Projects / Programmes
Gene electrotransfer of muscle - from studies on single cells to numerical optimization of parameters in tissue
| Code |
Science |
Field |
Subfield |
| 4.06.00 |
Biotechnical sciences |
Biotechnology |
|
| Code |
Science |
Field |
| B001 |
Biomedical sciences |
General biomedical sciences |
| Code |
Science |
Field |
| 3.04 |
Medical and Health Sciences |
Medical biotechnology |
gene therapy, skeletal muscle, numerical modelling, electrotransfection, microscopy
Researchers (20)
Organisations (3)
Abstract
In the last decades, in vivo gene transfer with electroporation (gene electrotransfer) has emerged as a promising method of delivery for nonviral gene therapies and recently also for DNA vaccines. Locally delivered electric pulses increase membrane permeability and enable transfer of plasmid DNA (pDNA) or short strand RNA into the cells. Specifically, skeletal muscle was found to be an ideal target tissue for electrotransfer since high efficiency of transfer can be obtained, it represents large portion of body mass that is relatively easily accessible, and prolonged expression of genes can be achieved.
Currently, one of major obstacles for efficient use of in vivo electrogene therapy (EGT) in clinics is still relatively low efficiency due to poor mobility of pDNA inside extracellular matrix (ECM). While studies performed on cells in culture have very controlled conditions, the mobility of pDNA is not hindered and the results have limited use when relating to tissue level. In contrast, in vivo environment is poorly controllable and it is difficult to investigate the mechanisms of the electrotransfer. Therefore, development of in vitro 3D gel model of tissue represents an important step between in vitro and in vivo studies. Furthermore, only few studies were done on primary myoblast cells, even though muscle tissue is identified as a tissue of choice for the gene electrotransfer due to its specific characteristic that enable efficient, prolonged and stable gene expression. In addition, the processes which contribute to efficient and prolonged expression in muscle cells are not yet fully understood.
Another remaining challenge in final realization of the method for clinical treatment is scaling up the in vivo research to humans. In spite of a large number of in vivo studies on animals the parameters are always determined after extensive experimentation. Several important papers on in vivo EGT identified a need for more systematic optimization of the electroporation protocol as a crucial step toward more efficient EGT, scale up of the method to human studies, and faster translation to clinics. Therefore, important step for successful use of gene electrotransfer for biomedical applications is development of advanced numerical models for optimization of the electroporation parameters.
In last decade also short RNAs were shown to play important part in gene regulation and are interesting for future therapies. In gene electrotransfer mostly siRNA for silencing of transgene were analyzed, while there is only a single study where miRNA was delivered using electrotransfer.
The objectives of the proposed project are to theoretically and experimentally analyze mobility of plasmid DNA and electrotransfer efficiency in 3D collagen models with embedded cells; to analyze and optimize experimentally gene electrotransfer on primary human myoblast culture and to analyze role of satellite cells in long-term electrotransfection efficiency; to determine optimal pulsing conditions for delivery of short RNAs in primary human myoblasts; to analyze and visualize different steps of gene electrotransfer experimentally and theoretically; and finally, to develop a 3D numerical model of a skeletal muscle for optimization of electrode positions and applied voltages for more effective in vivo electrotransfer and scale up analysis from small animals to humans.
In the proposed project we aim to integrate theoretical analysis with experimental observations on all levels of complexity from single cells to optimization in tissue. Our experimental observation of different steps of gene electrotransfer together with theoretical description and advance numerical modeling will allow improved quantification and optimization of the gene electrotransfer for both in in vitro and in vivo systems.
Significance for science
Our experimental observation of different key steps of gene electrotransfer together with theoretical description of the processes gave new insights into relevant mechanisms. This is important for faster optimisation of the protocols for transfection of cell suspensions, attached cells from primary to cancer cells. The experimental results and theoretical analysis developed during the project range from single cells visualization experiments to broad analysis of electrotransfer effifiency in primary human mioblasts, myotubes, to optimization of electrotransfection efficiency in 3D numerical models of skeletal muscle tissue. In parallel a comprehensive study was done on CHO cells to systmatically analyse all relevant steps of electrotransfection. In the last decades several different siRNA and miRNA molecules were identified as very promising targets for regulation of gene expression and treatment of different diseases. We have sucesfully optimised protocols for transfer of siRNA in primary human myoblasts – in parallel the study analysed the relevant mechanisms and potential differences between siRNA and pDNA delivery. We have alsodeveloped a first multiscale three-dimensional model of tissue electroporation where a dynamic single-cell solution is coupled with a multicellular 3D model We investigated the temporal evolution of the electric conductivity of such cell system during application of an applied electric field. In parallel we have furtehr developed 3D model that enables analysation and optimisation of electrodes and electric field distribution in skeletal muscle coupled with thermal effects. In parallel some practical methodological solutions (electrodes designe, comparison of different quantification tehcniques, automatic cell counting of microscopical images) can be used by other researchers that use transfection techniques in the broad field of biotechnology and biomedicine. Understanding the process involved in successful and long-lived gene electrotransfer will lead to faster development and application of gene therapy without viral vectors, and to improvement of existing protocols, thus increasing efficiency of gene electrotransfer. Furthermore, developed in vitro 3D gels together with theretical models of DNA electromobility can aid to understanding of the processes and to faster optimisation of protocols in vivo. Use of advanced 3D numerical modeling for optimization of electric parameters and electrode configuration together with analysis of thermal effects can be used for further improvement of protocolsfor delivery in muscle tissue, and hopefully to reduce number of sacrificed animals. The results are expected to be used by other researchers in fields of biotechnological and biomedical applications of EGT in in vivo and preclinical studies.
Significance for the country
PI has during the project established strong collaboration with several Slovenian groups with complementary expertise: Laboratory for biotechnology - KI, Institute of cell biology MF-UL, Laboratory for molecular neurobiology MF-UL nad with Department of biotechnology IJS. This enables efficinet transfer of methodological knowledge and expertiese between different research groups. Transfer of new technologies and methods in fields of biotechnology and biomedicine in research institutions in Slovenia and facilitating transfer of these new technologies in applications. PI and team members lead development of scientific techniques (protocols, electrodes, high-voltage generator) needed for efficient electrotransfer of DNA and short RNA molecules for gene delivery and silencing in cells (normal, cancer) and in ex vivo tissue samples and their translation into applications. Implementation of advanced numerical methods - group has extensive expertise in 3D numerical modeling which is being implemented in field of gene electrotherapy (EGT). Based on developed 3D numerical models of EGT we have established collaboration with the leading research medical center (head. Prof. Julie Gehl, Department of experimental Oncology, Herlev Hospital, Copenhagen) in Europe for translation and application of electrogene transfer for cancer therapy. Connections between Slovenian research institutions and partners from abroad that opens possibility of participation in EU projects in fields of gene electrotransfer and vaccination. Based on aquired knowledge we were invited in praticipation in COST consortium application BIOENECA that connect groups working in fields of neurodegenrative diseases, biomedicine and and stem cells. Connection of state-of-the-art knowledge with educational system was performed through undergraduate and graduate student courses in the field of biomedical engineering and biotechnology and with involvment of seveal younger researchers in the project. 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 biology, biochemistry 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 s-o-a-t numerical modeling. Close collaboration of PI with industry will further enable transfer of knowledge of methods/procedures (e.g. solutions for optimization with numerical modeling) into industry. At the start of the project PI established a new young group which consisting from graduate and doctoral students in different fields from physics to biology and biochemistry, and the proposed project enabled consolidation and obtaining critical mass of researchers needed for interdisciplinary approach. Training of our younger researchers included visits of top laboratories in Europe, which is important for further development of high quality research in Slovenia. Health care: Currently we developed successfully protocol for electrotransfer of plasmid DNA into explants of mice spinal cord tissue in collaboration with prof. Boris Rogelj who is a PI of a project “Pathogenic mechanism of the C9orf72 expanded hexanucleotide repeat mutation in neurodegeneration” related with Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), both being devastating neurodegenerative diseases that form two ends of a complex disease spectrum. This project analyses what makes ‘normal’ TDP-43 aggregate in ALS and FTLD and what is the role of perturbed RNA metabolism in these diseases.
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
