7 research outputs found

    Transferencia génica hepática hidrodinámica en modelos de cerdo in vivo y segmento humano ex vivo

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    Introducci√≥n: La terapia g√©nica es una estrategia terap√©utica que pretende utilizar √°cidos nucleicos como medicamentos para el tratamiento temporal o permanente de patolog√≠as hereditarias o adquiridas. En base al veh√≠culo utilizado para entregar el gen, podemos clasificar la terapia g√©nica en dos grupos: la terapia g√©nica viral (vectores virales) y la terapia g√©nica no viral (vectores no virales o ADN desnudo). La terapia no viral resulta menos eficiente pero m√°s segura. La hidrofecci√≥n (transferencia g√©nica hidrodin√°mica) ha mostrado un gran potencial, con resultados terap√©uticos en rat√≥n. Sin embargo, cuando la hidrofecci√≥n se ha trasladado a animales de mayor tama√Īo, el procedimiento ha mostrado baja eficacia. Quedan por resolver los motivos de esta ineficiencia. Desarrollo: la presente tesis tiene como objetivo confirmar o refutar el potencial inter√©s traslacional de la hidrofecci√≥n en base a un an√°lisis molecular cuantitativo del proceso de decodificaci√≥n gen√©tica del gen transfectado (eGFP, hAAT y hIL10) en tejido. Adem√°s, se eval√ļa el mecanismo del proceso de entrega g√©nica mediante el an√°lisis ultraestructural del tejido transfectado con nanopart√≠culas de oro (4-15 nm de di√°metro). Para ello, se han desarrollado 3 modelos de transferencia g√©nica hep√°tica: 1) modelo quir√ļrgico en cerdo con cierre vascular hep√°tico completo; 2) modelos de cateterismo endovascular sin cierre (abierto) o con cierre (cerrado) de la vena porta y 3) modelo de segmentos hep√°ticos humanos estancos procedentes de resecciones quir√ļrgicas. La entrega y transcripci√≥n del gen se eval√ļan como n√ļmero de copias por c√©lula mediante PCR cuantitativa, utilizando una curva patr√≥n preparada con el propio gen. La expresi√≥n de la prote√≠na se determina en tejido de manera semicuantitativa (Fluorescencia, Western Blot, Inmunohistoquimia) y cuantitativa (ELISA), y se expresa como n√ļmero de copias por c√©lula. En sangre la prote√≠na se determina mediante ELISA. Los resultados en cerdo muestran que la hidrofecci√≥n media una entrega g√©nica igual o superior a la del patr√≥n oro (modelo murino) y una traducci√≥n proteica en tejido (directamente proporcional al grado de cierre vascular del √≥rgano) similar a √©ste y al humano. Sin embargo, los niveles plasm√°ticos de la prote√≠na son claramente inferiores a los esperados en base a la cantidad presente en el tejido. En los experimentos con segmentos humanos, la hidrofecci√≥n media una traducci√≥n de la prote√≠na en tejido de 100-1.000 copias por c√©lula. La interpretaci√≥n ultraestructural del proceso de entrega g√©nica se lleva a cabo mediante inyecci√≥n hidrodin√°mica de nanopart√≠culas. El estudio por microscop√≠a electr√≥nica muestra que las part√≠culas peque√Īas tienen f√°cil acceso al n√ļcleo mientras que part√≠culas de mayor tama√Īo s√≥lo son observadas en el interior de ves√≠culas de c√©lulas con actividad fagoc√≠tica pero no en el citoplasma del hepatocito. Conclusi√≥n: 1. La eficiencia est√° directamente relacionada con el grado de cierre vascular del √≥rgano. 2. Condiciones de perfusi√≥n violentas conducen a resultados m√°s pobres y m√°s riesgos para el paciente. 3. No se requiere una entrega g√©nica muy elevada para una traducci√≥n eficiente de la prote√≠na. 4. En el modelo porcino, existe dificultad de exportar la prote√≠na humana traducida al torrente sangu√≠neo. 5. Los resultados obtenidos en segmentos humanos confirman la eficiencia de traducci√≥n de la prote√≠na y el potencial inter√©s traslacional del procedimiento. 6. La entrega g√©nica a los hepatocitos se verifica por un proceso de difusi√≥n a trav√©s de la membrana sin soluci√≥n de continuidad de la misma. 7. Las membranas del tejido hep√°tico limitan el acceso de part√≠culas al n√ļcleo, estando el di√°metro m√°ximo permitido cercano a los 4 nm. De las conclusiones parciales enumeradas se deduce que la hidrofecci√≥n hep√°tica es un procedimiento de transferencia g√©nica seguro y eficiente con gran potencial de inter√©s cl√≠nico traslaciona

    Mitochondrial DNA replacement techniques to prevent human mitochondrial diseases

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    Background: Mitochondrial DNA (mtDNA) diseases are a group of maternally inherited genetic disorders caused by a lack of energy production. Currently, mtDNA diseases have a poor prognosis and no known cure. The chance to have unaffected offspring with a genetic link is important for the affected families, and mitochondrial replacement techniques (MRTs) allow them to do so. MRTs consist of transferring the nuclear DNA from an oocyte with pathogenic mtDNA to an enucleated donor oocyte without pathogenic mtDNA. This paper aims to determine the efficacy, associated risks, and main ethical and legal issues related to MRTs. Methods: A bibliographic review was performed on the MEDLINE and Web of Science databases, along with searches for related clinical trials and news. Results: A total of 48 publications were included for review. Five MRT procedures were identified and their efficacy was compared. Three main risks associated with MRTs were discussed, and the ethical views and legal position of MRTs were reviewed. Conclusions: MRTs are an effective approach to minimizing the risk of transmitting mtDNA diseases, but they do not remove it entirely. Global legal regulation of MRTs is required

    Multicompartmental Lipopolyplex as vehicle for antigens and genes delivery in vaccine formulations

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    Vector design and its characterization is an area of great interest in current vaccine research. In this article, we have formulated and characterized a multicompartmental lipopolyplex, which associates multiple liposomes and polyplexes in the same complex. These particles allow the simultaneous delivery of lipid or water-soluble antigens associated with genes to the same cell, in much higher amounts than conventional lipopolyplexes. The vector characterization and optimization were carried out using liposomes with entrapped carboxyfluorescein and adapted electrophoretic assays. Two types of lipopolyplexes (containing hydrophilic or lipophilic antigens) were employed to evaluate their interest in vaccination. The lipopolyplex loaded with an extract of water-soluble melanoma proteins proved to efficiently induce humoral response in murine melanoma model, increasing the levels of IgM and IgG. The specificity of the immune response induced by the lipopolyplex was demonstrated in mice with the lipopolyplex containing the GD3 ganglioside lipid antigen, abundant in melanoma cells. The levels of anti-GD3 IgG increased markedly without modifying the expression of humoral antibodies against other gangliosides

    Pharmacogenetics in Neuroblastoma: What Can Already Be Clinically Implemented and What Is Coming Next?

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    Pharmacogenetics is one of the cornerstones of Personalized Precision Medicine that needs to be implemented in the routine of our patients' clinical management in order to tailor their therapies as much as possible, with the aim of maximizing efficacy and minimizing toxicity. This is of great importance, especially in pediatric cancer and even more in complex malignancies such as neuroblastoma, where the rates of therapeutic success are still below those of many other types of tumors. The studies are mainly focused on germline genetic variants and in the present review, state of the art is presented: which are the variants that have a level of evidence high enough to be implemented in the clinic, and how to distinguish them from the ones that still need validation to confirm their utility. Further aspects as relevant characteristics regarding ontogeny and future directions in the research will also be discussed

    Foxp3 Silencing with Antisense Oligonucleotide Improves Immunogenicity of an Adjuvanted Recombinant Vaccine against Sporothrix schenckii

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    Background: In recent years, there has been great interest in developing molecular adjuvants based on antisense oligonucleotides (ASOs) targeting immunosuppressor pathways with inhibitory effects on regulatory T cells (Tregs) to improve immunogenicity and vaccine efficacy. We aim to evaluate the immunostimulating effect of 2‚Ä≤OMe phosphorothioated Foxp3-targeted ASO in an antifungal adjuvanted recombinant vaccine. Methods: The uptake kinetics of Foxp3 ASO, its cytotoxicity and its ability to deplete Tregs were evaluated in murine splenocytes in vitro. Groups of mice were vaccinated with recombinant enolase (Eno) of Sporothix schenckii in Montanide Gel 01 adjuvant alone or in combination with either 1 ¬Ķg or 8 ¬Ķg of Foxp3 ASO. The titers of antigen-specific antibody in serum samples from vaccinated mice (male C57BL/6) were determined by ELISA (enzyme-linked immunosorbent assay). Cultured splenocytes from each group were activated in vitro with Eno and the levels of IFN-ő≥ and IL-12 were also measured by ELISA. The results showed that the anti-Eno antibody titer was significantly higher upon addition of 8 ¬ĶM Foxp3 ASO in the vaccine formulation compared to the standard vaccine without ASO. In vitro and in vivo experiments suggest that Foxp3 ASO enhances specific immune responses by means of Treg depletion during vaccination. Conclusion: Foxp3 ASO significantly enhances immune responses against co-delivered adjuvanted recombinant Eno vaccine and it has the potential to improve vaccine immunogenicity

    Ahora / Ara

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    La cinquena edici√≥ del microrelatari per l‚Äôeradicaci√≥ de la viol√®ncia contra les dones de l‚ÄôInstitut Universitari d‚ÄôEstudis Feministes i de G√®nere ¬ęPurificaci√≥n Escribano¬Ľ de la Universitat Jaume I vol ser una declaraci√≥ d‚Äôesperan√ßa. Aquest √©s el moment en el qual les dones (i els homes) hem de fer un pas endavant i eliminar la viol√®ncia sist√®mica contra les dones. Ara √©s el moment de denunciar el masclisme i els micromasclismes comen√ßant a construir una societat m√©s igualit√†ria. Cadascun dels relats del llibre √©s una den√ļncia i una declaraci√≥ que ens encamina cap a un m√≥n millor

    La b√ļsqueda de la unidad (la estructura de todas las cosas)

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    Dada la enorme variedad de objetos y materiales, no tiene mucho sentido investigar sobre la existencia de una estructura com√ļn si, al menos, no encontramos algunas propiedades comunes a todas las cosas. Vamos a indagar, pues, si existen propiedades generales, comunes, a todos los materiales, por muy distintos que sean
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