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)

Galactomannan-Decorated Lipidic Nanocarrier for Gene Supplementation Therapy in Fabry Disease

Gene supplementation therapy with plasmid DNA (pDNA) represents one of the most promising strategies for the treatment of monogenic diseases such as Fabry disease (FD). In the present work, we developed a solid lipid nanoparticles (SLN)-based non-viral vector with a size below 100 nm, and decorated with galactomannan (GM) to target the liver as an α-Galactosidase A (α-Gal A) production factory. After the physicochemical characterization of the GM-SLN vector, cellular uptake, transfection efficacy and capacity to increase α-Gal A activity were evaluated in vitro in a liver cell line (Hep G2) and in vivo in an animal model of FD. The vector showed efficient internalization and it was highly efficient in promoting protein synthesis in Hep G2 cells. Additionally, the vector did not show relevant agglutination of erythrocytes and lacked hemolytic activity. After the systemic administration to Fabry mice, it achieved clinically relevant α-Gal A activity levels in plasma, liver, and other organs, importantly in heart and kidneys, two of the most damaged organs in FD. This work shows the potential application of GM-decorated lipidic nanocarries for the treatment of FD by pDNA-based gene augmentation.This research was funded by MCIU/AEI/FEDER, UE (RTI2018-098672-B-I00) and by the University of the Basque Country UPV/EHU (GIU20/048)

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

MMP-9 Downregulation with Lipid Nanoparticles for Inhibiting Corneal Neovascularization by Gene Silencing

Gene silencing targeting proangiogenic factors have been shown to be a useful strategy in the treatment of corneal neovascularization (CNV). Among interference RNA (RNAi) molecules, short-hairpin RNA (shRNA) is a plasmid-coded RNA able to down-regulate the expression of the desired gene. It is continuously produced in the host cell, inducing a durable gene silencing effect. The aim of this work was to develop a solid lipid nanoparticle (SLN)-based shRNA delivery system to downregulate metalloproteinase 9 (MMP-9), a proangiogenic factor, in corneal cells for the treatment of CNV associated with inflammation. The nanovectors were prepared using a solvent emulsification-evaporation technique, and after physicochemical evaluation, they were evaluated in different culture cell models. Transfection efficacy, cell internalization, cell viability, the effect on MMP-9 expression, and cell migration were evaluated in human corneal epithelial cells (HCE-2). The inhibition of tube formation using human umbilical vein endothelial cells (HUVEC) was also assayed. The non-viral vectors based on SLN were able to downregulate the MMP-9 expression in HCE-2 cells via gene silencing, and, consequently, to inhibit cell migration and tube formation. These results demonstrate the potential of lipid nanoparticles as gene delivery systems for the treatment of CNV-associated inflammation by RNAi technology.This research was funded by the Ministerio de Economía y Competitividad (SAF2014-53092-R), by FEDER funds from the EU, and by the UPV/EHU (PPG17/65, GIU17/032). J Torrecilla and I Gómez-Aguado thank UPV/EHU for their research grants