Non-viral delivery of plasmid DNA has traditionally relied upon physical forces applied directly to target tissues. These physical methods typically involve contact between an applicator and the target tissue and often cause transient patient discomfort. To overcome the contact-dependent limitations of such delivery methodologies, an atmospheric direct current plasma source was developed to deposit ionized gas molecules onto localized treatment sites. The deposition of charged species onto a treatment site can lead to the establishment of an electric field with strengths similar to those used for traditional electroporation. In vitro experiments proved that this technology could transiently permeabilize cell membranes and that membrane restabilization followed first order kinetics. Optimum delivery of tracer molecules to cell suspensions occurred after 10 minutes of plasma exposure and was attained without adversely effecting cell viability. In vivo testing of the plasma discharge demonstrated the capability of this system to deliver plasmid DNA to murine skin. Initial experiments involved the injection of plasmid DNA encoding luciferase into the dermis of C57BL/6J mice and then exposing the tissue to plasma discharge for 10 mintues. Delivery by this method resulted in increased luminescence that was as much as 19-fold greater than DNA injection alone. Follow-up optimization experiments demonstrated it was possible to obtain luminescence results that were similar in magnitude to those obtained using electroporation, which under optimum conditions resulted in about a 40-fold increase in peak luminescence. Finally, optimum conditions were used to deliver a plasmid DNA encoding for the 120 kilodalton glycoprotein present on the surface of a macrophage tropic HIV. Results from this vaccination experiment indicated this method was capable of producing antigen specific humoral immune responses at similar levels as when electroporation was utilized as the delivery method.

Gemini surfactants are versatile gene delivery agents because of their ability to bind and compact DNA and their low cellular toxicity. The aim of my dissertation work was to develop non-invasive mucosal formulations of novel amino acid-substituted gemini surfactants with the general chemical formula C12H25(CH3)2N+-(CH2)3-N(AA)-(CH2)3-N+(CH3)2-C12H25 (AA= glycine, lysine, glycyl-lysine, lysyl-lysine). These compounds were formulated with a model plasmid DNA, encoding for interferon-γ and green fluorescent protein, in the presence of helper lipid, 1,2 dioleyl-sn-glycero-phosphatidyl-ethanolamine. Formulations were assessed in Sf 1 Ep epithelial cells. Among the novel compounds, plasmid/gemini/lipid (P/G/L) nanoparticles formulated using glycine- and glycyl-lysine substituted gemini surfactants achieved significantly higher gene expression than the parent unsubstituted compound. The key physicochemical properties, e.g. size, surface charge, DNA binding, and toxicity of P/G/L complexes were correlated with transfection efficiency. The presence of amino-acid substitution did not interfere with DNA compaction and contributed to an overall low toxicity of all P/G/L complexes, comparable to the parent gemini surfactant. A cellular uptake mechanistic study revealed that both clathrin- and caveolae-mediated uptake were major uptake routes for P/G/L nanoparticles. However, amino acid substitution in the gemini surfactant imparted high buffering capacity, pH-dependent increase in particle size, and balanced DNA binding properties. These properties may enhance endosomal escape of P/12-7NGK-12/L resulting in higher gene expression. Finally, the P/G/L complexes were incorporated into an in-situ gelling dispersion containing a thermosensitive polymer, poloxamer 407, and a permeation enhancer, diethylene glycol monoethyl ether (DEGEE). A 16% w/v poloxamer concentration produced a dispersion that gelled at body temperature and exhibited sufficient yield value to prevent formulation leakage from the vaginal cavity. The formulations were prepared with a model plasmid, encoding for red fluorescent protein, and administered topically to rabbit vagina. In agreement with our in vitro results, confocal microscopy revealed that glycyl-lysine substituted gemini surfactant exhibited higher gene expression compared to the parent unsubstituted gemini surfactant. This provided proof-of-concept for use of amino acid-substituted gemini surfactant in non-invasive mucosal (vaginal) gene delivery systems with potential therapeutic applications. These formulations will be developed with therapeutically relevant genes to assess their potential as genetic vaccines. In addition, new gemini surfactants will be developed by grafting other amino acids via glycine linkage to retain conformation flexibility and enhance endosomal escape of DNA complexes for higher transfection efficiency.

No description available.

Linear covalently closed (LCC) DNA minivectors serve to be superior to conventional circular covalently closed (CCC) plasmid DNA (pDNA) vectors due to enhancements to both transfection efficiency and safety. Specifically, LCC DNA minivectors have a heightened safety profile as insertional mutagenesis is inhibited by covalently closed terminal ends conferring double-strand breaks that cause chromosomal disruption and cell death in the low frequency event of chromosomal integration. The development of a one-step, E. coli based in vivo LCC DNA minivector production system enables facile and efficient production of LCC DNA minivectors referred to as DNA ministrings. This novel in vivo system demonstrates high versatility, generating DNA ministrings catered to numerous potential applications in gene therapy and vaccine development. In the present study, numerous aspects pertaining to the generation of gene therapeutics with LCC DNA ministrings have been explored with relevance to both industry and clinical settings. Through systematic assessment of induction duration, cultivation strategy, and genetic/chemical modifications, the novel in vivo system was optimized to produce high yields of DNA ministrings at ~90% production efficiency. Purification of LCC DNA ministrings using anion exchange membrane chromatography demonstrated rapid, scalable purification of DNA vectors as well as its potential in the separation of different DNA isoforms. The application of a hydrogel-based strong Q-anion exchange membrane, with manipulations to salt gradient, constituted effective separation of parental supercoiled CCC precursor pDNA and LCC DNA. The resulting DNA ministrings were employed for the generation of 16-3-16 gemini surfactant based synthetic vectors and comparative analysis, through physical characterization and in vitro transfection assays, was conducted between DNA ministring derived and CCC pDNA derived lipoplexes. Differences in DNA topology were observed to induce differences in particle size and DNA protection/encapsulation upon lipoplex formation. Lastly, the in vivo DNA minivector production system successfully generated gagV3(BCE) LCC DNA ministrings for downstream development of a HIV DNA-VLP (Virus-like particle) vaccine, thus highlighting the capacity of such system to produce DNA ministrings with numerous potential applications.

Orientadores: Adriano Rodrigues Azzoni, Anete Pereira de Souza / Texto em português e inglês / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T01:06:03Z (GMT). No. of bitstreams: 1 Favaro_MariannaTeixeiradePinho_M.pdf: 18876970 bytes, checksum: 4953fcf4a875c09d8246f6deb457b544 (MD5) Previous issue date: 2012 / Resumo: Entrega gênica é uma estratégia muito promissora com grande potencial médico, que consiste na introdução de ácidos nucléicos exógenos, e pode ser aplicada tanto para terapia gênica quanto para vacina de DNA. Contudo seu uso ainda é limitado pela falta de um vetor de entrega ideal, que seja ao mesmo tempo eficiente e seguro. Embora muito mais eficientes, os vetores virais ainda despertam preocupações a respeito de sua segurança. Por outro lado, vetores não-virais são muito mais seguros e facilmente manipuláveis, ainda que menos eficientes. Neste contexto, "vírus artificiais" são uma opção interessante, uma vez que são vetores não-virais desenvolvidos para explorar a arquitetura celular de uma forma eficiente, superando uma série de barreiras físicas, enzimáticas e difusionais, mas mantendo a segurança do DNA plasmidial (pDNA). O principal objetivo da abordagem estudada é explorar os motores moleculares, como dineína, para transportar cargas da periferia para o centrossoma de células de mamíferos através da rede de microtúbulos. Para isso, a cadeia leve Rp3 da dineína foi fusionada a um domínio de interação com DNA (DNA-binding) no N-terminal, e ao peptídeo membrano-ativo TAT no C-terminal. A proteína, nomeada T-Rp3, tem ainda um His-Tag. Esta proteína recombinante construída contém então diferentes domínios para promover condensação do pDNA (DNA binding), para facilitar a entrada na célula e no núcleo (TAT) e para aumentar o escape endossomal (His- Tag), além da própria Rp3 que deve assistir no tráfego intracelular, agindo assim diretamente na maioria dos principais obstáculos intracelulares enfrentados pelos vetores. Estudos de expressão indicam que a proteína recombinante é corretamente expressa em E. coli BL21(DE3). Experimentos de mobilidade em gel de agarose ("gel retardation assay") combinados com estudos de espalhamento de luz e potencial zeta indicam que a proteína efetivamente interage com o pDNA, formando complexos que são pequenos (~95 nm) e positivamente carregados (+28 mV na relação molar de pDNA:proteína 1:8000). Ensaios de transfecção em cultura de células HeLa indicam que T-Rp3 atinge uma eficiência de transfecção muito maior que a proteína nuclear Protamina (aqui usada como controle), chegando a ser 900 vezes maior a expressão relativa do gene repórter na relação molar de pDNA:proteína 1:8000. Na comparação com Lipofectamina 2000TM, um reagente bem caracterizado de transfecção aqui usado como controle positivo, a T-Rp3 demonstrou atingir níveis similares de eficiência, com a vantagem adicional de ser menos citotóxica, conforme evidenciado em ensaios de viabilidade celular. Transfecções realizadas na presença da droga Nocodazol indicam que a eficiência da T-Rp3 depende fortemente da rede de microtúbulos, uma vez que a eficiência é reduzida em 92% quando os microtúbulos estão despolimerizados. A partir das transfecções na presença da droga Cloroquina, pudemos observar que o aprisionamento no endossomo ainda é um fator limitante. Finalmente, ensaios de cromatografia de afinidade realizados com o domínio da cadeia intermediária de dineína imobilizado indicam que a cadeia leve recombinante T-Rp3 mantém a capacidade de interagir com o complexo da dineína. Analisados em conjunto, os resultados apontam para uma grande participação da rede de microtúbulos na eficiência de transfecção de T-Rp3, objetivo inicial deste trabalho / Abstract: Gene delivery is a promising technique with great medical potential that consists in the introduction of exogenous nucleic acids, and can be applied for gene therapy as well as DNA vaccination. However, its use is still limited by the lack of an ideal delivery vector, which is both safe and efficient. Although much more effective, viral vectors still raise several concerns about its safety. On the other hand, non-viral vectors are safer and easier to manipulate, but less efficient. In this context "artificial viruses" are an interesting option, since they are non-viral vectors intended to explore the cell's architecture in an efficient way, to overcome a series of physical, enzymatic and diffusional barriers, while still preserving the safety of plasmid DNA (pDNA) vectors. The main objective herein is to exploit molecular motors, like dynein, to transport cargoes from the periphery to the centrosome of mammalian cells via the microtubule network. For that, human dynein light chain Rp3 was fusioned to a N-terminal DNA binding domain and a C-terminal membrane active peptide, TAT. The protein, named T-Rp3, has additionally a His.Tag. The shuttle protein built contains therefore different domains to promote pDNA condensation (DNA binding), to increase cell and nucleus penetration (TAT) and to enhance endosomal escape (His.Tag), besides the Rp3 to assist in the cytosol trafficking, thus covering most of the major obstacles to the vectors in intracellular level. Expression studies indicate that the fusion protein was correctly expressed in soluble form using E. coli BL21(DE3) strain. Gel retardation assays, dynamic light scattering and zeta potential studies indicate an efficient complex formation between pDNA and the fusion protein, resulting in a particle that is both small (~95 nm) and potivelly charged (+28 mV in the molar ratio of pDNA:protein 1:8000) Transfection of cultured HeLa cells indicates that T-Rp3 has a much higher transfection efficiency when compared to the nuclear protein Protamine (here used as a control), reaching a 900-fold increase in expression of transfected reporter gene, both in the same molar ratio of pDNA:protein 1:8000. When compared to Lipofectamine 2000TM, a well-known transfection reagent here used as a control, T-Rp3 showed to reach similar levels of efficiency, but with the further advantage of being less cytotoxic, as observed in cell viability assays. Transfections performed in the presence of the drug Nocodazole indicate that T-Rp3 efficiency largely depends on the microtubule network, since its efficiency is reduced by 92% when microtubules are depolymerized. From transfections in the presence of Choroquine we can deduce that endosomal entrapment remains a limiting factor. Finally, affinity chromatography experiments performed with the immobilized domain of dynein intermediate chain demonstrate that the recombinant light chain T-Rp3 retains the ability to interact with the dynein complex. Taken together, these results point to a strong participation of the microtubule network in the enhanced efficiency of T-Rp3 / Mestrado / Genetica de Microorganismos / Mestre em Genética e Biologia Molecular

L’objectif repose sur l’élaboration de vecteurs d’ADN fonctionnalisés par des séquences peptidiques, DLC8-AS, ciblant les chaînes légères LC8 de la dynéine cytoplasmique, pour obtenir un transport actif jusqu’au noyau le long des microtubules (MTs). Des travaux précédents, menés sur des fluosphères fonctionnalisées par des DLC8-AS, ont montré une efficacité remarquable à condition de travailler avec de hauts taux de ligands. De tels niveaux de ligands ne sont pas transposables à des nanoparticules (NPs) d’ADN car ils affectent grandement leur stabilité colloïdale. Pour compenser cela, nous avons développé dans cette thèse, des NPs d’ADN faiblement fonctionnalisées (2-10 mol %) portant des dimères de DLC8-AS afin de bénéficier d'un effet dimérique vis-à-vis de la dynéine qui augmente l'affinité. Parmi les systèmes testés, 2 ont montré un gain lié à l’effet dimérique des DLC8-AS. Le 1er est basé sur un amphiphile cationique dimérisable de la cystéine, utilisé avec son homologue pegylé portant un motif DLC8-AS, pour produire, via l’oligomérisation des thiols, une population monodisperse de petites NPs d’ADN décorées (~60 nm). Les expériences menées sur cellules HeLa ont montré que les NPs décorées par les dimères de DLC8-AS avaient des efficacités de transfection améliorées (~250 fois) grâce à un mécanisme dépendant du système dynéine/MTs. Dans l’autre système, la surface de polyplexes de PEI a été décorée avec des amphiphiles octaarginine mono- ou bis-DLC8-AS. De façon remarquable, l’efficacité de transfection des polyplexes portant les ligands dimériques a été améliorée d’un facteur 50 par rapport au JetPEI standard. Ici encore, le mécanisme dépend des MTs. / The aim consists in engineering DNA carriers functionalized by peptide sequences, DLC8-AS, targeting the LC8 light chains of cytoplasmic dynein, to promote active transport towards the nucleus along the microtubules (MTs).Dépôt de thèseDonnées complémentairesPrevious works based on polystyrene fluospheres functionalized with DLC8-AS, showed a noteworthy transfection enhancement but as a cost of high levels of ligands. Such levels of functionalization are unsuitable for maintaining sufficient colloidal stability of DNA nanoparticles (NPs). In order to compensate for this, we developed in this thesis weakly functionalized DNA NPs (2-10 mol %) bearing dimers of DLC8-AS to benefit from a dimeric effect toward the dynein which increase the affinity. Among our designed systems, two revealed the benefit from taking advantage from the dimeric effect of DLC8-AS. The 1st one relies on a cationic and dimerizable cysteine based amphiphile, which was used with its dimerizable pegylated homologue containing DLC8-AS, to produce, through a thiol-disulfide oligomerisation process, a monodisperse population of small sized functionalized DNA NPs (~60 nm). Experiments carried out onto HeLa cells, showed that DNA NPs functionalized with DLC8-AS dimers exhibited enhanced transfection properties (~250 times) through a dynein/MTs dependant mechanism. The second consists in functionalizing the surface of PEI polyplexes with octaarginine amphiphiles carrying a mono- or bis-DLC8-AS. Remarkably, the transfection efficiency of polyplexes bearing the dimeric ligands was increased by a 50 times factor compared to the JetPEI golden standard. Here too, the mechanism strongly depends on MTs.

Ce travail consiste à utiliser deux systèmes microfluidiques de gouttes pour incorporer d'une part des acides nucléiques dans des liposomes cationiques et d'autre part étudier la dynamique de transfection dans des cellules mammifères. La première micropuce permet d'insérer de l'ADN dans des liposomes cationiques afin d'obtenir de manière reproductible des lipoplexes appropriés à la transfection de cellules dendritiques (DC). Plusieurs paramètres expérimentaux sont tout d'abord étudiés, tels que les débits d'entrée, l’entretien des propriétés des liposomes après leur traitement dans des micro-gouttes, les caractéristiques des lipoplexes (taille, polydispersité et charge) en fonction du rapport molaire de charge (R+/-) et de la géométrie de la puce. Ensuite, les lipoplexes produits dans des conditions optimisées: une micropuce avec un grand canal en serpentin et une région de division des gouttes qui diminuent la polydispersité des lipoplexes, fonctionnant à un rapport de débit eau/huile 0,25 et R+/- 1,5; 3; 5; 7 et 10; sont utilisés pour transfecter des DCs in vitro. Tous les lipoplexes transfectent les DCs, tout en offrant une activation des DCs. La seconde étape consiste à utiliser une micropuce à l'échelle de la cellule unique afin de contrôler les conditions de transfection et d'optimiser le rendement de production de protéines recombinantes. Ainsi, des cellules ovariennes de hamster Chinois (CHO-S) sont transfectées dans la micropuce avec différents types de lipoplexes (R+/- 1,5; 3; 5) dont la dynamique de transfection est suivie par la production de protéines vertes fluorescentes (GFP) et par la viabilité cellulaire. Cette micropuce a permis d'évaluer l’hétérogénéité des cellules transfectées, révélant la présence d'une sous-population produisant des niveaux particulièrement élevés de GFP. Ces hautes productrices (HP) ont une taille cellulaire plus importante que celle de la population moyenne. La charge des lipoplexes montre un rôle important pour transfecter CHO-S, puisque l’unique lipoplex chargé positif R+/- 5 produit plus de HPs. La quantité d’ADN délivrée influe sur la production de protéine, puisque R+/- 1,5 avec plus d’ADN augmente la productivité spécifique de GFP des HPs. Cette thèse est réalisée dans le cadre d'un programme de co-tutelle entre l'Université de Campinas, au Brésil, et l'École Polytechnique, en France. Ce travail a principalement contribué aux domaines de microfluidique et de délivrance de gènes. / This work aims to use one droplet-based microfluidic systems to incorporate nucleic acids into cationic liposomes and another one to study the mammalian cell transfection process. For this, the first step uses a droplet-based microfluidic system to complex cationic liposomes with pDNA in order to obtain reproducible and suitable lipoplexes to dendritic cells (DCs) transfection. For this purpose, some experimental parameters are investigated, such as inlet flow rates, the maintenance of liposomes’ properties after microfluidic processing, lipoplex characteristics (size, polydispersity and zeta potential) as function of molar charge ratio (R+/-) and microchip design. Then, lipoplexes produced in selected conditions: a microchip with large serpentine channel and split region, which decreases lipoplex polydispersity, operating at ratio aqueous/oil flow rate 0.25 and R+/- 1.5, 3, 5, 7 and 10; are used to transfect DCs in vitro. All lipoplexes transfect DCs while providing cells activation. The second step uses a single-cell microfluidic platform to investigate and control over the transfection conditions, in view of optimizing the recombinant protein production by transfected cells. In this context, Chinese hamster ovary cells (CHO-S) are transfected in microchip with different types of lipoplexes (R+/- 1.5, 3, 5) and monitored by green fluorescent protein (GFP) production and cell viability. The single-cell platform enables to assess the heterogeneities of CHO-S population, revealing the presence of a subpopulation producing significantly high levels of GFP. These high producers (HP) show increased cell size in comparison to the average population. Moreover, the charge of lipoplexes shows an important role to transfect CHO-S, since the unique positive charged lipoplex R+/- 5 produces more HPs. Additionally, the amount of pDNA delivered affects the protein production, since R+/- 1.5 with more pDNA increase GFP specific productivity of HPs. This thesis is a co-supervised program between University of Campinas, Brazil and École Polytechnique, France. In general, this work contributes to microfluidics and gene delivery areas.

Utilizing the diverse functionality of amino acids, a new class of amphiphilic graft copolymers has been synthesized, characterized, and explored for applications in biomaterials and nucleic acid delivery. This thesis research focused on the syntheses of oligopeptide-functionalized polyesters and polyolefins. Polyester functionalization was geared towards applications in biomaterials, tissue engineering, and drug delivery by incorporating sequences that promote cell-adhesion. These polyester- graft -oligopeptide materials were prepared by a 1,3-Huisgen cycloaddition reaction, "click" chemistry, of an azide-terminated oligopeptide (prepared by Fmoc-based solid phase peptide synthesis (SPPS)) and alkyne-containing polyester (synthesized by ring-opening polymerization). Following the syntheses of these materials, they were analyzed by nuclear magnetic resonance (NMR) and organic gel permeation chromatography (GPC). The oligopeptide-functionalized polyolefins were designed for nucleic acid complexation, and therefore the oligopeptide sequences were intended to incorporate positively-charged moieties ( e.g. , oligolysine) for DNA and short interfering RNA (siRNA) complexation. These graft copolymers, prepared by SPPS followed by ring-opening metathesis polymerization, have highly tunable structures that enable control over charge density and polymer backbone rigidity. Moreover, non-ionic hydrophilic grafts such as polyethylene glycol were integrated into these polyelectrolytes such that the charges along the polymer backbone are spaced accordingly while maintaining the hydrophilicity of the polymer. While numerous applications for such charged, "bio-tailored" materials can be envisioned, this work is geared towards positively-charged polyelectrolytes for their potential application in nucleic acid therapy, specifically the delivery of plasmid DNA and siRNA. These graft copolymers were characterized ( 1 H, 13 C NMR, organic and aqueous GPC), studied for their solution properties (static and dynamic light scattering), and investigated as polyplexes with plasmid DNA.

No description available.

No description available.