Duration of the project:
Collaborating parties / partners:
Institute of Oncology Ljubljana
R&D Center for Nanotechnologies in the Field of Magnetic Materials and Composites Ljubljana
University of Primorska Faculty of Health Sciences
University of Ljubljana, Faculty of Electrical Engineering
The aim of the project is the development of new gene therapy approaches, based on anti-angiogenic mechanisms, for treatment of malignant melanoma and sarcoma. The new plasmids coding for anti-angiogenesis of tumors will be tested in experimental tumors by non-viral delivery systems; electroporation and magnetic nanoparticles. For better translation into the clinics, combined modality treatment with radiotherapy will be tested.
The project is based on the construction of new plasmids coding for miRNA against endoglin (CD105) and melanoma cell adhesion molecule (MCAM/CD146), both specific for endothelial cells in tumors and melanoma cells capable of invasion. These specific targets will be used for the treatment of two variants of melanoma tumors in experimental animals, B16F1 melanoma with low metastatic potential and B16F10 with high metastatic potential. Because of some cross-specificity with other tumor type’s sarcoma SA-1 model will also be used. In addition to that, plasmid AMEP already prepared and validated to some extent by our industrial partner will be used. It also has anti-angiogenic and direct cytotoxic effect to melanoma cells and tumors.
As plasmid delivery systems, non viral techniques will be used; electroporation and magnetic nanoparticles. Electroporation is already established gene delivery method for transfection of tumors, muscle and skin (electrogene therapy). Nanotechnology for gene delivery is in its early developmental phase for use of magnetic nanoparticles (magnetofection). This approach is being developed in our research group, and we want to test and implement it in cancer treatment.
With electrogene therapy and magnetofection the transfection efficiency of constructed plasmids as well as plasmid AMEP in cells in vitro and the effect of the plasmids on cell’s proliferation, adhesion and invasion will be performed. Anti-angiogenic potential of constructed plasmids will be tested in vitro by angiogenesis assay (tube formation assay). Based on these results we will proceed in the investigation of anti-angiogenic and antitumor effects by intravital microscopy in window chamber. These data will provide evidence on anti-angiogenic effectiveness of the constructed plasmids coding for miRNAs in vivo, as well as their antitumor effectiveness on melanoma and sarcoma tumor models.
In vivo, on experimentally induced tumors and metastases, antitumor effectiveness of the constructed plasmids coding for miRNAs against CD105 and CD146 as well as plasmid AMEP will be tested. By intratumoral electrogene therapy or magnetofection, direct effects on established tumors will be tested. Also intramuscular transfection of plasmid AMEP is foreseen in order to test the possibility of systemic shedding of the protein in circulation and its effect on local tumor growth and metastasis. Again, both transfection methods will be used; electrogene therapy and magnetofection. All these experiments will provide starting point for further experiments combined with radiotherapy.
Anti-angiogenic therapies are already being combined with radiotherapy in treatment of cancer, however anti-angiogenic gene therapy as radiosenzitizing therapy is still in early stage of development. Therefore the constructed plasmids and plasmid AMEP will be tested as adjuvant gene therapy to radiotherapy. The best treatment combinations with either single or repetitive gene therapy combined with either single or fractionated radiation regimen will be explored. The predominant tumor model used will be melanoma.
The results will provide evidence on newly constructed plasmids coding for miRNA against CD105 and CD146 and plasmid AMEP on their antitumor effectiveness on melanoma and sarcoma tumors. Their antitumor effectiveness with or without combined modality treatment with radiotherapy will provide basis for translation of the knowledge into clinical trials.