191 research outputs found
CRISPR-ERA for Switching Off (Onco) Genes
Genome-editing nucleases like the popular CRISPR/Cas9 enable the generation of knockout cell lines and null zygotes by inducing site-specific double-stranded breaks (DSBs) within a genome. In most cases, when a DNA template is not present, the DSB is repaired by nonhomologous end joining (NHEJ), resulting in small nucleotide insertions or deletions that can be used to construct knockout alleles. However, for several reasons, these mutations do not produce the desired null result in all cases, instead generating a similar protein with functional activity. This undesirable effect could limit the therapeutic efficiency of gene therapy strategies focused on abrogating oncogene expression by CRISPR/Cas9 and should be taken into account. This chapter reviews the irruption of CRISPR technology for gene silencing and its application in gene therapy
CRISPR/Cas9-generated models uncover therapeutic vulnerabilities of del(11q) CLL cells to dual BCR and PARP inhibition
[EN]The deletion of 11q (del(11q)) invariably comprises ATM gene in chronic lymphocytic leukemia (CLL). Concomitant mutations in this gene in the remaining allele have been identified in 1/3 of CLL cases harboring del(11q), being the biallelic loss of ATM associated with adverse prognosis. Although the introduction of targeted BCR inhibition has significantly favored the outcomes of del(11q) patients, responses of patients harboring ATM functional loss through biallelic inactivation are unexplored, and the development of resistances to targeted therapies have been increasingly reported, urging the need to explore novel therapeutic approaches. Here, we generated isogenic CLL cell lines harboring del(11q) and ATM mutations through CRISPR/Cas9-based gene-editing. With these models, we uncovered a novel therapeutic vulnerability of del(11q)/ATM-mutated cells to dual BCR and PARP inhibition. Ex vivo studies in the presence of stromal stimulation on 38 CLL primary samples confirmed a synergistic action of the combination of olaparib and ibrutinib in del(11q)/ATM-mutated CLL patients. In addition, we showed that ibrutinib produced a homologous recombination repair impairment through RAD51 dysregulation, finding a synergistic link of both drugs in the DNA damage repair pathway. Our data provide a preclinical rationale for the use of this combination in CLL patients with this high-risk cytogenetic abnormality
Splice donor site sgRNAs enhance CRISPR/Cas9-mediated knockout efficiency
[EN]CRISPR/Cas9 allows the generation of knockout cell lines and null zygotes by inducing site-specific double-stranded breaks. In most cases the DSB is repaired by non-homologous end joining, resulting in small nucleotide insertions or deletions that can be used to construct knockout alleles. However, these mutations do not produce the desired null result in all cases, but instead generate a similar, functionally active protein. This effect could limit the therapeutic efficiency of gene therapy strategies based on abrogating oncogene expression, and therefore needs to be considered carefully. If there is an acceptable degree of efficiency of CRISPR/Cas9 delivery to cells, the key step for success lies in the effectiveness of a specific sgRNA at knocking out the oncogene, when only one sgRNA can be used. This study shows that the null effect could be increased with an sgRNA targeting the splice donor site (SDS) of the chosen exon. Following this strategy, the generation of null alleles would be facilitated in two independent ways: the probability of producing a frameshift mutation and the probability of interrupting the canonical mechanism of pre-mRNA splicing. In these contexts, we propose to improve the loss-of-function yield driving the CRISPR system at the SDS of critical exons
Dissecting the role of TP53 alterations in del(11q) chronic lymphocytic leukemia
[EN]Background
Several genetic alterations have been identified as driver events in chronic lymphocytic leukemia (CLL) pathogenesis and oncogenic evolution. Concurrent driver alterations usually coexist within the same tumoral clone, but how the cooperation of multiple genomic abnormalities contributes to disease progression remains poorly understood. Specifically, the biological and clinical consequences of concurrent high-risk alterations such as del(11q)/ATM-mutations and del(17p)/TP53-mutations have not been established.
Methods
We integrated next-generation sequencing (NGS) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 techniques to characterize the in vitro and in vivo effects of concurrent monoallelic or biallelic ATM and/or TP53 alterations in CLL prognosis, clonal evolution, and therapy response.
Results
Targeted sequencing analysis of the co-occurrence of high-risk alterations in 271 CLLs revealed that biallelic inactivation of both ATM and TP53 was mutually exclusive, whereas monoallelic del(11q) and TP53 alterations significantly co-occurred in a subset of CLL patients with a highly adverse clinical outcome. We determined the biological effects of combined del(11q), ATM and/or TP53 mutations in CRISPR/Cas9-edited CLL cell lines. Our results showed that the combination of monoallelic del(11q) and TP53 mutations in CLL cells led to a clonal advantage in vitro and in in vivo clonal competition experiments, whereas CLL cells harboring biallelic ATM and TP53 loss failed to compete in in vivo xenotransplants. Furthermore, we demonstrated that CLL cell lines harboring del(11q) and TP53 mutations show only partial responses to B cell receptor signaling inhibitors, but may potentially benefit from ATR inhibition.
Conclusions
Our work highlights that combined monoallelic del(11q) and TP53 alterations coordinately contribute to clonal advantage and shorter overall survival in CLL
Pharmacogenetics of Immunosuppressants in Solid Organ Transplantation: Time to Implement in the Clinic
Our aim in this chapter is to present the state of the art, including our own group research, in the field of immunosuppressant pharmacogenetics in the four main types of solid organ transplantation: kidney, heart, lung, and liver. The main focus will be on those findings in the field that have been widely investigated and then in those that are close to clinical implementation, mainly CYP3A5 genotyping for the adjustment of the initial tacrolimus dose. This recommendation will be discussed in more detail, explaining its clinical potential as well as its limitations. To end, a short opinion about the feasibility of implementation in the health systems as well as discussion about private companies selling pharmacogenetic tests will be presented
Biochemical, ultrastructural, and reversiblity studies on Huntingtin filaments isolated from mouse and human brain
Huntington's disease (HD) and eight additional inherited neurological disorders are caused by CAG triplet-repeat expansions leading to expanded polyglutamine-sequences in their respective proteins. These triplet-CAG repeat disorders have in common the formation of aberrant intraneuronal proteinaceous inclusions containing the expanded polyglutamine sequences. These aggregates have been postulated to contribute to pathogenesis caused by conformational toxicity, sequestration of other polyglutamine-containing proteins, or by interfering with certain enzymatic activities. Testing these hypotheses has been hampered by the difficulty to isolate these aggregates from brain. Here we report that polyglutamine aggregates can be isolated from the brain of the Tet/HD94 conditional mouse model of HD, by following a method based on high salt buffer homogenization, nonionic detergent extraction, and gradient fractionation. We then verified that the method can be successfully applied to postmortem HD brains. Immunoelectron microscopy, both in human and mouse samples, revealed that the stable component of the inclusions are mutant huntingtin-containing and ubiquitin-containing fibrils. Atomic-force microscopy revealed that these fibrils have a 'beads on a string' morphology. Thus, they resemble the in vitro assembled filaments made of recombinant mutant-huntingtin, as well as the Abeta and alpha-synuclein amyloid protofibrils. Finally, by shutting down transgene expression in the Tet/HD94 conditional mouse model of HD, we were able to demonstrate that these filaments, although stable in vitro, are susceptible to revert in vivo, thus demonstrating that the previously reported reversal of ubiquitin-immunoreactive inclusions does not simply reflect disassembling of the inclusions into their constituent fibrils and suggesting that any associated conformational or protein-sequestration toxicity is also likely to revert
Effect of Applying an Organic Amendment on the Persistence of Tebuconazole and Fluopyram in Vineyard Soils
Human Bone Marrow Stromal Cells Differentiate Into Corneal Tissue and Prevent Ocular Graft-Versus-Host Disease in Mice
Clinical trials have assessed the use of human bone marrow stromal cells (hBMSCs) for the treatment of immune-related disorders such as graft-versus-host disease (GVHD). In the current study, we show that GFP+-transduced hBMSCs generated from bone marrow migrate and differentiate into corneal tissue after subconjunctival injection in mice. Interestingly, these hBMSCs display morphological features of epithelial, stromal, and endothelial cells and appear at different layers and with different morphologies depending on their position within the epithelium. Furthermore, these cells display ultrastructural properties, such as bundles of intermediate filaments, interdigitations, and desmosomes with GFP- cells, which confirms their differentiation into corneal tissues. GFP+-transduced hBMSCs were injected at different time points into the right eye of lethally irradiated mice undergoing bone marrow transplantation, which developed ocular GVHD (oGVHD). Remarkably, hBMSCs massively migrate to corneal tissues after subconjunctival injection. Both macroscopic and histopathological examination showed minimal or no evidence of GVHD in the right eye, while the left eye, where no hBMSCs were injected, displayed features of GVHD. Thus, in the current study, we confirm that hBMSCs may induce their therapeutic effect at least in part by differentiation and regeneration of damaged tissues in the host. Our results provide experimental evidence that hBMSCs represent a potential cellular therapy to attenuate oGVHD
Use of maraviroc in patients with undetectable viral load: efficacy, tolerance and predictors of viral response in MARAVIROC-cohort study
Introduction: No controlled clinical trials had studied the role of maraviroc (MRV) in fully suppressed patients [1].
Methods and Materials: MRV-cohort is an observational, retrospective, multicentric (27 sites) large cohort study of patients
starting MRV in clinical practice under different circumstances, with at least 48 weeks of follow-up. For the present analysis we
selected all those patients starting with an HIV-RNAB50 copies/mL. Demographics, baseline CD4 cell count, past history of
antiretroviral treatment (ART), tropism, reasons for MRV use, MRV based therapy and change/end of MRV use were assessed.
Paired analysis of lipid, hepatic and kidney profile changes and univariate and multivariate analyses of HIV-RNAB50 copies/mL
at 48 weeks were explored.
Results: We included 247 out of 667 subjects from the entire cohort. At study entry, their median age was 47 years, 23% were
women, 31% MSM, 49% had CDC category C, median CD4 counts were 468 cells/mm3
, 46% were HCV and 4.5% AgHBs.
Tropism information was available in 197 (94% R5). Median length of prior ARTV was 10.7 years, with exposure to a median of
three drug families. Main reasons for prescribing MRV were: toxicity 38%, inmunodiscordance 23%, simplification 19% and
admission in a clinical trial 10.4%. MRV based therapies used were MRV2NRTIs 9%, MRVPI 46%, MRVPIother 40% and
MRVother 5%. At 48 weeks, 23% of patients had changed or finished MRV therapy due to toxicity 2.4%, virological failure 2%,
immunological failure 1.2%, simplification 3,2%, trial requirement 9.7%, medical decision 2.8%, treatment suspension 1.2% and
unknown 0.4%. At 48 weeks, no significant changes were observed in lipid, hepatic or kidney profiles, and 85% of patients
remained with HIV-RNAB50 copies/mL. Focusing on viral response univariate and multivariate models did not show any
significant baseline variable explaining viral failure.
Conclusions: In clinical practice MRV was used, mostly in R5 positive patients, with adequate efficacy and tolerance, but
important number of patients changed due to non-clinical reasons. In this scenario neither reason for use of MRV nor MRVbased
therapy explained viral failure
Metodología de la ecoinnovación PDCA, aplicada al prototipo de fachada vegetal aljibe Naturpanel en el Proyecto SOS-Natura
Este artículo expone la metodología de trabajo desarrollada en las investigaciones del Departamento de Construcción y Tecnología Arquitectónicas de la UPM, basada en el concepto de ecoinnovación, y en la normativa AENOR de Ecodiseño (UNE-EN ISO 14006), en normativas relacionadas como la UNE-EN ISO 9001 y 14001, entre otras. Dicha metodología considera los aspectos medioambientales desde las primeras fases de la investigación, para aumentar la ecoeficiencia de las propuestas producidas, de manera que satisfagan las necesidades humanas y proporcionen una mejor calidad de vida, a la vez que reduzcan progresivamente los impactos ambientales y la intensidad de consumo de recursos a lo largo de su ciclo de vida, hasta un nivel, al menos, en línea con la capacidad de asimilación de la Tierra. El objetivo de esta comunicación es explicar y dar a conocer, mediante ejemplos concretos, dicha metodología; los beneficios de la misma y cómo influye su utilización en el diseño arquitectónico de fachadas vegetales
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