La thérapie génique est une approche thérapeutique proposant d’utiliser des acides nucléiques (ADN,ARN, oligonucléotides) comme médicament. Les poly(éthylènimine)s linéaire (lPEI) et branchée (bPEI) sont des vecteurs de références pour le transfert de gènes, dans le domaines des polymères cationiques. L’objectif de ces travaux est de modifier la lPEI par des dérivés de la L-histidine-N-acryloyle, la L-arginine-Nacryloyleou le 1-adamantane méthylamine-N-acryloyle, en utilisant la réaction de Michaël. Les études physico-chimiques de ces polymères mettent en évidence le comportement de ces vecteurs en solution aqueuse et tamponnée, dont les effets peuvent avoir une influence non négligeable sur la complexation de l’ADN et le transfert de gène. La poly(éthylènimine-co-éthylènimine-N-éthylamide-N-2(3(3H-imidazol-4-yl) propionate de sodium) (lPEI-N-his) comportant 10 à 20% de greffons histidine a permis d’obtenir d’excellents résultats en transfection in vitro. Ces polymères ont montrés une très faible cytotoxicité sur les différentes lignées cellulaires utilisées pour cette étude. / Gene therapy is a therapeutic approach aiming at introducing corrective genetic materials into a cellin order to alleviate the symptoms of a disease. The linear (lPEI) and branched (bPEI) poly(ethylenimine) arethe gold standard among the polymeric vectors. The aim of this work is to modify the lPEI by N-acryloyl-Lhistidine,N-acryloyl-L-arginine and N-acryloyl-1 adamantane methylamine residue using the Michaelreaction to improve the transfection efficiency of cationic polymers. The physicochemical studies of thesepolymers are undertaken in aqueous and buffer solutions, and a higher buffering property of these polymersis emphasized. The polyplexes formation is found to be influenced by the behaviour of the macromolecules in the buffer solution. Transfection experiments are conducted in vitro, His-lPEI bearing 10 to 20% histidine residues allowed remarkable transfection efficiency (up to 95%) compared to unmodified lPEI. Moreremarkably, this new kind of cationic polymers showed very low cytotoxicity on cell lines.

Nucleic acid therapy holds real promise to offer less severe (lower side effects) as a treatment for life threatening and difficult to treat diseases such as cancer, heart disease or Alzheimer's disease. Theranostic nanomaterials that combine diagnostic imaging and therapeutic delivery, have potential to minimize the amount of time and dosage required for the treatment. This is achieved via delivery of nanoparticles that carry therapeutic payload as well as imaging agents; these agents need to circulate in the body longer due to its (larger) size and selectively accumulate in the tumor regions through the enhanced permeability and retention (EPR) effect. We have designed novel lanthanide (Gd, Tb or La) containing polymers with oligoethyleneamine and lanthanide chelating units to incorporate DNA binding and imaging agent functionality. Protonable amines along the polymer backbone electrostatically interact with DNA and compact it into a nanoparticle. These nanoparticles can be imaged both in vivo (Gd analogues, magnetic resonance imaging) and intracellularly (Tb chelation, fluorescence spectroscopy). Polymers were synthesized via step-growth polymerization to achieve a degree of polymerization of 18-24 for different analogues with varying amine number (three to six, N3-N6) along the backbone. Dynamic light scattering performed on the polyplexes (polymer-DNA complexes) indicate that they are in nanometer size range (50-80nm). All the polymers used to form polyplexes exhibited low toxicity to cultured human Glioblastoma cells (U-87) and showed variable transfection efficiency dependent on structure, comparable to G4 (sold as Glycofect"), a commercial transfection agent previously developed in our lab. This dissertation describes the first studies by the Reineke lab to monitor polyplex formation and destabilization using lanthanide resonance energy transfer (LRET). Polyplexes were formulated with Tb chelated N5 polymer and tetramethyl rhodamine (TMR) labeled pDNA, which are "LRET pairs". We observed decrease in luminescence intensity of Tb polymer (donor) in close proximity of TMR DNA (acceptor) in an intact polyplex at different N/P ratios. However, upon destabilization of polyplexes by addition of salt or heparin solution, the increase in distance between donor and acceptor resulted in increase in the luminescence intensity of Tb polymer. With the LRET technique, we are able to monitor formation and destabilization of polyplexes by monitoring change in luminescence of the donor chromophore (Tb). Polyplexes formulated with non-paramagnetic analogues (La chelated) of N4, polyethyleneimine (PEI) and G4 were studied using NMR to quantify free vs. bound polymer in a formulation. The amount of free polymer was measured by integrating the broad resonances from nanometer-sized particles (polyplexes) with narrow peaks from free polymer chains. This was supported by using an internal reference method to quantify free polymer amount from known internal reference concentration. We observed an increase in the amount of free polymer with N/P ratio for all three systems and both the methods showed comparable results. / Ph. D.

Gene therapy is the process of delivering genetic material, such as DNA (encoding for an important protein) into a patient’s cells in order to treat a particular disease such as a genetic disorder or heart disease. This process of DNA delivery into cells is known as “transfection” and it is important that the efficiency of transfection be optimized such that a patient can obtain maximum therapeutic benefit from such a treatment. DNA is susceptible to being destroyed by harsh physiological environments prior to reaching its target. This problem can be diminished with the use of vectors that not only protect against harsh conditions but also encourage entry into cells. By mixing 1) DNA with 2) positively charged polymers, “polyplexes” form which protect DNA from degradation and increase transfection efficiency. The development of effective polyplex formulations requires optimization. In the work presented here, it was discovered that when polyplexes contained specific sequences within the DNA called “CpG”, this lowered transfection efficiencies and increased inflammatory responses compared to DNA without CpG, as measured using a mouse lungs model. Thus, DNA composition played an important role in influencing DNA transfection efficiency of polyplexes. Another aspect to take into account is the degree of positive charge of the polymer. We tested a new polymer called poly(galactaramidoamine) or PGAA. We found that this PGAA can form polyplexes with DNA and could be used in gene therapy. At the present time, mechanisms by which the polyplexes get inside and transfect the cells are still unclear. We also introduced a new system called high-content screening to the gene delivery field. This system offers automated measurements of transfection efficiency and cytotoxicity and could be used to reveal the polyplexes trafficking inside cells.

Several approaches have been employed for directing the intracellular trafficking of DNA to the nucleus. Cationic polymers have been used to condense and deliver DNA and a few specific examples using chitosan as cationic polymer have been described. The concerted efforts in gene therapy to date have provided fruitful achievements toward a new era of curing human diseases. A number of obstacles, however, still must be surmounted for successful clinical applications. Therefore, chitosan-plasmid and quaternised chitosan-plasmid complexes (polyplexes) were investigated for their ability to transfect COS-1 cells and the results were compared with Transfectam/DNA lipoplexes for transfection efficiency. All of the chitoplexes utilised in this study proved to transfect COS-1 cells, however to a lesser extent than the Transfectam/DNA lipoplexes, which served as a positive control. Complexes formed with quaternised trimethyl and triethyl chitosan oligomers, specifically TMO L and TEO L, proved to be superior transfecting agents compared to other chitosans. The molecular mass of chitosan is considered to influence the stability of the chitosan/DNA polyplex, the efficiency of cell uptake and the dissociation of DNA from the complex after endocytosis. In literature it was shown that the toxicity of the chitosan1DNA polyplexes is relatively low compared to viral gene and lipid non-viral delivery vectors. This study showed that the percentage viable COS-1 cells when transfected with the chitosan polymers, oligomers, quaternised chitosan polymers and quaternised chitosan oligomers (chitoplexes) was higher than the percentage viable cells when transfected with lipoplexes prepared with Transfectam with the MTT assay. The Transfectam/DNA lipoplexes induced cell damage and a decreased viability of COS-1 cells were found. Chitosan/DNA and quaternised chitosan/DNA complexes did not affect the viability of the cell line. The degree of quaternisation of the polymers and oligomers and molecular size proved to be two important factors when considering effective non-viral gene delivery. It can be concluded that chitosan, especially quaternised oligomeric derivatives are polysaccharides that demonstrate much potential as a gene delivery system. The high solubility and low toxicity of chitosan allow its use in a wide variety of applications in the pharmaceutical industry and, as shown in this study, in gene delivery. / Thesis (Ph.D. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2006.

Several approaches have been employed for directing the intracellular trafficking of DNA to the nucleus. Cationic polymers have been used to condense and deliver DNA and a few specific examples using chitosan as cationic polymer have been described. The concerted efforts in gene therapy to date have provided fruitful achievements toward a new era of curing human diseases. A number of obstacles, however, still must be surmounted for successful clinical applications. Therefore, chitosan-plasmid and quaternised chitosan-plasmid complexes (polyplexes) were investigated for their ability to transfect COS-1 cells and the results were compared with Transfectam/DNA lipoplexes for transfection efficiency. All of the chitoplexes utilised in this study proved to transfect COS-1 cells, however to a lesser extent than the Transfectam/DNA lipoplexes, which served as a positive control. Complexes formed with quaternised trimethyl and triethyl chitosan oligomers, specifically TMO L and TEO L, proved to be superior transfecting agents compared to other chitosans. The molecular mass of chitosan is considered to influence the stability of the chitosan/DNA polyplex, the efficiency of cell uptake and the dissociation of DNA from the complex after endocytosis. In literature it was shown that the toxicity of the chitosan1DNA polyplexes is relatively low compared to viral gene and lipid non-viral delivery vectors. This study showed that the percentage viable COS-1 cells when transfected with the chitosan polymers, oligomers, quaternised chitosan polymers and quaternised chitosan oligomers (chitoplexes) was higher than the percentage viable cells when transfected with lipoplexes prepared with Transfectam with the MTT assay. The Transfectam/DNA lipoplexes induced cell damage and a decreased viability of COS-1 cells were found. Chitosan/DNA and quaternised chitosan/DNA complexes did not affect the viability of the cell line. The degree of quaternisation of the polymers and oligomers and molecular size proved to be two important factors when considering effective non-viral gene delivery. It can be concluded that chitosan, especially quaternised oligomeric derivatives are polysaccharides that demonstrate much potential as a gene delivery system. The high solubility and low toxicity of chitosan allow its use in a wide variety of applications in the pharmaceutical industry and, as shown in this study, in gene delivery. / Thesis (Ph.D. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2006.

Charles University, Faculty of Pharmacy in Hradec Králové, Department of Biochemical Sciences University of Vienna, Faculty center for Pharmacy, Department of Pharmaceutical Chemistry, Laboratory of MacroMolecular Cancer Therapeutics Candidate: Dajana Mihaličoková Supervisor (Charles University): PharmDr. Anna Jirkovská, Ph.D. Supervisor (University of Vienna): Univ.Prof. Dipl. Ing. Dr. Manfred Ogris Co-supervisor (University of Vienna): Dr. Haider Sami, Ph.D. Title of diploma thesis: In vitro assays for investigating nucleic acid assay Keywords: transfection, splice correction, BCA assay, polyplexes One of the most important tasks of biochemical research is to find out the right way how to cure cancer, genetic disorders and other illnesses which are still not curable. Towards this, gene therapy is emerging as a potential treatment owing to its ability to deliver genetic material inside the cell. Reporer gene based transfection process can be used to study gene expression. Transfection is mediated by vectors, either of viral or non-viral origin. Non-viral vectors offer several advantages over the viral counterparts like easier to synthesize, relatively cheap and the most important is their non-immunogenicity. Cationic polymers based on polyethylenimine form complexes with plasmid DNA reffered to as...

Orientador: Prof. Dr. Fernando Carlos Giacomelli / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biotecnociência, 2016. / A internalização celular de acidos nucleicos requer um agente de transfeccao, uma vez que o DNA isolado nao e capaz de atravessar membranas lipidicas. Embora diversos agentes polimericos de transfeccao tenham sido estudados, nenhum dos veiculos de entrega de gene propostos preenche todos os requisitos necessarios para uma terapia eficaz (capacidade de condensar e compactar o DNA, estabilidade em ambiente plasmatico, baixa toxicidade, eficiencia de internalizacao, escape endossomal e liberacao intracelular de material genetico). Nesta direcao, foram produzidos poliplexos a partir da complexacao entre copolimeros cationicos e DNA. Os poliplexos foram preparados a partir de DNA de timo de bezerro (ctDNA) dissolvido em tampao fosfato. Medidas de espectroscopia de correlacao de fluorescencia (FLCS), potencial-¿ê e eletroforese em gel de agarose indicaram complexacao eficiente entre as cadeias de DNA e os copolimeros em bloco PEO113-b-PDEA50 e POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47). Os copolimeros em bloco foram capazes de condensar as cadeias de DNA em particulas com tamanho desejado para absorcao celular por meio das vias de endocitose (RH ~ 65-85 nm). Os resultados obtidos por AFM, SAXS e SLS sugerem que as estruturas supramoleculares sao esfericas, porem de elevado grau de hidratacao. Os dados de ITC revelaram que a ligacao polimero/DNA e endotermica, e consequentemente o processo e governado por efeitos entropicos. A combinacao dos resultados sugerem que POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47) condensa as cadeias de DNA de forma mais eficiente do que PEO113-b-PDEA50. Mostrou-se tambem por medidas de espectroscopia de dicroismo circular que a conformacao do DNA permaneceu estavel, mesmo apos a complexacao e os poliplexos se mostraram estaveis em meio plasmatico. Os resultados obtidos de viabilidade celular mostram que ambos os sistemas apresentam baixa citotoxicidade, entretanto ensaios de microscopia e citometria de fluxo mostram que os sistemas nao sao funcionais do ponto de vista de expressao de GFP. Porem, quando estes sistemas sao avaliados na presenca de cadeias polimericas livres de polietilenoimina (PEI) se obtem sucesso nas etapas da internalizacao e transfeccao genica indicando que a presenca de cadeias livres de PEI tem papel fundamental nos processos. / PThe intracellular delivery of nucleic acids requires a vector system as they cannot diffuse across lipid membranes. Although polymeric transfecting agents have been extensively investigated, none of the proposed gene delivery vehicles fulfills all the requirements needed for an effective therapy namely ability to bind and compact DNA into polyplexes, stability in serum environment, endosome-disrupting capacity, efficient intracellular DNA release and low toxicity. Accordingly, investigations aiming at safe and efficient therapies are still essential to achieve clinical success of gene therapies. Polyplexes based on the complexation between DNA and cationic copolymers have been produced. The polyplexes were prepared by previously dissolving DNA from calf thymus (ctDNA) in phosphate buffer saline. Measurements of FCS, ¿ê-potential and agarose gel electrophoresis indicated the complexation of DNA and some of block copolymer systems, namely PEO113-b-PDEA50 and POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47). The block copolymers were evidenced to properly condense DNA into particles with desirable size for cellular uptake via endocytic pathways (RH ~ 65-85 nm). The shape and surface morphology of the polyplexes were investigated using SLS, SAXS and AFM and the data suggest that the supramolecular structures are spherical, however, highly swollen by water. The ITC data revealed that the polymer/DNA binding is endothermic and therefore the process in entropically driven. The combo of results supports that POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47) condenses DNA more efficiently and with higher thermodynamic outputs as compared to PEO113-b-PDEA50. Finally, circular dichroism spectroscopy indicated that the conformation of DNA remained the same after complexation and that the polyplexes are highly stable in serum environment. The cell viability results show that both systems have low cytotoxicity, however microscopy and flow cytometry assays showed that the polymers are poor tranfecting agents themselves. Nevertheless, in the presence of PEI chains, cellular uptake and transfection efficiency are subtantially enhances point out the fundamental importance of positive charges in the processes.

No description available.

Le sujet de cette thèse concerne l'élaboration de vecteurs fonctionnels de transfert de gènes destinés à la thérapie génique de tumeurs.<br />Deux familles de vecteurs synthétiques fonctionnels ont été synthétisées afin d'améliorer d'une part la biodisponibilité des vecteurs dans les applications in vivo et d'autre part leur spécificité de ciblage des tissus tumoraux. <br />La première famille de composés est constituée de télomères et de cotélomères « furtifs » issus des réactions de (co)télomérisation. Le design des télomères est basé sur un squelette « diblock » composé d'une partie polyaminée de degré de télomérisation aléatoire et d'une partie hydrophile (polyéthylène glycol). Le design des cotélomères est basé sur un squelette aléatoire de motifs aminés et de structures hydrophiles (tétraéthylène glycol et trishydroxyméthyl). La deuxième famille de vecteurs synthétiques est issue de la synthèse de lipides et polymères cationiques conjugués à un ligand dérivé de l'antagoniste des récepteurs à chimiokines CXCR-4, l'AMD3100 ou bicyclame.<br />Le potentiel des (co)télomères « furtifs » en tant qu'agents de condensation et de transfert de gènes a été évalué in vitro sur des cellules de carcinome pulmonaire humain A549.<br />Le potentiel des conjugués ciblés lipidiques et polymériques à compacter l'ADN et à transfecter de manière spécifique des cellules par la voie récepteur médiée a été évalué à la fois sur des cellules n'exprimant pas le récepteur CXCR-4 (A549, T98G) et sur des cellules l'exprimant (Jurkat).<br />Certains des (co)télomères « furtifs » testés (PEG2000-[NH2]n) ont démontré des efficacités de transfection comparables à des formulations lipidiques de référence (pcTG90/DOPE) tout en étant moins toxiques. Parmi les conjugués ciblés, le cotélomère iBu-[NH]80-[AMD]4 s'est révélé être un agent de transfert de gènes spécifique sur les cellules Jurkat sous certaines conditions de formulations, par rapport aux lipides et polymères conventionnels.<br />Mots clés : thérapie génique, transfert de gènes, lipoplexes, polyplexes, polyéthylène glycol, biodisponibilité, AMD-3100, ciblage, transfection.<br />Discipline : Chimie.

The present work describes a selection of strategies for the synthesis of functional aliphatic polycarbonates. Using an end-group functionalization strategy, a series of DNA-binding cationic poly(trimethylene carbonate)s was synthesized for application as vectors for non-viral gene delivery. As the end-group functionality was identical in all polymers, the differences observed in DNA binding and in vitro transfection studies were directly related to the length of the hydrophobic poly(trimethylene carbonate) backbone and the number of functional end-groups. This enabled the use of this polymer system to explore the effects of structural elements on the gene delivery ability of cationic polymers, revealing striking differences between different materials, related to functionality and cationic charge density. In an effort to achieve more flexibility in the synthesis of functional polymers, polycarbonates were synthesized in which the functionalities were distributed along the polymer backbone. Through polymerization of a series of alkyl halide-functional six-membered cyclic carbonates, semicrystalline chloro- and bromo-functional homopolycarbonates were obtained. The tendency of the materials to form crystallites was related to the presence of alkyl as well as halide functionalities and ranged from polymers that crystallized from the melt to materials that only crystallized on precipitation from a solution. Semicrystallinity was also observed for random 1:1 copolymers of some of the monomers with trimethylene carbonate, suggesting a remarkable ability of repeating units originating from these monomers to form crystallites. For the further synthesis of functional monomers and polymers, azide-functional cyclic carbonates were synthesized from the bromo-functional monomers. These were used as starting materials for the click synthesis of triazole-functional cyclic carbonate monomers through Cu(I)-catalyzed azide–alkyne cycloaddition. The click chemistry strategy proved to be a viable route to obtain structurally diverse monomers starting from a few azide-functional precursors. This paves the way for facile synthesis of a wide range of novel functional cyclic carbonate monomers and polycarbonates, limited only by the availability of suitable functional alkynes.