Navegando por Palavras-chave "Terapia Gênica"

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    Desenvolvimento de nanopartículas magnéticas de sílica mesoporosa como vetores não virais em magnetofecção
    (Universidade Federal de São Paulo (UNIFESP), 2020-05-15) Francisco, Giorgio Fernando De Avelar [UNIFESP]; Bizeto, Marcos Augusto [UNIFESP]; Universidade Federal de São Paulo
    Gene therapy is based on introduction of exogenous genetic material into the cells aiming the treatment of genetic diseases from the inactivation/substitutions of defective genes or by the introduction of genes which can express therapeutic proteins. Incompetent recombination virus based vectors are the most utilized at this transfer, but instead of the high efficiency, there are many risks associated to the possibility of the expression of remaining viral genes, promoting intense immunologic response and generation of competent replication virus by recombination which can promote oncogenesis. Thence, the development of non-viral vectors, mainly based on inorganic nanoparticles has become an interesting technological and scientific topic. The transfection process is complex, and many cell barriers must be transposed by the vector. The cell entry, generally, occurs by endocytosis, which requires ideals charge and size. After passing through cell membrane, the vector is imprisoned in cell compartments which interior has acidic medium and rich in nucleases that degrade the genetic material. Nucleic acids released at cytosol in this stage can be transferred to the cell nucleus, passing through the nucleus membrane where it will be transcript. In this work, capacity (or efficiency) of magnetofection was evaluated in order to improve the rate of cell uptake by endocytosis of vectors based in mesoporous silica nanoparticles. The magnetofection process consists in cell transfection with magnetic nanoparticles by applying an external magnetic field. Initially, it was synthesized different types of mesoporous silica nanoparticles combined with superparamagnetic nucleus of magnetite (Fe3O4), which were chemically modified with propyldiethylenotriamine to enable the conjugation with molecules of plasmid DNA responsible to promote the expression of green fluorescent protein (GFP) in HeLa cells. Among the as-prepared magnetic vectors, just one of them presented propitious characteristics to transfection study. This vector was prepared by hydrolysis of silica inorganic precursor using sodium hydroxide as catalyst around magnetic nanoparticles in the presence of micelle aggregates of surfactant hexadecyltrimethylammonium bromide, to shape the formation of silica wall pores. The as-prepared nanomaterial infrared spectrum presented the main bands of the vibrational modes of magnetite, silica and propyldiethylenotriamine group. The N2 physissortion isotherms showed an improvement on the surface area of 14 m2 /g to 254 m2 /g after the covering, with an isotherm of type IV, which is characteristic of mesoporous material. The X ray diffractometry identified the presence of magnetite as a crystal at face centered cubic form and the amorphous silica. The transmission electron microscopy showed an agglomerate of spherical particles with a well defined core-shell structure. The colloidal stability of the propyldiethylenotriamine modified nanoparticles was evaluated in dispersion made in sodium chloride solution by zeta potential (26 ± 1mV) and by determination of hydrodynamic size (516 ± 74nm) by dynamic light scattering. Finally, the magnetofection of HeLa cells with the as-prepared magnetic nanoparticles showed a higher transfection rate (5.4 ± 3.2%) when compared to the control groups presenting a good perspective of its use in gene therapy.
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    Desenvolvimento de um microgel de alginato carreado com vetores adeno-associados para liberação sustentada
    (Universidade Federal de São Paulo (UNIFESP), 2020-01-30) Cinel, Victor Dal Posolo [UNIFESP]; Han, Sang Won [UNIFESP]; http://lattes.cnpq.br/0069955147703693; http://lattes.cnpq.br/6845346545995430; Universidade Federal de São Paulo
    Gene therapy consists in manipulating and inserting exogenous genetic material in a patient to treat diseases. Although gene transfer technologies have greatly improved, there are still several limitations such as uncontrolled vector release that leads to immediate and excessive release of vectors at the site of administration. The consequence of this uncontrolled release is the leakage of vectors to other tissues, reducing gene transfer efficiency in the desired tissues and increasing the transfer of vectors to unwanted tissues. One solution to this problem is the use of vectors encapsulated in a biodegradable gel for slow and continuous release. Droplet microfluidics enabled the production of small and homogeneous microgels as carriers. Using alginate in a microfluidic device, it is possible to produce biocompatible alginate microgels with a sustained release kinetics of molecules. Despite being a promising carrier for gene therapy, there are no studies in the literature on the encapsulation of non-integrative viral vectors using microfluidics. In this work, 1.2% alginate microgel delivery system with type 2 adeno-associated vectors (AAV) was evaluated for its use for gene therapy. To this end, microgels encapsulate with model nanoparticles and AAVs were produced by microfluidics using the competitive ligand exchange crosslinking (CLEX) method. The microgels were produced and their encapsulation and controlled release capacity were evaluated by fluorescence spectroscopy, realtime quantitative PCR and fluorescence microscopy techniques, the latter to analyze the in vitro transduction efficiency of encapsulated AAVs. For the characterization, techniques of phase contrast microscopy, scanning electron microscopy and atomic force microscopy were used. Results showed that the pure 1.2% alginate microgels, microgels loaded with nanoparticles and microgels loaded with AAVs presented an average size of 125 µm, 106 µm and 116 µm, respectively. While pure and AAV microgels were monodisperse and with a regular topography, microgels with nanoparticles were monodisperse and showed an irregular and porous topography. The average encapsulation efficiency was 70.9% for nanoparticles and 13.2% for AAVs. Release kinetics studies have shown that the microgels produced can release encapsulated nanoparticles and AAVs in a continuous manner. In vitro transduction studies with HeLa cells using microgels with encapsulated AAVs showed that despite the low encapsulation efficiency, the AAVs released from the microgels were able to transduce cells, validating this delivery system for use in gene therapy studies.
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    Estratégias de targeting para terapia gênica baseadas em um gene antitumoral
    (Universidade Federal de São Paulo, 2025-01-17) Cinel, Victor Dal Posolo; Tavassi, Ana Marisa Chudzinski; http://lattes.cnpq.br/6899353560628046; http://lattes.cnpq.br/6845346545995430
    Objetivo: Este trabalho teve como objetivo investigar o gene do Amblyomin-X com diferentes targetings intracelulares como um candidato a terapia gênica antitumoral. Neste estudo foi avaliado o impacto da expressão e direcionamento do Amblyomin X, uma molécula antitumoral derivada de uma biblioteca de cDNA da glândula salivar do carrapato Amblyomma sculptum, para diferentes compartimentos subcelulares e os possíveis efeitos citotóxicos, com foco em melanoma humano. Métodos: Foram desenvolvidos plasmídeos para expressar o Amblyomin-X em células humanas e direcioná-lo para diferentes destinos: secreção; translocação e retenção no retículo endoplasmático; translocação para as mitocôndrias; e uma construção para a produção do Amblyomin X sem endereçamento específico. A expressão do Amblyomin-X foi avaliada em células de melanoma SK-MEL-28 e em células não tumorais HEK-293T. Para avaliar a produção da proteína, foram utilizadas as técnicas de RT-qPCR, Western Blot e espectrometria de massas. Para avaliação do potencial citotóxico, foram realizados os ensaios de MTT, detecção de atividade de caspases 3/7 e avaliação de morte celular por citometria de fluxo. Mutações sítio-dirigidas em lisinas foram realizadas para tentar estabilizar o Amblyomin X intracelular, e o inibidor do proteassoma comercial MG132 foi utilizado para investigar o mecanismo de degradação do Amblyomin X. Além disso, foram realizadas tentativas de adaptar células HEK-293 para cultivo em meios com baixa concentração de FBS para viabilizar a purificação e avaliação do Amblyomin X secretado. Resultados: Embora todas as formas do Amblyomin X tenham sido detectadas em células HEK-293T, apenas as formas endereçadas para o retículo endoplasmático e secretadas foram identificadas em células SK-MEL-28. Não foi observado efeito citotóxico do Amblyomin X em nenhuma das duas linhagens celulares. Além disso, nas células tumorais de melanoma, foi identificada a degradação do Amblyomin X sem endereçamento e do Amblyomin X mitocondrial via ação do proteassoma. As mutações sitio-dirigidas não foram capazes de estabilizar o Amblyomin X intracelular, e os ensaios de inibição do proteassoma indicam que essas formas do Amblyomin X são degradadas de maneira independente de ubiquitinação e SUMOilação. A adaptação das células HEK-293T para meio sem FBS foi ineficaz, e a redução de FBS levou à parada da expressão do Amblyomin X secretado. Conclusões: O estudo indicou que a expressão do Amblyomin X diretamente nas células humanas enfrenta desafios, como a degradação proteassomal em células tumorais e a ausência de atividade antitumoral. Estudos para desvendar em detalhes a interação do Amblyomin X com o proteassoma são necessários para o desenvolvimento eficiente de uma terapia gênica antitumoral com o gene do Amblyomin X.
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    Terapia gênica de isquemia de membro de camundongo com os genes Gm-Csf e M-Csf
    (Universidade Federal de São Paulo (UNIFESP), 2017) Mansano, Barbara Sampaio Dias Martins [UNIFESP]; Han, Sang Won [UNIFESP]; http://lattes.cnpq.br/0069955147703693; http://lattes.cnpq.br/2545468105494605; Universidade Federal de São Paulo (UNIFESP)
    Cancer is the world's largest public health problem and the second leading cause of death in the United States. It is a disease associated with aging. Infections are a serious cause of morbidity and mortality in cancer patients, especially infections related to health care. The platelets play several important functions in homeostasis that go beyond their role in coagulation and thrombosis, to the immune system already described in cancer and infection, interrelated with co-stimulatory molecules of the immune response, such as OX40 and CD40L, and by adhesion to other cell groups forming the platelet aggregates (AGP).Understanding these molecules in this group of patients may help in clinical management. The objective of the study was to analyze the circulating platelet aggregate profile and the prevalence of OX40 and CD40L in elderly cancer patients with acute bacterial infection. A cross - sectional and translational study was performed with 91 (45 patients with infection - CI and 46 patients without infection - SI) and 45 controls. Serum levels sCD40L was performed by enzyme immunosorbent assay (ELISA). The determination of platelets (CD41a +), AGP / CD62P +, AGP / CD40L+ and OX40 on the cell surface was performed by flow cytometry. Mann-Whitney and Kruskal-Wallis tests were used for analysis of medians between two and three groups, respectively. Values of p <0.05 were considered significant. For statistical analysis, GraphPadPrism v6.0 was used. Elevated basal levels of CD3 + /OX40 + T lymphocytes werw higher in patients (p <0.0001) when compared to controls. Patients with infection had reduced percentage levels of monocytes / OX40 + and neutrophils / OX40 + (p <0.0001, p = 0.01 respectively). Lymphocyte / CD40L + and platelet / CD40L + levels were higher during infection (p = 0.02, p = 0.0003, respectively). Elevated basal levels of AGP-lymphocytes, and reduced AGP-monocytes when compared to controls (p = 0.003, p = 0.04, respectively) were found. The levels of AGP-monocytes and AGPneutrophils were reduced in IC patients (p <0.004 and p = 0.009, respectively) and SI (p =0.001 and p = 0.01, respectively) when compared to controls, and similar between CI groups and SI. (P = 0.03). Levels of AGP-lymphocytes (p= 0,03), AGP-monocytes (p = 0.04), and AGP-neutrophils (p = 0.009) in the paired analysis before (baseline) and during infection were higher in baseline when compared to levels during infection. Levels of platelets /CD62P +, AGP-monocytes / CD62P + and AGP-neutrophils / CD62P + in IC and SI patients were lower when compared to controls. In the analysis of AGP-neutrophils / CD62P +, IC patients presented decreased levels when compared to SI. In the paired analysis, IC patients had elevated baseline levels of activated platelets and AGP-lymphocytes / CD62P + when compared to levels during infection (p <0.0001 and p = 0.02, respectively). Baseline levels of AGP-lymphocytes / CD40L + and AGP-monocytes / CD40L + patients were reduced when compared to controls (p <0.0001). Percentage levels of AGP-lymphocytes / CD40L + were reduced in patients during infection compared to controls (p <0.03). CI patients had elevated levels of AGP-neutrophils / CD40L + when compared to SI (p = 0.006) and similar to controls. SI patients presented reduced levels of this population when compared to controls (p <0.0001). In the analysis of sCD40L, there was no statistical difference between the groups. Changes in the expression of OX40 +, CD40L + and platelet aggregates may be associated with regulation of the immune response to infections in elderly cancer patients, as well as correlations with tumor microenvironment and immunosenescence secondary to carcinogenesis and cytoreductive treatments. All this may explain, in part, the frequency and presentation of severe infectious conditions in the elderly with cancer.