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In the biopharmaceutical industry, essentially, there are three components that play the main role in producing biopharmaceutical products, the host cell, the expression vector and the bioreactor and/or production environment. To produce the highly valued and desired products, the choice of a suitable host is one of the most important aspects. The host required is not only required to produce the desired product, but also needs to demonstrate robustness in a bioreactor system. Constantly facing challenges in a bioreactor, cells often undergo apoptosis, a well-known limiting factor in biopharmaceutical production, which ultimately leads to low yield of valuable protein(s). We have genetically engineered a CHO-K1 cell line to constitutively express human insulin-like growth factor-1 (IGF-1) and murine polyoma large T-antigen (PyLT-Ag) to generate Super-CHO and CHO-T respectively, two cell lines that can potentially serve different niches in the biopharmaceutical industry. In the first part of the project, we hypothesised that suspension-adapted Super-CHO and CHO-T cells are both resilient cell lines relative to the suspension-adapted CHO-K1 (designated as CHO-XL-99) when facing nutrient depletion, one of the most common problems in a bioreactor. Furthermore, in the second part of this project, the suspension-adapted CHO cell lines were also tested against a cytotoxic heavy metal, cadmium. Without the protection of the metal-resistance element, metallothionein, both Super-CHO and CHO-T cells were also challenged with cadmium to demonstrate their robustness over the parental cell line, CHO-XL-99. In the subsequent study, this project also focussed on the transfection efficiency of each parental and engineered CHO cell lines. Different strategies have been employed in the past in an attempt to improve productivity in the biopharmaceutical industry, from alterations in vector construction, improved culture condition, down to enhanced product recovery. However, the transfer and expression of the gene-of-interest (GOI) has still proven to be the limiting factor for achieving increased specific productivity. In an effort to improve transfection efficiency, strategies including cell cycle synchronisation and various transfection methods to deliver the GOI into the cells have been employed. Thus, the third part of this project has used synchronising agents in conjunction with commercially available lipid- and polymer-based reagents as delivery vehicle for the model protein, EGFP. The combination of cell synchronisation and transfection vehicle on transfection efficiency is studied here, in addition to their individual or collective effect on cell growth, apoptosis and viability. In summary, this project demonstrates the incidence of apoptosis in the cell culture induced by nutrient depletion and heavy metal, and that the use of transfection reagents solely, or in combination with synchronising agents also correlates with the increase of apoptotic indices in the cell culture. The use of the robust cell lines for transfection is an important aspect, and the balance between cell viability and the effort for augmenting transfection efficiency has to be met in order to achieve the maximum biopharmaceutical yields.

Malaria is a widespread parasitic disease, causing 300-500 million infections per year and resulting in over 1 million deaths. There is widespread resistance of the parasite to most of the antimalarial treatments available, indicating the need for a vaccine (http://www.rbm.who.int/wmr2005/). The translationally controlled tumour protein (TCTP) family are highly conserved eukaryotic proteins that have been assigned a variety of functions. While most studies have focused on the intracellular functions of TCTP, human, malarial and other parasitic TCTPs have also been reported to have extracellular functions in the induction of histamine release from immune cells (e.g. MacDonald et al., 2001; Rao et al., 2002). Malarial TCTP has been detected in the sera of malaria-infected individuals (MacDonald et al., 2001) and is also known to bind to the antimalarial drug artemisinin (Bhisutthibhan et al., 1998). In this study, TCTP was investigated as a malarial vaccine candidate due to a previously observed protective effect in mice infected with Plasmodium yoelii YM. In that study, PfTCTP immunisation conferred a significant delay in disease progression, as judged by reduced parasitemia and prolonged survival (Taylor, 2002). It was thought that the protective effect might have been due to the inhibition of the extracellular actions of malarial TCTP by the acquired host immune response. P. falciparum and P. yoelii TCTP were initially expressed in S. cerevisiae, as in the previous study. The recombinant proteins were used to vaccinate mice, which were then challenged with two strains of P. yoelii. No protective effect was observed for either vaccine, and so the previous results using PfTCTP could not be confirmed. The TCTP of P. yoelii and P. berghei were then expressed in E. coli, which increased yield and decreased proteolysis. The recombinant proteins were used as vaccines in mice challenged with P. yoelii YM, P. c. chabaudi AS, or P. berghei ANKA. A significant delay in disease progression was observed in PyTCTP-immunised mice challenged with the non-lethal P.c. chabaudi, as determined by a significantly reduced parasitemia at each day post-infection leading up to a delayed peak parasitemia. A significant reduction in parasitemia was also observed in the early stages of P. yoelii YM infection in PyTCTP-immunised mice. P. berghei ANKA was used to challenge C57BL/6 mice to determine whether PbTCTP immunisation could protect mice from cerebral malaria development, no protective effect was observed. P. berghei ANKA was also used as a second lethal malaria challenge model in BALB/c mice, no significant differences in disease progression were observed in immunised mice. To further assess the functions of malarial TCTP, several attempts were made to create a TCTP-knockout strain of P. berghei ANKA. A TCTP-knockout malaria strain could be assessed for alterations in morphology, infectivity and artemisinin sensitivity compared with wild-type parasites. Initial genotype analysis of parasites resulting from several transfection experiments indicated that TCTP disruption had been successful, however TCTP-disrupted parasites were strongly selected against, and stable knockout strains could not be obtained. This indicates that TCTP performs an important role within the malaria parasite.

Notre travail concerne la transfection génique. Celle-ci nécessite l'envoi au noyau cellulaire d'ADN exogène, c’est-à-dire, d'une grosse molécule hydrophile, qui, normalement ne peut pénétrer dans la cellule que par endocytose. Or, un tel processus conduit habituellement à une dégradation de la molécule, en grande partie dans les lysosomes. Dans notre introduction, après un bref rappel sur l'endocytose, nous présenterons les moyens qui permettent à l'ADN exogène d'échapper à une hydrolyse intracellulaire et d'atteindre le noyau sous une forme fonctionnelle. Les résultats que nous avons présentés nous apportent des informations sur l’influence de l’injection hydrodynamique sur l’endocytose par le foie. Rappelons que notre travail avait deux buts principaux : contribuer à l’élucidation du mécanisme de la transfection hydrodynamique et rechercher si l’injection hydrodynamique pouvait présenter des avantages pour introduire des protéines dans les cellules hépatiques.

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Keung, Yiu Cheung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 58-59). / Abstracts in English and Chinese. / Contents --- p.i / Acknowledgement --- p.iii / Abstract --- p.iv / Abbreviations and Acronyms --- p.vi / Chapter Chapter 1 - --- Amphiphilic Dendrons and Gene Transfection / Chapter 1.1 --- Introduction to Dendrimers and Gene Transfection --- p.1 / Chapter 1.2 --- Overview of Gene Transfection Vectors in the Literature --- p.4 / Chapter 1.2.1 --- Viral Vectors --- p.5 / Chapter 1.2.2 --- Non-viral Vectors --- p.5 / Chapter 1.2.2.1 --- Cationic Lipids --- p.6 / Chapter 1.2.2.2 --- Cationic Polymers --- p.7 / Chapter 1.2.2.3 --- Cationic Dendrimers --- p.8 / Chapter Chapter 2 - --- En Route to Dendritic Amphiphilic Vectors / Chapter 2.1 --- Introduction to the Design of Dendritic Amphiphiles --- p.13 / Chapter 2.2 --- Barriers to Gene Transfection --- p.13 / Chapter 2.2.1 --- Cellular Barriers --- p.14 / Chapter 2.2.2 --- Extracellular Barriers --- p.16 / Chapter 2.2.3 --- Material Design Considerations --- p.17 / Chapter 2.3 --- Amphiphilic Vector Design Strategy --- p.18 / Chapter 2.4 --- Summary --- p.21 / Chapter Chapter 3 - --- "Design, Synthesis and Structural Characterization of New Amphiphilic Dendrons for Gene Transfection" / Chapter 3.1 --- Design of the New Amphiphilic Dendrons . --- p.23 / Chapter 3.1.1 --- Incorporation of Hydrocarbon Dendrons --- p.24 / Chapter 3.1.2 --- Synthesis Feasibility --- p.24 / Chapter 3.2 --- Synthesis of the New Amphiphilic Dendrons --- p.25 / Chapter 3.3 --- Characterization of the Intermediates and Targeted Amphiphilic Dendrons --- p.28 / Chapter 3.3.1 --- 1H NMR Spectroscopy --- p.28 / Chapter 3.3.2 --- I3C NMR Spectroscopy --- p.30 / Chapter 3.3.3 --- Mass Spectrometry --- p.33 / Chapter 3.3.4 --- Critical Micelle Concentration --- p.35 / Chapter 3.4 --- Conclusion --- p.38 / Chapter Chapter 4 - --- Determination of Transfection Efficiency and Cytotoxicity / Chapter 4.1 --- Preparation of the Amphiphilic Dendrons for Transfection --- p.39 / Chapter 4.2 --- Transfection Efficiencies of the Amphiphilic Dendrons 1-10 --- p.39 / Chapter 4.3 --- Cytotoxicity Assay --- p.42 / Chapter 4.4 --- Conclusions --- p.44 / Chapter Chapter 5 - --- Experimental Procedures / Chapter 5.1 --- General Information --- p.46 / Chapter 5.2 --- Experimental Procedures --- p.47 / References --- p.58 / Appendix / NMR Spectra --- p.60

Hepatocyte growth factor (HGF) is a multifunctional mitogen, stimulating cell proliferation, motility, angiogenesis and morphogenesis via activating its receptor, c-Met tyrosine kinase. Overexpression of HGF and c-Met has been shown as a characteristic of cancer transformation and metastasis. Inhibition of HGF/c-Met signaling may abrogate the malignant and metastatic states of cancer cells and offer a useful therapeutic approach for treating cancers. Thus with the aim of creating inhibitors to HGF-cMet signaling, we constructed plasmids containing truncated N-terminus of HGF, NK1, NK2, NK3 and NK4, respectively, and transfected into MDA MB 435S cells by electroporation. After selection with antibiotics, stable transfectants were obtained. Proliferation assay showed that the truncated NKs significantly inhibited the growth of the cells. Moreover, wound healing assay showed that migration of the NK-transfected cells were also significantly inhibited in comparison with GFP-transfected and nontransfected cells. These results therefore suggest that truncated NKs may be good inhibitors to HGF/c-Met signaling in the proliferation and migration of human breast cancer cells in vitro. In the future, the in vivo animal model is needed to be carried out to further clarify the clinical values of truncated NKs for application to cancer therapy.

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<p>Electric field mediated gene delivery (EFMGD) or electrotransfection is a popular, non-viral gene delivery method that has been used in a variety of studies and applications ranging from basic cell biology research to clinical gene therapy. Yet, the mechanism(s) by which electrotransfection facilitates DNA delivery across the cell membrane into the cell and its subsequent intracellular transport across the cytosolic space towards the nucleus have been insufficiently studied and still remain controversial. Understanding these mechanisms and characterizing the intracellular journey of pDNA is important for understanding the physiological barriers of EFMGD within the cell, which can be used to engineer better solutions to overcome these barriers with the ultimate goal of improving the transfection efficiency of this technology. </p><p>Conventional thought in the field assumes that such transport modes as diffusion, electrophoresis, and electro-osmosis, which govern the entry of small molecules into cells through electric field-generated transient membrane pores, also apply to electric field-mediated delivery of therapeutic DNA. We propose that electrically-induced gene transfer into cells is governed by an alternative, more active mode of transport that entails the involvement of cellular endocytic processes. It is our hypothesis that pulsed electric field generate these membrane pores which interact with nearby DNA molecules; but that actual DNA translocation across the membrane is driven by endocytosis, which consequently, then, also plays a role in the intracellular transport of the DNA. To this end, we first investigated the dependence of electrotransfection efficiency (eTE) on binding of plasmid DNA (pDNA) to plasma membrane. Binding concentrates DNA molecules in the vicinity of the cell membrane, which should theoretically result in a greater number of DNA-membrane interactions during pulsed electric field, more internalized DNA, and ultimately, higher eTE values. We demonstrated that supplementing the electrotransfection buffer with divalent cations (Ca2+ and Mg2+) is an effective method of promoting pDNA adsorption to the cell membrane. This cation-mediated increase in DNA adsorption to the cellular membrane resulted in a consequent increase in eTE, up to a certain threshold concentration for each cation. To determine the timeframe for completion of pDNA internalization following pulse treatment, trypsin treatment was applied to cells at different timepoints after electrotransfection to strip off any residual, membrane-bound pDNA that had not been internalized. Trypsin treatment at 10 min post electrotransfection still resulted in a significant reduction in eTE, indicating that the time period for complete cellular uptake far exceeded the lifetime (~ 10 msec) of electric field-induced transient pores. The role of endocytosis was further probed by noting the effect on eTE when cells were treated with three endocytic inhibitors (chlorpromazine, genistein, dynasore) targeting different internalization mechanisms or silenced of dynamin expression using specific, small interfering RNA (siRNA). siRNA silencing and all three pharmacological inhibitors yielded substantial and statistically significant reductions in the eTE. Taken together, these findings suggest that the mechanism of electric-field mediated DNA internalization entails: (i) binding of pDNA to cell membrane and (ii) endocytosis of membrane-bound pDNA.</p><p>The same strategies of pharmacological endocytic inhibition and siRNA silencing was used to further explore and compare electric field-induced pDNA internalization in additional cell lines that differ in terms of cell type, proliferation rates, proliferative capacity (i.e. primary versus immortalized/cancer line), etc. in order to determine whether endocytosis is a universally implicated mechanism across many cell lines. Results showed different endocytic pathways to be recruited for pDNA uptake in a cell-dependent manner and that one or multiple pathways may contribute to uptake within a cell line. </p><p>Taken together, the studies presented in this dissertation provide both indirect and direct evidence suggesting an endocytic role in the translocation of pDNA across the cell membrane and its intracellular routing towards the nucleus for EFMGD. These seminal findings could potentially lead to better understanding of the intracellular barriers encountered by EFMGD, more strategic optimization of electrotransfection parameters than the trial-and-error approach currently used, and enhanced transfection efficiencies.</p> / Dissertation