Project coordinator: dr. Maja Čemažar, UP FVZ
Coordinator for NIB: prof. dr. Metka Filipič
Background. Gene therapy is becoming increasingly more important in cancer therapy. In recent years, non-viral approaches for transfection have become very attractive. One of the goals of researchers dealing with gene therapy or DNA vaccination is to develop safe and efficient systems for gene transfer into the target cells. Namely, this topic is still one the major hurdles in the progress of different approaches in gene therapy and is crucial factor for success of the therapy. Other factors that should be considered for development of gene therapy are the choice of appropriate therapeutic gene, regulation of its expression and administration route. In the proposed project, two of these factors will be addresses for the use in cancer gene therapy: safety of plasmid DNA by preparation of plasmid DNA without marker for antibiotic resistance and regulation of therapeutic gene expression by linking the therapeutic gene to cellular promoters to achieve either stress induced (p21 and radiation) or physiological regulation (tissues specific promoters for endothelial and muscle cells and fibroblsats) of expression. In addition, possible horizontal gene transfer between recombinant plasmid DNA or transgenic bacteria and commensal naturally occuring bacteria will be evaluated. Therefore, our hypothesis is that prepared plasmids encoding therapeutic genes under the control of tissue specific and inducible promoters without gene for antibiotic resistance are safe vectors for gene therapy with also regulated expression. Methods. Plasmids encoding reporter or therapeutic genes under the control of tissue specific or inducible promoters will be prepared by standard molecular biology techniques and according to the instructions of the producer of ORT® plasmid in order to prepare plasmids without gene for antibiotic resistance. Appropriate bacterial strains will be transformed with the plasmids to produce larger quantities of endotoxin free plasmid to be used in further experiments. The correctness of plasmids will be evaluated by gel electrophoresis and sequencing. To evaluate horizontal gene transfer, PCR assays and other standard laboratory procedures used for studying horizontal gene transfer as well as standard laboratory procedures used for preparing biofilm cultures will be used. To determine the effects of prepared plasmids on cell in cultures, standard test for proliferation, antiangiogenic and antimetastatic potential will be used. Antitumor effectiveness and histological analysis will be determined on tumor models in vivo by tumor growth delay assay, tumor control probability assay and immunohistochemistry. Execution and management of the project. The experiments will be performed at the laboratories of 5 partners participating in the project. All necessary equipment for molecular biology, cell cultures, immunocytochemistry, and animals experiments is available. The researchers involved in the project have vast experience in the field of plasmid preparation, electroporation as a drug and gene delivery method and radiobiology. The management and coordination of the project will be assured by regular meetings and constant communication with partners on the project. Relevance and potential impact of the results. The proposed project is designed as a preclinical project that addresses important issues regarding safe use of cancer gene therapy in clinical setting. The results of the project, if successful, may have impact on design and execution of cancer gene therapy trials in Slovenia and worldwide.
Significance for science
In the development of gene therapy one of the major focuses of research is the efficient and safe transduction/transfection of target cells. An ideal gene transfer method would allow introduction of sufficient concentration of DNA into the desired target cells with minimal side effects. Gene therapy using electroporation has proved to be a good alternative to viral methods and is already approved for treatment of canine melanoma in veterinary oncology. However, further improvements on construction of safer plasmid DNA need to be addressed for progression of electrogene therapy into broader clinical trials. Electroporation is among nonviral gene delivery systems, one of the most advanced, since several clinical trials are on-going for cancer treatment and also in DNA vaccination, later especially in veterinary medicine. Our research group was one of the pioneering groups in this field, the research on electroporation started 20 years ago, while on gene electrotransfer 12 years ago. The proposed research is a continuation of our work. Our previous studies were mostly concentrated on optimization of parameters of gene electrotransfer in tumors as well as in muscles. We showed that the histological properties of different tumors influence the transfection efficiency and that at least in part we can predict on the basis of known histological type of tumor the level of transfection efficiency and also treatment outcome. In the part of the research devoted to electrogene transfer to muscle, we demonstrated that with appropriate selection of parameters of electric pulses, dose of plasmid DNA and timing of DNA injection, one can control the level of expression of transgene and also the duration of expression. Recently, we proved that electroporation is also suitable for transfection of siRNAs and demonstrated that siRNA against CD105 and CD146 have antiangiogenic and antiproliferative effects on melanoma and endothelial cells in vitro. Furthermore, we demonstrated that electrogene therapy with plasmid encoding IL-12 given either locally or systemically is successful antitumor treatment for sarcomas also in combination with irradiation. The proposed studies address basic safety concerns of plasmid DNA and by combining the expertise of all involved partners it is foreseen that we will be able to prepare a safe therapeutic plasmid DNAs with great clinical potential on one hand and on the other contribute to the risk assessment for currently used plasmid DNA preparations by determination of possible horizontal gene transfer into naturally occurring commensal bacteria. These results are especially important also for different authorities that are involved in risk assessment as well as granting permission for clinical trials.