In collaboration with the Institute for pathophysiology (Faculty of medicine, University of Ljubljana) we developed the electroporation protocol and analyzed the mechanisms of introduction of short RNAs into cultured primary human myoblast cells. The protocols were optimized using siRNA against HIF-1α (Hypoxia Inducible factor 1α) mRNA. HIF is a transcription factor and key regulator of cellular oxygen homeostasis and has an important role in several pathologies. We optimized the electroporation medium and the parameters of electric pulses, which enabled us to achieve 80% silencing of HIF-1α mRNA. In parallel, we performed viability studies for the same electroporation conditions. Suppression of HIF-1α expression was first estimated by measuring HIF-1α mRNA with qPCR. Depletion of HIF-1α protein was subsequently confirmed with Western Blot. Due to involvement of HIF-1α in several muscle pathologies, the effect of HIF-1α silencing was confirmed also on two HIF-1α’s downstream gene targets; PGK (phosphoglycerate kinase) and VEGF (vascular endothelial growth factor). Manuscript of this research for the publication in an international journal is in preparation. The developed protocol will not only serve as a mean to study the mechanisms of introduction of gene material, but also for therapeutic purposes. HIF, PDK and VEGF (involved in angiogenesis) are involved in cellular metabolism and were identified as targets for cancer treatment. The research on primary human myoblast cells is performed in collaboration with the Laboratory for molecular neurobiology with the purpose of using electroporation as a method for introduction or silencing of therapeutic genes as a mean to treat specific disorders. Based on this work we were also invited to join the network of groups preparing COST proposal BIONECA.
F.01 Acquisition of new practical knowledge, information and skills
COBISS.SI-ID: 9892180We have developed a first 3D numerical model for optimization of gene electrotransfer in vivo of muscle tissue. It is the first study that presents optimization of electric parameters and electrode positions for gene electrotransfer in skeletal muscle. Furthermore, our numerical model is generic and can be applied to other tissues. The results can be used as a guideline for researchers in selecting optimal parameters for in vivo studies of electro-gene therapy and electro-DNA vaccination and could advance translation into clinical environment. Based on this we have established a collab oration with the leading research medical center (Herlev Hospital, Copenhagen) in Europe for translation and application of electrogene transfer for cancer therapy. We are preparing mutual publication where numerical modeling will be combined with data of muscles electrotransfer from in vivo study on pigs and on humans (clinical trial).
F.18 Transfer of new know-how to direct users (seminars, fora, conferences)
COBISS.SI-ID: 9805396