J3-4259 Preparation and validation of therapeutic plasmids without selection gene for antibiotic resistance for cancer gene therapy using inducible and tissue-specific promoters (Basic project ARRS)

Duration of the project:

1. 7. 2011 – 30. 6. 2014

Collaborating parties / partners:

University of Primorska, Faculty of Health Sciences
Institute of Oncology Ljubljana
National Institute of Biology Ljubljana
University of Ljubljana, Biotechnical Faculty

Principal investigator / researcher:

Prof. Maja Čemažar, PhD (SICRIS, ResearchGate)



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.