Projects / Programmes
Mechanisms of DNA delivery with electrogene transfer
| Code |
Science |
Field |
Subfield |
| 2.06.07 |
Engineering sciences and technologies |
Systems and cybernetics |
Biomedical technics |
| Code |
Science |
Field |
| T115 |
Technological sciences |
Medical technology |
electrogene transfer, electroporation, cell membrane, DNA
Researchers (18)
Organisations (2)
Abstract
Electroporation is a versatile biotechnology technique that among other allows transfer of DNA into biological cells. High-voltage pulses induce (electroporation of a cell membrane) pores in the cell membrane, that thus becomes transiently permeable for ions, molecules and macromolecules, which otherwise cannot permeate through the membrane. The so-called electrogene transfer is already an established method, where electroporation is applied for gene transfer in vitro and in vivo. However, several recent researches in vivo suggest that electrogene transfer could become important in gene therapy of various illnesses as an alternative method for DNA transfer to the viral transfection. Currently, majority of research is focused on improving in vivo transfection efficiency and in clinical trials while transfection mechanisms of electrogene transfer are not yet established.
Main goal of the proposed project is to clarify the DNA transfer mechanisms through a cell membrane during electroporation on the basis of existing data from literature and experimental data obtained within project. We are going to experimentally analyze the effect of various parameters on the efficiency of DNA transfer in case of different protocols on attached cells and cells in suspension. We will develop appropriate theoretical models of DNA permeation in the cell during the electrogene transfer and evaluate and analyze existing models from the literature. Further, we are going to evaluate possible discrepancies between results from in vitro systems and literature data from in vivo experiments. On the basis of our experimental data and theoretical models we will investigate new protocols for more efficient DNA transfer and examine the possibility of upgrading the existing electroporation apparatus, Cliniporator, with algorithm for on-line optimization of voltage pulses during electroporation (electrogene transfer). As a main result the proposed project would allow identification and understanding of mechanisms of DNA transfer and optimization of protocols and particularly electrical parameters for electrogene transfer.
Significance for science
Gene electrotransfer has in last years emerged as the most promising non-viral method for transfer of genetic material into biological cell. Recently it was also identified as an ideal method for DNA vaccination, which has show great potential for treatment of cancer. Still, the mechanisms are not fully understood and direct visualization of the process is very difficult. The main focus of this project was elucidating the mechanisms of gene electrotransfer on different level of complexity from giant lipid vesicles, analysis on different cell lines in vitro to analysis and optimization of parametres on collagen gels and with 3D numerical modeling.
In vitro experiments were combined with theoretical description of DNA mobility and interaction with the cell membrane. Obtained knowledge of the mechanisms combined with presented 3D numerical modelling of electrotransfection in tissue will enable optimization of parameters of in vivo EGT and faster translation into clinics. Sofar numerical models were applied only for oprimization of electrochmotherapy while for gene no such study existed. The results of the project are important for gaining basic knwoldge of the process as well as to serve as a connection between in vitro and in vivo studies.
We first clearly demonstrated experimentally that electrophoresis is crucial for efficient gene electrotransfer in vivo, where concentration of plasmid DNA is relatively low, while in in vitro environment electrophoresis is not crucial and efficient electropermeabilization is sufficient. Furthermore, our results showed that if we want to transfer or compare the conclusions from in vitro results in in vivo environment it is crucial that we use sub-optimal plasmid concentrations. This and other results are important for understanding the processes and mechanisms of gene electrotransfer in vitro and in vivo, as well as for improving the protocols.
The experimental results obtained during the course of the project are supported with theoretical calculations, which so far was done only in few studies in this field of research. This provided new insights in the process of gene electrotransfer, new knowledge and possibility to explain involved mechanisms .
Furthermore, we developed new high-voltage pulse generator, which enables us to apply different combinations of high- and low- voltage pulses for different sets of parameters. Possibility to switch the time-course of high- and low- voltage pulses enabled us to specifically study the role of each type of pulse and from this to deduce role of electrophoresis, electropermeabilization and process of contact between DNA and the cell membrane. This generator represents a unique prototype, which allows us to perform experiments which can not be done in other laboratories.
Our conclusions were so far presented in several international conferences and already published in two papers in one of the leading journals in the field of bioelectrochemistry. In preparation is the publication which will present the most important and newly gained results in one of leading journals in field of gene therapy, and which will be of ineterst for the researchers which design in vitro and in vivo studies of gene therapy and DNA vaccination.
Interdisciplinary project group and combination of theoretical models and experiments enabled us to transfer relevant knowledge from diverse scientific fields. Understanding the mechanisms and development of numerical models for optimization of EGT will enable faster translation into clinics. We expect that our results will be essential to research community which uses EGT for gene therapy and DNA vaccination. The final goal of the principal investigator is to gain knowledge and a range of advanced tools for analysis and application of EGT for different therapeutic targets, which would lead to new treatments.
Significance for the country
The project included interdisciplinary group of Slovenian researchers (physicists, biologists and electrical engineers) and covers fields of technology, biomedicine and biotechnology. Gene electrotransfection itself is currently most promising alternative to the virus transfection for use in gene therapy and DNA vaccination.
Within the project we successfully collaborated with researchers from France (CNRS, Toulouse) and transferred to Slovenia state of the art technology and latest findings in the field of electrogene transfection. We are one of the few groups, which successfully combines experimental gene electrotransfection in vitro on cell lines and model 3D gel systems, and experimental results are upgraded with analytical calculations and numerical modeling.
Health protection
Electrogene transfer is a promising method that allows transfer of genetic material in biological cell by means of electrical pulses. In contrast to viral vector transfer, the use of electric pulses allows safe transfer of DNA. Electrogene transfer has great potential for application in gene therapy for a series of diseases (autoimmune, cronical) and first clinical trials are already under way. Understanding the meachanisms of electrogene transfer will lead to faster development and application of gene therapy without viral vectors, and to improvement of existing protocols, thus increasing efficiency of electrogene transfer.
Establishing new technology in the field of biotechnology and medicine
We successfuly transferred new methods and protocols for analysis of gene electrotransfer and developed a series of protocols which enables us state-of-th –art research. Gained knowledge enabled us to establish collaboration with the group of Molecular Neurology (Faculty of Medicine, UL) with possibility of broader collaboration of partners from European Union and implementation of gene electrotransfer in clinical environment.
Development of new products for biotechnology and biomedical engineering with possibility of new EU research projects and cooperation with industry that would encourage development of high-tech industry in the field of biotechnology. This could open also additional connections between Slovenian research institutions and industrial partners from Slovenia and abroad.
One of the project activities was also development of new device for generating of high- and low-voltage pulses that also allows generation of arbitrary combination of HV and LV pulses of different amplitudes, lengths, pauses and number of pulses. The device is unique in the world and allows cooperation with other research groups both in Slovenia and internationally, patent is in preparation.
Transfer of advanced numerical methods in biomedicine – we successfully developed 3D numerical models for optimization of gene electrotransfer parameters in tissues
Connection of state-of-the-art knowledge with educational system and knowledge transfer in industry through undergraduate and graduate student courses in the field of biomedical engineering and biotechnology. PI and heads of collaborating groups have extensive expertise in interdisciplinary teams and were mentors of several graduate and doctoral students in different fields from electrical engineering to medicine. Involvement of graduate students, young researchers and postdoctoral researchers in the project enabled them hands-on approach in high-tech biophysical and biomedical methods and also possibility of direct knowledge transfer into industry through development of state-of-the-art numerical modeling and possible new devices. Close collaboration of PI with industry will potentialy enable transfer of knowledge of methods/procedures (e.g. solutions for optimization procedures in numerical modeling) into industry.
Most important scientific results
Annual report
2008,
final report,
complete report on dLib.si
Most important socioeconomically and culturally relevant results
Annual report
2008,
final report,
complete report on dLib.si
