Gene Delivery with Hyperbranched Polylysine

The delivery of exogenous nucleic acids into mammalian cells is a valuable technique for basic research studies on the expression, regulation and function of genes and proteins. At the same time, gene delivery to cultivated mammalian cells has become fundamentally important as a technology for the production of therapeutic proteins for preclinical and clinical applications. Alongside with the applications of gene delivery in biotechnology, gene transfer emerged in the early 1990s as a potential therapeutical method in which a functional copy of the defective gene is introduced to provide the missing function. Polymeric cations are widely used as nonviral transfection agents. The efficiency of these vectors, however, is lower than that of viral vectors and has until now precluded their extensive use in applications where stable and high level gene expression is required. Therefore a better understanding of the parameters that govern transfection efficiency of polycationic vectors is crucial for rational design of novel gene carriers. Thus, the main motivation behind this research was to investigate the role of the architecture and molecular weight of the polycation on the efficiency of transfection. The efficiency of transfection is determined by the cytotoxicity of the gene carrier, its reversible complexation with DNA, and proper intracellular trafficking of the resulting complex. For this purpose the 17 member library was synthesized, containing linear (LPL), hyperbranched (HBPL) and dendritic polylysine (DPL) analogues covering a broad range of molecular weights. HBPL, a new class of cationic polymers prepared by polycondensation of L-lysine, was developed to explore the influence of very high molecular weights and randomly branched structures on cytotoxicity, transfection efficiency, and internalization. The thesis is divided into four chapters. Chapter 1 reviews the current state-of-the-art on gene carriers and the future prospects of the field. Additionally, the current use of polycationic gene carriers for the production of recombinant proteins (r-proteins) via a large-scale transient gene expression (TGE) is discussed. The main goal of the Chapter 2 was to determine the influence of the architecture and molecular weight of the polycations on their in vitro biocompatibility. The extent of acute cell death after short exposure was found to be molecular weight dependent. The acute cytotoxicity of high molecular weight polycations was triggered by the permeabilization of the cell membrane. At high concentrations of small molecular weight polycations, membrane destabilization was caused by increased osmotic pressure. In contrast, delayed cell death was correlated to the collapse of mitochondrial transmembrane potential, triggering caspase-dependent apoptosis after intracellular accumulation of the polycation. The onset and extent of the delayed mode of cell death was shown to be dependent on the molecular weight and degree of branching of the polylysine analogues. Based on these results, the differential long term cytotoxicity reflects the resistance of branched polylysine analogues to proteolytic degradation. Thus the degradability and cumulative cytotoxicty can be predetermined by fine-tuning the frequency of branching points in the architecture of peptidic polycation. Chapter 3 demonstrates the evolution of transfection activity with the molecular weight and degree of branching of polylysine analogues. In summary, the results show that under identical cell culture and transfection conditions, the most critical parameter for high gene expression is the structure of the gene carrier. HBPL is the most efficient gene carrier in comparison to LPL and DPL and results in gene expression levels comparable to polyethyleneimine (PEI). Physico-chemical characterization of complexes revealed that those derived from plasmid DNA and HBPL contained a high quantity of the free polycation in comparison to those formed by LPL and DPL. The presence of the free polycation in the course of endocytosis of the complex may provide an explanation for the high transfection efficiency of HBPL. Chapter 4 describes a feasibility study aimed at proving whether HBPL is potentially applicable to large-scale r-protein production via TGE. Gene delivery efficiency and yield of r-protein were evaluated in serum-free, suspension-adapted CHO-DG44 cells under conditions mimicking the industrial manufacture of r-protein by TGE. High molecular weight HBPL mediates transfection and r-protein expression at levels comparable to the ones obtained with PEI. The main advantage of HBPL is its partial enzymatic degradability, potentially decreasing its cumulative cytotoxicity in the course of TGE. Also, HBPL efficiently transfects cell lines and primary cells in the presence of serum, thus broadening the potential applications of HBPL as a novel gene carrier for basic research applications in molecular and cellular biology. In conclusion, it is possible to obtain an efficient gene carrier by simple rearrangement of the topology of poly-L-lysine.

Transient Gene Expression in HEK-293E Cells for Recombinant Protein Production

Sophie Nallet

The growing demand of biopharmaceutical products is boosting the market for therapeutic recombinant proteins (r-proteins). More than half of the 140 r-proteins that have gained approval for human therapeutic use are manufactured in mammalian cells that make possible complex post-translational modifications, like glycosylation. In the industry, r-proteins are manufactured by stable gene expression (SGE) following Good Manufacturing Practice (GMP) standards. Transient gene expression (TGE) in mammalian cells is an alternative method for the expression of r-proteins whose main advantages are rapidity and flexibility. TGE involves the expression of the protein of interest from plasmids, which are transfected into the cells with a non-viral DNA transfecting agent, usually polyethylenimine (PEI). DNA is transcribed from the plasmids without the latter having been integrated into the host genome. Despite the recent improvements in r-protein titers by TGE and the scale up of this method to the 100-L scale, TGE remains so far a tool for protein production for research purposes. Therefore, to determine if TGE can be used for manufacturing therapeutic r-protein following GMP standards, different features related to reproducibility and product quality from TGE of a recombinant anti-rhesus D immunoglobulin G in HEK-293E cells using linear PEI were characterized. To test the effects of the size distribution and chemical structure of PEI on TGE, different commercially available PEIs were characterized and their impact on transfection and r-protein expression were assessed. The results showed that both the molecular weight distribution and the chemical structure of the PEI had an effect on TGE. However, the small variations in size distribution and structure between the batches of PEI from the same supplier did not affect significantly the transfection and r-protein production by TGE. Then, the fate of PEI and plasmid DNA in cell culture over time and their removal from the final r-protein during purification were determined and measured. Most of the PEI and pDNA remained in the cell culture medium after transfection. However, both PEI and plasmid DNA, which are additional components in TGE compared to processes based on recombinant cell lines, could be reduced to a concentration below the limit of detection of the assays after Protein A affinity purification of the antibody. To test the reproducibility of a TGE process, 10 batches of transfected cells were run in 500-mL orbitally shaken bottles. The cell specific productivity of the antibody was 20.2 ± 2.6 pg.cell-1.day-1 on average for the 10 batches. The purity and size consistency of the recombinant antibody produced in those 10 batches was demonstrated by gel electrophoresis and size exclusion chromatography. Then, the glycosylation profile of the antibody produced by TGE in HEK-293E cells was determined by mass spectrometry and was reproducible over the 10 batches. Moreover, it was similar to the glycosylation profile of the antibody produced by 3 stable HEK-293E cell lines. Finally, as a proof of concept, TGE was applied for the development of a vaccine against the respiratory syncytial virus. The fusion protein of the respiratory syncytial virus was produced by TGE and used as an antigen for the development of a recombinant vaccine. TGE enabled the rapid evaluation of the candidate vaccine in animal trials. Overall, the results of this study suggest that TGE is a reliable and reproducible method for manufacturing r-proteins of a consistent quality, provided that some particular features, such as reagents quality and process parameters, are well defined and controlled. Since TGE makes possible the rapid production of virtually any protein, even toxic, it could be used to provide GMP approved biopharmaceuticals for clinical trials in a short time, reducing development costs of biological therapeutic products.

EPFL2010

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

Establishment of recombinant mammalian cell lines

Madiha Derouazi

Recombinant proteins are gaining in importance for therapeutic applications. The proteins are expressed in stable cell lines, within which recombinant DNA has incorporated into the host cells genome. Identification and isolation of extremely high producers from transfected cell populations is a tedious and time-consuming effort. We proposed two different approaches for the establishment of recombinant cell lines: affinity cell sorting and microinjection. Even with the help of single cell sorting and analysis systems (flow cytometry) rarely more than thousand different cell clones, stably expressing a desired gene can be evaluated. We are considering a new approach in which polyclonal cell populations will be established which co-express a protein of interest and an epitope-tagged cell surface receptor. A microbead fixed antibody specific to the epitope tag is hoped to assist in the isolation of rare, but highly productive cells from large suspensions of transfected cells. For this reason we have also developed an efficient method for the bulk transfection of CHO cells. On the side of vector-development for this approach, we have chosen to investigate the utility of a mouse serotonin 5-HT3 receptor as a cell surface marker. This receptor can be expressed at very high levels (up to 1 x 107 receptors per cell) in heterologous systems. The receptor is detectable on the cell surface by means of fluorescent ligands, which allows the determination of transfection efficiencies in suspension cultures. A Flag-tag was fused to the amino terminal end of the receptor without adverse effects on receptor function or ligand binding. Only 5 % of receptor DNA in the transfection cocktail was sufficient to detect positive cells in a population. The specific interaction of the epitope-tagged receptor with the cognate antibody fixed to microbeads was tested in comparison to non-transfected control cells using laser scanning confocal microscopy. A 80% enrichment of cells expressing GFP and the 5-HT3 receptor was achieved in 30 minutes. We have investigated microinjection as a gene transfer method for the establishment of recombinant mammalian cell lines. The conditions for the injection of plasmid DNA into either the nucleus or the cytoplasm of CHO-DG44 cells were established using pMYK-EGFP, an expression vector with the enhanced green fluorescent protein (EGFP) gene under the control of the murine CMV promoter and the puromycin resistance gene for selection. To estimate plasmid copy number during microinjection, it was first necessary to determine the injection volume by fluorescence quantification of injected FITC-dextran. For microinjection into the cytoplasm the volume ranged from 200 to 350 fl and for nuclear microinjection it was about 180 fl. We then investigated the impact of DNA concentration and injection pressure on transient EGFP transient expression following microinjection into the cytoplasm or nucleus. As a general rule, we observed that concentration of the DNA solution injected into the cells was the most important parameter regardless of the pressure or the volume of injection. Recombinant cell lines expressing intracellular EGFP were established following microinjection into the nucleus and the cytoplasm with linear and circular DNA. Selection with puromycin, clonal expansion, and cell banking were completed within three to four weeks post-microinjection. In parallel, recombinant cell lines were established after calcium-phosphate transfection with linear and circular DNA. The number of plasmid copies integrated was determined by Southern blot and the number and localization of integration sites was determined by fluorescence in-situ hybridization (FISH). Growth of the cells and stability of EGFP expression were investigated by cultivation over a two-month period. This approach presents several advantages over other DNA delivery methods such as control of the quantity and localization of DNA delivered into the cell and speed of recovery of recombinant cell lines. This approach may be relevant for the establishment of recombinant cell lines from cells that are not easily transfected by classical methods.

EPFL2005

Transient Gene Expression in HEK-293E Cells for Recombinant Protein Production

Sophie Nallet

EPFL2010

Transient gene expression for rapid protein production

Natalie Muller

EPFL2005

Establishment of recombinant mammalian cell lines

Madiha Derouazi

EPFL2005