therapy; Gene Transfer Techniques; Transduction, Genetic; Genetic Vectors; Viruses; Cell Death; Bystander Effect; Immunity; Interferon Type I; Drosophila Proteins; Antennapedia Homeodomain Protein; Gene Products, tat; Peptide Fragments; Transcription Factors; Genes, Transgenic, Suicide; Thymidine Kinase; Ganciclovir