GC-03. Increasing transfection level during delivery of genetic material by polymer carriers of complex composition
Anastasia S. Bukreeva1, Anna S. Rogova1, Tatiana V. Machel3, Darya R. Akhmetova1, Alexander S. Timin1,2, Albert R. Muslimov1,2
1 Peter The Great St. Petersburg Polytechnic University, St. Petersburg, Russia
2 Pavlov University, St. Petersburg, Russia
3 ITMO University, St.Petersburg, Russia
Anastasia S. Bukreeva, phone: +7 (912) 578-67-96, email: firstname.lastname@example.org
Currently, gene therapy is a promising approach to the treatment of a wide range of diseases. Low efficiency of genetic material delivery is the main limiting factor for development of gene therapy, since the use of “naked” nucleic acids does not provide the desired result. Polymeric carriers protect the genetic material during its transport to the target cells, but it is also necessary to protect it after its entrance to the cell. Background activity of nucleases is considered a significant obstacle to efficient gene delivery using non-viral vectors. To resolve this problem, it is necessary to select an inhibitor that protects the genetic material from degradation and has a positive effect on the efficiency of transfection. The aim of this work was to evaluate the effectiveness of the peptide DNase II inhibitor (SLRLLQWFLWAC) in increasing the efficiency of transfection of mammalian cells.
Materials and methods
In this work, polyelectrolyte particles have been used, being obtained by applying layers of differently charged polymers (Polyarginine/Dextran sulfate) on calcium carbonate cores prepared in advance by co-precipitation of aqueous sodium carbonate solutions and calcium chloride salts. The model genetic material was plasmid DNA encoding green fluorescent protein. Variants of pDNA encapsulation into the core, between polymer layers and simultaneous packing into the core and into the layer were considered. The peptide inhibitor was added to the DNA at the 1:1 ratio. The in vitro experiments were carried out with HEK 293 cell culture. Different cell inoculation techniques were tested with the particles: they were added either to adherent cells, or were incubated with suspended cells, followed by seeding on culture plastic surface. Effects of fetal bovine serum (FBS) upon transfection efficiency was studied. The particles were added to the cells at a ratio of 100:1. Efficiency of transfection was assessed qualitatively by means of laser scanning confocal microscopy as well as quantitatively, using flow cytometry.
Analysis of the experimental results showed that the use of DNase II peptide inhibitor significantly increases the efficiency of transfection. The best results were obtained by incorporating genetic material both into the core and as a layer of a polymer particle. We have found that addition of particles to adherent cells, as well as usage of FBS-free media exerted positive effects upon the level of transfection.
In summary, the effect of a peptide inhibitor of DNase II upon transfection efficiency was investigated, and optimal conditions were determined for inoculation of mammalian cells with the particles containing genetic material.
This work was supported by the project of the Russian Science Foundation No. 19-75-10010.
Gene therapy, DNase inhibitor, transfection efficiency.