|Noninvasive noncontact electroporation for efficient gene therapy and DNA vaccination (2021-2024)|
|Programme head:||Prof. Damijan Miklavčič, University of Ljubljana, Faculty of Electrical Engineering|
|Funding:||Slovenian Research Agency (ARRS), Slovenia|
The project is funded by the Slovenian Research Agency (ARRS).
- Member of University of Ljubljana
- Noninvasive noncontact electroporation for efficient gene therapy and DNA vaccination
- 1.7.2021 - 30.6.2024
- Amount of financing
- 0.7 FTE
- Research activity
- Engineering sciences and technologies - Systems and cybernetics
- Research Organisation
Gene therapies in general carry the potential to tackle most critical and difficult health issues, including rapid vaccine development against SARS-CoV-2 and rare debilitating hereditary diseases. Recent successes in clinical studies have increased interest in gene therapies, but bottlenecks persist including the lack of safe and efficient methods for gene delivery.
While viral vectors are a natural choice to deliver Nucleic Acids into the target cells, their use is limited: they have limited cargo capacity and carry major safety concerns. Electroporation is one of the most promising nonviral alternatives however current conventional electroporation relies on contact and invasive electrodes which inflict trauma to tissue, induce pain and discomforting muscle contractions, and struggle to produce clinically relevant titres in humans. The method’s suitability for clinical implementation is thus limited and development is hindered by a lack of understanding of fundamental mechanisms.
The proposed project emfDNA lays the bases for developing a noncontact, painless, and noninvasive electroporation approach, which promises to overcome the outlined deficiencies. emfDNA builds on our exciting preliminary data which show successful gene electrotransfer achieved with pulsed electromagnetic fields (emf) eventhough that induce electric fields up to three orders of magnitude lower than in classical electroporation enable cell membrane permeabilization. emfDNA investigates electroporation from the perspective of its underlying electromagnetic, biological, biochemical and biophysical mechanisms and provides the fundamental understanding necessary to theoretically ground and develop this gene delivery approach. With its demonstration in vitro, emfDNA will provide a gentle yet efficient gene delivery, with the potential to radically change the field of gene therapy.
Link to SICRIS.
- The phases of the project and their realization
The project will be implemented through specific tasks (T) organized in work packages (WP) and will enable delivering deliverables (D) described below. Throughout the duration of the project, special care will be given to disseminate results of the project to the scientific community through different communication channels and to identify and protect intellectual property if and when deemed necessary.
WP 1 – Membrane permeabilisation mechanisms of nnEP and EP in vitro: Based on accumulated theoretical evidence and current understanding of nucleic acids transport across the membrane we will first focus on lipid oxidation–membrane permeabilisation as plausible mechanism of nnEP. This hypothesis will be tested in vitro on cells (T1.1 and T1.2) and on planar lipid bilayers (T1.3). The results from WP1 will serve in developing membrane transient conductivity increase and molecular transport models developed in WP 3.
M1: Confirmation of hypothesis – lipid oxidation is responsible for increased membrane permeability (12)
D1.1: Exposure parameters leading to consistent membrane permeabilisation for cells in vitro (12)
D1.2: Correlation between duration/magnitude of membrane permeability and level of lipid oxidation (18)
D1.3: Changes of electrical properties of lipid bilayer as a result of lipid oxidation (12)
WP 2 – Mechanisms linking electroporation, tissue damage, cell injury/repair, and cell death: Cell stress, injury, cell membrane repair and cell death following nnEP (using nnEP parameters determined in WP1) will be investigated in vitro (T2.1) by determining Damage-Associated Molecular Patterns (DAMP) molecules release from cells, stress and cell death related gene expression and other markers of different pathways of cell death (T2.2). Reducing inflammation and stress to cells will form the basis for sustained and high transgene expression.
M2: Confirming reduced injury, cell stress and secretion of DAMPs in comparison to classical EP (24)
D2.1: Determining DAMP release and transcription following nnEP in comparison to classical EP (18)
D2.2: Determining cell death pathway due to nnEP in comparison to classical EP (24)
WP 3 – Evaluation of nucleic acids transport and gene expression with nnEP and EP: We will investigate the efficiency of nnEP in introducing various NA (e.g. plasmid DNA – pDNA, messenger RNA – mRNA, self amplifying mRNA – saRNA and silencing RNA – siRNA) into cells. Research in vitro on different cells and parameters of nnEP identified in WP1 and WP2 will provide relevant answers with respect to overcoming identified barriers in gene delivery. In this WP we will quantify in vitro pDNA/RNA uptake and gene expression achieved by nnEP into different cells in comparison to classical EP with respect to the magnitude and duration of transgene expression (T3.1). Within this WP we will also develop numerical models for in vitro exposure systems to compare calculations with experimental results obtained in WP1 and WP2 and thus validate numerical results (T3.2).
M3: Achieving successful transfection of different cells by nnEP in vitro using pDNA
D3.1: Dosimetry of in vitro nnEP exposure systems used in experiments in WP1-2 (36)
D3.2: Identification of nnEP parameters achieving gene transfection for different cells in vitro (36)
- Citations for bibliographic records
Link to SICRIS.