Projects / Programmes
Antiangiogenic gene therapy of cancer using electroporation and magnetic nanoparticles as targeted delivery systems
| Code |
Science |
Field |
Subfield |
| 3.04.00 |
Medical sciences |
Oncology |
|
| Code |
Science |
Field |
| B200 |
Biomedical sciences |
Cytology, oncology, cancerology |
| Code |
Science |
Field |
| 3.01 |
Medical and Health Sciences |
Basic medicine |
gene therapy, anti-angiogenesis, melanoma, sarcoma, electroporation, electrogene therapy, nano technology, magnetofection, radiotherapy
Researchers (30)
Organisations (3)
Abstract
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.
Significance for science
The majority of anti-angiogenic therapies used in clinical practice is based on the inhibition of signaling pathways of vascular endothelial growth factor. These therapies are related to severe side effects and resistance of certain types of tumors. Therefore, there is a need on searching for new targets, which would be independent of the vascular endothelial growth factor. This project was focused on three new target molecules, namely endoglin (CD105), melanoma cell adhesion molecule (CD146 / MCAM), and integrins. Endoglin is a co-receptor of transforming growth factor ß and is expressed in specific activated endothelial cells in tumors, which can be activated by the vascular endothelial growth factor-independent signaling pathway, and therefore represents a potential and very suitable target. CD146 plays an important role in the development of melanoma and its progression, including invasion, angiogenesis, and metastatic potential, and therefore represents a potential target for gene therapy of melanoma, which incidence and mortality rate are increasing. Binding of protein AMEP on targeted ?5ßI and ?VßIII integrins, which are expressed on melanoma and activated endothelial cells, respectively, would inhibit their function and thus result in antitumor and antiangiogenic effectiveness. For targeting CD105 and CD146 we used a new approach in molecular biology, namely RNA interference. We have constructed new plasmids, one encoding shRNA against CD105 and another encoding shRNA against CD146. Integrins were targeted by AMEP protein, encoded by a plasmid AMEP. The majority of gene therapy clinical trials utilize viral delivery systems and due to the safety issues we decided for developing of non-viral ones. For the introduction of plasmids into the cells and tumors electroporation as already developed and optimized method, and magnetofection as a developing method were used. Thus, we tested the anti-angiogenic and anti-tumor effect in cell cultures and tumor models after gene electrotransfer and magnetofection with plasmids, designed specifically for targeting new therapeutic targets. We have demonstrated that the therapies are feasible, effective, specific and without side effects. The results obtained from the project contribute to the implementation of anti-angiogenic gene therapy, which could be in the future used in the combination with radiotherapy or standard cytotoxic cancer therapies because it would hinder the growth of tumors and the development of a malignant phenotype. We have also contributed to the development of magnetofection, as in vivo experiments demonstrated magnetofection as an effective, non-invasive and painless non-viral gene delivery method into tumors, and it certainly has a high potential for further studies and translation into clinical practice. By using recently developed molecular biology technique, such as siRNA interference technology, which regulates gene expression at the post-transcriptional level of mRNA, we have contributed to its improvement and potential future upgrade of gene therapy approaches. All three studied molecules, CD105, CD146 and the integrins, have proved to be potential targets, against which targeted therapies are worth developing.
Significance for the country
Anti-angiogenic gene therapy is relatively new approach for cancer treatment worldwide. A research on new targets for anti-angiogenic therapy was necessary, as so far established anti-angiogenic therapies have many side effects and targets involved in other signaling pathways are required. A research in the field of gene electrotransfer and magnetofection contributed in the development of other non-viral approaches for gene delivery into target cells and tumors. By optimizing the use and investigating magnetofection, we contributed to the knowledge of relatively young field of biomedical nanotechnology, which is important not only for Slovenia, but also in the world. The project also involved young PhD researchers from the University of Ljubljana, who learned new approaches of cancer gene therapy, gained also on the basic knowledge in this field, learned the preparation, bacterial cultivation and isolation of plasmids, learned different tests on cell cultures and tumor models for the determination of anti-tumor and anti-angiogenic effect, and examined the therapy mechanisms.
Most important scientific results
Annual report
2011,
2012,
2013,
final report,
complete report on dLib.si
Most important socioeconomically and culturally relevant results
Annual report
2011,
2012,
2013,
final report,
complete report on dLib.si
