The aim of this research was to formulate a novel biodegradable biocompatible cationic microparticle vector for the delivery of DNA vaccines. The work builds upon previous research by Singh et al which described the adsorption of DNA to the surface of poly (D,L-lactide-co-glycolide) (PLG) microparticles stabilised with the surfactant cetyltrimethyl ammonium bromide (CTAB). This work demonstrated the induction of antibody and cellular immune responses to HIV proteins encoded on plasmid DNA adsorbed to the particle surface in mice, guinea pigs and non-human primates. The present research aim was to develop an adsorbed DNA vaccine with enhanced potency and increased safety compared to CTAB stabilised PLG microparticles (PLG/CTAB) by replacement of the surfactant CTAB with an alternative cationic agent. The cationic polymers chitosan and poly (N- vinylpyrrolidone/2-dimethylaminoethyl methacrylate), dimethyl sulfate quaternary (PVP-PDAEMA) were investigated as alternative stabilisers to CTAB. From a variety of initial formulations, the most promising vector(s) for DNA vaccination were selected based on physiochemical data (chapter 3) and in vitro DNA loading and release characteristics (chapter 4). The chosen formulation(s) were analysed in greater depth (chapters 3 and 4), and gene expression was assessed by in vitro cell transfection studies using 293T kidney epithelial and C2C12 myoblast non-phagocytic cell lines (chapter 5). The cytotoxicity of the microparticles and their constituents were also evaluated in vitro (chapter 5). Stability and suitability of the formulation(s) for commercial production were assessed by cryopreparation and lyophilisation studies (chapters 3 and 4). Gene expression levels in cells of the immune response were evaluated by microparticle transfection of the dendritic cell (DC) line 2.4 and primary bone marrow derived DCs (chapter 6). In vivo, mice were injected i.m. with the formulations deemed most promising on the basis of in vitro studies and humoral and cellular immune responses were evaluated (chapter 6).

Extensive investigations have been made to try and understand the physical properties and structures of condensed DNA phases in vitro during the past decades (Bloomfield, 1991, Marquet and Houssier, 1991, Bloomfield, 1996). The packing pathways of DNA molecules are three dimensional processes, and are not yet fully understood (Yoshikawa et al., 1997). Distinguishing different single DNA molecules at different locales in time in the presence of condensating or dissociating agents is crucial for understanding the mechanisms of packing and unpacking of DNA molecules. The aim of this study is to provide an increased understanding of the some of the pathways of packing and unpacking of DNA. This aim was achieved by monitoring in time and at molecular scale the interaction between the DNA and polyamidoamine dendrimers, as condensating poly cations, and by observing the dissociation of some of these condensates in time when they were exposed to DNase I enzyme, as a dissociating in vivo agent. The main techniques used were atomic force microscopy (AFM) and gel electrophoresis. We believe that the results could be beneficial to the understanding of the in vivo condensation and dissociation process of certain DNA morphologies. Chapter 1 will focus on providing an overview of the single molecule techniques used, atomic force microscopy as a means of detecting individual biomolecules in near physiological conditions with time and its application in monitoring non-viral gene delivery systems on surfaces. Methods used for gene delivery, and the PAMAM dendrimers as one of the recently applied polymer in non-viral gene delivery are also reviewed. The materials and methods used in this thesis were considered in chapter 2. Chapter 3 will concentrate on the factors effecting the interactions of generation 4, 6 and 8 PAMAM dendrimers on surfaces. An understanding of these interfacial interactions is important to understand their effects on the individual DNA molecules. This aim was achieved by using AFM as an imaging and force measuring tool to visualize and characterize the adsorption of these dendrimers on mica, gold and on alkanethiol self assembled monolayers (SAMs). Developing a deep understanding of the adsorption of DNA onto oppositely charged substrates would be of fundamental importance in understand the packing and unpacking pathways of these molecules. This philosophy is demonstrated in Chapter 4 in which the ability of the monovalent cations to facilitate imaging of DNA, and the effect of these monovalent cations in the partial condensation of DNA is explored. In addition, Chapter 4 introduces DNA imaging in the presence of divalent cations in liquid and in air. The folding pathways of dendrimer-induced DNA condensation with time on the surface of mica in aqueous environment were the targets of Chapter 5. In addition, the surface-influenced DNA condensation in the presence and absence of sufficient soluble cations and the ionic strength dependence were also studied. Structural volume and hence information regarding the number of plasmid molecules in each condensate was explored. Furthermore, the effect of loading ratio and generation type on the complex retardation in gel electrophoresis was investigated. Chapter 6 investigates the different mechanisms of the DNA-PAMAM dendrimer condensate relaxation and fragmentation by DNase I with time and explores the mechanisms of wrapping and unwrapping of the DNA on the larger generations of dendrimers. The final chapter, Chapter 7, discuses the progress made towards the aims of this thesis. Interestingly, this investigation is one of the first to apply atomic force microscopy operating in liquid to visualize at the molecular scale and in real time DNA molecules in the absence of multivalent cations, to explore the formation of DNA complexes with ethylene di amine PAMAM dendrimers that have real potential as gene delivery vectors and to investigate the different condensation and dissociation pathways of individual DNA molecules. Overall it is hoped that the work described in this thesis provides a step forward in the methods applied for AFM based nano-force biomolecular imaging with time and provides a valuable information that aid in developing a successful non-viral gene delivery system.

Gene therapy has a great potential for the treatment of a wide range of diseases. However, the development of a safe and efficient delivery vector is the major obstacle for gene therapy. Recently synthesized 2 - (dimethylamino) ethyl methacrylate 2-(methacryloxloxyethyl phosphorylcholine) (DMA-MPC) diblock copolymer was investigated in this work as a novel non-viral vector for gene delivery. It has been previously demonstrated that the cationic DMA block can condense DNA efficiently. The zwitterionic PC head groups are found naturally in the outer leaflet of biomembranes and are extremely biocompatible. It is thus proposed here that the MPC can act as a new steric stabilizer to the system. Different compositions of DMA-MPC diblock copolymers were evaluated. The MPC block with minimum length 30 monomeric units can successfully provide steric stabilization to the system, and reduce nonspecific cellular interaction by providing a steric barrier to the DNA complexes. However, long MPC chain can hinder the interaction between cationic DMA and DNA, leading to the formation of loosely condensed complexes which were more susceptible to enzymatic degradation. Therefore the composition of the copolymer must be carefully adjusted so that the DNA condensing and steric stabilization effect are well balanced. In order to investigate the cellular uptake mechanism DMA homopolymerDNA complexes, the effect of different endocytosis inhibitors was examined. Microtubules and actin filaments were involved in the uptake of DNA complexes, suggesting that the complexes were internalised by endocytosis. Both the clathrin- and caveolae- mediated pathway were responsible for the uptake of DNA complexes, and the former appeared to be the main route of entry. Finally, folic acid ligand was incorporated into the DMA-MPC copolymer in order to improve the specific targeting. Initial data showed that there was selective uptake of the folate conjugated system in folate receptor expressing cells possibly via receptor mediated endocytosis. However, parameters such as the optimum length of MPC component, number of ligands per DNA complex and the composition of the system need to be further investigated in order to maximize the specificity and transfection efficiency.

Στη γονιδιακή θεραπεία, οι κλινικές μελέτες μέχρι τώρα χρησιμοποιούν ιϊκούς φορείς για την μεταφορά του διαγονιδίου στα κύτταρα στόχους. Το DNA των ιϊκών φορέων ενσωματώνεται στο γενετικό υλικό των κυττάρων, και αυτό εμπεριέχει τον μεγάλο κίνδυνο της παρεμβολής στο ενδογενές πρόγραμμα γονιδιακής έκφρασης (μεταλλαξιγένεση λόγω ένθεσης). Μια λύση σε αυτή την ανεπιθύμητη κατάσταση είναι η χρήση επισωματικών φορέων και ιδιαίτερα όσων φέρουν χρωμοσωμικά στοιχεία. Παλαιότερα είχε αναφερθεί ότι ο πρότυπος επισωματικός φορέας pEPI που βασίζεται στο Scaffold /Matrix Attachment Region (S/MAR), παραμένει ως σταθερό επίσωμα για πολλές γενεές σε κυτταρικές σειρές ανθρώπου και ποντικού, αλλά δεν παραμένει για πολλές γενεές σε ανθρώπινα CD34+ κύτταρα. Για να ενισχυθεί η ικανότητα του φορέα να υποστηρίξει την γονιδιακή μεταφορά και συγκράτηση του σε πρωτογενή αρχέγονα/προγονικά αιμοποιητικά κύτταρα, πρώτον ενισχύθηκε η μεταγραφή του S/MAR χρησιμοποιώντας τους ισχυρούς υποκινητές EF1/HTLV ή SFFV για το διαγονιδίο της eGFP και δεύτερον προστέθηκε μια αλληλουχία έναρξης της αντιγραφής (IR) από το γενετικό τόπο των β σφαιρινικών γονιδίων. Στην εργασία αυτή έγινε μεταφορά των νέων αυτών φορέων με την μέθοδο της πυρινικής ηλεκτροδιάτρησης σε κύτταρα CD34+ που απομονώθηκαν από κινητοποιημένο περιφερικό αίμα δοτών μυελού των οστών τα οποία διαμόλυναν επιτυχώς. Στην συνέχεια τα διαμολυσμένα κύτταρα CD34+ επιλέχθηκαν με FACS και καλλιεργήθηκαν σε θρεπτικό υλικό μεθυλοκυτταρίνης. Μετά την πάροδο 14 ημερών, ανιχνεύτηκε, με μικροσκοπία φθορισμού, έκφραση της eGFP στις τελικά διαφοροποιημένες αιμοποιητικές αποικίες που προέκυψαν. / Gene therapy clinical trials are currently based on integrating viral vectors; this approach presents the major risk of insertional mutagenesis. A solution to this side effect could be the use of episomal vectors and particularly the ones carrying chromosomal elements.We previously reported that the prototype episomal vector pEPI, based on a Scaffold /Matrix Attachment Region (S/MAR), functions as a stable episome for many generations in human and murine hematopoietic cell lines, but mediates very low long term retention in human CD34+ cells. To enhance the vector’s potential for gene transfer into primary hematopoietic stem/progenitor cells, (a) was enforced transcription through the S/MAR by using the strong hybrid EF1/HTLV or SFFV promoters to drive expression of the upstream transgene (eGFP) and (b) was included the replication initiation region (IR) from the β-globin gene locus. In this thesis the new vectors where delivered by nucleofection in CD34+ cells isolated from mobilized peripheral blood of healthy donors; th cells were efficiently transfected. Moreover the the transfected CD34+ cells were separated with FACS and cultured in methylocyttarine containing medium. After 14 days, eGFP expression was readily detected by fluorescence microscopy in the differentiated hematopoietic colonies.

Η ποσοτική ή ποιοτική ανεπάρκεια του λυσοσωματικού ενζύμου γλυκοκερεβροσιδάση οδηγεί στην παθολογία της νόσου Gaucher με κεντρικό το ρόλο της συσσώρευσης του υποστρώματός της, του γλυκοκερεβροσιδίου, στα μακροφάγα. Για περισσότερες από δύο δεκαετίες χρησιμοποιούνται γ-ρετροϊικά οχήματα στην ερευνητική προσέγγιση της διόρθωσης του ελλείμματος μέσω γονιδιακής μεταφοράς του γονιδίου της γλυκοκερεβροσιδάσης. Όπως έχει αναδειχθεί και σε κλινικό επίπεδο, είναι επιτακτική η ανάγκη για το σχεδιασμό γ ρετροϊικών οχημάτων, τα οποία θα είναι όχι μόνο αποτελεσματικά αλλά και ασφαλή, με κύριο στόχο τον περιορισμό της πιθανότητας εξαλλαγής του κυττάρου από τις συνέπειες της ενσωμάτωσης («μεταλλαξιγένεση κατά την ενσωμάτωση»). Η παρούσα μελέτη έχει ως στόχο τον έλεγχο της αποτελεσματικότητας νέων και θεωρητικά ασφαλέστερων σε σχέση με προηγουμένως χρησιμοποιηθέντα σε προκλινικό και κλινικό επίπεδο γ-ρετροϊικών οχημάτων για τη γονιδιακή μεταφορά του γονιδίου της γλυκοκερεβροσιδάσης σε προγονικά αιμοποιητικά κύτταρα του ανθρώπου. Στο πρώτο μέρος παρουσιάζεται η μελέτη έξι νέων και θεωρητικά ασφαλέστερων γ ρετροϊικών οχημάτων με το γονίδιο της γλυκοκερεβροσιδάσης, τα οποία είναι Self Inactivating (SIN) οχήματα και φέρουν αλληλουχίες για τη βελτίωση της ολοκλήρωσης της μεταγραφής, είτε από τον ιό WHV (WPRE, Woodchuck hepatitis virus Post-transcriptional Regulatory Element) είτε από τον ιό SV40 (2xSV40 USE, Simian Virus 40 Upstream Sequence Element σε δύο διαδοχικά αντίγραφα). Καταδεικνύεται ότι αυτά τα νέα SIN γ ρετροϊικά οχήματα είναι ικανά να μεταφέρουν ένα ενεργό φυσιολογικό γονίδιο γλυκοκερεβροσιδάσης σε CD34+ ανθρώπινο πρωτογενή προγονικό αιμοποιητικό κυτταρικό πληθυσμό. Τα οχήματα αυτά εμφάνισαν διαφορές στην αύξηση της ενζυμικής δραστικότητας, που προέκυψε από τη διαγονιδιακή έκφραση, οι οποίες οφείλονταν εν μέρει σε διαφορές στην αποδοτικότητα της διαμόλυνσης. Το δεύτερο μέρος της μελέτης περιλαμβάνει τέσσερα νέα SIN γ-ρετροϊικά οχήματα με το γονίδιο της γλυκοκερεβροσιδάσης με αντίστοιχο αριθμό οχημάτων-μαρτύρων, τα οποία είναι σχεδιασμένα με στόχο την κυτταροειδική έκφραση στα μονοκύτταρα / μακροφάγα, ώστε να περιοριστούν οι κυτταρικοί πληθυσμοί που εκτίθενται στην πιθανότητα ενεργοποίησης πρωτογκογονιδίων από τη δραστικότητα ενός εσωτερικού υποκινητή. Τα οχήματα φέρουν τους υποκινητές των ανθρώπινων γονιδίων CD11b και CD68 σε αλληλουχίες που είχαν επιδείξει στο παρελθόν μυελοειδοειδική έκφραση σε πειράματα γονιδιακής μεταφοράς. Τα νέα αυτά οχήματα ελέγχθηκαν ως προς την ικανότητά τους να παρουσιάζουν ισχυρότερη έκφραση του διαγονιδίου μετά από μονοκυτταρική / μακροφαγική διαφοροποίηση διαμολυσμένου CD34+ ανθρώπινου πρωτογενή προγονικού αιμοποιητικού κυτταρικού πληθυσμού σε σχέση με την έκφραση σε ένα λιγότερο διαφοροποιημένο διαμολυσμένο κυτταρικό πληθυσμό. Τα αποτελέσματα του δεύτερου μέρος της μελέτης σε συνδυασμό με τα ήδη υπάρχοντα βιβλιογραφικά δεδομένα ενθαρρύνουν την περαιτέρω διερεύνηση του υποκινητή του CD68 στο πλαίσιο των προσπαθειών για μυελοειδοειδική γονιδιακή μεταφορά του γονιδίου της γλυκοκερεβροσιδάσης, χωρίς να ενθαρρύνουν την περαιτέρω διερεύνηση του υποκινητή του CD11b. / Quantitative or qualitative deficiency of the lysosomal enzyme glucocerebrosidase results in the accumulation of its substrate, glucocerebroside, in macrophages, leading to the pathology of Gaucher disease. For more than two decades of research, gammaretroviral vectors have been used for gene transfer of the glucocerebrosidase gene for the correction of the enzyme’s deficit. It has been shown, even on clinical level, that the design of efficient as well as safe gammaretroviral vectors, aiming mainly at eliminating “insertional mutagenesis”, is an imperative need. The present study aims at the determination of the efficiency of new and theoretically safer, gammaretroviral vectors, compared to previously used ones on preclinical and clinical level, for the gene transfer of the glucocerebrosidase gene into human hematopoietic progenitor cells. In the first part of the study, six new and theoretically safer gammaretroviral vectors of the glucocerebrosidase gene have been evaluated. All six vectors are Self-Inactivating (SIN) and bear sequences for the improvement of transcriptional termination. These are either the WPRE (“Woodchuck hepatitis virus Post-transcriptional Regulatory Element”) from the virus WHV or the 2xSV40 USE (two copies of “Simian Virus 40 Upstream Sequence Element” in tandem repeat) from the virus SV40. It is shown that these new gammaretroviral vectors are efficient in transferring an active wild type glucocerebrosidase gene copy into a CD34+ human primary hematopoietic progenitor cell population. All six vectors showed increased enzyme activity, as compared to the untransduced cells, albeit with differences amongst them, partly due to differences in the transduction efficiency. The second part of the study focuses on the analysis of four new SIN gammaretroviral vectors of the glucocerebrosidase gene and the corresponding number of control vectors, designed for cell-specific expression in monocytes / macrophages. The aim is to thus limit the range of cell populations to be exposed to possible proto-oncogene activation from the internal promoter activity. In these vectors the expression of a glucocerebrosidase gene is driven by one of the promoters of the human genes for CD11b and CD68, which have exhibited myeloid specific expression in gene transfer experiments in the past. These new vectors were studied for their effectiveness in leading to stronger transgene expression after monocytic / macrophagic differentiation of a transduced CD34+ human primary hematopoietic progenitor cell population, compared to a less differentiated transduced cell population. The results of this study, taken together with the current bibliographical data, are encouraging for further study mainly of the CD68 promoter in the context of the research approach to myeloid specific gene transfer of the glucocerebrosidase gene.