Core-shell Structured Chitosan-polyethylenimine Nanoparticles for Gene Delivery: Improved Stability, Cellular Uptake, and Transfection Efficiency

Gene therapy emerged as a promising treatment option for acquired and inherited diseases. The delivery of nucleic acids relies on vectors that condense and encapsulate their cargo. Especially non-viral gene delivery systems are of increasing interest. However, accomplishing therapeutic transgene expression levels and limited tolerability of these systems remain a challenge. Therefore, we investigate in the improvement of nucleic acid delivery using depolymerized chitosan – polyethylenimine DNA complexes (dCS-PEI/DNA). These core complexes are further entrapped into chitosan-based shells, functionalized with polyethylene glycol and cell penetrating peptides. This modular approach allows to evaluate the effect of functional shell components on physico-chemical particle characteristics and biological effects. The optimized ternary complex combines a core-dCS-LPEI/DNA complex with a shell consisting of dCS-PEG-COOH, which resulted in improved nucleic acid encapsulation, cellular uptake, transfection efficiency, and transfection potency in human hepatoma HuH-7 cells and murine primary hepatocytes. Effects on transgene expression are confirmed in wild-type mice following retrograde intrabiliary infusion. After administration of only 100 ng complexed DNA, ternary complexes induce a high reporter gene signal for three days. We conclude that ternary core-shell structured particles comprising functionalized chitosan are a promising gene delivery technology for both in vitro as well as in vivo applications.

Engineering of chitosan-based derivatives for non-viral gene delivery

Laura Camille Louise Nicolle

Gene therapy offers the possibility to treat or even cure diseases originating from genetic defects by introducing a therapeutic gene in target cells or correcting the initial defective gene. Amongst different options, non-viral vectors rely on the delivery of such genes using vehicles composed of lipids, inorganic nanoparticles or polymers. These systems are designed to protect the genetic material, while efficiently delivering it to the cells of interest. The initial backbone can be complemented with various components to help it overcome the many physiological barriers it may encounter during its journey. Amongst polymers, chitosan is a polysaccharide presenting many advantages for such purpose. Besides its biocompatibility and biodegradability, its potential for functionalization, thanks to numerous primary amines and hydroxyl groups, make it a backbone of choice for the preparation of a polymeric delivery vehicle. This work describes the covalent functionalization of chitosan with other polymers, peptides, and small molecules to later form polymeric nanoparticles condensing therapeutic DNA. Each component brings additional features to the initial system, thus finely tuning it for biological applications. The final system aims at treating liver inherited diseases such as ornithine transcarbamylase deficiency and phenylketonuria.This manuscript first exposes the preparation of a chitosan-based cationic system able to properly condense anionic DNA through electrostatic interactions. Due to its excellent DNA condensation properties, polyethylenimine was grafted to chitosan via a succinyl linker. The first in vitro investigations confirmed the potential of the resulting core system, but in vivo experiments highlighted toxicity coming from the remaining cationic charge on the nanoparticles. In order to better shield this cationic charge, bifunctional polyethylene glycol chains terminated by carboxylic acid moieties were grafted to chitosan amines. The resulting polymer was used to coat the first polymeric system through electrostatic interactions between the carboxylate groups and the remaining free amines of the chitosan-polyethylenimine derivative. The newly formed core-shell complex showed sustained cell viability in vitro and induced higher transfection efficiency of the gene as compared to the core system alone. However, its in vivo evaluation via retrograde intrabiliary infusion did not confirm such results: although transfection efficiency was retained, the core-shell complex did not significantly decrease the toxicity previously reported.The last part of this work includes the decoration of the core-shell system with targeting ligands making possible to perform in vivo assays via intravenous injection, for which the developed polymeric systems did not induce toxicity in preliminary experiments. So far, only a proof-of-concept with folic acid, an anti-cancer targeting ligand, was achieved. To do so, the shell polymer was further functionalized with folic acid-derived polyethylene glycol chains through strain-promoted azide-alkyne cycloaddition. The same strategy shall be applied in the near future with a liver-specific targeting ligand. Lastly, the improvement of the system's cell-penetration features was tackled by the grafting asparagusic acid-derived polyethylene glycol chains following the same strategy than for folic acid.

EPFL2022

Transient gene expression for rapid protein production

Natalie Muller

Recombinant proteins are important for biomedical research and for the treatment of human disease. Therefore it is necessary to develop reproducible bioprocesses to rapidly produce proteins of adequate quality and quantity. Expression in mammalian cells is preferred if the proteins are to be properly folded and post-translationally modified. For the rapid production of milligram to gram quantities of a protein in mammalian cells, large-scale transient gene expression is an attractive option. The two most important components of this technology are the cell cultivation system and the gene delivery method. For this reason the goal of this thesis was to develop novel cost-efficient cell cultivation systems and gene delivery methods for the large-scale transfection of mammalian cells in chemically defined media free of any animal-derived components including serum. Cultivation of mammalian cells in suspension is essential for large-scale transient gene expression. A superior system for the cultivation of mammalian cells based on the agitation of cells in "square-shaped" glass bottles (square bottles) was developed. It is an inexpensive but efficient means to grow cells to a high density without instrumentation. The growth and viability of cultures of both human embryo kidney (HEK) 293E and Chinese hamster ovary (CHO) DG44 cells in agitated one-liter square bottles exceeded that in spinner flasks, reaching cell densities 1.5 times higher on average. Furthermore, an efficient and reproducible method to transfect cells in agitated square bottles was developed for both HEK 293E and CHO DG44 cells. A novel gene delivery method termed calfection that relies on the addition of DNA and CaCl2 directly to a suspension culture was developed. Calfection has a number of advantages over existing gene delivery methods such as calcium phosphate-DNA coprecipitation in that there are no time-dependent steps. The DNA/CaCl2 solution can be stored for long periods and is filterable. It is suitable for both large-scale transfections in bioreactors and for high-throughput transfections in microtiter plates. One drawback, however, is its dependence on the presence of serum in the culture medium. A second gene delivery method, serum-free transfection with polyethylenimine (polyfection), proved to be highly efficient for the transfection of both HEK 293E and CHO DG44 cells. A reproducible method for polyfection in microtiter plates, 50 ml centrifuge tubes, agitated square and round bottles, spinner flasks, and bioreactors was optimized for these two cell lines. Polyfection proved to be a simple and successful gene transfer method in both instrumented and non-instrumented cultivation systems. Importantly, it could be performed in serum-free chemically defined media. With HEK 293E cells, 80 mg/l of antibody was produced in agitated square bottles and with CHO DG44 cells, more than 2 g of antibody were produced in one week in a 150-liter bioreactor.

EPFL2005

Accelerating the development time of recombinant mammalian cell lines producing high titers of protein therapeutics

Sébastien Chenuet

The forthcoming arrival on the market of numerous protein therapeutics that require high clinical doses will foster the need for high-producing mammalian cell lines. Furthermore, pressures to reduce the development time for new therapeutics has meant more emphasis on efforts to accelerate the process that yields such cell lines. The objective of this thesis work was to develop methods to decrease the time needed to generate stable and high producing mammalian cell lines. This could be achieved by overcoming some of the major limitations that hamper the process of cell lines development. The host systems chosen for this study were Chinese hamster ovary (CHO DG44) and baby hamster kidney (BHK-21). The recombinant proteins used as reporters were the green fluorescent protein (GFP) and a human anti-Rhesus D IgG. One limitation hampering the development of cell lines producing high recombinant protein titers is the extensive variability of transgene expression within a transfected population as a result of the gene delivery process. This decreases the reproducibility of the generation of high producing cell lines. A direct comparison of two gene chemical delivery methods, polyethylenimine (PEI)- and calcium phosphate (CaPO4)-mediated transfection, with a mechanical method (microinjection) provided some insights into ways to overcome this limitation. Indeed, a correlation was observed between the amount of plasmid DNA delivered into cells and the specific productivity of the recovered recombinant cell lines. Hence, by increasing the amount of DNA delivered per cell, IgG-producing clones with specific productivities up to 22 pg/cell/day were recovered with a high efficiency. A second limitation is the time needed to assure the stability and clonality of cell lines by current analytical methods. This was overcome by using GFP as a reporter for the level of production of a co-expressed therapeutic protein. The co-transfection of plasmids individually carrying the GFP and IgG heavy (Hc)- and light chain (Lc) genes, resulted in integration of all three plasmids at the same site in the cell's genome. As a consequence, the stability of GFP expression correlated with that of IgG expression. This proved to be advantageous finding and eliminating unstable cell lines early in the process of selection. As a consequence, it was possible to derive in serum-free medium clones with a specific productivity almost 6 times higher than that of clones derived from transfection performed with the IgG Hc and Lc vectors only. A third limitation to the rapid recovery of stable cell lines is the time devoted to the cultivation of transfected cells in selective medium, normally one month. Here, the time of selection was shortened to one week by increasing the stringency of the selective pressure. The results from studies of plasmid integration kinetics by fluorescence in situ hybridization (FISH) showed that the increased stringency of selection decreased the time needed to enrich a transfected population with recombinant cells. Furthermore, stable clones were shown to be present in this population after one week of selection and could be uncovered by identifying patterns of GFP expression predictive of production stability. By limiting the screening to clones exhibiting these stable patterns of GFP expression, the number of clones needed in order to isolate stable cell lines producing high IgG titers could be dramatically reduced. In conclusion, these results combined together provided a new procedure to isolate within a week of the start of selection, stable and high-producing clones. 30-40% of the clones isolated using this procedure were stable for at least three months and their average specific productivity was 29 pg/cell/day.

EPFL2008