Design of a lentiviral vector as a therapeutic strategy against alzheimer´s disease

Alzheimer’s disease (AD) is a chronic neurodegenerative disorder characterized by a progressive loss of cognitive functions. One of the hallmarks is the formation of amyloid plaques, composed mainly by Aß peptide oligomers (AβOs). Neprilysin (NEP) is the most important endopeptidase and crucial for the degradation of Aß in the brain, avoiding amyloid plaques formation. Because NEP is decreased in brains of patients with AD, we aim to develop a lentiviral vector (LV) capable of specifically expressing in hippocampal neurons, to study its role in AD and therefore its possible therapeutic function.First, a construct containing the complete cDNA of NEP downstream of the hippocampal-specific promoter human synapsin (SYN-1) was performed. NEP cDNA was obtained from pBOB-NEP plasmid and was cloned under the SYN-1 promoter to obtain SYN-NEP plasmid. SYN-NEP also contains the ubiquitous CMV promoter, located outside the sequence to be packaged in LV. The correct cloning was checked by BamHI/KpnI digestion followed by 1% agarose gel electrophoresis and was verified by sequencing. To test NEP expression under the CMV promoter, 293T cells were transfected with SYN-NEP. SYN-RFP plasmid expressing the reporter red fluorescent protein (RFP) and pBOB-NEP were used as transfection and positive controls, respectively. After 48 hours NEP expression was evaluated by western blot (WB) and RFP by fluorescence microscopy.Electrophoresis of SYN-NEP digestion resulted in two bands of 3392pb and 7644pb, which coincided with the molecular weights of the insert containing NEP and backbone, respectively. This in turn was validated by sequencing. Transfection efficiency calculated by expression of RFP was 80%. WB showed a 85kDa band only in those lanes corresponding to transfection with pBOB-NEP and SYN-NEP.In conclusion, NEP was successfully cloned downstream of SYN-1 promoter and is expressed correctly under a ubiquitous promoter. This construction is the first step for the production of LV.Fil: Abrey Recalde, Maria Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional e Ingeniería Biomédica - Hospital Italiano. Instituto de Medicina Traslacional e Ingeniería Biomédica.- Instituto Universitario Hospital Italiano de Buenos Aires. Instituto de Medicina Traslacional e Ingeniería Biomédica; ArgentinaFil: Gonzalez Hermida, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional e Ingeniería Biomédica - Hospital Italiano. Instituto de Medicina Traslacional e Ingeniería Biomédica.- Instituto Universitario Hospital Italiano de Buenos Aires. Instituto de Medicina Traslacional e Ingeniería Biomédica; ArgentinaFil: Baez, Veronica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencias; ArgentinaFil: Jerusalinsky, Diana Alicia. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Frecha, Cecilia Ariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional e Ingeniería Biomédica - Hospital Italiano. Instituto de Medicina Traslacional e Ingeniería Biomédica.- Instituto Universitario Hospital Italiano de Buenos Aires. Instituto de Medicina Traslacional e Ingeniería Biomédica; ArgentinaLXI Reunión anual de la Sociedad Argentina de Investigación Clinica; LXVI, Reunión anual de la Sociedad Argentina de Inmunología; XLVIII Reunión Anual de la Sociedad Argentina de Farmacología Experimental; VII Reunión Anual de la Sociedad Argentina de Nanomedicina y V Congreso Nacional de la Asociación Argentina de Ciencia y Tecnología de Animales de LaboratorioArgentinaSociedad Argentina de Investigación ClinicaSociedad Argentina de InmunologíaSociedad Argentina de Farmacología ExperimentalSociedad Argentina de NanomedicinaAsociación Argentina de ciencia y tecnología de animales de Laboratori

Epstein-Barr virus down-regulates tumor suppressor DOK1 expression

The DOK1 tumor suppressor gene encodes an adapter protein that acts as a negative regulator of several signaling pathways. We have previously reported that DOK1 expression is up-regulated upon cellular stress, via the transcription factor E2F1, and down-regulated in a variety of human malignancies due to aberrant hypermethylation of its promoter. Here we show that Epstein Barr virus (EBV) infection of primary human B-cells leads to the down-regulation of DOK1 gene expression via the viral oncoprotein LMP1. LMP1 alone induces recruitment to the DOK1 promoter of at least two independent inhibitory complexes, one containing E2F1/pRB/DNMT1 and another containing at least EZH2. These events result in tri-methylation of histone H3 at lysine 27 (H3K27me3) of the DOK1 promoter and gene expression silencing. We also present evidence that the presence of additional EBV proteins leads to further repression of DOK1 expression with an additional mechanism. Indeed, EBV infection of B-cells induces DNA methylation at the DOK1 promoter region including the E2F1 responsive elements that, in turn, lose the ability to interact with E2F complexes. Treatment of EBV-infected B-cell-lines with the methyl-transferase inhibitor 5-aza-2′-deoxycytidine rescues DOK1 expression. In summary, our data show the deregulation of DOK1 gene expression by EBV and provide novel insights into the regulation of the DOK1 tumor suppressor in viral-related carcinogenesis.Fil: Siouda, Maha. World Health Organization; FranciaFil: Frecha, Cecilia Ariana. World Health Organization; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Accardi, Rosita. World Health Organization; FranciaFil: Yue, Jiping. World Health Organization; FranciaFil: Cuenin, Cyrille. World Health Organization; FranciaFil: Grufat, Henri. Inserm; Francia. Université Claude Bernard Lyon 1; Francia. Centre National de la Recherche Scientifique; Francia. Ecole Normale Supérieure; FranciaFil: Manet, Evelyne. Inserm; Francia. Université Claude Bernard Lyon 1; Francia. Centre National de la Recherche Scientifique; Francia. Ecole Normale Supérieure; FranciaFil: Herceg, Zdenko. World Health Organization; FranciaFil: Sylla, Bakary S.. World Health Organization; FranciaFil: Tommasino, Massimo. World Health Organization; Franci

Diseño y producción de vectores lentivirales para modular la expresión de genes involucrados en la inmunosupresión

La proteína STUB1 fue recientemente caracterizada como una enzima de tipo E3 ligasa, capaz de promover la degradación del factor de transcripción FOXP3 de manera específica. Es ampliamente conocido que FOXP3 tiene un rol clave en la diferenciación de células T regulatorias y por lo tanto en procesos de inmunosupresión. Además, se ha visto que STUB1 está sobreexpresada en algunas células tumorales cuya función en ellas es aún desconocida. Nos planteamosinvestigar si la administración de STUB1 en células T podría tener un efecto en los niveles de inmunosupresión. Entre las herramientas de transferencia genética más adecuadas para la expresión de genes en células hematopoyéticas se encuentran los vectores lentivirales. El objetivo de este trabajo es diseñar un vector lentiviral de expresión de STUB1 humana. En primer lugar, se estudió la expresión de STUB1 en las líneas celulares tumorales HEK293T y HeLa. Por RT-PCR se comprobó la presencia de RNA mensajero de STUB1 en células derivadas de cáncer cérvico-uterino. A partir del diseño de primers específicos con sitios de enzimas de restricción se aisló el cDNA de STUB1 y se clonó en un plásmido lentiviral autoinactivable (SIN), río abajo del promotor ubicuo EF1alfa, seguido por una secuencia IRES y la secuencia del gen reportero GFP (LV-STUB1). Los plásmidos se amplificaron por transformación en bacterias E. Coli competentes (TOP10), seleccionándose los clones positivos por PCR colony. Posteriormente, se comprobó la presencia del inserto mediante corte con enzimas de restricción específicas y se confirmó la correcta secuencia y orientación del cDNA de STUB1 por secuenciación. Los vectores lentivirales se produjeron en células productoras HEK293T. Se realizó una cotransfección de tres plásmidos: plásmido empaquetador portando los genes gag-pol, plasmido que codifica para la envuelta del virus de la stomatitis vesicular (VSVG), y aquél con la secuencia de STUB1IRESGFP. Se optimizó el método de transfección utilizando el sistema PEI, en preferencia a otros sistemas testeados (CaCl2, Lipofectamina). Los vectores lentivirales fueron recolectados al día 2 de la transfección y preservados -80 grados para su posterior utilización en células target. Se testeó la eficiencia de transducción de las partículas lentivirales en células Jurkat E6-1. Los niveles de GFP se midieron por Citometría de Flujo, 72h post transducción. Los resultados obtenidos demuestran que el vector LV- STUB1 es capaz de transducir más del 35% de las células a una multiplicidad de infección de 0.5 vectores/célula (MOI: 0.5), sin afectar la viabilidad celular. Estos resultados sugieren que sería posible obtener una mayor expresión del transgén a MOIs más altas. Los próximos experimentos incluyen la validación funcional del transgén y la optimización delvector para su utilización en modelos de inmunosupresión.Fil: Salcedo, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional e Ingeniería Biomédica - Hospital Italiano. Instituto de Medicina Traslacional e Ingeniería Biomédica.- Instituto Universitario Hospital Italiano de Buenos Aires. Instituto de Medicina Traslacional e Ingeniería Biomédica; ArgentinaFil: Gonzalez, Hermida P.. Hospital Italiano; ArgentinaFil: Abrey Recalde, J.. Hospital Italiano; ArgentinaFil: Oliver, Javier. Hospital Italiano; ArgentinaFil: Frecha, Cecilia Ariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional e Ingeniería Biomédica - Hospital Italiano. Instituto de Medicina Traslacional e Ingeniería Biomédica.- Instituto Universitario Hospital Italiano de Buenos Aires. Instituto de Medicina Traslacional e Ingeniería Biomédica; ArgentinaXXXVIII Reunión Científica Anual de la Sociedad Argentina de VirologíaCordobaArgentinaSociedad Argentina de Virologi

Usefulness of myeloid dendritic cells in cellular in vitro assays for evaluating immediate hypersensitivity reactions to betalactams.

Introduction Dendritic cells (DCs) are the most potent antigen presenting cells (APCs) with an important role detecting, processing, and presenting antigens to T cells. The analysis of maturation after in vitro stimulation with the culprit drug, and their ability to trigger the proliferation of specific T cell populations in patients with immediate drug hypersensitivity reactions (IDHRs) could serve to prove the sensitization to a drug. Most approaches used monocyte-derived DCs (moDCs), although their sensitivity is not optimal. The different nature of moDCs and myeloid DCs (mDCs), main DC population involved during the in vivo development of IDHRs, could influence the sensitivity of these in vitro tests. Therefore, we evaluated the effect of two betalactams (BLs), amoxicillin (AX) and clavulanic acid (CLV) in moDCs and mDCs from selective-allergic patients (AP) to each BL, as well as to assess their capacity to stimulate different T cell populations. Methods mDCs and moDCs were obtained from 14 AX-AP, 14 CLV-AP and 10 Healthy controls (HC). After stimulating with the culprit BL, maturation and their capacity to stimulate different T cell populations was assessed by flow cytometry. Results Higher maturation was observed in both AX- and CLV-AP compared with HC when using BL-stimulated-mDCs, whereas with moDCs, only higher was observed in AX-AP, but not in CLV-AP. The % of positive maturation cases was higher with mDCs than moDCs, with higher % with AX compared to CLV. The most relevant T cell population proliferative response was obtained in CD4+Th2 cells, reaching to 67% of positivity when using mDCs, followed by 50% with the traditional LTT, and only of 22% with moDCs from AX-AP. The specificity was higher than 80% in all cases. Conclusions mDCs from selective AP efficiently recognised the culprit drug and triggered the proliferation of T-cells, mainly those with a Th2 cytokine pattern, although these responses depended on the drug.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Viral derived “nanoblades” loaded with cas9/ sgrna ribonucleoproteins and aav6 for donor dna cassette delivery

Programmable nucleases have enabled rapid and accessible genome engineering in cells andliving organisms. However, their delivery into target cells can be challenging, specially intoprimary cells. Here, we have designed Nanoblades, a new technology to deliver a genomiccleaving agent into cells. These are murine leukemia virus-derived virus like particle (VLP),which are loaded with Cas9 protein through fusion with the gag viral structural protein, andwith guide RNAs. Cas9 together with gRNAs introduces site specifics double strand break(DSBs) in target gene which can be repaired by non-homologous end-joining (NHEJ) or byhomology-directed repair (HDR) introducing a new sequence from an exogenous templateDNA bearing homology to the sequences flanking the DSBs (donor-DNA).Previously, we demonstrated that Nanoblades were extremely efficient in delivery of theirCas9/sgRNA cargo into K562 human cell line and human T, B , HSCs and HSCs derivedprogenitors T cells (pro-T cells ), thanks to their surface co-pseudotyping with baboonretroviral and VSV-G envelopes.The objective of this work was to edit Wiscott Aldrich Syndrome (WAS) gene locus by HDRusing Nanoblades and AAV6 carrying a donor-DNA, which consists in GFP reporter geneflanking by homologous arms of the WAS gene.AAV6 were added to K562 cells at different times points with respect to Nanobladesaddition, in order to find optimal time that maximizes HDR. Different multiplicities ofinfection (MOI) of AAV were tested. HDR-mediated gene editing was determined by PCRand GFP expression by FACS, 7 days after Nanoblades addition.Our results shows that HDR-mediated edition of WAS gene occurred in a 50% of cells, whennanoblades and AAV6 (MOI 100000 vg/cell) were added at same time. We are currentlytesting this protocol of AAV6 and Nanoblades in HSCs and pro-T cells.In summary, Nanoblades in combination with AAV6 carrying donor-DNA are efficienttools for gene editing and have important prospects for basic and clinical translation forgene therapy.Fil: Abrey Recalde, Maria Jimena. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Hospital Italiano. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Instituto Universitario Hospital Italiano de Buenos Aires. Instituto de Medicina Traslacional E Ingenieria Biomedica.; ArgentinaFil: Gutierrez Guerrero, Alejandra. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Hospital Italiano. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Instituto Universitario Hospital Italiano de Buenos Aires. Instituto de Medicina Traslacional E Ingenieria Biomedica.; ArgentinaFil: Mangeot, Phillipe. Inserm; FranciaFil: Costa, Caroline. Inserm; FranciaFil: Bernandin, Ornelie. Inserm; FranciaFil: Froment, Giselle. Inserm; FranciaFil: Molina, Francisco Martin. No especifíca;Fil: Karim, Bellabdelah. No especifíca;Fil: Frecha, Cecilia Ariana. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Hospital Italiano. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Instituto Universitario Hospital Italiano de Buenos Aires. Instituto de Medicina Traslacional E Ingenieria Biomedica.; ArgentinaFil: Ricci, Emiliano. Inserm; FranciaFil: Cosset, Francose. Inserm; FranciaFil: Verhoeyen, Els. Inserm; FranciaLXIII Reunión Anual de la Sociedad Argentina de Investigación Clínica;LXVI Reunión Anual de la Sociedad Argentina de Inmunologia y Reunión anual de la Sociedad Argentina de FisiologíaMar del PlataArgentinaSociedad Argentina de Investigación ClínicaSociedad Argentina de InmunologiaSociedad Argentina de FisiologíaSociedad Argentina de VirologíaAsociación Argentina de Nanomedicin

Diagnosis of immediate reactions to amoxicillin: Comparison of basophil activation markers CD63 and CD203c in a prospective study

Amoxicillin (AX) combined or not with clavulanic acid (CLV) is frequently involved in IgE-mediated reactions. Drug provocation test (DPT) is considered as the gold standard for diagnosis, although contraindicated in high-risk patients. Basophil activation test (BAT) can help diagnose immediate reactions to beta-lactams, although controversy exists regarding the best activation marker. We have performed a real-life study in a prospective cohort to analyze the real value of BAT as diagnostic tool and the best activation marker, CD63 and CD203c, for the evaluation of immediate reactions to these drugs.We thank Claudia Corazza for her invaluable English language support; Verónica Prados and Ana Molina for their help in technical support in flow cytometry methods. This work has been supported by Institute of Health “Carlos III” (ISCIII) of the Ministry of Economy and Competitiveness (MINECO; grants co-funded by European Regional Development Fund: PI15/01206, PI17/01237, PI18/00095, PI20/01734, RETICS ARADYAL RD16/0006/0001, and RICORS REI (RD21/0002/0008); Andalusian Regional Ministry of Health (grants PI-0241-2016, PE-0172-2018, and PI-0127-2020); Spanish Ministerio de Ciencia e Innovación Proyectos de I + D + I «Programación Conjunta Internacional», EuroNanoMed 2019 (PCI2019-111825-2)). AA holds a Senior Postdoctoral Contract (RH-0099-2020) with the Andalusian Regional Ministry of Health (cofunded by European Social Fund (ESF): "Andalucía se mueve con Europa". GB holds a “Juan Rodes” contract (JR18/00054) by ISCIII of MINECO (cofounded by ESF). ID holds a clinical research stabilization contract by Andalusian Regional Ministry of Health (RB-0001-2022). ML holds a “Rio Hortega” contract (CM20/00210) by ISCIII of MINECO (cofounded by ESF). CF holds a Marie Skłodowska-Curie Individual Fellowship by the European Union's Horizon 2020 research and innovation program (agreement n° PI-0241-2016101027955). CM holds a “Nicolas Monardes” research contract by Andalusian Regional Ministry of Health (RC-0004-2021). // Funding for open access charge: Universidad de Málaga / CBUA

Latin American study of hereditary breast and ovarian cancer LACAM : a genomic epidemiology approach

Q2Q1Artículo original1-13Purpose: Hereditary Breast and Ovarian Cancer (HBOC) syndrome is responsible for ~5–10% of all diagnosed breast and ovarian cancers. Breast cancer is the most common malignancy and the leading cause of cancer-related mortality among women in Latin America (LA). The main objective of this study was to develop a comprehensive understanding of the genomic epidemiology of HBOC throughout the establishment of The Latin American consortium for HBOC-LACAM, consisting of specialists from 5 countries in LA and the description of the genomic results from the first phase of the study. Methods: We have recruited 403 individuals that fulfilled the criteria for HBOC from 11 health institutions of Argentina, Colombia, Guatemala, Mexico and Peru. A pilot cohort of 222 individuals was analyzed by NGS gene panels. One hundred forty-three genes were selected on the basis of their putative role in susceptibility to different hereditary cancers. Libraries were sequenced in MiSeq (Illumina, Inc.) and PGM (Ion Torrent-Thermo Fisher Scientific) platforms. Results: The overall prevalence of pathogenic variants was 17% (38/222); the distribution spanned 14 genes and varied by country. The highest relative prevalence of pathogenic variants was found in patients from Argentina (25%, 14/57), followed by Mexico (18%, 12/68), Guatemala (16%, 3/19), and Colombia (13%, 10/78). Pathogenic variants were found in BRCA1 (20%) and BRCA2 (29%) genes. Pathogenic variants were found in other 12 genes, including high and moderate risk genes such as MSH2, MSH6, MUTYH, and PALB2. Additional pathogenic variants were found in HBOC unrelated genes such as DCLRE1C, WRN, PDE11A, and PDGFB. Conclusion: In this first phase of the project, we recruited 403 individuals and evaluated the germline genetic alterations in an initial cohort of 222 patients among 4 countries. Our data show for the first time in LA the distribution of pathogenic variants in a broad set of cancer susceptibility genes in HBOC. Even though we used extended gene panels, there was still a high proportion of patients without any detectable pathogenic variant, which emphasizes the larger, unexplored genetic nature of the disease in these populations

Diseño de vectores lentivirales regulados fisiológicamente para terapia génica del síndrome de Wiskott-Aldrich

Incluye resumen y conclusiones en inglésTesis Univ. Granada. Departamento de Bioquímica y Biología Molecular. Leída el 26 de junio de 200