Desarrollo de nanopartículas sólidas lipídicas como sistemas de administración de ADN para terapia génica

290 p. (Bibliogr. 257-290) Correo electrónico de la autora: [email protected][EN] This work includes the development, characterization and optimization of a gene delivery system based on solid lipid nanoparticles (SLNs) . Formulations were characterized in terms of particle size , surface charge and the ability to protect the DNA from nucleases . Likewise, the efficiency of transfection and cell viability in vitro was assessed in culture cells and transfection capacity in vivo after intravenous administration in BALB/c mice . Lyophilization process was also optimized, and a short- and medium-term stability study was carried out with the lyophilized formulations. The results demonstrated that the in vitro transfection efficiency was dependent on the ratio of cationic lipid DOTAP in SLNs and on the DOTAP tp DNA ratio used to prepare SLN – DNA complexes. Likewise, the ability of the transfection in vitro depends on cell line; it is lower in ARPE-19 cells than in HEK293 . Moreover, the incorporation of the cell-penetrating peptide SAP resulted in an increase in the level of transfection in both cell lines . Furthermore, it is possible to obtain lyophilized SLNs stable for 6 to 9 months when stored at 30 ° C/65 % RH and 25 ° C/60 % RH , respectively. Finally, the study showed that SLNs are able to transfect in vivo after intravenous administration in BALB/c mice, leading to the expression of green fluorescent protein in the liver and spleen, which is maintained for at least 7 days.[ES] Este trabajo recoge el desarrollo, caracterización y optimización de un sistema de administración de genes basado en nanopartículas sólidas lipídicas (SLNs). Las formulaciones se caracterizaron determinandose el tamaño de partícula, la carga superficial y la capacidad de protección del ADN frente a nucleasas. Así mismo, se evaluó la eficacia de transfección y la viabilidad celular in vitro en cultivos celulares y la capacidad de transfección in vivo tras su administración intravenosa en ratones Balb/c. También se optimizó el proceso de liofilización y se llevó a cabo un estudio de estabilidad a corto y medio plazo de las formulaciones liofilizadas. Los resultados demostraron que la eficacia de transfección in vitro dependía de la proporción de lípido catiónico DOTAP en las SLNs y de la relación DOTAP:ADN utilizada para elaborar los complejos SLN:ADN. Así mismo, la capacidad de transfección in vitro de los vectores dependía de la línea celular, siendo menor en las células ARPE-19 que en las HEK293. Por otro lado, la incorporación del péptido de penetración celular SAP en los vectores produce un incremento en el nivel de transfección en las dos líneas celulares. Además, es posible obtener SLNs liofilizadas estables durante 6 y 9 meses cuando se almacenan a 30 ºC/65%HR y 25 ºC/60%HR, respectivamente. Finalmente, este trabajo demostró que las SLNs son capaces de transfectar in vivo tras su administración intravenosa en ratones Balb/c, dando lugar a la expresión de proteína verde fluorescente en el hígado y en el bazo, que se mantiene durante al menos 7 días

Legislación y Deontología - Aprendizaje Basado en Problemas

Duración (en horas): Más de 50 horas. Destinatario: EstudianteEl objetivo general de la asignatura Legislación y Deontología es que el alumnado conozca e interprete los aspectos de la legislación sanitaria que tienen implicación directa en el área farmacéutica. Las distintas actividades se centrarán en el manejo, consulta e interpretación de normativas y leyes relacionadas con el ámbito sanitario, y más específicamente con el ejercicio farmacéutico. Los estudiantes conocerán la legislación relativa, por un lado, a la adquisición de una oficina de farmacia, y por otro, a la correcta dispensación de un medicamento y los pasos posteriores para que la administración sanitaria abone al farmacéutico la cantidad económica correspondiente

Development of nucleic acid vaccines: use of self-amplifying RNA in lipid nanoparticles

Self-amplifying RNA or RNA replicon is a form of nucleic acid-based vaccine derived from either positive-strand or negative-strand RNA viruses. The gene sequences encoding structural proteins in these RNA viruses are replaced by mRNA encoding antigens of interest as well as by RNA polymerase for replication and transcription. This kind of vaccine has been successfully assayed with many different antigens as vaccines candidates, and has been shown to be potent in several animal species, including mice, nonhuman primates, and humans. A key challenge to realizing the broad potential of self-amplifying vaccines is the need for safe and effective delivery methods. Ideally, an RNA nanocarrier should provide protection from blood nucleases and extended blood circulation, which ultimately would increase the possibility of reaching the target tissue. The delivery system must then be internalized by the target cell and, upon receptor-mediated endocytosis, must be able to escape from the endosomal compartment into the cell cytoplasm, where the RNA machinery is located, while avoiding degradation by lysosomal enzymes. Further, delivery systems for systemic administration ought to be well tolerated upon administration. They should be safe, enabling the multiadministration treatment modalities required for improved clinical outcomes and, from a developmental point of view, production of large batches with reproducible specifications is also desirable. In this review, the concept of self-amplifying RNA vaccines and the most promising lipid-based delivery systems are discussed.This work was supported by the Basque Government's Department of Education, Universities and Investigation (IT-341-10)

α-Galactosidase A Augmentation by Non-Viral Gene Therapy: Evaluation in Fabry Disease Mice

Fabry disease (FD) is a monogenic X-linked lysosomal storage disorder caused by a deficiency in the lysosomal enzyme α-Galactosidase A (α-Gal A). It is a good candidate to be treated with gene therapy, in which moderately low levels of enzyme activity should be sufficient for clinical efficacy. In the present work we have evaluated the efficacy of a non-viral vector based on solid lipid nanoparticles (SLN) to increase α-Gal A activity in an FD mouse model after intravenous administration. The SLN-based vector incremented α-Gal A activity to about 10%, 15%, 20% and 14% of the levels of the wild-type in liver, spleen, heart and kidney, respectively. In addition, the SLN-based vector significantly increased α-Gal A activity with respect to the naked pDNA used as a control in plasma, heart and kidney. The administration of a dose per week for three weeks was more effective than a single-dose administration. Administration of the SLN-based vector did not increase liver transaminases, indicative of a lack of toxicity. Additional studies are necessary to optimize the efficacy of the system; however, these results reinforce the potential of lipid-based nanocarriers to treat FD by gene therapy.This research was funded by Merck Salud Foundation, MCIU/AEI/FEDER, UE (RTI2018-098672-B-I00) and by the University of the Basque Country UPV/EHU (GIU17/032)

Novel Golden Lipid Nanoparticles with Small Interference Ribonucleic Acid for Substrate Reduction Therapy in Fabry Disease

Substrate reduction therapy (SRT) has been proposed as a new gene therapy for Fabry disease (FD) to prevent the formation of globotriaosylceramide (Gb3). Nanomedicines containing different siRNA targeted to Gb3 synthase (Gb3S) were designed. Formulation factors, such as the composition, solid lipid nanoparticles (SLNs) preparation method and the incorporation of different ligands, such as gold nanoparticles (GNs), protamine (P) and polysaccharides, were evaluated. The new siRNA–golden LNPs were efficiently internalized in an FD cell model (IMFE-1), with GNs detected in the cytoplasm and in the nucleus. Silencing efficacy (measured by RT-qPCR) depended on the final composition and method of preparation, with silencing rates up to 90% (expressed as the reduction in Gb3S-mRNA). GNs conferred a higher system efficacy and stability without compromising cell viability and hemocompatibility. Immunocytochemistry assays confirmed Gb3S silencing for at least 15 days with the most effective formulations. Overall, these results highlight the potential of the new siRNA–golden LNP system as a promising nanomedicine to address FD by specific SRT.This research was funded by MCIU/AEI/FEDER, UE, grant number RTI2018-098672-B-I00; by the UNIVERSITY OF THE BASQUE COUNTRY UPV/EHU, grant number GIU20/048; and by the BASQUE GOVERNMENT, grant number IT1587-22, GIC21/34

Nucleic Acid Delivery by Solid Lipid Nanoparticles Containing Switchable Lipids: Plasmid DNA vs. Messenger RNA

The development of safe and effective nucleic acid delivery systems remains a challenge, with solid lipid nanoparticle (SLN)-based vectors as one of the most studied systems. In this work, different SLNs were developed, by combination of cationic and ionizable lipids, for delivery of mRNA and pDNA. The influence of formulation factors on transfection efficacy, protein expression and intracellular disposition of the nucleic acid was evaluated in human retinal pigment epithelial cells (ARPE-19) and human embryonic kidney cells (HEK-293). A long-term stability study of the vectors was also performed. The mRNA formulations induced a higher percentage of transfected cells than those containing pDNA, mainly in ARPE-19 cells; however, the pDNA formulations induced a greater protein production per cell in this cell line. Protein production was conditioned by energy-dependent or independent entry mechanisms, depending on the cell line, SLN composition and kind of nucleic acid delivered. Vectors containing 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) as unique cationic lipid showed better stability after seven months, which improved with the addition of a polysaccharide to the vectors. Transfection efficacy and long-term stability of mRNA vectors were more influenced by formulation-related factors than those containing pDNA; in particular, the SLNs containing only DOTAP were the most promising formulations for nucleic acid delivery.This research was funded by the MCIU/AEI/FEDER, UE (RTI2018-098672-B-I00) and by the UPV/EHU (GIU17/032)

Analysis of pattern recognition and dimensionality reduction techniques for odor biometrics

In this paper, we analyze the performance of several well-known pattern recognition and dimensionality reduction techniques when applied to mass-spectrometry data for odor biometric identification. Motivated by the successful results of previous works capturing the odor from other parts of the body, this work attempts to evaluate the feasibility of identifying people by the odor emanated from the hands. By formulating this task according to a machine learning scheme, the problem is identified with a small-sample-size supervised classification problem in which the input data is formed by mass spectrograms from the hand odor of 13 subjects captured in different sessions. The high dimensionality of the data makes it necessary to apply feature selection and extraction techniques together with a simple classifier in order to improve the generalization capabilities of the model. Our experimental results achieve recognition rates over 85% which reveals that there exists discriminatory information in the hand odor and points at body odor as a promising biometric identifier

Seed Silhouettes as Geometric Objects: New Applications of Elliptic Fourier Transform to Seed Morphology

Historically, little attention has been paid to the resemblance between seed silhouettes to geometric figures. Cardioid and derivatives, ellipses, heart curves, lemniscates, lenses, lunes, ovals, superellipses, waterdrops, and other figures can be used to describe seed shape, as well as models for quantification. Algebraic expressions representing the average silhouettes for a group of seeds are available, and their shape can be described and quantified by comparison with geometric models. Bidimensional closed-plane figures resulting from the representation of Fourier equations can be used as models for shape analysis. Elliptic Fourier Transform equations reproduce the seed silhouettes for any closed-plane curve corresponding to the contour of the image of a seed. We review the geometric properties of the silhouettes from seed images and discuss them in the context of seed development, plant taxonomy, and environmental adaptation. Silene is proposed as a model for the study of seed morphology. Three groups have been recently defined among Silene species based on the structure of their seed silhouettes, and their geometric properties are discussed. Using models based on Fourier Transform equations is useful in Silene species where the seeds are homogenous in shape but don’t adjust to described figures.Project “CLU-2019-05-IRNASA/CSIC Unit of Excellence”, funded by the Junta de Castilla y León and co-financed by the European Union (ERDF “Europe drives our growth”)

Gene-terapia: ikuspegi terapeutiko berria begietako gaitzen tratamenduan

Gene-terapia etorkizun handiko tresna bezala sortu da tratamendurik ez duten asaldurentzat. Azido nukleiko terapeutikoen administrazioan oinarritzen da gaixotasunak tratatzeko. Gene-terapia arrakastatsua izan dadin material genetikoaren askapen eraginkorra bermatu behar da itu-zeluletan. Geneen administrazio-sistemen artean, bektore biralak asko erabili dira ahalbidetzen duten transferentzia genikorako gaitasun onarengatik. Hala ere, horien arrisku nagusien ondorioz (immunogenizitatea eta mutagenesia), bektore ez-biralen diseinua sustatu da. Bektore ez-biralak seguruagoak dira eta ekoizpena errazagoa da, baina hauen muga nagusia transfekzio-eraginkortasun baxua da. Gene-terapiarako organo interesgarri bat begia da, eskuragarria eta aztertzeko erraza baita. Gainera, immunitate-sistematik babestuta dago. Gene-terapia entsegu kliniko guztien % 1,3 bakarrik tratatzen dituzte begietako gaitzak, baina etorkizun handia aurkeztu dute azken urteetan. Izan ere, 2018an Estatu Batuetan eta Europan gene-terapian oinarritutako begirako lehen medikamentuaren komertzializazioa onartu zen, Luxturna®, Sortzetiko Leberren Amaurosiaren tratamendurako. Berrikuspen honetan, begiko administrazio-bideak, gene-terapia estrategiak eta transferentzia geniko eraginkorrerako gainditu behar diren begiko mugak aurkezten dira. Halaber, gene-terapiaren bidez tratatzeko hautagai diren begietako gaitz ezberdinak ere biltzen dira. Gene-terapiaren inguruan egindako ahaleginei eta aurrerapenei esker, begietako gaitzak tratatzeko medikamentu berriak garatu dira. Horietako asko entsegu klinikoetan ebaluatzen ari dira oraindik, baina beste batzuk jada merkatura eta pazienteetara iritsi dira.; Gene therapy has emerged as a promising tool for disorders that have no cure. It consists in the administration of therapeutic nucleic acids into patients for treating diseases. The success of gene therapy relies on the efficient delivery of the genetic material to target cells. Among gene delivery systems, viral vectors have been widely used due to their good gene transference efficacy. However, their potential risk associated with immunogenicity and mutagenesis has promoted the design of non-viral vectors. Non-viral vectors are safer and easier to produce, but their main limitation remains lower transfection efficacy. An attractive candidate for gene therapy is the eye, since it is easily accessible, easily examined and relatively immune privileged. Only 1,3% of all gene therapy clinical trials treat ocular disorders, but they have shown great potential in recent years. In fact, in 2018 the Food and Drug Administration and the European Medicine Agency approved the commercialization of the first gene therapy based ocular drug, Luxturna®, for the treatment of Leber Congenital Amaurosis. In this review, we present the main administration routes to eye, gene therapy strategies and ocular barriers to overcome for successful gene transfer. Different ocular disease candidates to be treated by gene therapy are also reviewed. The efforts and advances made in the field of gene therapy have led to the development of new drugs to treat eye diseases. Many of them are still being evaluated in clinical trials, but some have already reached the market and patients