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

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)

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)

mRNA-Based Nanomedicinal Products to Address Corneal Inflammation by Interleukin-10 Supplementation

The anti-inflammatory cytokine Interleukin-10 (IL-10) is considered an efficient treatment for corneal inflammation, in spite of its short half-life and poor eye bioavailability. In the present work, mRNA-based nanomedicinal products based on solid lipid nanoparticles (SLNs) were developed in order to produce IL-10 to treat corneal inflammation. mRNA encoding green fluorescent protein (GFP) or human IL-10 was complexed with different SLNs and ligands. After, physicochemical characterization, transfection efficacy, intracellular disposition, cellular uptake and IL-10 expression of the nanosystems were evaluated in vitro in human corneal epithelial (HCE-2) cells. Energy-dependent mechanisms favoured HCE-2 transfection, whereas protein production was influenced by energy-independent uptake mechanisms. Nanovectors with a mean particle size between 94 and 348 nm and a positive superficial charge were formulated as eye drops containing 1% (w/v) of polyvinyl alcohol (PVA) with 7.1–7.5 pH. After three days of topical administration to mice, all formulations produced GFP in the corneal epithelium of mice. SLNs allowed the obtaining of a higher transfection efficiency than naked mRNA. All formulations produce IL-10, and the interleukin was even observed in the deeper layers of the epithelium of mice depending on the formulation. This work shows the potential application of mRNA-SLN-based nanosystems to address corneal inflammation by gene augmentation therapy.This research was funded by MCIU/AEI/FEDER, UE (SAF2014-53092-R), the UPV/EHU (GIU 20/048) and by the Università degli Studi di Torino (Ricerca Locale 2019)

Analysis and performance of lumped-element kinetic inductance detectors for W-band

Lumped-element superconducting resonators are a promising technology for their use in millimeter-wave observations and quantum computing applications that require large arrays of extremely sensitive detectors. Among them, lumped-element kinetic inductance detectors (LEKIDs) have shown good performance in the submillimeter band in several earth-based telescopes. In this work, LEKIDs for their use as millimeter-wave receivers of astronomical applications are presented. LEKID arrays using a thin bilayer of superconducting titanium/aluminum (Ti/Al), deposited on the silicon substrate, have been designed and fabricated. The design of a dual-polarization LEKID with the goal of detection at the W -band for two orthogonal polarizations is described and a fabricated array has demonstrated absorption at ambient temperature. Also, an approximate design methodology of the coupling parameter for LEKIDs' readout, essential for dynamic range optimization of the detector under millimeter-wave radiation, is proposed. In addition, the resonance characteristics and coupling factor of the fabricated superconducting resonators using high-quality internal factor Qi under cryogenic temperatures have been analyzed. The design guidelines in this work are applicable to other LEKID arrays, and the presented superconducting Ti/Al thin-film LEKIDs can be used in future receiver arrays in the millimeter bands.This work was supported by Ministry of Science, Innovation and Universities under Grants ESP2017-83921-C2-2-R, ESP2017-86582-C4-1-R, ESP2017-86582-C4-3-R, ESP2017-92706-EXP, AYA2017-92153-EXP, “Severo Ochoa” Programme for Centres of Excellence in R&D (MINECO, Grant SEV-2016-0686). By Comunidad de Madrid under Grant P2018/NMT-4291. D.G. and A.G also acknowledge Grant DEFROST N62909-19-1-2053 from ONRGlobal. A.G. acknowledges IJCI-2017-33991

Bi-layer kinetic inductance detectors for W-band

An array of superconducting kinetic inductance detectors (KID) has been fabricated and it has demonstrated absorption at W-Band. The use of a bi-layer structure based on aluminum (AI) and titanium (Ti) shows a lower superconducting critical temperature (T c ), which allows the detection at W-band. A design methodology is presented taking into account the KID geometry in order to maximize the absorption and a dual-polarization KID has been designed using the proposed methodology. Two prototypes of KID on Silicon substrate have been fabricated showing a good agreement between measurement and simulation results. The measurements at room temperature from 65 to 110 GHz show the matching at the frequency band, while dark cryogenic characterization demonstrated the low frequency design.The authors acknowledge financial supports: Ministry of Science, Innovation and Universities Grants ESP2017-83921-C2-2-R, ESP2017-86582-C4-1-R, ESP2017-86582-C4-3-R, MAT2017-85617-R, ESP2017-92706-EXP, AYA2017-92153-EXP and from Comunidad de Madrid through Grant P2018/NMT-4291 TEC2-SPACE-CM. A.G. acknowledges IJCI-2017-33991; IMDEA Nanociencia acknowledges support from the “Severo Ochoa” Programme for Centres of Excellence in R&D (MINECO, Grant SEV-2016-0686). D.G. and A.G also acknowledge Grant DEFROST N62909-19-1-2053 from ONR-Global

Learning ethical, environmental and professional responsibility at Universitat Politècnica de València. Where are we?

[EN] This paper presents a study on the development of the cross-curricular learning outcome (CCLO) "Ethical, environmental and professional responsibility" for students of different Bachelor's Degrees taught at Universitat Politecnica de Valencia (Spain). The work involved in the development of this learning outcome entails great complexity, given the double dimension of responsibility that it involves. At the end of their training at the university, students are expected to show ethical, environmental, and professional responsibility towards themselves and others. Interviews have been conducted with lecturers who work and assess this outcome in their subjects, most/all of them related to science and engineering. The objective was to identify the learning approach used in the different subjects to guarantee the acquisition of this CCLO by the students. A focus group has also been carried out with students to determine the importance they give to this learning outcome, and to know their degree of satisfaction with the training received. The methodology used to obtain the data from lecturers and students and to process the information to get a precise diagnosis is fully described in the paper. Results are satisfactory to some extent: most of the lecturers carry out appropriate activities and most students achieve the expected proficiency level. Finally, recommendations are given to improve the development of this cross-curricular learning outcome.This innovative educational project and the APC of this paper were funded by Universitat Politecnica de Valencia, through the project PIME/20-21/219 "Evaluacion del nivel de adquisicion de la CT07 Responsabilidad etica, medioambiental y profesional en los estudios de grado de la UPV. Propuestas de mejora".Gimenez-Carbo, E.; Gómez-Martín, ME.; Fenollosa Forner, EJ.; Cabedo Fabres, M.; Coll-Aliaga, E.; Andrés-Doménech, I.; Sebastiá-Frasquet, M.... (2021). Learning ethical, environmental and professional responsibility at Universitat Politècnica de València. Where are we?. Sustainability. 13(17):1-18. https://doi.org/10.3390/su13179991S118131

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

Evaluation of machine learning algorithms and structural features for optimal MRI-based diagnostic prediction in psychosis

A relatively large number of studies have investigated the power of structural magnetic resonance imaging (sMRI) data to discriminate patients with schizophrenia from healthy controls. However, very few of them have also included patients with bipolar disorder, allowing the clinically relevant discrimination between both psychotic diagnostics. To assess the efficacy of sMRI data for diagnostic prediction in psychosis we objectively evaluated the discriminative power of a wide range of commonly used machine learning algorithms (ridge, lasso, elastic net and L0 norm regularized logistic regressions, a support vector classifier, regularized discriminant analysis, random forests and a Gaussian process classifier) on main sMRI features including grey and white matter voxel-based morphometry (VBM), vertex-based cortical thickness and volume, region of interest volumetric measures and wavelet-based morphometry (WBM) maps. All possible combinations of algorithms and data features were considered in pairwise classifications of matched samples of healthy controls (N = 127), patients with schizophrenia (N = 128) and patients with bipolar disorder (N = 128). Results show that the selection of feature type is important, with grey matter VBM (without data reduction) delivering the best diagnostic prediction rates (averaging over classifiers: schizophrenia vs. healthy 75%, bipolar disorder vs. healthy 63% and schizophrenia vs. bipolar disorder 62%) whereas algorithms usually yielded very similar results. Indeed, those grey matter VBM accuracy rates were not even improved by combining all feature types in a single prediction model. Further multi-class classifications considering the three groups simultaneously made evident a lack of predictive power for the bipolar group, probably due to its intermediate anatomical features, located between those observed in healthy controls and those found in patients with schizophrenia. Finally, we provide MRIPredict (https://www.nitrc.org/projects/mripredict/), a free tool for SPM, FSL and R, to easily carry out voxelwise predictions based on VBM images